Book - Sex and internal secretions (1961) 13

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I have decided to take early retirement in September 2020. During the many years online I have received wonderful feedback from many readers, researchers and students interested in human embryology. I especially thank my research collaborators and contributors to the site. The good news is Embryology will remain online and I will continue my association with UNSW Australia. I look forward to updating and including the many exciting new discoveries in Embryology!
Young WC. Sex and internal secretions. (1961) 3rd Eda. Williams and Wilkins. Baltimore.
Section A Biologic Basis of Sex Cytologic and Genetic Basis of Sex | Role of Hormones in the Differentiation of Sex
Section B The Hypophysis and the Gonadotrophic Hormones in Relation to Reproduction Morphology of the Hypophysis Related to Its Function | Physiology of the Anterior Hypophysis in Relation to Reproduction
The Mammalian Testis | The Accessory Reproductive Glands of Mammals | The Mammalian Ovary | The Mammalian Female Reproductive Cycle and Its Controlling Mechanisms | Action of Estrogen and Progesterone on the Reproductive Tract of Lower Primates | The Mammary Gland and Lactation | Some Problems of the Metabolism and Mechanism of Action of Steroid Sex Hormones | Nutritional Effects on Endocrine Secretions
Section D Biology of Sperm and Ova, Fertilization, Implantation, the Placenta, and Pregnancy Biology of Spermatozoa | Biology of Eggs and Implantation | Histochemistry and Electron Microscopy of the Placenta | Gestation
Section E Physiology of Reproduction in Submammalian Vertebrates Endocrinology of Reproduction in Cold-blooded Vertebrates | Endocrinology of Reproduction in Birds
Section F Hormonal Regulation of Reproductive Behavior The Hormones and Mating Behavior | Gonadal Hormones and Social Behavior in Infrahuman Vertebrates | Gonadal Hormones and Parental Behavior in Birds and Infrahuman Mammals | Sex Hormones and Other Variables in Human Eroticism | The Ontogenesis of Sexual Behavior in Man | Cultural Determinants of Sexual Behavior
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Section D Biology of Sperm and Ova, Fertilization, Implantation, the Placenta, and Pregnancy

Biology Of Spermatozoa

David W. Bishop

David W. Bishop, Ph.D.

Staff Member, Department of Embryology, Carnegie Institution Of Washington. Baltimore. Maryland

I. Introduction

In no way is the present review intended to represent a renovation of the comparable section in the second edition of Sex and Interjial Secretions (Hartman, 1939) . It would be both presumptions and impracticable to attempt to update Professor Hartman's discussion of the physiologic role of spermatozoa in reproduction; this stands as a landmark now two decades old. In his review many problems were noted, some since solved, others still in the course of solution, and many even yet ignored.

The major advances in sperm biology during the intervening years have been world-wide and substantial. Stimulated in large measure by the exigencies of the animal-breeding industry. Lardy and co-workers at Wisconsin developed biochemical methods and concepts pertaining to spermatozoa, particularly those of the bull. Mann and his many able Cambridge colleagues have elucidated major aspects of the metabolic and enzymatic activities of spermatozoa in several domestic species. Significant contributions have appeared from various laboratories, too numerous to designate, from the basic demonstrations, by the Engelhardt school, of the role of adenosine triphosphate (ATP) and adenosinetriphosphatase (ATPase) in the motility of sperm, to the apparently unique metabolic characteristics of human spermatozoa reported principally by MacLeod.

A second major stride in the study of the male gamete has been provided by the development of the electron microscope. By virtue of the increase in magnification, up to 1000-fold, cells can be visually dissected down to elements on the order of 10 A in size. Not the least of its accomplishments, the electron microscope has made possible the demonstration that all sperm flagella and all cilia throughout the plant and animal kingdoms possess the same basic pattern of longitudinal filaments, the well known 2x9 + 2 array. The full significance of this structural constancy is yet to be realized, but inasmuch as these filaments are generally assumed to represent the motile organelles of the cell, the physicochemical basis for motility may eventually be resolved. Likewise, the electron microscope has facilitated the study of spermatozoa during their maturation and in the initial stages of fertilization. Significant alterations in the acrosome, for example, seem to be related to the processes involved in the union of gametes.

The sperm has, in fact, been more closely scrutinized and is now recognized as something more than a uniform, finished product of the spermatogenic process. Mammalian spermatozoa from the same gonad may well differ with respect to phenotypic and antigenic characteristics. They are, moreover, far from functionally mature as they leave the testis; structural and biochemical changes occur during their sojourn and transport through both the male and female genital tracts such that their capacity for fertilization is enhanced with time and migration. Investigation of these processes constitutes a very active area of research in current studies of reproductive physiology.

Another important advance in recent years, which may here be singled out for comment, concerns the mechanism of transport of spermatozoa through the female genital tract. One of the earliest features of mammalian reproduction to be studied, only recently has the full weight of experimental attack demonstrated the important endocrinologic role involved in the process.

These, among other, developments in sperm biology are considered in some detail in the pages that follow. No attempt is made to survey completely the available literature, which is enormous; rather, what seem to be significant current principles and processes are discussed within the scope and space allotments of the present volume. The general characteristics of whole semen and its jiroduction, reviewed elsewhere (see Mann, 1954; chapters by Albert and by Price and Williams-Ashman) , are necessarily slighted in favor of a fuller discussion of the internal environment of the male and female genital tracts and the probable conditions surrounding the sperm in vivo. Fortunately, a number of recent reviews cover many of the principal, broad points noted above and serve as background for the material reported here (MacLeod, 1943b; Ivanov, 1945; Mann, 1949, 1954; Austin and Bishop, 1957; Colwin and Colwin, 1957; Fawcett, 1958; Mann and Lutwak-Mann, 1958; Bishop, 1961; Tyler and Bishop. 1961).

The function of the male gamete is to serve as activator of the ovum and contributor of paternal hereditary components to the zygote. The sperm thus stimulates an otherwise relatively inert egg and initiates a new course of development. In Weissmannian terms, it represents a continuity, of part at least, of the germ plasm from one generation to the next. In a very real phylogenetic sense, the gamete is one haploid generation momentarily sandwiched between two extended diploid generations.

Sperm, unlike most cells, are designed to function outside of their native environment. Where fertilization is external, spermatozoa may be shed into an aqueous medium of different ionic strength which offers little shelter, scant buffering capacity, toxic ions, and a lack of energy substrate essential for extended metabolic activity. In the case of internal fertilization, on the other hand, these conditions are generally obviated, and the seminal plasma, the vehicle for transport, affords additional security features beneficial to sperm survival. However, the introduction of sperm into the female animal places them, even here, in foreign surroundings which, although natural, may not always prove hospitable. There is evidence to indicate, for example, that the normal protective and immune responses of the female against foreign invasion reach even to the oviduct and uterus and to their luminal secretions.

The motility of typical spermatozoa is certainly their most striking characteristic. Indeed, the degree of motility is frequently equated to fertilizing capacity and survival. The sperm of many nonmammalian species, however, may appear quite immotile, although they are fully capable of fertilizing normal eggs. The sperm of the herring {Clupea) , for example, are immotile as shed and remain so until brought into the vicinity of homologous eggs (Yanagimachi, 1957). The giant sperm of the hemipteran insect, Xotonecta glauca, show no movement until activated by fluids from the female genital system (Pantel and de Sinety, 1906). The sperm of many invertebrate animals, moreover, are nonflagellated, a specialization particularly common among decapod Crustacea. Amoeboid spermatozoa are found among ascarids, and in the sponge, Grantia, it is claimed that the sperm lose their flagella and are engulfed by modified collar cells which transform into amoeboid forms and transport the parasite-like sperm to the oocytes (Gatenby, 1920). It thus appears that, whereas nature has endowed the male gametes with fiagella from the lowest protistan to the highest mammal, she has developed secondary modifications toward less specialized conditions among numerous organisms between the evolutionary extremes.

II. Functional State of Gametes after Spermatogenesis

The gross structure and organization of spermatozoa are generally considered complete when the gametes leave the testis. The statement is frequently seen that spermatozoa, as found in the male efferent ducts, are ready for fertilization, unlike the egg cells which often, and in all mammals, are ovulated in a cytogenetically incomplete state of development, later to be activated by the sperm. In a general way, this contrast in the functional activity of the gametes is realized, and the most cogent evidence in behalf of the fertilizing capacity (although less than normal) of testicular sperm is the record of conceptions resulting from insemination by sperm removed from the gonads of chickens and men (Munro, 1938; Adler and Makris, 1951). Normally, however, the process of sperm maturescence is not complete until some time after sperm formation, and fertilizing capacity is fully realized only after a period of sojourn in the male and female genital tracts (Redenz, 1926; Young, 1929a, 1931; Munro, 1938; Bishop, 1955).

A. The Maturation of Spermatozoa

A number of structural and physical changes, here only briefly noted, occur in spermatozoa during transit through the ducts. Morphologically, the most obvious modification is the loss of the "kinoplasmic droplet," a cytoplasmic residuum of dubious function characteristic of immature cells and only rarely found in sperm of a normal ejaculate (Merton, 1939a; Gresson and Zlotnik, 1945; Mukherjee and Bhattacharya, 1949). Less obvious changes are a concomitant decrease in free-water content and an increase in specific gravity of sperm as they mature, as in the bull (Lindahl and Kihlstrom, 1952). Salisbury (1956) found evidence to indicate that changes occur in permeability to water and in intracellular concentrations of monovalent cations (Na+ and K+), modifications which might account for the increase in capacity for movement of sperm at this stage in their developmental history. Unpublished observations of electron micrographs of human sperm by Fawcett indicate that the midpiece may undergo further significant alterations after spermatogenesis, changes which involve particularly the mitochondrial sheath and the annulus at the junction of the midpiece and principal piece of the flagellum.

In the female genital tract, further sperm modifications occur which appear to be necessary for fertilization. A period of incubation in the tubal fluids is required during which changes (capacitation) occur that seem to involve both enzymatic and structural properties of the sperm (Austin and Bishop, 1958a; Chang, 1958; Noyes, 1959b t. During this 2- to 6-hour interval, the sperm, at least of the rat, hamster, and rabbit, undergo certain changes in the head, which include loss of the "galea capitis" and partial dissolution of the acrosome.

What other changes occur in spermatozoa, in vivo, during their transport through the genital tracts, and of what consequence such modifications are to either survival or fertilization can only be surmised. In some respects, the metabolic properties of epididymal and seminal sperm differ, as studied in vitro (see Metabolism). Pronounced changes, of course, follow activation at the time of ejaculation, changes associated with energy production and motility. Other biochemical activities are believed to occur, moreover, which may be regarded as part of the "resting" metabolism of sperm. This will be discussed in a later section. On the other hand, certain deleterious changes may also take place, particularly during sperm storage, to such an extent that large molecular moieties, such as cytochrome c, are apparently lost from both bull and ram spermatozoa (Mann, 1954).

B. Cytogenetic Differences In Sperm

As the result of meiosis and segregation, spermatozoa are haploid in chromosome number and bear one-half of the hereditary complement which is carried into the next generation at fertilization. The two main types of sperm, X- and Y-bearing (in mammals), are responsible for female and male offspring, respectively, on union with the X-chromosome egg. Attempts have indeed been made, with questionable success, to separate these two kinds of sperm both by electrophoretic (Schroder, 194:0a, b, 1941a, b, 1944; Gordon, 1957) and by countercurrent centrifugal methods (Lindahl, 1956).

Genetically distinct spermatozoa were long ago demonstrated by Landsteiner and Levine (1926), who showed that the A and B blood-group antigens occur in human sperm, without, however, making clear whether the specific phenotype of a given sperm is determined by its haploid set of genes or by the diploid set of the spermatocyte from which it is derived. GuUbring (1957) has recently revived this issue and claims that the A and B antigens occur on separate sperm produced by a heterozygous AB blood-group male. Further evidence of gene-induced sperm heterogeneity is afforded by the work of Beatty (1956), who studied the 3,4-dihydroxyphenylalanine (DOPA) reaction in sperm from pigmented and pale rabbits. A high correlation was found between the melanizing activity of the spermatozoa and the depth of coat color of the rabbits from which they came. It will be remembered that Snell (1944) found significant antigenic differences in the sperm of inbred strains of mice, those of strain C being readily distinguishable from those of strain C57 on a basis of their agglutination with specific antisera. Braden (1956, 1958a, 1959) has recently made an intensive study of sperm variation in pure strains of mice. Statistically significant differences in size and shape of the sperm head were demonstrated in the four inbred lines, CBA, C57BL, A, and RIII. IVIoreover, at fertilization, strain differences become apparent in the tendency for more than one sperm to penetrate the egg membranes, sperm of strain C57BL, for example, showing a significantly higher percentage (26 per cent) than those of other strains (12 to 14 per cent). Further, Braden (1958b) has found abnormalities in the segregation ratio of mice which tend to indicate that the actual allele present (e.g., at the T-locus) in the sperm determines certain of its properties including its relative fertilizing capacity. The precise nature of the impairment is unknown but seems to involve the ability of the sperm to traverse the uterotubal junction (Braden and Gluecksohn-Waelsch, 1958). This is the same gene which Bryson (1944) found to affect both sperm morphology and motility in the heterozygous (^V^^) male. These results are yet fragmentary but are strongly indicative of the fact that spermatozoa reflect their haploid genotype and that when they bear an unfavorable allelic constitution they may display a decreased fertilizing potential. The possibility exists that subviable mutants, recessive in the heterozygous condition, might have profound detrimental effects when segregated into particular gametes.

C. Requirements for Large Sperm Numbers

Any suggestion at the present time to justify the large number, or excess," of sperm ordinarily involved in insemination is at best a hazardous supposition. The earlier speculations which presupposed a sperm swarm" to supply hyaluronidase for the dissipation of cumulus cells (McClean and Rowlands, 1942) are inconsonant with the facts, since only a very few sperm, on the order of 25 to 250, are to be found in the presence of fertilized eggs of rats, rabbits, and ferrets, for example, while the cumulus cells are still clustered about the eggs (Braden and Austin, 1953; Chang, 1959). That many more sperm are produced and inseminated than are necessary for fertilization is not be to denied. A phenomenon implicating survival of the species can be expected to have built-in safety factors, and sperm production is no exception, particularly if the male is to be capable of frequent ejaculation. Very probably, the pattern for high gametic production was set long ago among animals which reproduced by means of external fertilization where sperm, egg, and larval loss are very high. In fact, obstacles to successful fertilization are present in mammals as well; definite blocks to sperm transport, for example, occur at the cervix, uterotubal junction, and tubal isthmus in many animals. But it is to be emphasized, in the light of evidence cited in


the two preceding sections, that the waste of healthy spermatozoa may be less than previously conjectured. The exigencies of the complex series of cellular and functional changes which ensue during the passage of sperm through the genital tracts and the possibility of genetic variation with consequent differences in fertilizing capacity suggest that the number of physiologically effective sperm in the ejaculate may be but a fraction of the total inseminated.

III. Sperm Transport and Storage in the Male Tract

Aside from the accessory reproductive glands that supply, in large measure, the constituents of the seminal plasma (see chapter by Price and Williams-Ashman), the male genital tract of vertebrates is essentially a collection and transport system, designed to convey the spermatozoa from the testis to the ejaculatory duct (Fig. 6.1). It does more than this, however, in that the gametes, on the one hand, are altered in their capacity for fertilization and, on the other hand, are stored, motionless, often for long periods of time, preparatory to ejaculation. The intrinsic changes within the maturing sperm and the interrelations between the gametes and the various segments of the male duct system are only just beginning to be appreciated. The cytologic integrity and the functional activity of the male reproductive ducts are directly influenced by the androgen output of the testicular interstitium and presumably vary in their influence on the spermatozoa within the tract. Spermiation, the release and shedding of spermatozoa from the testes of amphibians, is, of course, hormonally induced (Van Oordt, Van Oordt and Van Dongen, 1959; Witschi and Chang, 1959) ; the mechanism of release is discussed elsewhere in this volume (chapter by Greep) . In the cock, which has no glands analogous to the seminal vesicles or prostate, "seminal" fluids are contributed by the seminiferous tubules and vasa efferentia (Lake, 1957).

A. Sperm Transport

It can be stated with reasonable assurance that sperm migration within the male tract is, from the sperm's viewpoint, in the main a passive process (Simeone, 1933). The mechanism by which they are moved along the duct system, however, is not well understood; the mechanics may well vary in different segments. Certain workers have emphasized the currents of fluid which could sweep the sperm out of the seminiferous tubules and into the efferent ducts and epididymis (see Young, 1933; Macmillan, 1953). Resorption of fluid by the efferent ducts (Young, 1933; Ladman and Young,

1958) or cpididymal epithelium (Mason and Shaver, 1952; Cleland, Jones and Reid,

1959) would complete the fluid circuit and simultaneously concentrate the sperm mass in the distal reaches of the duct system. Certain ligation experiments in which the male ducts were occluded at various levels tend to support this concept of transport by fluid currents, and circumstantial evidence is further afforded by the presence of motile cilia in the upper segment of the genital tract. On the other hand, other experiments which involved separation of the testis from the efferent ducts, thereby cutting off the supply of fluid, demonstrate unequivocally that the sperm, under these circumstances, are carried distally by some other means of tubal transport (Young, 1933; Macmillan and Harrison, 1955).


More recently acquired evidence indicates that muscular activity may play the predominant role in sperm transport through the male ducts. Roosen-Runge (1951) has observed movement in the seminiferous tubules of the dog and rat, both in the intact testis and in vitro in physiologic saline at 36° C. The undulating motion was attributed, by Roosen-Runge, to the contraction and relaxation of the Sertoli cells within the tubules. A more plausible explanation may rest in Clermont's recent (1958) electron micrographic demonstration of fibrous elements which lie in the wall of the seminiferous tubule of the rat and seem to resemble smooth muscle cells.

The ductuli efferentes of the adult rat can be cultured successfully in roller-tube tissue-culture preparations (Battaglia, 1958). Tubules maintained as long as 12 days show spontaneous movement, presumably due to muscular contractions. This activity could provide a mechanism whereby spermatozoa are carried along these ducts, in vivo.

Migration of spermatozoa through the epididymis proper is mainly, although perhaps not exclusively, brought about by spontaneous peristaltic and segmental movements of the duct. Such activity was first clearly shown in the guinea pig by Simeone (1933) and in the rat by Muratori (1953) and has been confirmed and recorded cinematographically by Risley (1958, 1960). Rhythmic contractions sweep along the adult tubule at regular intervals of 7 or 8 seconds. After gonadectomy, contractions continue in the mature duct for two more weeks. Hypophysectomy results in the loss of activity within 10 days in the head, and within 13 days in the body and tail of the epididymis. In tissue-culture preparations, the spontaneous movement of the epididymis also continues for some time (12 days), the activity being the same whether the ducts are excised from normal or from gonadectomized rats (Battaglia, 1958). It is of some historic interest to note that Moore and Quick, as early as 1924, suggested a neuromuscular mechanism for epididymal sperm transport as a result of their studies on vasectomized rabbits; at the same time they refuted the then hotly contested claims of Steinach and others that vasectomy reults in seminiferous atrophy and interstitial hypertrophy. Complete occlusion of the rat vasa eff'erentia, on the other hand, is claimed to lead invariably to si^crmatogenic destruction (Harrison, 1953).

Transport through the epididymis requires 2 to 4 days in the fowl, 4 to 7 days in the rabbit, 9 to 14 in the ram, 14 to 18 in the guinea pig, 8 in the mouse, about 15 in the rat, and 19 to 23 days in man (Toothill and Young, 1931; Munro, 1938; Edwards, 1939; Brown, 1943; MacMillan and Harrison, 1955; Asdell, 1946; Dawson, 1958; Oakberg and DiMinno, 1960). The guinea pig determinations of Toothill and Young (1931 ) made use of the migration of India ink particles, injected into the head of the epididymis, and not of sperm transport per se. The apparently rapid rate of migration of sperm in the fowl may be attributed to the relatively short length of the epididymis (Munro, 1938). Isolation of the testis from the epididymis of the guinea pig increases transport time by 1 to 2 weeks, possibly as a result of interruption of flow of fluid through the excurrent ducts, or perhaps as a consequence of operational disturbances which involve changes in the local vascularization and nerve supply.

The vas deferens serves mainly for the accumulation and storage of sperm, but what sperm migration does occur seems primarily dependent upon the muscular activity of the duct. The vasa of the rat and dog are normally quiescent in sexually inactive males, but are capable under experimental conditions, in vitro, of a high degree of muscular activity (Martins and do Valle, 1938; Valle and Porto, 1947). Belonoschkin (1942) claimed that peristaltic activity of the vas deferens aids in sperm transport in man.


B. Sperm Survival

Spermatozoa may reside in the genital tract for considerable periods of time before being discharged at ejaculation. They generally lose their capacity for fertilization before their capacity for motility during storage in the ducts. Survival times vary from several weeks to many months in different species. Bats normally store spermatozoa over the winter months, and this may be typical of certain other hibernating mammals as well. Knaus (1933) claimed that epididymal spermatozoa remain viable and fertile for a year in vasectomized rabl)its, but the process of sperm renewal was not eliminated in his experiments. Mouse spermatozoa in the excurrent ducts maintain their capacity for fertilization for 10 to 14 days after spermatogenesis has been inhibited by x-ray irradiation (Snell, 1933). Rat epididymal spermatozoa, in animals with ligated vasa, remain capable of motility for about 6 weeks, but lose their ability to fertilize eggs within 3 weeks (White, 1933b); castration further reduces sperm survival to approximately 2 weeks (Moore, 1928). Likewise, in the guinea pig, after ligation of the efferent ducts, epididymal spermatozoa retain their capacity for motility some 60 days and for fertility 20 to 35 days; castration reduces motility to about 3 weeks (Moore, 1928; Young, 1929b). Translocation of the epididymis to the abdominal cavity further limits sperm survival to about 2 weeks. When the rabbit epididymis is anchored in the abdomen, sperm motility and fertility are reduced from about 60 and 38 days in the controls to 14 and 8 days, respectively. Demonstrations such as these seem to indicate that body temperature may have a pronounced effect even on relatively mature spermatozoa (Knaus, 1958) ; however, the translocation procedure may primarily affect the epididymis, which in turn alters the longevity of the spermatozoa. In at least one type of natural experiment we have evidence that excessive body temperature is seasonally avoided by the gametes. In certain passerine birds during the active breeding season a transient thermal adaptation provides lower temperatures for the storage of morphologically mature spermatozoa (Wolfson, 1954). The sperm-engorged, distal ends of the vasa deferentia here increase prominently in size and become tortuously coiled, so as to result in a cloacal, scrotum-like swelling, the internal temperature of which is about 4°C. less than body temperature.

When the testes have been separated from the epididymides and time allowed for recovery, the potential sperm capacity of the duct system can be determined. Young (1929b) found that guinea pigs, prepared in this manner, can copulate successfully as many as 20 times over a 2-month period. The relative storage facilities of the major segments of the ducts can be determined by actual sperm count. Chang (1945) diligently counted the sperm in the vasa and epididymides of several ram genital tracts and found the greatest accumulation in the tail of the epididymis (Table 13.1). By frequent ejaculation of the ram, approximately twice a day, he further was able to estimate the average rate of sperm production to be about 4.4 X 10^ cells per day. In the bull the rate is less, about 2.0 X 10^ sperm daily (Boyd and VanDemark, 1957) ; most of the epididymal sperm storage here is also in the tail (45 per cent) compared with that in the head (36 per cent) (Bialy and Smith, 1958).

TABLE 13.1

Distribution of spermatozoa in male genital tract of ram (From M. C. Chang, J. Agric. Sc, 35, 243-246, 1945.)

Segment


Sperm Count

(X 109)


Percentage of Total


p]pididymis

i. Caput

Corpus


17.3

8.4 104.3

1.5 0.3


13.1

6.4 79.1


Efferent duct

Vas deferens

Ampulla ....


1.1 0.2




C. The Functional Microanatomy of the Epididymis

The epididymis has received considerable attention from microscopists bent on the elucidation of the role this part of the duct system plays in the reproductive physiology of the male. A number of recent papers have contributed to our understanding of the segmental organization of the epididymis, the cytochemistry of the mucosa, and the response of the duct to steroid influences. For many histochemical details, and historical surveys of much of the earlier literature, the following papers should be consulted: Reid and Cleland (1957), Cavazos (1958), Maneely (1958, 1959), and Reid (1958, 1959) concerning the rat; Ladman and Young (1958) on the guinea pig; Nicander (1957) for the rabbit; and Nicander (1958) concerning the stallion, ram, and bull. Although exquisite in detail and extensive in scope, these papers, with few exceptions, have added little to the earlier contributions concerning the function of the epididymis vis-a-vis the physiology of the spermatozoa within the lumen (c/. Young, 1933; Mason and Shaver, 1952). With the cytochemical background now available, however, and the current interest in epididymal physiology, the expectations to be derived from a more functional approach should now be fulfilled.

Emphasis has again been placed on the epididymal mucosa, and particularly on the vacuolar and endoplasmic reticular system as a site for the reabsorption of fluid (Nicander, 1957; Reid and Cleland, 1957; Ladman and Young, 1958) , in contrast to its function as a secretory organ (Hammar, 1897; Henry, 1900; Benoit, 1926; Maneely, 1954; Goglia and Magh, 1957). The old question as to the cause of increasing sperm density has apparently been resolved recently by ligation experiments in the rat (Cleland, Jones and Reid, 1959) ; a specialized region of the epididymis absorbs fluid from the lumen at the point where sperm concentration suddenly increases. Virtually nothing is known about the transport of substances, other than water and possibly inorganic ions, across the mucosal boundary, despite the elaborate cytochemical reports, which include data for acid and alkaline phosphatase activitv (Bern, 1949a, b, 1951; Wislocki, 1949; Maneely, 1955, 1958; Montagna, 1955; Allen and Slater, 1957, 1958; Cavazos, 1958; Allen and Hunter, 1960), metachromatic substances (Cavazos, 1958), glycogen (Leblond, 1950; Montagna, 1955; Nicander, 1957, 1958; Cavazos, 1958; Maneely, 1958), lipids (Christie, 1955; ]\Iontagna, 1955; Cavazos and Melampy, 1956; Nicander, 1957, 1958), glycoprotein (Cavazos, 1958) , and nucleic acids (Nicander, 1957, 1958; Cavazos, 1958). It would be of interest to know how these cytochemical characteristics vary, if indeed they do, with sexual activity, on the one hand, and, on the other hand, with certain functional processes, such as the reabsorption of fluid from the duct, the possible transfer of tagged molecules across the limiting membrane, the elaboration and secretion of, for example, glycerylphosphorylcholine present in the epididymis (Dawson and Rowlands, 1959) , and the uptake of large molecular moieties into the mucosa from the lumen, as demonstrated with trypan blue, pyrrhol blue, fuchsin, and India ink particles (von Mollendorf, 1920; Young, 1933; Mason and Shaver, 1952; Shaver, 1954).

Nicander's studies have the added merit that cytochemical demonstrations are correlated with regional differentiation of the epididymis; the duct is divided into 6 to 8 cytologically distinct segments. Such division includes the efferent ducts as part of the epididymis, whether they appear to be nested within a depression of the testis, as in the guinea pig, or quite external to it as in the stallion, ram, bull, and rabbit. All told, the epididymis is an imposing duct, a single continuous tube about 10 feet long in the guinea pig and up to 280 feet in the stallion (Ghetie, 1939; Maneely, 1959).

An impressive series of contributions pertaining to the regional differentiation and histology of the rat epididymis has been published by Reid (1958, 1959) and Reid and Cleland (1957), of the University of Sydney. They divide the rat epididymis into six discrete zones, plus the rete and efferent ducts, on the basis of cell type. The efferent ducts and zones 1 to 4 constitute the head, part of zone 4, the isthmus, and zones 5 and 6, the tail of the epididymis (Fig. 13.1). The relative lengths and diameters of the successive zones and the cellular types are represented in Figure 13.2. Six major cell types are discernible: principal, basal, ciliated, apical, halo, and clear cells (Fig. 13.3). Ciliated cells are confined to the efferent ducts — "the most beautiful ciliated cells of the vertebrate body" <von Lenhossek, 1898). Much of the remainder of the epididymis is lined with prominent, nonmotile stereocilia (Reid and Cleland, 1957). Fluid resorption from the lumen is pronounced in zone 4 (Cleland, Jones and Reid, 1959). The principal features of epididymal histogenesis in the rat are summarized in Table 13.2. During the first three weeks of postnatal development, the epididymis remains in an vmdifferentiated state. At about four weeks, differentiation is first noted in the head of the epididymis and is completed by day 37. Differentiation of the tail begins later than that of the head and is completed only at 14 weeks of age. Sperm are first found in the testis at 8 weeks and appear in the epididymis 2 weeks later.

The epididymis, like the accessory reproductive glands, responds to changes in circulating androgen, and its normal histologic integrity also is apparently dependent on the male hormone (Maneely, 1959) . Castration is followed by reduction in tubal diameter and loss of specific cellular components (Cavazos, 1958; Maneely, 1958). Acid and alkaline phosphatase activities of the mouse epididymis decrease after gonadectomy (Allen and Slater, 1957, 1958). Intracellular polysaccharides, visualized by the periodic acid-Schiff (PAS) reaction, are also reduced in the rat by castration (Maneely, 1958). All such responses can be corrected entirely or in part by the administration of testosterone propionate in adequate doses (Cavazos, 1958, and others). Maraud and Stoll ( 1958) have presented evidence to show, in the chicken at least, that epididymal morphogenesis from the undifferentiated Wolffian duct is dependent on a factor, presumably androgen, elaborated by the primitive testis; in the absence of the gonad, the epididymis remains in an undifferentiated state. The rat epididymis also seems to depend on testicular androgen for its early neonatal differentiation (Cieslak, 1944). Posterior hypothalamectomy of the male guinea pig is followed by extensive degeneration of the reproductive organs, including the epididymis (Soulairac and Soulairac, 1959) ; subsequent administration of chorionic gonadotrophin (25 I.U.) and testosterone propionate (2.5 mg.) results in only a slight recovery of the epididymal epithelium at sacrifice 8 days after injection.



Fig. 13.1. Right testis and epididymis of rat viewed from (A) lateral and (B) medial aspects; cranial end uppermost. Small circles, efferent ducts; crosses, coni vasculosi ; coarse dots, zone 1 ; oblique hatching, zone 2; fine horizontal hatching, zone 3; coarse horizontal hatching, zone 4 ; fine dots, zones 5 and 6A; unmarked, zone 6B and deferent duct. (After B. L. Reid and K. W. Cleland, Australian J. Zool., 5, 223-246, 1957.)



Fig. 13.2. Diagrammatic representation of rat epididymis, (a) Scaled diagram of efferent and epididymal ducts in longitudinal section ; luminal diameter scaling one-half that of epithelial height, (b) Cross-sections of ducts; epithelial height and luminal diameter drawn to the same scale; sperm arrangement indicated, (c) Relative sperm density, id) General representation of epithelium, (e) Specific types of cells: a, ciliated cells; b, apical cells; c, basal cells; d, halo cells: e, clear cells. (After B. L. Reid and K. W. Cleland, Australian J. Zool., 5,223-246,1957.)


Seasonal changes in the epididymis have been demonstrated by Wislocki (1949) and others and can be interpreted as representing periodic fluctuations in androgen output.


D. The Epididymis in Relation to Sperm Physiology

Until more adequate and precise information is forthcoming concerning the physiology of the epididymis, its relation to the changes undergone by the spermatozoa within the tract can only be surmised. Little enough, indeed, is known about the chemical composition of the fluids surrounding the sperm in the rete and efferent ducts, and virtually nothing is known concerning the contributions made by the epididymis as a whole to sperm welfare. Fluid is most likely resorbed from the proximal and intermediate portions of the duct, and some secretion may be contributed in exchange (Macmillan and Harrison, 1953; Macmillan, 1953). The nature of this secretion is obscure. No mechanism has been suggested to account for the significant quantity of glycerylphosphorylcholine present in the epididymis and epididymal fluid of various mammals (boar, bull, rats, and guinea pigs) , although the concentration of this component is known to be androgen-dependent (Dawson, Mann and White, 1957; Dawson and Rowlands, 1959), and is presumably secreted in the proximal portion of the duct. It is unlikely that glycerylphosphorylcholine or its degradation products serve the sperm in a metabolic capacity, but its presence suggests a possible function in the further maturation of the gametes (Dawson and Rowlands, 1959). No metabolic substrates, e.g., glucose or fructose, have been detected in the fluids of the epididymis. A PAS-positive glyco]iroteinaceous secretory product has been demonstrated in the lumen of the rat epididymis (Maneely, 1958), but its function and relation to sperm transport and survival are obscure. The most complete analyses of inorganic ions in the fluids of the male tract are those by Cragle, Salisbury and Muntz (1958) for the testicular and ampullar fluids of the bull. The values are compared with those of seminal plasma and seminal vesicular fluid in Table 13.3.


Fig. 13.3. Representative sections of rat epididymis, approx. 750 X- (D Zone lA; basal, principal, and apical cells visible. (£) Zone 4B; principal cells with perinuclear vacuoles. (3) Zone 3; clear cytoplasm in supranuclear areas. (4) Zone 4A (late); prominent vacuoles m supranuclear areas. (5) Zone IC: basal, principal, apical, and halo cells. (6') Zone 5A ; low columnar principal cells. (7) Zone 5A ; as in (6'); note clear cell at C. iS) Zone 4A (early); strings of small vacuoles in supranuclear regions. (From B. L. Kcid and K. \^ . Cleland, Australian J. Zool., 5, 223-246, 1957.)



TABLE 13.2

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720


SPERM, OVA, AND PREGNANCY


TABLE 13.3

Mineral concentrations in male reproductive fluids

(From R. G. Cragle, G. W. Salisbury and J. H.

Muntz, J. Dairy Sc, 41, 1273-1277, 1958.)



Testicular


Ampullar Fluid


Seminal Ve

Seminal


Ele

Fluid


sicular Fluid


Plasma


ment


(average


(average of


(average of


(average of



of 12 samples)


3 samples)


10 samples)


10 samples)



mg. per


mg. per


mg. per


mg. per



100 ml.


100 ml.


100 ml.


100 ml.


B


0.80


0.59


0.73


1.48


P


229.00


328.00


10.00


55.00


Mg


14.90


8.50


17.50


11.60


Ca


4.60


32.00


70.00


51.00


Fe


2.59


1.08


0.38


0.35


Cu


1.36


2.10


0.95


1.36


Na


178.00


137.00


251.00


273.00



One type of epididymal reaction which may be of considerable importance, although the mechanism of the process is little understood, is that concerning ionic exchange, alluded to above. Salisbury and Cragle (1956) showed that shifts in the sodium-potassium ratio occur in the luminal contents of the goat and bull when sampled at different levels of the tract. The combined "semen" (sperm and fluid) tends to show a relative increase in sodium ion and an increase in K+ + Na+ when comparisons are made of tubal contents from successively lower regions of the tract. Freezing-point determinations indicated that the fluid is initially hypertonic (— 0.600°C.), with respect to blood, and decreases in tonicity with passage through the tube. Determinations of epididymal plasma and seminal plasma of ejaculated bull semen tended to confirm these results with respect to increase in Na+ and the combined K+ -lNa+ values (S0renson and Andersen, 1956).

In a general way, the capacities for motility and fertility seem to be acquired about the same time, but in neither case is this brought about by a sudden change. The capacity for fertilization increases as the gametes are taken from more distal regions of the tract. In the fowl, for example, in


semination with sperm from the testis, epididymis, and vas deferens, respectively, gave 1.6, 18.8, and 65.3 per cent fertile eggs (Munro, 1938). Similarly, in the guinea pig, sperm removed from the proximal and distal portions of the epididymis and used in artificial insemination resulted in 33.4 and 68.0 per cent pregnancies (Young, 1931). After ligation of the vasa deferentia and aging of the sperm, the percentage of fertility from proximal and distal sperm shifted to 44.2 and 32.5 per cent, respectively, for 20-day postligation sperm, and to 49.0 and 25.0 per cent for 30-day stored sperm. It seems clear that, with storage, the maturation of the sperm is followed by a process of senescence. This was further suggested by Young's experiments, since the percentage of aborted and resorbed fetuses increased apparently when fertilization was accomplished by aged spermatozoa.

Whether or not the relative fertility rates of spermatozoa from different levels of the male genital tract can be explained entirely on the assumption that motility and fertilizing capacity go hand in hand remains to be seen, since other aspects of sperm behavior also change with transit through the ducts. Young (1929c) pointed out, for example, that the heat resistance (to 46°C.) of guinea pig, rat, and ram sperm decreases as they migrate through the tract, and Lasley and Bogart ( 1944) showed that the resistance of boar sperm to "cold shock" is likewise reduced.

E. THE FATE OF NONUTILIZED SPERM IN THE MALE

In the absence of ejaculation, the question arises as to the fate of the millions of gametes which are continuously generated during spermatogenesis. It hacl been previously assumed that sperm elimination is by "insensible ejaculation"; sperm have been detected in the urinary outfiow (Wilhelm and Seligmann, 1937). It was shown, however, by Young and Simeone (1930; Simeone and Young, 1931), and since confirmed by others, that the sperm of the guinea pig, for example, undergo degeneration and dissolution within the epididymis. The disposal of the degradation products of the sperm, on the other hand, is not clear from these experiments. They could very possibly be


BIOLOCiY OF SPERMATOZOA


721


voided tliroii^li the vas deferens or he ab.sorl)ed by the duct iniicosa and i)hagocyto.scd, as suggested l)y Mason and Sliaver (1952) and Montagna (1955). The possibihty of absorption of si)enn and of sperm products by the epithelium poses a significant problem relating to self -immunization whicli is discussed in a later section.

F. ACQUISITION BY SPERM OF THE CAPACITY FOR MOTILITY

By the time spermatozoa are primed for union with the eggs they must be sufficiently activated to undergo independent movement, since motility, with rare exceptions, is a prerequisite for fertilization. Sperm activation is delayed in many species until the gametes are in intimate association. Among l)oth invertebrate and vertebrate animals, instances are known in which sperm are shed in a nonmotile condition and are activated only when passively brought into association with homologous eggs as noted above. Frequently the gametes are transferred in large bundles or packets, enclosed in spermatophores, and l)ecome motile only after the casings are rui)tured when in contact with the female I Drew, 1919). Generally, however, the gametes are stimulated when shed externally or ejaculated into the female genital tract. This event corresponds to a spectacular moment in the metabolic life of the cell when tlie exergonic processes are shifted into high gear by the abrupt supply of oxygen, substrate, or cofactors, in mammals copiously provided by the secretions of the accessory reproductive glands.

Before ejaculation, and for much of the time during their storage in the ducts, sperm are quite capable of motility but, so far as can be determined, remain, in vivo, in a (|uiescent state (Simeone, 1933). The reproductive advantage of this is obvious since, before activation, sperm survival is estimated in terms of months; after activity has been acquired, survival is a matter of days or hours (sec Table 13.8). The blocks both to the excessive utilization of energy and to motility, in vivo, are regarded as largely of a pliysical nature — the relative or absolute absence of oxygen which otherwise would encourage aerobic respiration, and the lack of glycolytic substrate, such as glucose or


fi'uctose, which when present fosters anaerobic processes (Mann, 1954; Walton, 1956). Infrequent reports of transient motility by sperm immediately after removal from the genital tract, thereby implying that the cells are motile in vivo (White, Larsen and Wales, 1959), must be confirmed and may be attributable to the admission of oxygen during the sam]>ling procedure. Earlier supi^ositions that sperm immobilization within the ducts is due to high CO2 concentration or low pH level (Redenz, 1926) have been contraindicated (Bishop and Mathews, 1952a). Other physiologic factors, involving both intrinsic features of the gametes and exchange reactions between them and the luminal fluids, may play a role, but if so, their nature and action are unrecognized.

The capacity for motility on a general scale is first attained by sperm during transit through the epididymis (Redenz, 1926). Cells removed from the tail of the duct, of the bull for example, immediately become highly active when suspended in physiologic saline and given access to oxygen; under anaerobic conditions, glucose, fructose, or mannose initiates vigorous flagellation. Sperm removed from the head or the isthmus of the epididymis, on the other hand, rarely become motile and at best show only a slow nonprogressive type of undulation. Other mammals present a similar picture, the precise epidiclymal region where motile capacity is attained varying among species.

Some degree of flagellation, albeit of a leisurely, low-frequency type, can be observed in sperm recovered from the testes of various mammalian species (Tournade, 1913; Young, 1929a; Bishop, 1958d). These gametes are incapable of activation to full motility by the addition of oxygen, glycolytic substrate, divalent cations (Mg+ + , Ca+ + ), or ATP (Bishop, unpublished data). Austin and Sapsford (1952) have observed that the axial filament of the living rat spermatid undergoes mo^-ement even before the flagellum begins to push out from the margin of the roughly sjiherical cell. In lower forms as well, particularly among insects, sperm motility can be seen during the period when the gametes are still attached to their nurse cells within the gonad


722


SPERM, OVA, AND PREGNANCY


(Anderson, 1950). In conclusion, then, it would seem that spermatozoa must develop much of the machinery for movement while undergoing spermiogenesis in the testis, that subtle changes occur while they are in the mammalian epididymis such that the full capacity for motility is here acquired, and finally that this ability for flagellation is realized normally only on activation at the time of ejaculation.

Although the problem has been recognized for many years, only recently has serious attention been paid to the nature of the possible changes in spermatozoa that are responsible for the acquisition of the capacity for motility within the epididymis. In setting forth a hypothesis to account for this phenomenon, Salisbury (1956) has focused needed attention on the problem. His suggestion follows from determinations of cation concentration and freezing point depression values of fluids of the genital tract of the ram and the bull, noted above (Salisbury and Cragle, 1956). The supposition is that a decrease in K+/Na+ and an absolute increase in K+ -t- Na+ concentration, nevertheless accompanied by a total reduction in tonicity of the fluids, bring about permeability changes such that the sperm become hydrated and, as a result, capable of full metabolic activity (Salisbury, 1956). An ingenious theory, it is, nevertheless, not easily reconciled with the generally accepted demonstration that sperm lose water rather than gain it as they mature (Lindahl and Kihlstrom, 1952; IVIann, 1954). Until more precise information is available concerning such details as the sodium and potassium concentrations of the sperm vis-avis those of the fluid of the ducts, the actual water- and cation-permeability of sperm at various stages, and the effect of shifts in the sodium-potassium ratio on motility of epididymal sperm, in vitro, this problem cannot be fully resolved.


IV. Insemination

At the time of mammalian sperm transfer, many millions of vigorously motile spermatozoa are introduced into the female genital tract. Mixed with the fluid component of the semen only at the moment of ejaculation, the sperm normally are activated by their sudden access to both oxvgen and the hexose energy substrate of the plasma. The source and composition of the seminal fluid have been reviewed elsewhere (Mann and Lutwak-Mann, 1951 ; Mann, 1954) and are further discussed in the chapter by Price and Williams-Ashman. Only certain characteristics of semen, relevant to sperm transport and welfare, need be noted here.

What is the normal function of seminal plasma, and to what extent is it dispensable? The fluid component contributed by the accessory glands can conceivably serve several functions which include its role as (1) a vehicle for sperm transport, (2) a medium containing essential inorganic ions and of adequate buffering capacity, (3) a satisfactory osmotic milieu, and (4) a source of energy substrate. Seminal plasma, by virtue of its very complex composition, also performs other duties. It supplies, for example, the enzyme and substrate responsible for vaginal-plug formation; it contains certain substances, unique to the reproductive fluids, such as antagglutin which ostensibly prevents undue sperm agglutination; it provides such ingredients as ascorbic acid, ergothioneine, and possibly glutathione, which may play a role in the adjustment of the oxidation-reduction potential. On the other hand, some components of seminal plasma may indeed be by-products with no obvious beneficial role and even, perhaps, with harmful effects on the gametes; both alcohol and sulfonamides, for example, are excreted into the plasma (Farrell, 1938; Osenkoop and MacLeod, 1947).

Since the vital process of sperm activation is accomplished by their admixture with plasma, and since a fluid vehicle is essential for sperm transport, it goes without saying that seminal plasma normally is necessary for the reproductive process. To suggest that artificial insemination with epididymal sperm suspended in saline, successful as it is, proves the dispensability of seminal plasma, is to ignore the normal biologic accomplishments of natural insemination. Nevertheless, it is a fact that artificial insemination has proved highly successful in the reproduction of many types of animals. Moreover, the collection and analysis of the ejaculate, coupled with artificial insemination by natural or modified semen, constitute the basis for much of our knowledge concerning the entire field of i'e|)r()(liu'tive physiology and animal breeding.

A. Ejaculation

The ejaculatory response in many mammals occurs seasonally, corresponding to periodic activity of the testes and accessory glands, and is dependent on a variety of neurohumoral factors. In some animals, including man, potency continues throughout the period of reproductive maturity. Volume of semen and s})erm concentration, being contingent on both secretory activity of the accessory glands and spermatogenic activity of the gonads, vary with successive collections. Repetitive ejaculation can "exhaust" the sperm supply (Table 13.4), and >uch procedures have been used, albeit without great practical success, to test the spermatogenic productivity of man and of various domestic animals.

The potential for ejaculation of many animals is quite striking. Carpenter (1942) observed that free-ranging macaque monkeys are capable of ejaculating 4 times a day for 3 or 4 days, whereas the so-called "black ape" (a baboon, Cijnopithecus) , studied by Bingham (1928), ejaculated 3 times in 20 minutes. Domestic cats have been known to inseminate 10 females within 1 hour, and rabbits, 38 to 40 does in 8 hours (Ford and Beach, 1951). White rats can ejaculate 4 times in 15 minutes and as many as 10 times during a 3-hour period. Chang and SheafTer (1957) reported that a golden hamster copulated 50 times in an hour, with ejaculation occurring during most of the mounts. ]\IcKenzie and Berliner (1937) collected 20 ejaculates in 1 day from a ram, the 19th sample of 0.66 ml. containing over 1 l>illion sperm, compared with the first ejaculate of 0.7 ml. which contained 3.5 liillion cells.

There is relatively little correlation between the number of intromittent thrusts and the number of actual ejaculations, or between the duration of copulation and the volume of seminal discharge. In rodents, for example, intromission may occur as many as 80 to 100 times before ejaculation, or insemination may occur on the first intromission. Copulation in the macaque may involve several dozen mountings and well over a hundi-ed pelvic thrusts before ejaculation


TABLE 13.4

Chanyes in volume and sperm (lensitt/ of hull

ejaculates collected frequently throughout a

one-hour period

(From T. Mann, Advances EnzvmoL, 9,

329-390, 1949.)


Number of Ejaculate


Collection Time


Volume


Number of

sperm, mil.

per ml. semen



min.


ml.



1



4.2


1664


2


10


3.9


680


3


18


3.7


254


4


28


3.7


648


5


38


3.4


135


6


45


3.5


342


7


55


2.7


390


8


63


2.9


98


occurs. The prolonged copulation of the ferret and sable, as long as 3 hours, represents, not excessive ejaculation or insemination, but rather a functional adaptation to delayed ovulation (Ford and Beach, 1951). In the dog, however, the mounting time is roughly proportional to the duration of ejaculation, averaging in several breeds about 6V2 minutes (Perez Garcia, 1957). An important accomplishment of genital activity, at least in the rat, is the stimulation of sufficient corpus luteal function to support pregnancy (Ball, 1934).

Cerebral and constitutional influences incite and modify the physiologic processes of both erection and ejaculation (Rommer, 1952) under the direct innervation by luml)ar centers operating through muscular and vascular mechanisms. Spinal section in man does not necessarily prevent seminal emission (Ford and Beach, 1951). The relevant neural pathways in man have been summarized by Whitelaw and Smith wick (1951) from their observations on partially sympathectomized patients (Fig. 13.4, a and b) ; sympathetic fibers and the second, thii'd, and fourth sacral parasympathetic outflows are involved. The abolition of ejaculation through bilateral presacral sym})athectomy, without loss of erection, has been demonstrated in dogs (Van Duzen, Slaughter and White, 1947) and rodents (Bacq, 1931). In man and other mammals (rat, cat, and dog) , the cerebral cortex inlays an important role in male sexual activity (Ford and Beach, 1951), but an e\-aluati()n of the co-ordination between cerebral and spinal centers warrants considerable further study. The subject is further discussed in the chapter by ]\Ioney. Ejaculation in men and dogs is accompanied by pronounced cardiovascular intensification (Pussep, 1921; Boas and (ioldschmidt, 1932; Bartlett, 1956) affecting both blood pressure and pulse rate (Figs. 13.5, 13.6). Androgen administration increases libido and copulatory arousal (see cha])ter by Young ) , but the manner in which this is related to the preceding neurogenic factors is not well understood (Cheng and Casida. 1949).


4. Erection — normal


Sensory (stimulation of glans)

i via pudendal nerve


i Lumbar center


Psychic (higher centers of cerebral cortex)

i diencephalon

i cord


Inhibition of vasoconstrictor fibers (sympathetic) dorsal and lumbar


Parasympathetic sacral (S2 — S4)

i Vesical plexus

i Vascular supply of penis

i Dilatation of vessels

i Engorgement of sinuses

i Passive compression of veins and retardation of venous outflow


B. Ejaculation — normal


Sensory stimuli from glans

I via pudendal nerve


Psychic stimuli from higher centers

i diencephalon

i cord


i Summation of sensory and psychic stimuli producing so-called orgasm

i Lumbar center


Sympathetic motor

i Smooth-muscle contraction of prostate, seminal vesicles and vas deferens. Closure of internal sphincter

i Emission


Parasj-mpathetic motor

i Contraction of striated muscle, ischiocavernosus, bulbocavernosus and contractorurethrae muscles

i Ejaculation


Fig. 13.4. The probable neural pathways involved in (A) erection and (B) ejaculation in man, based on observations after partial sympathectomy. (From G. P. Whitelaw and R. H. Smithwick, New England J. Med., 245, 121-130, 1951.)


150


140


130


120


<


110


cr



h


en



<


100


LxJ



X



90


80


70






n



nP


/


■ ^


r

•\J


IIU

! t


^





INTROMISSION ORGASM 1 1 1 1 1 1 1 \


1 1


10


20


30


60


70


80


90


40 50

MINUTES


Fig. 13.5. Heart rate of man during coitus recorded by cardiotachometer. (From E. P. Boas and E. F. Goldschmidt, The Heart Rate, Charles C Thomas, 1932.)



B. Collection of Seminal Components

Various methods of seminal collection may be employed for cither whole or fractional analyses. Normal ejaculates are cxix'ctcd when induced l)y masturbation, electrical stinuilation, or discharge into an artificial vagina. Sperm samples without seminal plasma are readily obtainable from the epididymis and vas deferens of the excised tracts of many animals (e.g., the guinea pig, rat, boar, bull, and stallion) by backflushing the vas and cutting the epididymis. Relatively uncontaminated prostatic fluid is procurable, e.g., from men and dogs, and vesicular fluid from men, by manual massage of the appropriate glandular regions. Incomjilete ejaculates are produced after extirpation of such organs as the seminal vesicles and Cowper's glands; the prostatic isolation operation, perfected on dogs l)y Huggins, Masina, Eichelberger and Wharton (1939) and illustrated in Chapter 6. Figure 6.2, permits the collection of large amounts of uncontaminated prostatic fluid.

C. Seminal Volume And Successive Fractions

Seminal volume bears some relation to animal size but the individual contributions


Fig. 13.6. Blood pressure of dog during coitus. (After L. M. Pussep, Der Bhitkreidauj im Gehirn beini Koitus, Dorpat, 1921.)


TABLE 13.5

Volume and sperm density of mammalian ejaculates (From T. Mann, Advances Enzymol., 9, 329-390, 1949; S. A. Asdell, Patterns of Mammalian Reproduction, Comstock Publishing Co., 1946.)



Volume of Single Ejaculate


Density of Sperm in Semen


Species



Most





Range


common value


Range


Average



ml.


n,l.


cells per til.


cells per til.


Man


2-6


3.5


50,000-150,000


100,000


Dog


2-15


6


70,000-900,000


200,000


Rabbit ...


0.4-6


1


100,000-2,000,000


700,000


Boar


150-500


250


25,000-300,000


100,000


Bull


2-10


4


300,000-2,000,000


1,000,000


Ram


0.7-2


1


2,000,000-5,000,000


3,000.000


Goat


0.1-1.25


0.6


3,000,000-4,000,000


3,500,000


Stallion....


30-300


70


30,000-800,000


120,000


of the respective accessory glands determine the quantity of semen in the ejaculate. The volumes of seminal discharge from several mammals are presented in Table 13.5. The


enormous volume of the boar ejaculate, as much as half a liter, may be of importance in "washing" the sperm through the uterus, inasmuch as in the sow the semen is deposited directly into the cervix, and the ejaculate is so proportioned that the spermatozoa are concentrated in the earlier fractions and are followed by a copious flood of relatively sperm-free fluid (McKenzie, Miller and Bauguess, 1938) . When fractional collection is possible, the spermatozoa are found generally concentrated in the initial or middle portion of the ejaculate (ram, dog, boar, horse, and man). The ejaculate of the dog consists of a small, initial, clear, relatively sperm-free portion, followed by a milky fraction containing the bulk of the spermatozoa, and finally a slow but copious dribble largely derived from the prostate (Evans, 1933; Hartman, unpublished data). Collection by means of the electrically stimulated "split-ejaculation" technique has


BIOLOGY OF SPERMATOZOA


727


(Icinonstrated in the bull an abundant sperm-free initial portion which apparently is derived from the urethral glands and presumably serves to clear the urethral passage b(>fore the transport of spermatozoa (Lutwak-jNIann and Rowson, 1953). In man, however, approximately three fourths of the sjierm are present in the first 40 per cent of the ejaculate (MacLeod and Hotchkiss, 1 942 ) . Qualitative contributions to the total ejaculate by the several accessory glands liave been carefully studied and are discussed elsewhere (see chapter by Price and Williams- Ashman ) .

D. Effective Sperm Concentration

It is not a simple matter to determine what might be the minimal effective sperm count necessary to insure fertilization. The earlier standards of what constitutes a subfertile human seminal density have undergone considerable re-evaluation. The onceacceptable value of minimal concentration, 80 to 100 million cells per ml. of semen, has now been reduced to one half or less. On the other hand, the spotty records of pregnancies in women whose husbands' sperm counts consistently average 1,000,000 per ml., or less, may be viewed with some skepticism CMichelson, 1951; Sandler, 1952). The extensive studies of ]\IacLeod and Gold (1951) on human subjects of proved fertility, compared with men of infertile marriages, indicate a significant break between the two groups in the neighborhood of 20,000,000 cells per ml. of semen. Current trends in the evaluation of semen tend to minimize sperm density, as such, and to regard this property only with reference to other criteria, including volume, total sperm number, morphology, and, of course, motility.

A reasonable gauge of minimal effective si)erm count necessary to insure fertilization lias been provided by dilution tests and artificial insemination of domestic and laboratory animals. In cattle, the normal ejaculate, which contains some 4 billion sperm, can be reduced 500 to 1000 times without sacrificing high productivity (Salisbury and Bratton, 1948; Braden and Austin, 1953). Ral)l)it fertilization is unimpaired when the normal inseminate is decreased 500-fold (Cheng and Casida, 1948; Chang, 1951a; Chang, 1959; Braden and Austin, 1953).


That mere number of sperm is not the only factor was clarified by Chang (1946a, b) who showed that the concentration and nature of the diluent are also important. The percentage of fertile eggs recovered from does inseminated with a suboptimal number of sperm (ca. 40,000) decreases as a function of the volume of saline diluent (from 0.1 to 1.0 ml.). On the other hand, if rabbit seminal plasma is substituted for saline as the diluent, fertilizing capacity is enhanced (Chang, 1947b, 1949). The nature and the effect of the sperm diluent are further discussed below ; it is sufficient to point out here that many factors may determine the absolute number of sperm required for a high rate of fertilization.


E. Site of Insemination

The location of the deposition of semen during ejaculation differs in various animals and may account, in part, for the variations recorded for time of transport through the female genital tract. Intravaginal insemination predominates in the rabbit, dog, ewe, cow, and man, whereas intrauterine deposition occurs in the mouse, rat, sow, mare, and probably the hamster (Braden and Austin, 1953; du Mesnil du Buisson and Dauzier, 1955; Chang and Sheaffer, 1957).

Experimental insemination has been attempted by a number of routes. Administration of sperm into the ovarian bursa of receptive mice proved highly satisfactory (Runner, 1947). Intraperitoneal insemination has been accomplished in fowl (Van Drimmelen, 1945), guinea pigs (Rowlands, 1957), rabbits (Hadek, 1958b), and, with bare success, in the cow (Skjerven, 1955; McDonald and Sampson, 1957). In an extensive animal breeding investigation, Salisbury and VanDemark (1951) showed that artificial insemination was equally effective in cattle, as judged from the nonreturn rate, when semen was deposited in the vagina, the body of the uterus, or the uterine horns.


F. Artificial Insemination

Little more than a brief account of this special and applied subject seems appropriate at the moment. Excellent surveys of the development, techniques, and accomplishments of artificial insemination have appeared from time to time, two of the more general being those of Anderson ( 1944) and Emmens and Blackshaw (1956). Legal and ethical aspects of artificial insemination in man have been dwelt upon extensively in the semiclinical literature (Haman, 1947; Nicolle, 1949; Guttmacher, Haman and MacLeod, 1950; Ellis, 1952; Pope Pius XII, 1957).

According to various historic accounts, the Arabians have for centuries practiced, if not thoroughly understood, the art of artificial insemination in the breeding of their horses.^ In more recent times Spallanzani developed a method for the artificial insemination of amphibia and, in 1782, first successfully inseminated a dog. Shortly thereafter, the first successful insemination of a woman was recorded (Home, 1799). Now it is a common ])ractice, the world over, for the selective breeding of various species of mammals (Walton, 1958). The techniques have also been applied, both for academic and for practical aims, to other types of animals, including fowl (Quinn and Burrows, 1936; Van Drinnnolen, 1945), viviparous fish (Clark, 1950), and insects (Laidlaw and Eckert, 1950; Lee, 1950).

Constant efforts are being made to improve the dilution and storage media of sperm for routine use in artificial insemination (see Salisbury, 1957). At present, 5- or 6-day survival of bull semen, diluted with egg yolk-sodium citrate and stored in the presence of antibiotics at 2 to 3°C., is about all that can be expected. Most types of semen lose their fertilizing capacity much sooner than this. It is a curious fact that fowl sperm, which survive so well (2 or more weeks) in the female genital tract, cannot be iireserved in vitro more than a few hours without decline in fertilizing capacity (Garren and Shaffner, 1952; Carter, McCartney, Chamberlin and Wyne, 1957) .


  • Walter Heape ( 1898) recounted an interesting tale which probably has some basis in fact : "It is taken from a book written in the year 700 of the Hejira, and therein is described how an Arab of Darfour, the owner of a valuable mare on 'heat,' armed with a handful of cotton wool which had been saturated with the discharge from the vagina of his mare, approached by stealth a valuable stallion belonging to a member of a neighbouring hostile tribe, a stallion whose services for his favourite mare the owner was desperately anxious to obtain ; and having sufficiently excited the animal with the scent of the material he had brought, he obtained spermatic fluid from him on the same handful of cotton, and hastening back to his mare, which he had been obliged to leave some distance away,

pushed the whole into her vagina, and obtained by that means a foal."


The most significant advance — certainly the most striking — in the field of sperm preservation during the past decade is the remarkable success in maintaining cells in a viable condition at extremely low temperature. The very early history began with Mantegazza's (1866) and Davenport's ( 1897) successful demonstrations that deepfrozen ( — 17°C.) human sperm could regain motility. Despite other attempts to improve the degree of recovery by the addition of various substrates and by control of temperature changes, a marked measure of success was to await the discovery of Polge, Smith and Parkes (1949), who showed that eciuilibration of the semen with glycerol before freezing greatly enhances sperm recovery and motility after warming to room temperature. This work, on rooster and human spermatozoa, catalyzed many investigations of the problem M'ith the result that today there are few common mammals whose sperm have not been vitrified, stored at —79° or — 196°C., and warmed up for observation of motility or used in breeding experiments (Emmens and Blackshaw, 1956; Polge, 1957; VanDemark, Miller, Kinney, Rodriguez and Friedman, 1957; Martin and Emmens, 1958). The presence of glycerol is essential, in concentrations between 10 and 15 per cent, for bull sperm, to 20 per cent for those of fowl (Martin and Emmens, 1958). Bull spermatozoa have been stored successfully in this fashion for periods up to 6 years (Walton, 1958).

Artificial insemination with previously deep-frozen, thawed sperm has resulted in conception and viable young in a number of animals. The degree of fertility varies, being low in the rabbit and as high as in normal matings in the bull (Emmens and Blackshaw, 1956). Pregnancies have been reported for several women inseminated with spermatozoa treated in this manner (Bunge and Sherman, 1954).

The advantages of the perfection of the low-tempcrature method for the preservation of animal sperm are obvious. In the case of bull semen, for example, the procedure permits long-term storage and, in the long i-un. greater use of the sperm. An additional advantage is that the storage intervals allow for i)rogeny testing, a procedure which takes time and is of considerable importance in identifying the breeding value. As applied to man, on the other hand, the method would seem to have only limited usefulness in ex(■e|)tional instances. One might suppose, for example, that successive ejaculates of an ()lig()s|)erniic individual could be stored and pooled in this fasliion and give, upon insemination, a sufficiently high sperm count to insure fertilization. The advantage of transportability of frozen semen, i)ractical in animal husl)andry, would not be expected to play a significant role in matters concerning human fertility.

'The changes which may occur in cells during storage at such low temperatures, or during the freezing or thawing process, can only be surmised. Based on the resumption of motility at room temperature and fertilizing cajjacity, the alterations in bull spermatozoa must be minor. In other kinds of spermatozoa, those of the rabbit for example, metabolic and permeability changes may l)e more pronounced. Subtle changes, sucli as might be induced in the cytogenetic api)aratus, are unknown; there is the question of whether they have been sought.

The mechanism of the protective action of glycerol in maintaining the spermatozoa during the relatively slow freezing process and while in storage is obscure. The effect is probably not merely one of the prevention I if ice crystal formation, but rather one which involves the stability of the internal ionic concentration of the cell. One can suppose that without glycerol, the withdraw^al of fi-ee water would result in severe changes possibly involving an increase in ionic strength, alteration in jiH, the production of toxic concentrations of such substances as urea and dissolved gases, and an actual liliysical I'eorganization of intracellular components (Lovelock, 1957). One suggestion is that the elements sensitive to deep fieezing are lii)oprotein complexes which, in the ])resence of glycerol, aic pic\-ented fi'oni • lenaturation (Lovelock, 1907).

Although deep freezing and cold storage of sperm are currently receiving the greatest attention, othei- methods of controlling


metabolism and motility are being considered and may ultimately prove useful in the preservation of sperm for artificial insemination. Such metabolic blocking agents as tetrazolium compounds (Bishop and Mathews, 1952b) and carbon dioxide (Salisbury and VanDemark, 1957; VanDemark and Sharma, 1957; du Mesnil du Buisson and Dauzier, 1958) can reversibly inhibit the processes involved in the utilization of substrate and the expenditure of energy. Another api)roach has recently been suggested by the work of Petersen and Nordlund ( 1958) whose })reliminary experiments indicate that bull sperm can be subjected to 150 atmospheres of pressure, in nitrogen, and sur\-i\-e such treatment for two weeks, after which motility is regained. Whether such a procedure destroys fertilizing capacity has not yet been ascertained.


V. Sperm Transport and Survival in the Female Tract

The vigorous motility of seminal spermatozoa has long been a source of fascination and naturally gave strong support to early suppositions that migration in the female tract is due to the activity of the cells themselves. This is now known not to be generally true, and only in certain limited segments does active sperm motility seem of possible importance in transport from the vagina to the site of fertilization in the oviduct. Suggestions have been made, in fact, that sperm motility may be unnecessary even for egg penetration (Allen and Grigg, 1957) , but such has never been demonstrated in studies of fertilization of either invertebrate or vertebrate gametes.

The over-all transport system for mammalian spermatozoa is principally provided by muscular contractions of the walls of the tract, with a questionable role played by ciliary activity of the mucosa; under some circumstances, however, active flagellation of the gametes themseh^es is important (cf. Hartman. 1939).

A. Duration of Transport

The most striking evidence that sperm migration in the female tract cannot be attributed solely to sperm motility is afforded by the results of studies of the rate of transport and the time required to pass from the point of insemination to the site of fertilization or to intermediate levels of the reproductive system. Thus, for example, the mean velocity of bull sperm is on the order of 100 /x per sec. (Moeller and VanDemark, 1955; Gray, 1958 ) , and if a straight path were followed, it would require about IV2 hours for the gametes to cover the entire length of the tract; actually the time required after natural mating is less than 2V2 minutes (VanDemark and Moeller, 1951).

Rapid sperm migration through the uterus was first demonstrated by Hartman and Ball (1930) in the rat; within 2 minutes after copulation myriads of sperm had entered the tubes (Table 13.6 ) . A subsequent investigation showed that a few sperm were present at the periovarial sac within IVk minutes after copulation (Warren, 1938). Blandau and Money (1944) later indicated that at copulation, rat sperm are catapulted through the cervix into the uterine cornua, and within 15 minutes have entered the Falloi^an tubes in considerable numbers. By clamping the middle of the tubes at various times after copulation, the distribution of sperm could be determined. After 15 minutes, sperm were found in 42 per cent of the uterine (lower) segments of the oviducts examined and 21 per cent of the ovarian (upper) segments; after 30 minutes, 85 per cent and 62 per cent, respectively; after 45 minutes, 90 per cent and 96 per cent; and at 60 minutes, both the uterine and ovarian portions of oviducts of all animals studied contained sperm.

After insemination of the mouse, sperm reach the tubal infundibulum, the site of fertilization, within 15 minutes (Lewis and Wright, 1935). In the bitch, 20 minutes or

TABLE 13.6

Time of passage of rat spermatozoa into

female genital tract

(From C. G. Hartman and J. Ball, Proc. Soc.

Exper. Biol. & Med., 28, 312-314, 1930.)


Animal


Killed after Ejaculation


Uterus Clamped near Apex after Ejaculation


Sperm Located


1 2 3 4


1 min.

1 min. 30 sec. "Immediately"


2 min. 100 sec. 54 sec. 54 sec.


Apex of uterine cornua Apex of cornua Lower part of cornua Vagina


less are required (Evans, 1933; Whitney, 1937), and in the hamster about 30 minutes (Chang and Sheaffer, 1957). Rubenstein, Strauss, Lazarus and Hankin (1951) claimed that human sperm deposited at the cervix just before hysterectomy, can be recovered from the Fallopian tube 30 minutes later ; the nature of the operation, however, might seriously affect the rate of transport. Other reports of sperm-transport time in women range up to 3 hours (Chang and Pincus, 19511.

As part of a series of marvelously planned and executed experiments on cattle, VanDemark and co-workers have shown that sperm migration requires only 2 to 4 minutes whether the heifers are mated or artificially inseminated. Indeed, even dead sperm, after artificial insemination, were transported to the uj^pcr reaches of the oviduct within 4.3 minutes (VanDemark and Moeller, 1951). Sperm-migration times reported for the ewe have varied considerably, ranging from several hours (Green and Winters, 1935) down to 6 to 16 minutes (Starke, 1949; Schott and Phillips, 1941). This variation is to be accounted for less by changes dependent on the estrous cycle (Dauzier and Wintenberger, 1952) than by improvements in technique.

The rabbit, in many ways a domestic anomaly in reproductive matters, apparently requii-es several hours for transport of a significant number of sperm, although the "vanguard" may reach the ampulla within an hour after insemination (Chang, 1952; Adams, 1956). Heape's demonstration in 1905 of approximately 4 hours for migration seems to have stood the test of time. On the basis of recovery of spermatozoa from separate segments of the genital tract, Parker (1931) andBraden (1953) found that 2.5 to 3 hours are required for transport. Confirmation is afforded by experiments involving ligation of the tubes at various intervals after copulation (Adams, 1956; Gr,'enwald, 1956) ; whereas some eggs are fertilized when the tubal blocks are made prior to 2.5 hours, ligations made 3 to 5 hours after copulation do not prevent a high percentage of fertility. Whether this order of transport time is an adaptation to induced ovulation or is otherwise unique to the rabbit is not known ; comparable experimentation on the cat and ferret, for example, which also normally ovulate only when stimulated by copulation, might be instructive.

The time for sperm migration in fowl is of the same order of magnitude as that in most mammals (Mimura, 1941). Fowl sperm, labeled with inorganic P-^-, were recovered from the infundibulum within an hour after insemination; the number found depended on the site of administration, i.e., intravaginal or intrauterine (Allen and Grigg, 1957). Killed sperm also reached the infundibulum when placed in the uterus, but not when introduced intravaginally.

The study of seminal components, other than sperm, indicates that tubal transport must involve a muscular mechanism. In both the sow and the mare, certain natural seminal constituents, e.g., fructose, citric acid, and crgothioneine, are found in the uterine horns within an hour after mating (Mann, Polge and Rowson, 1955). Gunn and Gould ( 1958 ) produced a Zn*'^-labeled component of prostatic fluid in rats which served as a marker for tubal transport. In animals killed at intervals between 0.5 and 1.5 hours after mating, a significant quantity of the isotope had reached the uterotubal junction by 1 hour, and radioactive labeling was found throughout tlie oviduct at 1.5 hours.

B. Mechanism of Transport in the Uterus and Oviduct

The muscular contractility of the genital tract has been implicated in the process of sperm migration since the earliest studies of mating behavior and insemination (see Austin and Bishop, 1957). The normal activity of the uterus and Fallopian tube is well known (Westman, 1926; Parker, 1931; Reynolds, 1931, 1949). The contractions of the tract are not, however, peristaltic waves which might favor rapid, directed sperm transport, but rather segmentation waves which encourage dispersal from the source. Indeed, what peristalsis can be observed in the estrous oviduct (e.g., the rabbit) is directed from the fimbriated toward the uterine end (Reynolds, 1949j.

Both mechanical and psychic factors influence the contractility of the genital tract and appear to augment sperm migration. In the ral)bit (Heape, 1898; Krehbiel and Carstens, 1939), and probably in many other animals, stimulation of the external genitalia increases uterine activity. The mating response also enhances uterine action in the mare (Millar, 1952) and cow (VanDemark and Hays, 1952). According to VanDemark and Hays (1952) , the mere sight of the bull is sufficient to induce strong uterine contractions in the estrous and postestrous heifer (Fig. 13.7). The activity of the Fallopian tube of the rabbit also appears to be stimulated by the presence of a suitable buck (Westman, 1926).

In the oviducts of rabbits, Parker (1931) emphasized both the segmentation contractions and the local ab- and adovarian ciliary currents in accounting for dispersal of sperm, once they pass the uterotubal junction. In a recent series of interesting experiments, however. Black and Asdell (1958) tended to minimize ciliary activity, which is generally directed toward the uterus, and to attribute sperm distribution in the rabbit oviduct to the segmentation process brought about by the circular musculature of the tube. Tubal secretions, pronounced at the time of ovulation (Bishop, 1956a), serve as vehicle of transport for the sperm. The copious uterine fluid secreted in the rat during tlie proestrum performs the same role (Warren, 1938).



Fig. 13.7. Uterine responses in an estrous cow stimulated by various mating activities: A, bull brought within sight of cow ; B, bull allowed to nuzzle vulva ; C, bull mounts but does not copulate; D, bull copulates; E, bull ejaculates. (From N. L. VanDemark and R. L. Havs, Am. J. Physiol., 170, 518-521, 1952.)



It is probable that ciliary activity plays a greater role in some animals than in others in distributing sperm throughout the female tract. Thus, Parker ( 1931 ) stressed the importance of adovarian ciliary currents in the oviducts of the turtle, pigeon, and chicken. With respect to a6ovarian currents, moreover, it should be pointed out that these, too, could serve a function by orienting the sperm toward the infundibulum ; whereas unnecessary emphasis should not be placed on this as a transport mechanism, considerable evidence exists to show that sperm orient against a current and, when free-swimming, make considerable progress upstream (Adolphi, 1906a, b; Yamane and Ito, 1932; von Khreninger-Guggenberger, 1933; Brown. 1944; Sturgis, 1947).

The activity of the several segments of the female genital tract varies with phases of the ovarian cycle and, as a consequence, may alter the rate of sperm migration (see Austin and Bishop, 1957). The active motility of both the Fallopian tube and uterus, characteristic of estrus, is depressed by progestational conditions, although little change is found immediately after ovulation (Reynolds, 1949; Borell, Nilsson and Westman, 1957; Black and Asdell, 1958). Cyclic changes in sperm-transport time through the uterus and oviducts have been noted in the cow (Warbritton, McKenzie, Berliner and Andrews, 1937) and sow (du Mesnil du Buisson and Dauzier, 1955 ) . Recent work of Noyes, Adams and Walton (1959) suggests that estrogen enhances fertilization of rabbit ova transplanted into castrates by increasing the efficacy of sperm transport, i.e., by reducing the obstacles to sperm migration present in nonestrous does (Noyes, 1959a).

The most spectacular development involving endocrine control of sperm transport during the past decade has been the demonstration that oxytocin, as an important mediator of uterine activity, is essential, in some cases at least, for the rapid migration of sperm from the cervix to the site of fertilization (VanDemark and Moeller, 1951; VanDemark and Hays, 1952; Hays and VanDemark, 1951, 1953a). Excised and perfused cow uteri function as a transport system so long as oxytocin is present in the perfusate. Motile sperm, artificially inseminated into the cervix, are carried to the ovarian end of the oviduct in as few as 2.5 minutes. Even nonmotile sperm are transported throughout the tract within 5 minutes. In the absence of oxytocin, however, sperm migration does not occur; in fact, the cells do not even enter the fundus. Oxytocin is also apparently released during natural and artifical insemination of the cow (Hays and VanDemark, 1951, 1953b). and its administration, during mating, augments uterine contractility (Hays and VanDemark, 1953a ) . Oxytocin may have a general role in the uterine responses to mating and rapid transport of spermatozoa through the genital tracts of some other animals as well (Harris, 1951; Cross, 1958), although it is to be noted that coitus is claimed to abolish temporarily uterine contractions in women (Bickers and Main, 1941).

C. Critical Regions of Sperm Transport

The unrestricted passage of sperm, which is apparently characteristic of the heifer, is not, however, exhibited by all mammals. The cervix, the uterotubal junction, and, to a lesser degree, the isthmus of the Fallopian tube can each constitute an obstacle to free sperm transport. In these regions, active sperm motility may then assume some significance as a means of migration. In the rabbit only 1 sperm in about 50,000 reaches the site of fertilization; in the ewe and rat, the proportion is even smaller (Braden, 1953). According to Braden, of the total number of sperm deposited in the rabbit vagina during a normal insemination (about 60 X 10" cells), the proportions transmitted are roughly as follows: approximately 1 out of 40 traverses the cervix ; of these, one-third reach the uterotubal junction; 1 out of 160 passes the uterotubal junction and enters the Fallopian tube; and of these, one-fourth ultimately reach the ampulla. The distribution of spermatozoa throughout the rabbit genital tract at various times after copulation is presented in Figure 13.8.


BIOLOGY OF SPERMATOZOA


733



10 14 18 22

HOURS AFTER MATING

Fig 13 8 Changes in sperm number in various sections of the genital tract of rabbit after copulation. (From A. W. H. Braden, Australian J. Biol. Sc, 6, 693-705, 1953.)


734


SPERM, OVA, AND PREGNANCY


1. The Cervix

This portal connecting the vagina and the uterus is generally regarded as constituting a partial block to sperm transport in certain animals in which ejaculation occurs in the vaginal vault, e.g., the rabbit, ewe, and man (Warbritton, McKenzie, Berliner and Andrews, 1937; Chang, 1951b; Braden, 1953; Noyes, Adams and Walton, 1958). Sperm migration through the rabbit cervix is a gradual process (Florey and Walton, 1932; Braden, 1953) and, although the mechanism certainly is not definitely known, is possibly to be attributed to active flagellation of the sperm themselves, with little or no help from the cervical duct (Noyes, Adams and Walton, 1958; cf. Hartman, 1957). Dead cells, according to Noyes and colleagues, fail to negotiate the cervical passage, as do radiopacjue media. It should be noted that the latter finding is inexplicably at variance with a similar experiment of Krehbiel and Carstens (1939), who found that radiopaque medium does pass the rabbit cervix in significant amounts. Noyes, Adams and Walton (1959) also indicated that estrogen treatment facilitates cervical transport of spermatozoa, that is, decreases the resistance to migration shown by untreated animals, in this case castrated does. Whether the effect is actually on the cervical musculature, the secretion of cervical mucus, uterine motility, or some other system is not clear from these experiments.

The cervix should not be regarded as always constituting an obstacle to sperm transport. In at least two species with intravaginal insemination, namely, the heifer and dog, sperm transport is extremely rapid. The cervices in these animals, therefore, rather than retarding progress, must aid considerably in the migration of spermatozoa.

A frequently suggested theory to account for the passage of spermatozoa into the uterus envisages "insuck" of the semen through the cervix. Indeed, a transient negative uterine pressure of about 0.7 lb. per square inch has been demonstrated during coitus in the mare (Millar, 1952). However, the significance of such determinations in this animal is obscure since ejaculation normally occurs directly into the uterus (Braden and Austin, 1953) . Nevertheless, in consider


ation of the concept relevant to women, it is reasonable to assume that the uterus can aspirate sperm and mucus into the uterine cavity by virtue of the elasticity of that organ following contraction (Belonoschkin, 1949, 1957) . This subject is more extensively reviewed by Hartman (1957).

Much attention has been focused on the questions of the nature of cervical mucus, its cyclic changes, and its penetrability by spermatozoa in vitro (Shettles, 1949). The importance of cervical mucus is obvious if sperm reside in the cervix for considerable periods of time, as in women, or if the sperm have to negotiate the canal by their own motile faculties; it is of much less significance when the sperm are shot through the cervical canal at ejaculation, as in the sow, or are carried through rapidly by muscular contractions, as in the heifer.

The secretory activity of the cervix responds to variations in the ovarian cycle, and the physicochemical composition of the mucus changes accordingly. The response of the cervix to cyclic changes was first clearly stated by Allen (1922) for the mouse. Much of our current understanding stems from the important monograph of Sjovall (1938) concerning investigations of human and guinea pig cervices. There now exists adequate evidence that changes in human cervical mucus correlate well with ovulatory and with endometrial, vaginal, and other indications of estrogenic activity (Sjovall, 1938; Vicrgiver and Pommerenke, 1946; Shettles, 1949; Bergman, 1950; Cohen, Stein, and Kaye, 1952; Odeblad, 1959). Cervical mucus in women is most copiously secreted during the estrogenic phase; its dry weight at this time is minimal (Bergman, 1950), tonicity is low (Bergman and Lund, 1950 », and pH, as generally determined, is elevated. Estrogenic mucus is also claimed to be richer in glucose and polysaccharide, but these components may be derived less from the cervical secretion than from the uterine glands higher in the tract (Bergman and Werner, 1950). Lipid is present in lower amounts at ovulation. Benas (1958) found changes, as determined by paper electrophoresis, in the extractable protein, with a predominance of albumin in pre-ovulatory mucus and a prevalence of /?- and y-globulins


BIOLOGY OF SPERMATOZOA


735


in iiiucus collected after ovulation. No cyclical changes, however, were found by Bergman and Werner (1950) in carbohydrate hydrolysates of cervical mucus, which, when tested chromatographically, showed the presence of galactose, mannose, fucose, and hexosamine.

A recent investigation of cervical mucus from the cow (Gibbons, 1959a) has demonstrated the presence of glucose, glycogen, protein, alkaline phosphatase, lysozyme, antagglutin, and common inorganic ions. Isolation and relative purification of mucoid, prepared from bovine mucin, show that it changes in physical consistency with phases of the cycle; molecular configuration, as determined by sedimentation, viscosity, and flow-birefringence measurements, is altered and is probably due to changes in state of liydration (Gibbons and Glover, 19591. Chemically, bovine mucoid consists of about 75 per cent carbohydrate and 25 per cent amino acid residues and resembles human blood-group substances (Glover, 1959b).

The presence of glucose and hydrolyzable l)olysaccharide in cervical mucus suggests the availability of metabolic substrate for the spermatozoa, but the utilization of these energy sources can only be conjectured. Moricard, Gothie and Belaisch (1957) have indicated that inorganic S^^ is apparently taken up by human sperm from cervical mucus, but the significance of this uptake cannot at present be evaluated.

Many investigators have attempted to correlate cyclical changes in cervical mucus with capacity for sperm progression, in vitro (Fig. 13.9). Maximal penetration by human sperm, observed in capillary tubes, occurs in estrogenic cervical secretion when the mucus is most copious and least viscous (Lamar, Shettles and Delfs, 1940; Guttmacher and Shettles, 1940; Shettles, 1940; Pommerenke, 1946; Leeb and Ploberger, 1959). Very little or no penetration is observed in pre-ovulatory or postovulatory mucus. Just before menstruation penetral)ility sharply increases, a change probably correlated with the premenstrual rise in circulating estrogen. During pregnancy, cervical mucus is only slightly penetrable, although the endocervical glands are hyperactive at this time (Guttmacher and Shet


tles, 1940; Atkinson, Shettles and Engle, 1948). Postmenopausal mucus is relatively impenetrable by spermatozoa, but after adequate estrogenic administration, a mucus is secreted which is characteristic of that of the ovulatory phase. Ovariectomized women ordinarily produce a scant, viscous mucus which is increased upon estrogen administration (Moricard, 1936; Abarbanel, 1946, 1948; Pommerenke and Viergiver, 1946). It has been claimed (Gary, 1943), although not confirmed, that mucous secretion in women is enhanced by orgasm and that this facilitates sperm penetration.

These studies on sperm, in vitro, have in a general way largely confirmed the earlier work of Sjovall ( 1938 ) , whose investigations of sperm penetration through the guinea pig cervix were mainly confined to observations in vivo. The penetration of sperm through the cervical mucus, in vitro, however, is at best only an approximation to the normal process of insemination and cervical transport, and the meaning of these carefully compiled results is not easy to assess. Their full significance must await further correlation between sperm migration in vitro and transit in situ. Certain evidence, indeed, tends to suggest that the condition of the cervical mucus in women may be of relatively little importance in sperm transport. In the series of 51 women studied by Rubenstein, Strauss, Lazarus and Hankin (1951), spermatozoa were found to have passed rapidly through the cervix at all stages of the cycle. No particulars were given concerning the condition of the cervical mucus, the presurgical coital history of the patients, or the possible effect of the operation (hysterectomy) on sperm transport. Their re])ort, however, seems to conflict with many of the above-cited observations in vitro which indicate that sperm migration is limited to the ovulatory phase of the cycle.

2. The Uterotubal Junction

The speed of sperm transport through the upper genital tract is in general so rapid that in only two species, the rat and rabbit, is the junction between the uterus and the oviduct stressed in current literature as being an obstacle to sperm migration (Braden, 1953). Yet, for almost half a century, de


736


SPERM, OVA, AND PREGNANCY


mm/mm. 2.0

1.5

1.0

0.5

mg. 500

400

300

200

7 14

12

10

8

37.1 37.0 36.9 36.8 36.7



SPERM PENETRATION


DRY CONTENT OF MUCUS



BASAL TEMPERATURE



98.8 98.6 98.4 98.2 98.0


12 16

DAY OF CYCLE


20


24


28


Fig. 13.9. Variation in human sperm penetration in vitro through cervical mucus during a single cycle (from J. K. Lamar, in Problems oj Human Fertility, George Banta Publishing Company, 1943), correlated with cyclical changes in the mucus and in body temperature based on 35 cycles (from P. Bergman, Acta obst. et gjmec. scandinav., Suppl. 4, 29, 1-139, 1950).


scriptive and experimental investigations have pointed out the complexity of the junction and the high pressures often required to force an opening through the lumen in this region. Rubin's initial paper (1920) indicated that gas pressures of 40 to 100 mm. Hg could be considered a normal range for human tubal insufflation, the uterotubal junction being the major source of resistance. In


the cat, fluid pressures of 250 to 300 mm. Hg are incapable of "forcing" the opening when injections are made through the uterus (Lee, 1925a; Anderson, 1928). On the other hand, tubo-uterine injections of fluid, that is, those from tube to uterus, recjuire very little pressure to force the opening. Other species behave differently. With relatively little pressure, between 25 and 40 mm. Hg, fluid can


BIOLOGY OF SPERMATOZOA


737


l)e forced through the junction from the uterine to the ovarian side in both the cow and ewe (Anderson, 1928). The resistance to flow, in the cow at least, is greatest during estrus (Anderson, 1927; Whitelaw, 1933). During this early and important period of investigation, the structural aspects of the uterotubal junction of a wide variety of mammals were described, particularly the villi and folds which ajipear to guard the opening of the Fallopian tubes (Lee, 1925b; Anderson, 1928). Anderson's paper should be consulted for details of the comparative structure of the junction in 25 species of mammals and for her particularly thorough discussion of this region in the sow.

A general conclusion which arises from these considerations of the uterotubal junction is that the structure is sufficiently complex (Fig. 13.10) to render spurious many attempts to correlate forced-fluid determinations with sperm transport. It seems likely that in a case like the cat, for example, the fluid pressure applied would occlude the uterotubal orifices with villi or folds, and that the greater the pressure, the tighter the seal; under normal conditions the junction would remain more or less patent, at least between muscular contractions, and allow for sperm transport.

That migration through the uterotubal junction in the rat, under some circumstances, is probably accomplished by the gametes themselves was indicated by the ingenious investigation of Leonard and Perlman ( 1949). They injected live spermatozoa of one or more species, as well as dead sperm and India ink particles, into the rat uterus. Spermatozoa of the rat, mouse, guinea pig, and bull were injected singly, and combinations of rat-guinea pig, rat-mouse, and ratbull sperm were introduced together. Distribution throughout the reproductive tract was determined 1 to 14 hours later. Under these conditions icf. Table 13.6) motile rat si)ermatozoa freely penetrated the uterotubal junction in both estrous and diestrous animals, but dead spermatozoa and inert particles did not; foreign spermatozoa passed through only very rarely. A similar experiment on the rabbit, which also shows evidence of uterotubal blockade, should pro^•e rewarding.



Fig. 13.10. Uterotubal junction of the rabbit. (From D. H. Anderson, Am. J. Anat., 42, 255-305, 1928.)

3. The Isthmus

The lower segment of the oviduct constitutes a partial obstacle to sperm migration in both the rat and rabbit (Chang, 1951b; Braden, 1953). In the latter, transport of both sperm and eggs is slowed by a decrease in muscular activity, in contrast to the movements characteristic of the upper segment of the duct (Black and Asdell, 1958) . The small diameter of the lumen of the isthmus, along with its kinks and extensive mucosal folding, may also retard sperm transport.

In a recent extensive study to ascertain the source of the fluctuations in gas pressures during tubal insufflation of the rabbit, Stavorski and Hartman (1958) demonstrated that the isthmus is more important than the actual uterotubal union in the degree of resistance offered to applied pressure. Sphincters were observed at both the uterotubal and tubo-ampullar junctions, but the elbow-like kinks in the isthmus were found to be the major source of resistance. The pressures necessary to force an opening were of the same order of magnitude whether a uterotubal or a tubo-uterine approach was employed. A suddenly applied high jiressure


738


SPERM, OVA, AND PREGNANCY


was found to meet with great resistance ; the more slowly the pressure was built up, the lower the peak pressure required to open the isthmian and uterotubal constrictions. The reciuired pressures generally were higher in those animals receiving estrogen.

D. NUMBER OF SPERM AT THE SITE OF FERTILIZATION

In the few species subjected to careful investigation, the number of spermatozoa recovered from the ampulla, or what is regarded as the site of fertilization, at the approximate time of fertilization, is surprisingly low. A summary of available evidence is included in Table 13.7. Whereas these data represent, in some instances, only single determinations and, in others, mean values within a very wide range, they show quite clearly that only a minute fraction of the inseminate is present in the vicinity of the ova when fertilization occurs. In some of these studies (Moricard and Bossu, 1951; Blandau and Odor, 1949), search failed to reveal many more sperm than the number of eggs undergoing fertilization. The presence of so few sperm at this critical point is evidence enough against the once-popular view that a sperm "swarm" is essential for fertilization — either to denude the ova of their

TABLE 13.7

Number of spermatozoa found at the site of

fertilization in several mammals


TABLE 13.8 Sperm survival times in, the female tract


Species


Mean

No.

Sperm

Tube


Postcoital Time


Reference




hr.



Rat


43


?


Austin, 1948



12


12


Blandau and Odor, 1949



30


24




45


12


Braden and Austin, 1954; Moricard and Bossu, 1951


Mouse


17


10-15


Braden and Austin, 1954


Rabbit


500


?


Chang, 1951a



38


4


Braden, 1953



250


10



Ferret


200


6


Hammond and Walton, 1934



500


24



Sheep


184*


24-48


Braden and Austin, 1954



673 1


24-48




Maximal


Maximal



Animal


Duration


Duration


Reference



Fertility


Motility




hr.


hr.



Rabbit


30-32


_


Hammond and Asdell, 1926


Mouse.


6


13


Merton, 1939b


Guinea




Yochem, 1929; Soderwall an.l


pig


21-22


41


Young, 1940


Rat


14


17


Soderwall and Blandau, 1941


Ferret


36-48



Hammond and Walton, 1934


Sheep


30-48


48


Green, 1947; Dauzier and Wintenberger, 1952


Cow .


28-50



Laing, 1945; Vandeplassehe and Paredis, 1948


Horse.


144


144


Day, 1942; Burkhardt, 1949


Man


28-48


48-60


Farris, 1950; Rubenstein et al., 1951; Home and Audet, 1958


Bat


135 days


159 days


Wimsatt, 1944


  • Ovarian third of oviduct.

t Entire ampulla.


cumulous auras by hyaluronidase or to supply some ingredient for sperm penetration. Conversely, Braden and Austin (1954) have suggested that an accomplishment of the filtering out of the overwhelming majority of sperm during transport is to so limit the number of male gametes present that multiple sperm penetration of the ova is reduced, thereby preventing polyspermy and anomalous development.

E. DUR.\TIO\ OF FERTILIZING CAPACITY

The retention of fertilizing capacity by mammalian spermatozoa is relatively limited (Table 13.8). As in the male tract, the capacity for fertilization is lost more promptly than is their ability to move. In the female guinea pig, for example, motility of sperm continues for as long as 40 hours after mating, whereas fertilizing capacity is lost about 22 hours after copulation (Yochem, 1929; Soderw^all and Young, 1940); in the mouse these periods are approximately I3Y2 and 6 hours, respectively (Merton, 19391)). In the consideration of sperm survival in parts of the tract other than the fertilization site, sperm motility is the most convenient, although not necessarily the only, criterion of longevity.

The values presented in Table 13.8 are the most accurate available, but the degree of precision with which such data can be ob


BIOLOGY OF SPERMATOZOA


739


tained varies considerably among species, dependent as they are upon the estimates of the time of fertilization. Reliable figures may l)e expected in such forms as the guinea pig, which is known to ovulate some 10 hours after the onset of heat, or the rabbit which ovulates about 10 hours after copulation. But in women the exact time of ovulation cannot be determined with sufficient accuracy to permit a precise statement as to the duration of fertilizing capacity of the spermatozoa. The relatively long survival time reported for the mare may reflect a kind of thermal adaptation of the spermatozoa, because in the stallion the testicles are carried in shallow scrotal sacs, the temperature of which is probably close to that of the body.

Hil)ernating mammals which copulate in the autumn often show excessively long peI'iods of sperm survival in the female (Hartman, 1933). In bats of the genera Myotis and Eptcsicus, the spermatozoa inseminated in the fall are capable of motility and of fertilization at the time of ovulation in the spring (Wimsatt, 1942, 1944), even though subseciuent copulations may occur in nature during the spring mating season (Pearson, Koford and Pearson, 1952). Long-range sperm survival is, of course, well known in various poikilothermic animals, including arthropods and lower chordates (see Hartman, 1939). Custodians of reptiles, particularly, have recorded interesting breeding data relevant to the longevity of sperm in the female. Fertile eggs have been laid by the diamond-back terrapin and various snakes, 4 to 5 years after isolation; due to the unlikelihood of delayed development, this indicates sperm survival for periods of several years (Barney, 1922; Haines, 1940; Carson, 1945).

Some attention has been directed toward the possible deleterious effect of the aging of sperm in the female tract ; although still capable of fertilization, they might give rise to abnormal or nonviable embryos (Austin and Bishop, 1957). This change with senescence has been well established in fowl (Crew, 1926; Nalbandov and Card, 1943; Van Drimmelen and Oettle, 1949; Dharmarajan, 1950), and might be expected to occur in mammals; the evidence, however, does not support it. Young's early data


( 1931 ) indicated that guinea pig sperm, aged in the male tract, could lead to an increase in the percentage of abnormal embryos ; but no such "overaging" effect was demonstrated in sperm maintained in the female tract (Soderwall and Young, 1940; Soderwall and Blandau, 1941).

Somewhat more recently, another type of sperm behavior was discovered which involves the capacity for fertilization (Austin, 1951; Chang, 1951b). This concerns not the maximal limit of survival, but rather the initial attainment of full fertilizing competency, a continuation, in a sense, of the process of sperm maturation long since begun in the male genital tract. This phenomenon of "capacitation," demonstrated thus far only in rats and rabbits, requires 2 to 6 hours of conditioning of the male gametes and probably involves both physiologic and structural alterations in the cells which enable them to penetrate the zonae pellucidae of the eggs (Austin, 1952; Austin and Braden, 1954; Chang, 1955, 1959). Capacitation is assumed to occur normally in the female genital tract. Under experimental conditions, the injection of rat sperm into the periovarian sac (Austin), or the introduction of rabbit sperm into the Falloi)ian tube (Chang), accomplishes fertilization only after a delay of several hours, unless the sperm have been previously capacitated in another suitable reproductive environment. Such a milieu for rabbit sperm is afforded by the reproductive ducts of female rabbits under a variety of hormonal conditions, and by the reproductive tracts of both immature animals and castrates, with or without the addition of gonadotrophin or estrogen (Chang, 1958). The uteri of pseudopregnant rabbits, however, and those treated with progesterone, were found unsuitable for sperm capacitation. Some doubt has been cast upon the s]iecificity of the factors which bring about sperm conditioning by the demonstration, in the rabbit, that not only does capacitation occur in the uterus and Fallopian tube, but also in such unusual environments as the isolated bladder and colon of either male or female animals and in the anterior chamber of the eye as well (Noyes, Walton and Adams, 1958a, b; Noyes, 1959b).

As to the nature of the changes induced in


740


SPERM, OVA, AND PREGNANCY


the spermatozoa during capacitation, earlier suppositions leaned toward the view that something is lost or gained by the gametes which results in enzyme activation recjuired for fertilization (Austin and Bishop, 1957). It has since been suggested that the change, in rat sperm at least, involves processes leading to the disintegration or loss of the acrosome from the sperm head, thereby exposing structures responsible for egg penetration (Austin and Bishop, 1958a, b). The reversible counteraction of capacitation by rabbit seminal plasma, demonstrated by Chang (1957), casts some doubt, however, on the likelihood of pronounced structural changes occurring during this phase of si:)erm maturation. Until the physiologic changes responsible for the suggested morphologic alterations are clarified, the mechanism of capacitation will remain obscure.

F. DUR.\TION OF SPERM MOTILITY THROUGHOUT THE TRACT

The viability of spermatozoa in the ampulla, assessed by motility, outlasts their fertilizing capacity (Table 13.8). Elsewhere in the tract, motility serves as a criterion for sperm longevity, and considerable variation in the ability of separate segments of the tract to support it has been demonstrated. Rat spermatozoa, for example, survive in the cornua about 12 hours, compared with 16 or 17 hours in the oviducts (White, 1933a) . Sperm motility in the human fundus appears to be less than that in the Fallopian tube (Farris, 1950; Rubenstein, Strauss, Lazarus and Hankin, 1951).

In most mammals the alkaline cervical mucus sustains motility well, whereas the acidic vaginal depository is detrimental. Motile spermatozoa have been reported in human cervical mucus a week after coitus, although the average duration of motility here is closer to 2 days. The duration of motility in cervical mucus varies with the cycle, maximal motility coinciding with the time of ovulation (Beshlebnov, 1938; Cohen and Stein, 1951). Estrogen-induced hypersecretion of mucus is claimed to increase sperm viability as well as penetrability. Longevity in the cervical mucus of the estrous macaque is approximately 24 hours.

The primate vagina is notably inhospita


ble to spermatozoa, presumably because of its high acidity. Motility is sustained in the human vagina rarely longer than 3 to 4 hours (Weisman, 1939), and the duration is believed to vary inversely wdth changes in vaginal acidity (pH 4 to 5). The human vaginal pH, curiously enough, has been claimed to reach a minimum at the time of ovulation, an overt sign, according to Schockaert, Delrue and Ferin (1939), of high estrogenic activity (Fig. 13.11 ). On the other hand, a sharp rise in vaginal pH of approximately 0.5 unit was claimed by Zuck and Duncan (1939) to be coincident with ovulation; this elevation is inconstant and, when it does occur, may be due to the presence of alkaline cervical mucus. Normally, in the cow, the influx of mucus renders the vagina alkaline at estrus (Lardy, Pounden and Phillii)s, 1940). Under normal circumstances, the inseminate is only briefly, if at all, exposed to the vaginal medium. When not ejaculated directly into the cervix or uterus, the semen may be conducted rapidly toward the cervical canal by longitudinal contraction waves (Noyes, Adams and Walton, 1958). It is doubtful, therefore, whether the high hydrogen-ion concentration, characteristic of the vagina, is of any great significance in the reproductive economy of most mammals.

G. SPERM VIABILITY IX RELATION TO TUBAL PHYSIOLOGY

In view of the great wealth of information concerning uterine and tubal transport and sperm survival, on the one hand, and uterine function and hormonal responses, on the other, there has been an appalling lack of interest in the nature of the genital fluids and the immediate environment surrounding the spermatozoa during their sojourn within the female genital tract. The corresponding deficiency of our knowledge of the male genital tract was previously noted. Difficulties in technique exist, to be sure, but they are far from insurmountable, and rich rewards should result from exploration in this virgin, but obviously fertile, field.

A review of the extensive literature on the cytochemistry of the endometrium and tubal epithelium and on the changes with variations in the estrous cycle reveals consider


BIOLOGY OF SPERMATOZOA


741



14 7

DAYS BEFORE NEXT MENSES


Fig. 13.11. Cyclical changes in midvagina 37 normally menstruating women. (After A, Obst. & Gynec, 47, 467-494, 1944.)


I i)H. A\erage values of 632 determinations on E. Rakoff, L. G. Feo and L. Goldstein, Am. J.


able secretory activity (Joel, 1940; Hadek, 19oo, IQoSa; Borell, Gustavson, Nilsson and Westman, 1959; Fredricsson, 1959a, b), but little correlation with the behavior of the gametes within the lumen. When the secretory history of specific substances has l)een followed, the interest has generally l)een in postfertilization stages, as, for example, the mucopolysaccharides released into the oviduct of the rabbit several days after ovulation (Greenwald, 1957; Zachariae, 1958).

On the other hand, several studies of the genital fluids afford some data on pH, oxygen tension, potassium and sodium ratio, enzyme content, and possible metabolic substrates. Warbritton, McKenzie, Berliner and Andrews, (1937) reported that the pH levels of the Fallopian tube, uterine horns, cervix, and vagina of the ewe are, respectively, 6.4 to 7.3, 6.6 to 7.3, 6.1 to 7.5, and 6.5 to 7.8. The wide variations to be noted here are more striking than the actual determinations. More recently, Blandau, Jensen and Rumery (1958) recorded pH values for the fluid of rat periovarian sac, ampullae, and uteri as follows: 7.7. to 8.4, 7.3 to 8.5, and 7.4 to 8.3. There thus appeared little change throughout the tract, but all regions were alkaline with respect to the peritoneal fluid


and blood. These wide variations and the pronounced alkalinity suggest that the loss of carbon dioxide from the fluids may have been responsible for the high pH values reported.

The oxygen tension of rabbit genital fluids has been determined and found adequate to support aerobic respiration (Bishop, 1957). Uterine values, determined by equilibration, range from 25 to 45 mm. Hg (Campbell, 1932 ) . The oxygen tension of Fallopian tubal fluid, measured directly with an oxygen electrode, is approximately 40 mm. Hg (Bishop, 1956b). Birnberg and Gross (1958), however, claimed that changes in the human Fallopian tube during the ovulatory phase render it anaerobic (determined enzymatically) ; if this finding is confirmed, it bears significantly on the anaerobic preferences of hiunan sperm as studied in vitro (see below ) .

Ionic and organic components of the luminal fluids of the cow have been analyzed (Olds and VanDemark, 1957a. b, c; VanDemark, 1958). The data for follicular, tubal, uterine, and vaginal fluids are presented in Table 13.9. Reducing substances, possibly glucose, were found in uterine fluid but were not detected in oviductal fluid. Shih. Kennedy and Huggins (1940) iiave


742


SPERM, OVA, AND PREGNANCY


TABLE 13.9

Composition of bovine genital fluids

(From N. L. VanDemark, Internat. J. Fertil., 3,

220-230, 1958.)


Dry matter (per cent)

Ash (percentage of dry matter)

Sodium (mg. per 100 ml.)

Potassium (mg. per 100 ml.)

Calcium (mg. per 100 ml.)

Total N (gm. per 100 ml.)

Reducing substance (as mg. glucose per 100 ml.)


Source of Fluid


>


2


3

6


2.4


10.0


15.9


41.1


10.3


7.1


170


220


208 .


166


183


223


11


15


12


0.17


1.09



9


50



7.5 9.3 304 36 12 0.96


contributed extensive data concerning the chemical composition of the uterine fluids of the rabbit, rat, and dog (Table 13.10).

An interesting report on potassium and sodium concentrations of uterine fluid in the proestrous rat indicates that K is relatively high (37 niEci./l.) and remains constant after copulation with a vasectomized male; Na decreases, however, by about 11 per cent from the initial value of 115 niEq./l. (Howard and DeFeo, 1959). The shift may be due to the change from follicular to luteal phase, but because of the contributions of the several accessory glands, the significance of the change is not clear. Nonetheless, the high initial K/Na ratio (0.32) suggests a marked K-tolerance on the part of the spemi and, further, a secretory action of the genital mucosa leading to the accumulation of potassium within the lumen.

The paucity of data concerning enzymatic activity by the uterine fluids was indicated by Reynolds (1949). Since that time little has been added, except for two suggestive papers dealing with amylase activity of the tube and its fluids. Human tubal cysts con


tain high concentrations of such an enzyme and have led to the supposition that intraluminal glycogen — if any should exist — might be hydrolyzed to provide a substrate for sperm (Green, 1957). McGeachin, Hargan, Potter and Daus (1958) confirmed the presence of amylase in the cysts and found high activities also in tubal epithelium of man, rabbit, cow, and sheep, but not of other species studied. In an electrophoretic study of the cornual fluids of the estrous rat, low concentrations of 4 major proteid components were found, which appeared to differ in their mobility characteristics from serum proteins (Junge and Blandau, 1958).

It is clear that energy substrates and other biochemical components of seminal plasma are introduced into the tubes in animals in which intrauterine ejaculation occurs (Mann, Polge and Rowson, 1955). However, the significance of these constituents for tubal i)hysiology is highly doubtful after intravaginal insemination. Relatively little glycolytic substrate seems to be present in the fluids recovered from the tract. In the rabbit, for example, little or no hexose, and only traces of phospholipids, can be detected, either before or after cojmlation (Table 13.11) ; lactate is present in appreciable quantities and might conceivably serve as a metabolic substrate (Bishop, 1957; Mastroianni, Winternitz and Lowi, 1958). At the present time, it is not easy to ascertain which metabolic substrates and products are associated with the activities of the spermatozoa and which with the activities of the mucosal cells lining the tract. More work is necessary to fill in the metabolic and physiologic details of the sketch just barely outlined.

Abundant evidence indicates that the tubal contents are a product of active se


TABLE 13.10

Chemical composition of uterine fluids

(From H. E. Shih, J. Kennedy and C. Huggins, Am. J. Physiol., 130, 287-291, 1940.)



H2O


PH


CO2


Total N


NPN


Protein


CI


Na


Ca


K


Glucose


Inorganic P


Rabbit


979 982 984


7.78 7.55 6.09


mmoles

perl.

53.6 61.8 3.0


0.8 1.0 0.8


gm per

0.37 0.29 0.20


gm per

2.7 5.1 3.8


mmoles perl.

98 98 167


mmoles perl.

158 169 162


mmoles perl.

4.7 1.5 3.5


mmoles perl.

6.1 4.3 5.2


mg. perl.

0-160 0-150 0-80


mmoles perl.

0-0.20


Rat




0-0.03




BIOLOGY OF SPERMATOZOA


743


TABLE 13.11

Metabolic substrates in rabbit tubular fluid

(From D. W. Bishop, Internat. J. Fertil.,

2, 11-22, 1957.)


Condition of Animal


Substrate


Glucose


Fructose


Lactate


Phospholipid


Estroiis

Pregnant

Castrate


7of:L

0-2 0-2 0-1


mg. per 100 ml.

<1 <1

<1


mg. per

100 ml.

6.8 15.0 7.5


lo'otl

0-8

Trace



cretion and not merely a transudate from the vascular system or overflow from the peritoneal cavity. The presence of secretory cells in the tubal epithelium is well known; they undergo morphologic and apparent physiologic alterations which parallel changes in ovarian activity (Hadek, 1955, 1958a; Borell, Nilsson, Wersall and Westman, 1956). In the rabbit the secretion is regarded as essential for normal development of the egg (Westman, Jorpes and Widstrom, 1931), and it may be necessary for the normal functioning of spermatozoa and the process of fertilization as well (r/. Whitten, 1957).


The secretory activity of the rabbit Fallopian tube has been investigated and the volume of flow and secretory pressure in singly and doubly ligated tubes determined (Bishop, 1956a). Mean tubal secretion rates in lightly anesthetized estrous, pregnant, and castrate rabbits were 0.79, 0.37, and 0.14 ml. per 24 hours, respectively. Secretory activity was maximal at the time when the spermatozoa are in the ampulla. Active secretion, as opposed to passive diffusion or transudation, was demonstrated by manometric determinations of the pressures developed within a closed tubal system over a 36-hour period. Pressure maxima in estrous, pregnant, and castrate rabbits averaged 46.0, 15.6, and 11.8 cm. HoO, respectively (Fig. 13.12). Both secretory volume and pressure decreased from the 11th to the 21st day of pregnancy. Further indication that tubal secretion is an active process is shown by its sensitivity to pilocarpine; a single injection of 1 mg. of pilocarpine hydrochloride almost doubled the secretory pressure, to a value of 75 to 80 cm. HoO, in estrogen-dominated animals (Fig. 13.13). A program initiated by Clewe and Mastroianni (1959, I960) permits the continuous


cm. HgO



Fig. 13.12. Secretion pressures in rabbit oviducts. A, estrous; B, 14-da.v pregnant; C, 51day castrate, (from D. W. Bishop, Am. J. Physiol., 187, 347-352, 1956a.)


744


SPERM, OVA, AND PREGNANCY



Fig. 13.13. Effect of pilocarpine on tubal secretory pres.sure: right and left oviducts recorded. A, pilocarpine-HCI (1 mg. in 1 ml. saline, I.M.) injected 51/2 hours after catheterization; B, control estrous records. (From D. W. Bishop, Am. J. Physiol., 187, 347-352, 1956a.)

collection of oviduct secretion over a period of many weeks. Their values for secretion rate are somewhat higher than those noted above, for example, 1.29 ml. per 24 hours for the rabbit in estrus.

Although the present state of knowdedge permits only a fragile evaluation of the significance of these secretory products on the activity and viability of the gametes and fertilized eggs, tubal secretion can hardly be denied. Further chemical and physical analysis of the components of the fluids might profitably be attempted, not only in the rabbit and cow, but in other mammals as well. In the final analysis, the survival and fertilizing capacity of the sperm are functions of the relation between the cell's intrinsic properties and the environment in which it operates.

H. THE FATE OF NONFERTILIZING SPERMATOZOA

Relatively soon after insemination, excess sperm have disappeared from the lumen of the genital tract. Within 20 to 24 hours in the mouse and rat, little indication of the sperm mass can be found (Blandau and Odor, 1949; Austin, 1957) . In the sow uterus, a few sperm are present about 50 hours


after copulation, but none can be found 25 hours later (du Mesnil du Buisson and Dauzier, 1955). The general fate of the unsuccessful sperm, recently reviewed by Austin (1957), has long been held to be enzymatic dissolution and phagocytic engulfment in the lumen (Konigstein, 1908; Sol)otti, 1920). Except for a brief spate in the Russian literature (Kushner, 1954; Vojtiskova, 1955), little credence has been given to the many claims of Kohll)rugge ( 1910, 1913) that sperm and sperm products are incorporated into the genital epithelium and have profound effects on the maternal physiology (see Hartman, 1939). Indeed, a subsequent paper by Vojti?kova (1956) and others by Posalaky and colleagues in Prague (1956, 1957a, b) have been quite explicit in stating that the earlier histologic demonstrations of sperm in the epithelial mucosa can be explained on the basis of technical artifacts, principally incurred during the sectioning of tissues. Within the past year or two, however, a number of instances have come to light which make it amply clear that sperm do, under some circumstances, enter or are conducted into the uterine and tubal mucosa. Sperm in, or in association with, leukocytes have been found in the uterine glands of the guinea pig and in the tubal mucosa of other species, including the rat, rabbit, hedgehog, mole, stoat, mouse, and bat (Austin, 1959, 1960; Austin and Bishop, 1959; Edwards and Sirlin, 1959). How commonly this occurs and what its significance may be for the subsequent reproductive capacity of the female remain to be seen. The findings within seven groups of mammals indicate that the phenomenon may be widespread. The association of the spermatozoa with leukocytic infiltration further suggests that the genital tract may, under some circumstances, be regarded as a route of foreign cell invasion. A natural skepticism regarding the ability of spermatozoa to penetrate somatic tissues is somewhat lessened by the realization that the process is a normal feature of reproduction in certain invertebrate animals. Manton (1938) cites records of this among rotifers, turbellarians, leeches, and the bedbug {Cimex) ; in Peripatopsis (Onychophora) , the sperm are described as invading the body


BIOLOGY OF SPERMATOZOA


745


wall at the attachment site of the spermatophore and then passing into vascular channels through which they actively migrate to the ovary where sperm penetration and fertilization occur.

VI. Immunologic Problems Associated with Spermatozoa

A. ANTIGENICITY OF SPERM

The antigenic properties of spermatozoa have been recognized since the turn of the century through the pioneer studies of Landsteiner (1899), Metchnikoff (1899), and Metalnikoff (1900), who, almost simultaneously, discovered that guinea pigs produce antibodies against heterologous and homologous sperm. Landsteiner's work is of classic interest not only because it was barely the first, but also because he used an in vivo method to demonstrate an immune response against sperm. Bull spermatozoa, he found, remain active when injected into the peritoneal cavity of normal guinea pigs, whereas if the pigs have been l^reviously injected parenterally with bull sperm, the peritoneally administered sperm rapidly become immotile. These early discoveries were to be followed by a great wave of interest in sperm antigens, generally assayed by in vitro methods, and attempts to induce sterility in female animals by injection of suspensions of spermatozoa or testicular homogenate. After a lull in activity, interest was rekindled by the development of new immunologic procedures and concepts, and the awareness of the iml^lication of immune processes to problems of fertility and fertility control (Katsh, 1959a; Tyler and Bishop, 1961).

Specific antigens have been demonstrated in, or on, spermatozoa of many mammals, including the rabbit, rat, mouse, guinea pig, dog, ram, bull, and man. The methods used for their determination have generally involved the classical serologic procedures — agglutination, immobilization, precipitin, and complement fixation — and the more recently introduced Oudin and Ouchterlony agar gel-diffusion techniques. The results, in general, indicate a relatively high degree of species-specificity, but some cross-reactivity does occur (Mudd and Mudd, 1929;


Henle, 1938; Smith, 1949a). Tissue-specificity is also incomplete. The AB-blood group antigens, for example, as pointed out previously, are present in human sperm (Landsteiner and Levine, 1926; Gullbring, 1957), and a comparable similarity of sperm-erythrocyte agglutinins has been claimed in cattle (Docton, Ferguson, Lazear and Ely, 1952). Common antigenicity between brain and testicular tissue has been demonstrated (Lewis, 1934; Freund, Lipton and Thompson, 1953; Katsh and Bishop, 1958) and may relate to the mature germ cells themselves.

As routinely determined by means of agglutination or immobilization of fresh sperm in the presence of antisperm serum, the antigenicity of the gametes is customarily attributed to surface moieties and exposed reactive groups. Smith (1949b), however, called attention to the reactivity of the more deeply situated antigenic substances in her study of heterologous reactions among rodent sperm. In part, of course, the masking and unmasking of combining groups are a function of the technical procedures to which the cells are exposed and arc features wliich have to be circumvented or recognized in investigations of this kind. The surface properties of, and "leakage" from, spermatozoa are known to change with storage, dilution, washing, and centrifugation, which, when severe enough (Mann, 1954), can be expected to alter the apparent natural antigenicity of the cells (Smith, 1949b; cf. Pernot, 1956).

The number of antigenic sul)stancL's on the sperm surface is a moot ])oint and may prove merely a matter of definition, if not of semantics, depending on the techniques involved (see Table 3 in Tyler and Bishop, 1961). Henle, Henle and Chambers (1938) localized three distinct antigens in bull sperm by preparing, in rabbits, agglutinating and complement-fixing antibodies against the head and tail fractions. One antigen was found to be head-specific, another tail-specific, and the third common to both head and tail of the intact sperm. On the other hand, when the agar-diffusion method was applied to the study of sperm antigenicity, many reactive substances appeared which seemed to be surface antigens.


746


SPERM, OVA, AND PREGNANCY


In one series of experiments, 7 precipitin bands were observed with washed human sperm tested against rabbit antihuman sperm sermii (Rao and Sadri, 1959). This investigation further indicated that 4 of the sperm antigens were common to seminal plasma, but were not present merely as contaminants. A parallel investigation, employing essentially identical procedures, led to the conclusion that all of the human sperm antigens are also present in plasma and the two materials cannot, immunologically, be distinguished (Weil, Kotsevalov and Wilson, 1956). A similar conclusion grew out of a study of rabbit semen, and the suggestion was made that "the effective antigens found in seminal plasma and spermatozoa of semen appear to originate in the seminal vesicle" (Weil and Finkler, 1958). Because practically all large molecular moieties and cells are potentially antigenic, and because spermatozoa may safely be assumed to arise in the testis, this statement obviously oversimplifies the facts. The point is brought up merely to emphasize the caution that should be exercised in the use of and interpretations derived from various techniques. There are sperm antigenic differences in strains of animals and in individuals within strains, as Snell ( 1944 ) and Landsteiner and Levine (1926) have long since pointed out. More, rather than less, immunologic differentiation will probably be forthcoming in the future. Indeed, Weil (1960) has recently found that the antigenic properties differ in epididymal and seminal sperm of the rabbit; the spermatozoa apparently take up and bind antigenic material from the seminal plasma during ejaculation.

B. SPERM-INDUCED IMMUNE RESPONSES IN THE MALE

Antibodies against spermatozoa, both foreign and those of the same individual, are produced with facility by members of both sexes. Why an animal should so react against autologous antigen, i.e., a male against its own sperm, is not clear. According to the concepts put forward by Burnet and Tenner (1949), Billingham, Brent and Medawar (1955, 1956), and others, an organism undergoes a state of


"recognition" of its own native substances during the tolerant period before antibodies are produced. Thereafter, it does not consider these, or other substances initially introduced during the tolerant period, as foreign. The formation by an adult animal of antibodies against injected autologous spermatozoa, moreover, is generally attributed to the fact that sperm are not normally produced until late in development; thus they have not had a chance to be "recognized" as native and are treated as foreign material when injected. The further supposition must be made that spermatozoa in the testis are somehow normally insulated from the rest of the body, at least from the antibody-forming sites, and therefore fail to evoke antibody production and an immune response. Such speculations are tentative and must await further understanding of the general nature of antigenic stimulation, antibody production, and antigen-antibody complex formation, subjects which are currently undergoing rapid growth and ]icrplexing change (Talmage, 1957, 1959; Lederberg, 1959).

Because antibody production is evoked by autologous sj^erm, the question arises whether auto-immunization occurs and, further, whether it is of any biologic significance. Sperm agglutination occurs in otherwise normal ejaculates of rabbit, bull, and man, and the seminal plasma can be shown to contain agglutinating antibodies (Wilson, 1954; see Tyler and Bishop, 1961). The possibility exists that an antigenic stimulus for antibody formation may arise following sperm absorption or penetration into the epididymal mucosa during a period of inflammation, a process often associated with intense leukocytic infiltration (Mason and Shaver, 1952; Montagna, 1955; King, 1955). In man, such a reaction is claimed to be common after mild epididymal infection; it causes no impairment of testicular function, but produces a tissue response characterized by granulomatous lesions (Steinberg and Straus, 1946; Cronqvist, 1949; King, 1955). Similar lesions have been described in cases of granulomatous orchitis, in which spermatozoa were present in macrophage cells and in the lymphatic system (Friedman and Garske, 1949). Cruickshank


BIOLOGY OF SPERMATOZOA


747


and Stuart-Smith (1959) have recently described circulating antisperm antibodies in men who had previously suffered orchitis. It seems not unlikely, therefore, that certain cases of auto-agglutination of ejaculated sperm may have arisen from some kind of autosensitization and passage of the antibodies into the seminal plasma. If auto-immunization does occur by such means, it is assumed that tubal inflammation or infection must be present to effect the immune reaction; otherwise the condition should be much more common since resorption of nonejaculated sperm from the epididymis seems to be a normal process (see above). The seminal and follicular component, antagglutin, discovered by Lindahl and Kihlstrom (1954), which tends to prevent abnormal clumping of sperm, does not counteract agglutination by prepared antiserum (Lindahl, 1960) ; it seems rather to operate through another, nonserologic type of mechanism.

Bocci and Notarbartolo (1956) suggested that immunologic factors might contribute to a state of sterility on a basis of their finding of positive antisemen skin reactions in some men suspected of infertility.

Rumke (1954) and Riimke and Hellinga ( 1959) made extensive studies of sperm agglutinins in the sera of sterile men. In a series of over 2000 cases, they found a considerably higher incidence of sperm-agglutinating antibodies in the sera of childless men (4.1 per cent) than in those of normal fertile controls (1.0 per cent). Among a small group of 21 relatively aspermic patients, all of whose sera had sperm agglutinins, 16 showed occlusions or obstructions of the male tract. In the light of these demonstrations, the suggestion may be ventured that auto-immunization occurs in the male, that the mechanism may result from spermatozoal reactions involving the tubal epithelium, and that the antibodies produced may impair fertility. To what extent, if any, variations in androgen levels modify the epididymal reactivity in this regard is completely unknown.

An unusual syndrome, aspermatogenesis, can be readily induced in the guinea pig by injecting homologous spermatozoa or homogenized testis combined with adjuvant


(Freund, Lipton and Thompson, 1953; Freund, Thompson and Lipton, 1955;'.'Katsh and Bishop, 1958; Tyler and Bishop, 1961). The immune response is due to a delayed sensitization and is apparently not associated with the high levels of circulating antisperm antibodies which can be detected by such methods as sperm agglutination, immobilization, and complement-fixation (Freund, 1957; Katsh and Bishop, 1958). The testicular lesion, as observed 1 to 2 months after injection, is characterized by loss of germinal epithelium and decrease in gonadal weight and volume (Fig. 13.14). The Sertoli elements are affected very little, if at all. The interstitial tissue remains functional, as judged by the normal size and activity of the accessory glands. Since the induction of aspermatogenesis by the injection of spermatozoa has been established only in the guinea pig and rat, the implications for reproductive physiology may be limited; its occurrence and the possible mechanism, however, are of substantial importance to the general areas of delayed sensitization and the immune response (Katsh, 1958, 1959c; Voisin, Toullet and Mauer, 1958).

In contrast to these investigations of active immunization with sperm, the introduction of antisperm serum into male animals has been shown to affect fertility in a limited number of instances. Mice and rabbits both show reproductive impairment after injection of homologous antibody serum (de Leslie, 1901; Guyer, 1922). In rats, a considerable weight loss (24 per cent) of the testes is accompanied by sloughing of germinal epithelium after injection of rat sperm antiserum produced in the rabbit (Segal, 1961). The testicular reaction appears to be a specific response against the homologous sperm.

C. SPERM-INDUCED IMMUNE RESPONSES IN THE FEMALE

The memorable statement of Charles Darwin (1871) that the diminution of fertility may be explained in some cases by the profligacy of the women" may be taken to imply a sensitization against the male reproductive products, although another not unlikely explanation may involve the im


748 SPERM, OVA, AND PREGNANCY




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Fig. 13.14. Aspermatogenesis induced in the guinea pig by injection of testicular homogenate and adjuvant. A, normal adult guinea pig testis used as donor, approx. 65 X, B, same, approx. 260 X ; C, testis of semicastrate 2 months after injection of autologous testicular homogenate, 65 X ; £>, same, 260 X, note normal interstitial tissue; E, testis of guinea p-g injected at 1 week and sacrificed at 5 months of age, 65 X ,: F, same, 260 X. (From D. W. Bishop, unpublished photographs.)


BIOLOGY OF SPERMATOZOA


749


paired health of the subjects. Three decades after Darwin, the immunization of laboratory animals against spermatozoa suggested a mechanism by which sensitization could come about. During the following half century the pros and cons of this issue were to rage. The parenteral introduction of either homologous or heterologous spermatozoa was early claimed by many workers to induce some degree of female sterility in a wide variety of animals (see Parkes, 1944; Tyler and Bishop, 1961). Some experiments seemed so successful that a patent was once granted for an antisterility preparation based on this procedure (Baskin, 1937). Such experiments, however, are beset with difficulties of control and natural biologic variation. It is not surprising, therefore, that more recent investigations have tended to discredit the earlier reports of sterility induced by sperm injection, and to provide adequate explanation for many of the apl)arent positive results (Eastman, Guttmacher and Stewart, 1939; Hartman, 1939; Henle and Henle, 1940; Lamoreux, 1940). The ancient role of spermotoxins in inducing female sterility seemed thus to be laid at rest.

The issue was again raised with the ad\ent of adjuvants which have the ability of potentiating the effect of an antigenic stimulus. Quite recently, evidence has accrued indicating that reproductive capacity may indeed be impaired in female rabbits and guinea pigs when they are injected with sjierm or testis homogenate combined with adjuvant (Katsh and Bishoj), 1958; Isojima, (h-aham and Graham, 1959; Katsh, 1959b). In treated guinea pigs, the fertility (number bearing litters) was reduced to 24 per cent compared with 84 per cent for the controls. The rate of fetal death and resorption was high, but there seems to have been little effect on ovulation or fertilization. High titers of circulating antisperm antibodies were present, but their connection with the decreases in fertility is not clear. One reasonable explanation for the occurrence of these induced effects on reproductive capacity was suggested by Katsh (1957), who attributed the fetal loss to a possible anaphylactoid response of the uterus to foreign antigen. Other plausible mechanisms may involve the gametes or develoi)ing embryos


directly; circulating antibodies can pass into the uterine and tubal fluids and might impair development (McCartney, 1923).

These recent results, then, not only give some credence to the early claims for induced sterility, but also raise the question as to the possibility of naturally acquired sensitization in breeding females. Little direct evidence can be cited in support of such a hypothesis since only fragmentary immunologic studies have been made which indi'-.'ate sensitization or antisperm antibody titers in the sera of animals not previously inoculated (see Tyler and Bishop, 1961). However, in a series of over 200 women, Ardelt (1933) found a positive correlation between frequency of coitus and complement-fixation titer against human spermatozoa. Studies of this sort on various species should ]irove rewarding.

Whereas the evidence concerning the degree of sensitization of the female is scant, the means by which antigenic stimulation might occur seems adequate. The penetration of the tubal epithelium by sperm has been noted ; under some circumstances, this phenomenon may be relatively common, as when mild infections or lesions occur within the tubal mucosa. Another possible site of antigenic stimulation, particularly in animals like the rabbit, is the peritoneum, for not only do sperm pass through the tract and enter the body cavity (Hartman, 1939; Home and Audet, 1958) , but the peritoneum is an adequate site for antibody formation. Furthermore, repeated deposition of spermatozoa into the rabbit vagina results in high titers of circulating antisperm antibodies (Pommerenke, 1928). A comparable situation has also been demonstrated in heifers in which genetically tagged erythrocytes, rather than sperm, were introduced into the intra-uterine cavity, with the result that specific antibodies subsequently^ appeared in the blood (Kiddy, Stone, Tyler and Casida, 1959). These results have been interpreted as demonstrating the passage of antigen into the circulation where access is gained to the sites of antibody formation ; it is to be noted, however, that the tissues of the reproductive tract itself do on occasion produce antibodies (Kerr and Robertson, 1953). It is worth pointing out that, in other experiments, antibodies, rather than


750


SPERM, OVA, AND PREGNANCY


antigens, seem to be transported across the genital epithelium, or to migrate by way of the peritoneal cavity. Parsons and Hyde (1940), for example, fomid circulating antibodies after introducing antisperm serum into the vaginas of rabbits, and McCartney (1923) claimed that circulating antibodies, actively produced in rats against sperm, could be detected in the uterine and vagmal fluids. Antibodies are known, of course, to pass into the uterine lumen of rabbits during pregnancy (Brambell, Hemmings and Henderson, 1951).

Very little has been attempted in altering the fertility of female animals by means of passive immunization with spermatozoa, perhaps because the outstanding investigation of Henle, Henle, Church and Foster (1940) was so conclusive. Repeated injection of mice with antisperm serum, produced in rabbits, failed to modify reproductive capacity in any significant way.

The treatment of fresh sperm with specific antisperm serum has profound effects on the gametes, the basis, in fact, of the sperm-agglutination and sperm-immobilization test methods. The treatment generally renders sperm, both invertebrate and vertebrate, incapable of fertilizing eggs (Godlewski, 1926; Tyler, 1948; Kiddy, Stone and Casida, 1959). A significant contribution, moreover, has been the recent demonstration that if the exposure to antiserum is carefully controlled, surprising and subtle effects may occur when these sperm are used for artificial insemination. Rabbit sperm, treated for 15 minutes with high concentrations of bovine antirabbit antiserum before insemination, were incapable of effecting fertilization, as judged from the recovery of unfertilized ova. However, a 15-minute exposure of sperm to the same, but diluted, immune serum permitted fertilization, but resulted in a high percentage of embryonic deaths (Kiddy, Stone and Casida, 1959). No such fetal wastage occurred when rabbit sperm were similarly exposed to normal bovine serum. The antisera employed in these experiments were prepared against whole semen, rather than against washed sperm, but any additional antigenic components in plasma would not be expected to have altered the results. Various inter


pretations can be placed on these findings, including the possibility that the fertilizing sperm might have carried antibodies into the egg which impaired development, or, an alternative possibility, that the antibodies had a mutagenic action on the spermatozoa leading to abnormal development after fertilization (Kiddy, Stone and Casida, 1959). There seemed to be no injurious effect on the sperm that resulted in delayed fertilization; thus the effects cannot be attributed to aging of the ova.

An immunologic mechanism has been implicated by Gershowitz, Behrman and Neel (1958) to account for the variations from the expected ratio of offspring of couples with incompatible ABO-blood groups. These investigators found hemagglutinins in the cervical mucus of 17 out of 77 cases so distributed that they might be regarded as constituting a preconceptive selection mechanism by blood group antibodies of the uterine secretions acting on the sperm.

In conclusion, a brief survey of the immunologic literature relating to fertility indicates that spermatozoa may be deeply involved in both experimentally and naturally induced modifications in reproductive performance and capacity. Other immunelike interrcactions between specific substances, fertilizin and antifertilizin, extracted from invertebrate eggs and sperm, also have been demonstrated; the possible role of these reactions in the fertilization process is discussed in the following chapter.

VII. Morphology and Composition of Spermatozoa

A. STRUCTURAL FEATURES

As one of the first objects to be viewed microscopically (van Leeuwenhoek, 1678) , the spermatozoon has had a long morphologic history,- and still enjoys great popularity, particularly among cytochemists and electron microscopists. No exhaustive item ^Reimer Kohnz (1958) calls attention to a recent "find" in the library of the Cologne Cathedral which, if genuine, would shed revolutionary light on the history of microscopic science. A manuscript, purported to have been illuminated by monks of the Reichenau Monastery ca. 1000 A.D., is interpreted as showing an egg with eight spermatozoa attached !


BIOLOGY OF SPERMATOZOA


751


ization of sperm morphology is intended here, and even less is necessary by virtue of many extensive surveys which, over the years, have reviewed and collated the literature of the times, in the light of contemporary interests and in relation to other areas of biologic progress (Retzius, 1909; Wilson, 1925; Bradfield, 1955; Hughes, 1955, 1956; Franzen, 1956; Nath, 1956; Colwin and Colwin, 1957; Bishop and Austin, 1957; Anberg, 1957; Fawcett, 1958; Schultz-Larsen, 1958; Bishop, 1961). The two historical surveys of Hughes (1955, 1956) are of particular interest to anyone mindful of the past.

Wilson (1925), among others, drew attention to the great variation in animal sperm, including the existence of nonflagellated and nonmotile gametes among certain invertebrate groups. More recently, Franzen (1956), in an admirable survey of many kinds of invertebrate spermatozoa, has emphasized what he believes is a significant correlation between sperm morphology and physiologic demands of the particular type of reproductive process concerned. Considerable attention has been paid to sperm size, from the small, microscopic sea-ui'chin gamete, some 40 /j. long, to the relatively gigantic sperm of the hemipteran insect, Notonecta glauca, which is reputed to be about 12 mm. in length (Pantel and de Sinety, 1906; Gray, 1955). The claim was once current that, because of the difference in chromosome number, a sperm population displays a bimodal size-distribution curve, but careful biometric studies by van Duijn ( 1958) and others have shown this to be untenable. More recently, differences in size and shape of sperm have been demonstrated in different inbred strains of mice ; the characteristics seem to be genetically determined and, when intermingled, lead to extreme variation in hybrid crosses (Braden, 1959).

Gravimetric, interferometric, and refractometric methods have been applied to the study of sperm in an analysis of their physical properties. By such procedures, one can determine that bull sperm have a relative density of 1.280 (Lindahl and Kihlstrom, 1952), a dry mass averaging 7.1 X 10~^ mg. (Leuchtenberger, Murmanis, Murmanis, Ito and Weir, 1956), and a total


weight of about 2.86 X 10~^ mg. (see Bishop, 1961). Human sperm contain at least 45 per cent "solid material" in the head, and possibly 50 per cent "solids" in the tail, as assessed by the method of immersion refractometry (Barer, Ross and Tkaczyk, 1953; Barer, 1956).

Cytochemical procedures, frequently combined with extraction procedures, have proved useful in the investigation of sperm composition, particularly in tracing the differentiation of cellular elements, such as the Golgi apparatus and Nebenkern, through spermiogenesis, and in identifying the chemical nature of various structures in the mature gamete. By means of PAS-positive tests for 1 ,2-glycol groups, for example, the acrosome was found to consist of polysaccharide associated with some proteinaceous material, complexed possibly as mucopolysaccharide (Schrader and Leuchtenberger, 1951 ; Leblond and Clermont, 1952; Clermont and Leblond, 1955). Further, on extraction and hydrolysis, this material from guinea pig sperm proved to contain galactose, mannose, fucose, and hexosamine (Clermont, Glegg and Leblond, 1955). These are precisely the same coml)onents found by Bergman and Werner (1950) in carbohydrate hydrolysates of human cervical mucus (see above).

The electron micrographic studies of spermatozoa, of which there have been a great number, are well summarized by Anberg's fine treatise (1957) on human sperm and Fawcett's eloquent review (1958) of mammalian sperm in general (Fig. 13.15). Fawcett makes the historic point that in some instances the electron micrograph has confirmed details wdiich theoretically should be invisible with the light microscope, but were seen and described, nevertheless, by an earlier generation of able microscopists — the enumeration, for example, of the 11 tail filaments of the fowl sperm by Ballowitz in 1888. But many other features have been discovered by electi'on microscopy. The postnuclear cap and cytoplasmic sheath, previously described as parts of the human sperm' head, apparently do not exist (Fawcett, 1958). The acrosome system of the human sperm is less discrete than that observed in other types of gametes. The nu


752



SPERM,


OVA,


AND PREGNANCY





■B


Grnrsuie



i


E


^





I'K., 1:M.'i KliriMiii 1. 1,-1 I, M~ of mammalian >i>riiii. A.V> >tagcs m tlic lorniation of the

Lead cap of the humau sperm. B, late spermatids of the eat (i) and guinea pig {2) in roughly longitudinal section; note approximation of axial filament to centriole. C, principal piece of guinea pig sperm tail ; note that each peripheral doublet appears as one tubular and one solid element: 7 outermost fibers are present at this level (see Fig. 13.175). D, terminal region of human sperm tail; the doublets appear as hollow cylinders. E, midpiece of human sperm ; the electron-dense outermost array of filaments is surrounded by many mitochondrial bodies. {A and B from D. W. Fawcett, Internat. Rev. Cytol., 7, 195-234. 1958; C, courtesy of D. W. Fawcett; D and E from A. Anberg, Acta ob.st. et gvnec. scandinav., Sup])l. 2, 36, 1-133, 1957.)


BIOLOGY OF SPERMATOZOA


753


cleus, instead of occupying only the posterior portion, seems rather to extend the entire length of the head (cf. Bishop and Austin, 19571. During differentiation, the nuclear cln-omatin condenses into a homogeneous, electron-dense mass, but Yasuzumi, Fujinmra, Tanaka, Ishida and Masuda (19561 demonstrated in enzymatically treated bull sperm helical strands which may correspond to distinct chromosomes. During spermiogenesis in the guinea pig the four spermatids resulting from meiosis remain attached by intercellular bridges until late in the development of the gametes (Fawcett, 1959). Such connections may allow for significant interchange of materials and for mutual interaction among the members of the tetrad.

Electron microscopy has confirmed the traditional view that there are two centrioles present in the neck region of the sperm which are directly or indirectly associated with the axillary bundle extending into the flagellum (Fawcett, 1958). The homology of the centriolar body with the basal granule (blepharoplast) is assumed.

The spiral body, typical of the middle piece of the sperm, is made up principally of the mitochondrial elements, arranged spirally but not in a continuous helix. The distribution of the mitochondria, constituting in large measure the "power plant" of the cell by reason of their oxidative and phosphorylative activities, is in close association with the flagellar apparatus, particularly the fibrillar elements of the tail. The mitochondrial system is derived from or related to the Nebenkern, a prominent cell inclusion in spermatids of lower forms. In some insect sperm, in the absence of a true midpiece, the mitochondria extend far down into the flagellum (Rothschild, 1955). What had been considered the helical covering of the sperm tail might better be regarded as a "fibrous sheath" since the structure is neither continuous nor constituted of uniform successive gyres (Fawcett, 1958). The outer membrane, probably the true physiologic surface of the cell, is a continuous envelope and is apparently derived from the spermatid cell membrane.

Emanating from electron micrographic investigations, a universal fibrillar pattern in


flagella and cilia is generally acknowledgeu. ]\lodifications exist but incontestable evidence indicates that the basic arrangement, as seen in transverse sections, is the now familiar 2 X 9 -t- 2 array. Surrounding 2 central filaments is a ring of 9 double fibrils (Figs. 13.16, 13.17^1, B), all of which seem to extend, uninterrupted, from proximal to distal tip of the flagellum. On extensive, but nevertheless largely circumstantial evidence, the outer filaments are generally regarded as the motile organelles. Inoue (1959), however, in summarizing the evidence pertinent to ciliary movement, suggests that the outer fibrils may actually be conductile elements, whereas the two central filaments take a more active part in motility. Certain other features of the sperm tail, including the



Fig. 13.16. Electron micrographs of fowl sperm flagella. Of the 11 major filaments, two (M fibrils) are differentiated from the remainder and constitute the central pair. Sperm were exposed to distilled water, fixed in formalin, and shadow-cast with platinum. (From G. W. Grigg and A. J. Hodge, Australian J. Scient. Res., ser. B, 2, 271-286, 1949.)


754


SPERM, OVA, AND PREGNANCY


chemical nature of these longitudinal filaments, their proximal association with yet another array of 9 peripheral fibers, their relation to the matrix of the flagellum, and their relation to one another, are described elsewhere in considerable detail (Bishop, 1961 ) . As a general conclusion, the three main divisions of sperm into head, middle piece, and tail correspond roughly to their genetic, metabolic, and motile functions.

B. BIOCHEMICAL FEATURES

The availability and homogeneity of spermatozoa have long appealed to the biochemist in choosing a cell tyjie for study. Both chemical and histochemical methods have been employed in investigations of the composition of sperm, and recent developments in quantitative cytochemistry show good agreement in the results obtained by the two general procedures. Complete analyses of the chemical components of several types of spermatozoa are now available and include the full range of substances from ions to enzymes, many of which have been roughly localized within the major regions of the cells. For more extensive treatment concern




FiG. 13.17^. Highly diagrammatic representation of transverse sections of sperm flagellum and cilium ; 2 central and 9 double peripheral fibrils typical of all such motile organelles. Mitochondria (oblique hatching) present in midpiece. An additional array of 9 outermost filaments (solid) in the midpiece of mammalian sperm extends into the proximal portion of the flagellum. The fibrous sheath of the tail is frequently ribbed as indicated.




Fig. 13.17fi. Diagram of rat sperm tail at various levels from midpiece (A) to tip (G). Note bilateral symmetry of fibrillar arrangement and termination of outer longitudinal fibers at different levels of flagellum. (Courtesy of D. W. Fawcett.)

ing the functional composition of sperm, the reader is referred to several reviews (Marza, 1930; van Duijn, 1954; :Mann, 1954; Bishop, 1961 ) ; only selected features of the voluminous literature will be noted here.

Just short of a century ago, Miescher, and later Kossel, and their co-workers took up the study of the basic proteins — protamines and histones — of fish sperm nuclei, easily procurable by plasmolysis of the cytoplasm and collection of the heads by centrifugation. Progress was rapid and by the 1920's more was known, it was claimed, about the chemistry of the spermatozoon than about any other cell (Marshall, 1922). These early studies have expanded into investigations of the basic proteins as conjugates with desoxyribonucleic acid (DNA), and particular attention has been directed toward the significant and systematic changes from the histone- to the protamine-type protein during sperm differentiation (Miescher, 1897; Kossel, 1928; Mirsky and Pollister, 1942;


BIOLOGY OF SPERMATOZOA


755


Pollister and Mirsky, 1946; Stedman and Stednian, 1951 ; Felix, Fischer, Krekels and Mohr, 1951 ; Bernstein and Alazia, 1953a, b; Alfert, 1956; Vendrely, Knobloch and Vendrely, 1957; Ando and Hashimoto, 1958; Felix, 1958). Histone is regarded as typical of somatic chromosomes, whereas protamines characterize the nuclei of mature sperm (Daly, Mirsky and Ris, 1951). The two types of basic proteins differ in their solubility and physical properties and in their chemical composition as well; protamines are found to have fewer amino acids when compared to histones from the same animal (Daly, Mirsky and Ris, 1951 ) . Both are very rich in arginine. This polyamino acid is reported to constitute some 70 per cent of the protamine, "gallin," of fowl sperm (Fischer and Kreuzer, 1953), and about 50 per cent and 30 per cent, respectively, of the solid matter of bovine and human sperm nuclei ( Leuchtenberger and Leuchtenberger, 1958). Total amino acid composition and other chemical characteristics of sperm nuclcoprotein have been reported on numerous occasions (see Sarkar, Luecke and Duncan, 1957; Daly, Mirsky and Ris, 1951; Porter, Shankman and Melampy, 1951 ; Dallam and Thomas, 1953) .

Sperm DNA has been isolated from a variety of species and its nucleotide composition determined (Chargaff, Zamenhof and Green, 1950; Chargaff, 1951; Chargaff, Lipshitz. Green and Hodes, 1951 ; Elmes, Smith and White, 1952). According to Elmes, Smith, and White, the purine and pyrimidine bases of human sperm — guanine, adenine, cytosine, and thymine — are present in the molar ratio of 0.92:1.23:0.84:1.01, which is consistent with the "thymus-type" composition of nucleic acid. The absolute amount of DNA ])er sperm nucleus is measurable, both by direct chemical analysis and by ultraviolet microspectrophotometry (Vendrely and Vendrely, 1948, 1949, 1953; Mirsky and Ris, 1949, 1951 ; Leuchtenberger, Leuchtenberger, Vendrely and Vendrely, 1952; Walker, 1956; Knobloch, Vendrely and Vendrely, 1957; Leuchtenberger and Leuchtenberger, 1958). Bull sperm contain approximately 3.3 X 10-^ mg. of DNA per nucleus. Of particular significance was the Vendrelys' (1948) demonstration that the sperm nu


cleus contains half as much DNA as does the diploid nucleus of the corresponding somatic cell, thereby giving strong support to the theory that DNA is identical with the substance responsible for hereditary transmission. In a recent study of the sperm of bull and man, the Leuchtenbergers (1958) indicated that, whereas the amount of DNA is constant in gametes from fertile individuals, there is a tendency for DNA deficiency in the sperm from infertile individuals (see also Weir and Leuchtenberger, 1957). This finding is surely of great significance but its cause and meaning are at present obscure.

The amount of ribonucleic acid (RNA) in sperm nuclei is small, but sufficiently large to be detected. Leuchtenberger, Leuchtenberger, Vendrely and Vendrely (1952) gave a value for bull sperm of about 0.1 X 10~® mg. of RNA per nucleus.

C. THE LOCALIZATION OF ENZYMES

The mammalian spermatozoon has a full spectrum of enzymes which enables it to carry on the usual glycolytic and oxidative processes associated with the production of energy (Mann, 1954). In addition, there are relatively specific enzyme systems associated with movement, others related to fertilization, and still others (e.g., amino acid oxidase) possibly concerned with modification of the substrate wdth which the sperm come in contact. Some of these enzymes have been tentatively localized in specific regions of the sperm, thereby shedding some light on the intracellular activities of the gametes and their constituent structures.

Since both mechanically separated and naturally ejaculated sperm tails, free from the heads, are capable of motility, oxidation, and glycolysis, it is obvious that the key enzyme systems concerned with these processes are relatively self-contained within the flagcllum (Engelmann, 1898; Cody, 1925; Mann, 1958a). As used here, the term flagellum includes the mitochondria-containing middle piece, for without it the tail fragment rapidly loses its capacity for metabolism and motility (Bishop, 1961). The enzymes wdiich have, by direct or indirect means, been identified in the ram sperm fia


756


SPERM, OVA, AND PREGNANCY


gellum and are known to be involved in the Embden-Meyerhof glycolytic process, include hexokinase, phosphohexoisomerase, phosphohexokinase, aldolase, enolase, and lactic dehydrogenase (Mann, 1949, 1954). Cytochrome oxidase, determined both manometrically (Zittle and Zitin, 1942) and spectrophotometrically (Nelson, 1955a), is present in the tail fraction of bull sperm, and the complete cytochrome system can be demonstrated in flagellar preparations which include the midpieces as well (Mann, 1954). From what is known about mitochondrial activity in general, one assumes that most, if not all, of the enzyme systems associated with respiration, oxidative phosphorylation, and electron transport through the cytochrome system are concentrated in the sperm midpiece. Succinic dehydrogenase can be demonstrated in flagellar fractions both by biochemical and cytochemical methods (Mann, 1954; Nelson, 1955a; Kothare and De Souza, 1957). Nelson (1959) has further been able to show in frozen-dried sections of the rat sperm flagellum what seems to be succinic dehydrogenase activity in the outermost longitudinal fibers of the tail.

The sperm flagellum, at least in man and bull, when tested cytochemically, gives positive reactions for acid phosphatase, and the bull sperm tail shows alkaline phos])hatase activity as well (Wislocki, 1950; ]\lelampy, Cavazos and Porter, 1952). Both types of phosphatase have been cytochemically localized in the midpiece of the rat sperm (Friedlaender and Fraser, 1952; Melampy, Cavazos and Porter, 1952). The precise functions, however, of these enzymes in the sperm are not clear.

One or more adenosinetriphosphatases (ATPases) have been extracted from or demonstrated in the flagella of invertebrate and mammalian spermatozoa (Felix, Fischer, Krekels and Mohr, 1951; Nelson, 1954, 1955b; Engelhardt and Burnasheva, 1957; Burnasheva, 1958; Hoffmann-Berling, 1955; Bishop and Hoffmann-Berling, 1959). In frozen-dried sections of rat sperm flagella, ATPase has presumably been visualized in association with the outermost array of fibrils (Nelson, 1958a).

In the head of the mammalian sperm, only acid and alkaline phosphatases have


been reported and these determinations were achieved by cytochemical localization (Wislocki, 1949, 1950; Melampy, Cavazos and Porter, 1952; Friedlaender and Fraser, 1952).

Thus far, no enzymes have been identified in the mammalian sperm head which compare with the invertebrate sperm lysins, believed to play some role in egg penetration (Tyler, 1948). Hyaluronidase, which effectively disperses the cumulus cell mass around mammalian ova, is present on the sperm but has not been localized in any one region. Buruiana (1956) found that hyaluronidase activity is common to mammalian sperm, whereas trypsin activity is characteristic of bird sperm; of the species studied, only the rabbit sperm showed both types of enzymatic activity. Amylase has been demonstrated in bull sperm, but because of the violence of the extraction procedure, little is known as to its site of action (Lundblad and Hultin, 1952). Other enzymatic activities have been found in intact sperm or cell homogenates, such as aconitase in bull (Lardy and Phillips, 1945; Humphrey and Mann, 1948), cholinesterases in boar and guinea pig (Sekine, 1951; Sekine, Kondo and Saito, 1954; Grieten, 1956), and glycosidases and sorbitol dehydrogenase in ram (Conchie and Mann, 1957; King and Mann, 1958). Sorbitol deiiydrogenase may serve to convert the seminal plasma constituent, sorbitol, to fructose, a normal metabolic substrate for spermatozoa.

D. THE SPERM SURFACE

As far as can be determined from electron micrographs, the sperm cell membrane is identical with, or at least derived from, the spermatid membrane. In the mature ram sperm, as in many invertebrate sperm, the membrane was claimed to swell osmotically in response to hypotonic changes in the medium (Green, 1940). This is not true of bull sperm (Rothschild, 1959) ; in fact most mammalian sperm are resistant or indifferent to osmotic changes (Emmens, 1948; Pursley and Herman, 1950; Blackshavv, 1953a, b; M. W. H. Bishop, 1955). This feature is in contrast to the selective permeability with respect to many organic molecules, both charged and uncharged


BIOLOGY OF SPERMATOZOA


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(Mann, 1954). Rothschild (1959) investigated the anaerobic heat production in buffered suspensions of bull semen under various anisotonic conditions and found that an initial shock reaction, marked by reduced heat production and metabolic activity, was followed by gradual recovery or adaptation which in some cases was complete. Such adaptation seems particularly characteristic of bull sperm but the nature of the osmotic regulation is not entirely clear. Under severely unfavorable conditions the ])ermeability of ram and bull sperm is so altered as to permit the apparent leakage of large molecules such as cytochrome c (Mann, 1951a, 1954). Pronounced changes in permeal)ility accompany the phenomenon known as "cold-shock" (Mann and LutwakMann, 1955).

Chemical analyses of ram and bull sperm by Green (1940), Zittle and O'Dell (1941), and others indicate that the surface membrane contains lipid, probably bound as phosjiholipoprotein; the lipid-free meml)rane is high in nitrogen and cystine and bears a superficial resemblance to keratin (Mann, 1954). The toughness and the elastic properties of human sperm actually have been ciualitatively determined by dexterous microdissection technique (Moench, 1929).

The sperm surface at physiologic ionic strength and pH bears a negative charge which has been claimed to be higher on the tail than on the head (Joel, Katchalsky, Kedem and Sternberg, 1951 ) . The gametes thus tend to migrate electrophoretically toward the anode. According to Machowka and Schegaloff (1935), this movement is counteracted, at certain field strengths, by a galvanotropic tendency to swim actively toward the cathode. The negative charge on the sperm surface may be attributable to phosphate, carboxyl, and/or sulfate groups attached to organic components of the membrane.

Several attempts have been made to utilize the electrophoretic properties of sperm in order to separate X- and Y-bearing gametes. Schroder (1940a, b, 1941a, b, 1944) , in an interesting and apparently careful series of investigations, claimed to have accomplished this with rabbit sperm ; the two types of gametes thus separated, when arti


ficially inseminated into does, gave predominantly (78 to 80 per cent) male or female offspring. More recent work by Gordon (1957) suggests concurrence in these findings, but both the technicjue employed and the conclusions derived indicate the need for further confirmation. If such electrophoretic separation of the two cytogenetically distinct types of sperm is possible, it would be of interest to ascertain the reason for the behavior, whether, for example, the maleand female-producing gametes carry different ^-i:)otentials or otherwise vary in surface composition. Schroder's studies did indeed indicate that the electrophoretic response might be attributable to differences in the comjionents of the lipoprotein sheaths of the two tyi)es of spermatozoa.

VIII. Sperm Metabolism

A. SOURCES OF ENERGY

In biochemical investigations of spermatozoa the focus of attention has been on the metabolic processes associated with the production of chemical energy required for motility. Although the sperm of relatively few species have been extensively explored, a fairly consistent pattern of metabolic activities has been established. Mammalian sperm, in general, display extensive glycolytic activity under both aerobic and anaerobic conditions, and carry on oxidative respiration when conditions are appropriate (Mann, 1954). Invertebrate spermatozoa, on the other hand, rely almost entirely on oxidative processes and show little, if any, glycolysis (Rothschild, 1951a). Regardless, however, of the nature of the substrate and the pattern of metabolism, the importance of the chemical conversions lies in the coupling of these exergonic reactions with the synthesis of ATP as a utilizable source of chemical energy for the performance of work (Lardy, Hansen and Phillips, 1945; Lehninger, 1955, 1959). In active spermatozoa much of this energy source is consumed by the processes underlying motility ; an unknown fraction may be utilized in other activities, including possible synthetic processes, conduction, and membrane transport.

In mammalian spermatozoa, anaerobic glycolysis supplies sufficient ATP energy to


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SPERM. OVA, AND PREGNANCY


support motility for long periods of time; however, respiratory processes coupled with oxidative phosphorylation are far more efficient and can be assumed to furnish sperm, as other tissues, 8 to 10 times as much ATP for the same amount of initial substrate degraded (for general discussion, see Lehninger, 1955; Slater, 1958). Sperm motility is sustained so long as a minimal concentration of intracellular ATP persists ; with the exhaustion of ATP, motility ceases (Engelhardt, 1945; Lardy, Hansen, and Phillips, 1945). In 1945, Lardy and Phillips suggested the presence of ATP in bull sperm and Mann ( 1945) succeeded in isolating from ram sperm the nucleotide, as the barium salt, and characterizing it as ATP. Soon thereafter it was shown to be functionally identical with ATP isolated from muscle (Ivanov, Kassavina and Fomenko, 1946). ATP has since been extracted from sperm of the sea urchin. Echinus esculentus (Rothschild and Mann, 1950). A considerable body of evidence has suggested that phosphagen is present in mammalian sperm which might serve as a phosphorus donor for the reconstitution of ATP from adenosine diphosphate (ADP) (see Bishop, 1961, for review) ; recently, however. White and Griffiths (1958) re-examined the problem and failed to find any significant amount of creatine phosphate or the enzyme which might take part in transphosphorylation in the sperm of the ram, rabl)it, or bull.

B. INVERTEBRATE SPERM METABOLISM

The processes underlying motility and survival of invertebrate spermatozoa are oxygen-dependent and involve the utilization of endogenous reserves (Rothschild, 1951a). In sea urchin sperm, on which such investigations have almost exclusively centered, the oxidative substrate seems to be phospholipid, mainly situated in the midpiece (Rothschild and Cleland, 1952) . About 20 per cent of the intracellular phospholipid of the sperm of Echinus esculentus is depleted during incubation over a 7-hour period at 20°C. According to Rothschild, sea-urchin spermatozoa do not utilize glycolytic substrates (glucose or fructose), and there is scant evidence of a "sparing" of endogenous substrate by exogenous hexose.


Among certain other forms which, like the sea urchin, reproduce by external fertilization, the spermatozoa rely principally, if not entirely, on oxidative mechanisms. This is true, for example, of the starfish, Asterias (Barron, 1932) as well as the frog, Rana (Bernstein, 1954). On the other hand, some invertebrate sperm are less restricted in their metabolic capacity. The sperm of the oyster, Saxostrea, for example, normally depend on respiratory processes, but if these are inhibited by an oxidative inhibitor such as cyanide, and suitable substrate is present, glycolysis can occur (Humphrey, 1950). Barron (1932) indicated that sperm of various marine animals differ significantly in their tolerance for anaerobic conditions, as determined by the saf ranin test for oxygen ; sperm of Arbacia, Asterias, and Nereis retain their motility and fertilizing capacity when exposed to anaerobiosis for 1, 2, and 5 hours, respectively.

The importance of oxidative phosi)horylation, in contrast to oxygen consumption per se, to sperm motility has been clearly demonstrated in the sperm of the clam, Spisula (Gonse, 1959). Dinitrophenol, an uncoupling agent, inhibits sperm motility while increasing Oo uptake several fold. Amytal, on the other hand, at a concentration which severely depresses respiration, only slightly impairs motility.

Determinations of respiratory cjuotients (R.Q.) of invertebrate spermatozoa yield values approximating 1.0 (Barron and Goldinger, 1941; Hayashi, 1946; Barron, Seegmiller, Mendes and Narahara, 1948; Spikes, 1949; Humphrey, 1950). Such data suggest carbohydrate rather than lipid or phospholipid as substrate. Rothschild (1951a), however, has emphasized the technical difficulties besetting such determinations and the errors which may arise; in his view, loss of bicarbonate from the seawater diluent gives erroneously high R.Q. values. Yet, in support of the possible utilization of glucose or fructose by sea urchin sperm { Arbacia and Psaynmechinus) stands Wicklund's demonstration that exogenous hexose significantly prolongs motility and fertilizing capacity of sperm (in Runnstrom, 1949 ) , a point also suggested by the work of Spikes (1949).


BIOLOGY OF SPERMATOZOA


759


It seems that, although there may exist some variation in the ability of invertebrate sperm to withstand anaerobiosis or to utilize glycolytic substrates to a limited extent, these cells generally are dependent on respiratory i)rocesses for the major production of chemical energy. Since the conditions of external fertilization deny them ready access to glycolytic substrates in the environmental milieu, the sperm have failed to develop, or have secondarily lost, their glycolytic capacity, so characteristic of mammalian and avian spermatozoa. It is unlikely, although, of course, possible, that failure to utilize hexoses rests on the impermeability of the sperm to these substrates.

C. MAMMALIAN SPERM METABOLISM

As is the case with invertebrate spermatozoa, most of wdiat is known about the biochemical characteristics of mammalian sperm has been acquired from studies, in vitro. To the extent that experimental conditions may duplicate those within the genital tract, the behavior of sperm, in vivo, can only be surmised. Considerable variation is seemingly inherent in the metabolic characteristics of sperm of different species and in the gametes removed from different levels of the tract (see Dott, 1959). There is little doubt that such variation exists, but the causes may not be so distinctive as is generally claimed. Discounting differences in sperm behavior attributable to variations in handling and experimental procedure, it seems likely, without implying fundamental differences in metabolic patterns, that sperm, like most other types of cells, possess a lability of subcellular activity which enables them to regulate to external and intrinsic factors. The variations in sperm behavior, which at times seem so unique, are not likely to conflict with the conservative concept of the biochemical unity of living matter" (Fruton and Simmonds, 1959) .

The principal metabolic characteristics of mammalian spermatozoa have been extensively reviewed by Mann (1949, 1954) ; elsewhere special attention has been paid to human sperm (MacLeod, 1943b; Ivanov, 1945; Westgren, 1946; Lundquist, 1949). It is now well established that both glyco


lytic and oxidative processes provide energy for mammalian sperm and either one or both types of metabolic pattern can serve the sperm after insemination into the female genital tract. Motility of ram and bull sperm, in vitro, is enhanced by the presence of both hexose and oxygen together (Walton and Dott, 1956). Whereas fructose is the common natural substrate at ejaculation (see chapter by Price and Williams-Ashman), most mammalian sperm also utilize glucose and mannose with equal or greater facility (Mann, 1954). The j)rincipal steps in the degradation of sperm hexose to lactic acid occur by the well known Embden-Meyerhof scheme involving ATP as phosphate donor and diphosphopyridine nucleotide (DPN) as hydrogen carrier (electron transport system) ; this has been demonstrated in both ram and bull sperm, mainly by the identification of individual enzyme systems and glycolytic intermediates (Mann, 1954). The several components of the cytochrome-cytochrome oxidase electron transport system have been established by manometric and spectrophotometric methods in a variety of spermatozoa, including those of man (MacLeod, 1943a; Mann, 1951a). Less direct, but nevertheless adequate, evidence further indicates that the Krebs tricarboxylic acid cycle is involved in the oxidative processes (Mann, 1954; White, 1958). Indeed, there is no evidence to suggest that the over-all metabolic systems of sperm, at least of the ram and bull, are significantly different from those of muscle or of most other mammalian tissues. The rates of glycolysis and oxidation vary, but the mechanisms are basically the same. Moreover, it is probable that under many conditions, in vivo, there is considerable interaction between the glycolytic and oxidative processes (for general discussion, see Packer, 1959; Packer and Gatt, 1959). Both types of metabolic pathways, glycolytic and oxidative, are complete within the sperm flagellum. This is clear from the fact that in both the guinea pig (Cody, 1925) and bull (Mann, 1958) cases have been reported in which the fiagella are naturally separated from the heads at the time of ejaculation; such flagella are actively motile and show high rates of lac


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SPERM, OVA, AND PREGNANCY


tate production and oxygen consumption. Both turkey and cock sperm also utilize glycolytic and oxidative substrates, although at lower rates than those generally found in mammalian spermatozoa (Winberg, 1939; Pace, Moravec and Mussehl, 1952; Bade, Weigers and Nelson, 1956; Lorenz, 1958).

The range of substrates metabolized by mammalian sperm is extensive and includes carbohydrates, lipids, and amino acids. Of the three readily glycolyzable hexoses — glucose, fructose, and mannose- — glucose is preferentially utilized by sj^erm of the bull, ram, and man (Mann, 1951b; van Tienhoven, Salisbury, VanDemark and Hansen, 1952; Flipse, 1958; Freund and MacLeod, 1958). Hexosc degradation is such that one mole of glucose gives rise to two moles of lactate (Flipse and Almquist, 1955; MacLeod and Freund, 1958). Lactic acid tends to accumulate, since the rate of glycolysis, in bull sperm for example, exceeds the rate of pyruvate oxidation ( Melrose and Terner, 1951). Evidence bearing on the possibility of direct oxidation of glucose by way of the hexose monophosjihate shunt is fragmentary and thus far negative (Wu, McKenzie, Fang and Butts, 1959). Glycolysis can, of course, occur under l)oth aerobic and anaerobic conditions. The addition of exogenous hexose to a respiring system of sperm tends to "spare" the respiratory substrate (Lardy and Phillips, 1941; O'Dell, Almquist and Flipse, 1959); this partial inhibition of oxidation by glycolysis is a manifestation of the well known Crabtree effect ( Crabtree, 1929; Terner, 1959) and can be interpreted as a form of metabolic regulation.

Since the initial demonstration (Lardy and Phillips, 1941) that endogenous phospholipid seems to constitute the natural respiratory substrate of bull spermatozoa, many oxidizable substances have been shown to increase oxygen uptake or to support sperm motility (Mann, 1954; White, 1958). Considerable species variation occurs in the apparent facility with which such substances are oxidized, but some of this variation depends less on utilization than on the extent to which the substances penetrate specific kinds of sperm. Succinate and malate, for example, can increase the respi


ration and motility of washed ram sperm, but are without effect on bull sperm under similar conditions, presumably because of their failure to penetrate the cells (Lardy and Phillips, 1945; Lardy, Winchester and Phillips, 1945). Changes in cell permeability induced by rough handling, severe centrifugation, storage, or specific chemical treatment, such as exposure to surface-active detergents, can alter the rate and degree of substrate penetration and thereby produce profound changes in respiratory activity (Koefoed-Johnsen and Mann, 1954).

Among the oxidative substrates which increase respiration of mammalian sj)erm may be included the end products of anaerobic glycolysis — pyruvate and lactate — as well as acetate, butyrate, propionate, citrate, and oxaloacetate (Lardy and Phillips, 1944; Mann, 1954). Glycerol is oxidized to lactic acid by ram and bull spermatozoa (Mann and White, 1957; White, 1957), probably by entering the EmbdenMeyerhof jjathway as glycerol phosphate at the triose phosphate level. In experiments involving C"-tagged glycerol, it has been claimed that bull sperm can complete the oxidation to C^'*02 under anaerobic conditions (O'Dell, Flipse and Almquist, 1956), a point which requires confirmation. The glycerol moiety of the seminal constituent, glycerylphosphorylcholine, apparently is not made available to the sperm for respiratory activity (Mann and White, 1957).

In the early work on phospholipid oxidation, it was concluded that endogenous reserves are readily utilized and that egg phospholipid can serve as an exogenous source of energy (Lardy and Phillips, 1941). This finding is supported by the study of Crawford, Flipse and Almquist (1956) who determined the uptake by bull spermatozoa of P'^--labeled egg phospholipid. Bomstein and Steberl (1957), on the other hand, found a negligible decrease in intracellular phospholipid and an inappreciable utilization of exogenous lecithin during incubation of well washed preparations of bull sperm. Recent re-analysis of the nature of the lipids in ram sperm indicates that 55 to 60 per cent is in the form of choline-based acetal phospholipid or plasmalogen (Lovern, Olley, Hartree and ]\Iann, 1957).


BIOLOGY OF SPERMATOZOA


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Although this material can he oxidized hy sperm with an R.Q. of about 0.71, there is no detectable change in lij^d phosphorus (Hartree and Mann, 1959) ; rather, it is the fatty acid residue which is oxidized. Whatever the precise composition and nature of the intracellular oxidizable reserves, the supply nuist be fairly copious and the utilization efficient; some 20 years ago Moore and Mayer (1941) showed that ram sperm can remain motile in neutralized seminal j^lasma for 20 hours or more after the sugar, and presumably other exogenous stores, are exiiausted (see Lardy, Winchester and Phillips, 1945). The details of lipid oxidation in spermatozoa have not been elaborated, but it is assumed that, as in other tissues, the fatty acid residues react with acetyl-Coenzyme A and enter the tricarboxylic acid cycle to be ultimately oxidized to carbon dioxide and water.

Earlier work had established that the addition of amino acids, particularly glycine, to suspensions of fowl or bull sperm increases many fold the duration of motility and in fowl sperm stimulates oxygen consumption as well (Lorenz and Tyler, 1951; Tyler and Tanabe, 1952). No utilization of the amino acids was detectable and the phenomenon was interpreted on a basis of the chelation of heavy metal ions, such as occurs, for example, with ethylenediaminetetraacetate (Versene) (Tyler and Rothschild, 1951). More recent experiments involving the use of C^'*-labeled glycine have shown that this amino acid is actually taken up and metabolized by sperm of the bull, without, however, increasing oxygen consumption (Flipse, 1956; Flipse and Alm([uist, 1956; Flipse and Benson, 1957). (ilucose depressed but did not eliminate, the utilization of glycine; on the other hand, the addition of glycine had little or no effect on the utilization of glucose (Flipse, 1958). The principal pathway of glycine catabolism in sperm seems to involve glyoxylate, formate, and carbon dioxide. This is similar to the scheme of glycine oxidation in rat liver and kidney (Nakada and Weinhouse, 1953). Certain other amino acids, namely phenylalanine, tryptophan, and tyi-osine, also are metabolized by sperm of the bull and ram by a process of oxidative


deamination catalyzed by the enzyme, lamino acid oxidase (Tosic, 1947, 1951). Hydrogen peroxide is produced in this reaction and is toxic unless eliminated by catalase (Tosic and Walton, 1950). Thus it is clear that certain amino acids are oxidized by sperm, but the significance of these reactions to the total energy-producing metabolic processes of the cells cannot be regarded as great.

D. EPIDmVMAL SPERM AND METABOLIC REGULATION

Striking differences have been claimed for the metabolic behavior, in vitro, of bull sjx'rm, from different segments of the epididymis, suggestive of metabolic regulation in relation to sperm maturation in the male genital tract (Henle and Zittle, 1942). These differences are manifested by lower rates of endogenous respiration and aerobic glycolysis, and a higher rate of anaerobic glycolysis, by epididymal sperm as compared with the rates shown by washed sperm of semen (Lardy, Hansen and Phillips, 1945; Lardy, 1952). Inasmuch as the motility of the spermatozoa from both sources is essentially similar, such metabolic behavior indicates a higher biochemical efficiency of the epididymal sperm. One can indeed demonstrate an inhibition of glycolysis by oxygen (the Pasteur effect) in epididymal sperm which is less readily displayed by washed seminal sperm.

In a search for the cause of these differences. Lardy found evidence for a so-called metabolic regulator which is present in a bound or inactive form in epididymal sperm and which is released or becomes active at the time of ejaculation (Lardy, Ghosh and Plaut, 1949). The action of the regulator was thus considered to increase respiration and aerobic glycolysis to levels characteristic of semen. This regulating activity was tentatively identified with a sulfur-containing component extractable from semen and from testicular tissue; its action w^as found to be similar to that of cysteine and reduced glutathione (Lardy and Ghosh, 1952; Mann, 1954). Relatively little work has since been done to identify further the metabolic regulator or to demonstrate a similar agent in other species of sperm.


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SPERM, OVA, AND PREGNANCY


Further study would be desirable to demonstrate whether bull semen contains a specific metabolic substance which might account for these effects, or whether, on the other hand, the changes noted are part of a more generally applicable type of cell regulation. For example, both the low rate of endogenous respiration and the Pasteur effect, characteristic of epididymal sperm, indicate an efficient phosphorylating system; the metabolism of seminal sperm, on the other hand, suggests that uncoupling of respiration and phosphorylation may have occurred {cf. Bomstein and Steberl, 1959). The similarity of action of the sperm metabolic regulator and dinitrophenol, a known uncoupling agent, further supports this interpretation (Johnson and Lardy, 1950; Lardy, 1953). Lehninger (1955) has stressed the relationship between uncoupling and the inhibition of the Pasteur effect in other (mitochondrial) metabolic systems. A general type of metabolic regulation such as this, rather than a system unique to one type of cell, might account for some of the apparent discrepancies reported by different investigators in their studies of mammalian gametes (Melrose and Terner, 1951). White (1960), for example, working with ram sperm has failed to confirm the work of Lardy and associates; he found no significant difference in oxygen uptake or in fructolysis whether the sperm were from the epididymis or from the ejaculate. At first glance this seems to represent a marked metabolic difference in the sperm of closely related animals; considering, however, the delicate balance of cell regulation at the metabolic level (for general discussion, see Krebs, 1957; Packer and Gatt, 1959) the variation is not necessarily profound. The striking differences shown by Dott (1959) in the epididymal sperm of 5 species of domestic mammals may also represent subtle effects of metabolic control rather than overt manifestations of fundamentally different systems of cell metabolism. He found that the epididymal sperm of the bull, ram, and rabbit are activated, in vitro, either by oxygen or by fructose under anaerobic conditions; boar sperm, however, apparently require oxygen, and stallion sperm require fructose, to initiate motilitv. Once stimu


lated, boar sperm glycolyze hexose freely, indicating perhaps that the action of the oxygen is to metabolize an intracellular inhibitor of some process necessary for motility (Dott, 1959). The response of stallion sperm suggests that the main source of energy is derived from aerobic glycolysis and, further, that endogenous oxidative reserves are scant or that the respiratory processes are inhibited at some critical point. The oxygen uptake of seminal sperm from the stallion is generally low. In certain features this situation in stallion sperm corresponds to the metal)olic behavior of human seminal sperm.

E. HUMAN SPERM METABOLISM

Princij^ally through the investigations of MacLeod (1941-1946), the metabolic activities of sperm in the human ejaculate are generally considered to present a rather unique picture. Human sperm show a high rate of anaerobic glycolysis which is only slightly depressed by oxygen; the rate of oxygen consumption in the presence of glucose is extremely low — according to Terner (1960j, about one tenth that of bull sperm. When hexose is replaced by any one of a number of amino or fatty acids, sperm motility is gradually lost; it is not known, however, to what extent these exogenous substances do or do not penetrate the cell. Of the nonglycolyzable substrates employed, only succinate stimulated oxygen consumption, and this reaction was accompanied, when glucose was present, by a 40 per cent reduction in lactate production (MacLeod, 1946). MacLeod further claimed that oxygen, even at low tensions, is detrimental to human sperm suspended in Ringer glucose; after several hours at 38°C., aerobic motility is seriously impaired. Motility is also suppressed by glycolytic inhibitors (iodoacetate and fluoride), but is claimed to be unaffected by respiratory poisons (cyanide, azide, and carbon monoxide). These considerations led MacLeod to the conclusion that human sperm rely entirely on the energy of glycolysis for motility and are unable to utilize effectively oxidizable substrate, despite the fact that the cells contain the main components of the cytoclirome system and tricarboxylic acid cycle.


BIOLOGY OF SPERMATOZOA


76a


The I'c'latively high glycolytic activity of Imiuaii sperm was compared by MacLeod 1 1942) to the metabolism of certain types of tumor cells (see Warburg, 1956a, b).

The apparent toxicity of oxygen on human sperm in vitro was attributed to the production of hydrogen peroxide, inasmuch as the effect could be eliminated by the addition of catalase. The enhanced respiration induced by succinate, noted above, was also found to be accompanied by an increase in HoOo formation. As a possible mechanism of peroxide formation, the autooxidation of a flavo-like compound was suggested (MacLeod, 1943b, 1946).

These investigations on human sperm cmpiiasize the preferential utilization of glycolytic substrates, under the conditions of the experiments. The relative failure, however, of respiratory substrates to support motility might well bear further scrutiny. This is particularly true in light of the rapid oxidation of succinate, as shown by MacLeod, and the recent report of Terner ( 1960) that saline suspensions of human sperm oxidize both pyruvate and acetate, as shown by C^^Oo production from pyru


ger, 1959; Hacker, 1959). In human sperm, however, the rates of oxidative respiration and phosphorylation are low and appear to be initially metabolically suppressed. Thus the oxidative inhibition is not induced by high glycolytic activity itself (Crabtree effect), but rather, glycolysis is favored by the previous suppression of respiration. The inhibition of oxidation, in turn, can be attributed, if MacLeod is correct, to the production of toxic amounts of hydrogen peroxide, and this seems to be the relatively unique feature of human sperm metabolism. A plausible explanation for both the source of the peroxide and the failure of respiration and oxidative phosphorylation can be formulated following the suggestion by MacLeod (1942). Thus, it is characteristic of flavoi)rotein (FAD) that, as a "pacemaker" in the oxidative chain (Krebs, 1957), it can either transfer hydrogen atoms from reduced diphosphopyridine nucleotide (DPNH + ) to the cytochrome system or, by auto-oxidation, noncatalytically combine with molecular oxygen to form hydrogen peroxide (Fruton and Simmonds, 1958) (see schema).



vate-2-C^'* and acetate-1-C^^. Dinitrophenol stimulated C^'*02 production from both glucose-C^-* and pyruvate-2-C^*.

The peculiar metabolic behavior of human sperm may be partially clarified l)y reference to the principles of intracellular regulation and alternative metabolic pathways, characteristic of other cellular and subcellular systems. Of the two main types of energy-producing pathways, which in a sense are normally in competition (Krebs, 1957; Racker and Gatt, 1959 (, the process of glycolytic phosphorylation in human s])erm dominates oxidative phosphorylation. This imbalance could be brought about by the unequal distribution of such rate-limiting substances as ADP or inorganic phosphorus (for general discussion, see Lehnin


Unlike most respiring cells which follow the first of these alternative pathways, human sperm seem to be shunted off into the nonphosphorylative peroxide-producing route. Succinate is known to bypass DPN and to donate hydrogen directly to FAD (Krebs, 1957). As previously mentioned, in human sperm succinate causes increases in both oxygen uptake and peroxide formation and a decrease in lactic acid accumulation (MacLeod, 1946). But whether this represents a shift from glycolytic to oxidative pathways or merely an inhibition of glycolysis, possibly by the poisoning of sulfhydryl-containing enzymes by excessive amounts of i)eroxide (MacLeod, 1951), is not known.

Speculative as these interpretations con


764


SPERM, OVA, AND PREGNANCY


cerning human sperm may be, they have some merit. New avenues of investigation are opened by a broader approach. Moreover, some advantage is gained by attempts to relate certain aspects of the apparently exotic behavior of human sperm to the metabolic patterns and principles common to other mammalian tissues. Many issues are yet to be resolved, including the question of the utilization of oxidative substrates vis-dvis their jiermeability, and the recently announced difference in sensitivity of human sperm to endogenous versus exogenous hydrogen jieroxide (Wales, White, and Lamond, 1959 ». Tests might be applied to determine whether peroxide is produced in accordance with the scheme noted above or whether it may arise from endogenous nitrogenous sources, comparable to its formation from exogenous aromatic amino acids, as previously noted (Tosic, 1947; VanDemark, Salisbury and Bratton, 1949; Tosic and W^alton, 19501.

F. METABOLIC-THERMODVX.\MIC INTERRELATIONS

Underlying much of the above discussion are many quantitative data pertaining to the metabolic and thermodynamic properties of sperm. Rates of oxygen consumption

TABLE 13.12 Vital statistics of hull spermatozoa (Data obtained in buffered saline, 37°C.; calculations based on free-energy change of hydrolysis of —8 kcal. per mole of adenosine triphosphate.)


Anaerobic fructolysis (Mann,



1954)


1.7 mg./lO' sperm/hr.


Energy liberated


6.27 X 10-6 erg/sperm /sec.


Energy trapped as ATP


1.76 X 10-6 erg/sperm/sec.


Endogenous oxygen uptake



(Lardy, 1953)


200 fi\./W sperm/hr.



1000 meal./ 10' sperm/hr.



1.5 X IQ-s erg/sperm/sec.


Anaerobic heat production



(Clarke and Rothschild,



1957)


220 nical./109 sperm/hr. 2.55 X 10-6 erg/sperm/sec.


ATP phosphorus liberated


aerobically* (Nelson, 1954,



1958b)


21 Mgm. P/mg. sperm N/min.



2.5 X 10-'9m ATP/sperm /sec.



2 X 10-12 meal. /sperm /sec.



8.4 X 10-8 erg/sperm/sec.


Energy required for motility



(Nelson, 1958b)


3.15 X 10-8 erg/speim/sec.


(Rothschild, 1959)


2.11 X 10-' erg/sperm/sec.


  • Based on fragmented cells and expressed as

net result of balance between hydrolysis and synthesis of ATP.


and of fructolysis have been determined for sperm of a wide variety of species (Mann, 1954). Values have also been obtained for heat production (Bertaud and Probine, 1956; Clarke and Rothschild, 1957; Rothschild, 1959), ATP hydrolysis, and the energy requirements for flagellar movement. Some of these properties for one species are tentatively summarized in a table of vital statistics for bull spermatozoa (Table 13.12). Expressed on a per sperm basis, the energy, in ergs, calculated for substrate utilization and heat production indicate a wide thermodynamic safety factor in the balance sheet between energy generated and that required.

In Rothschild's exacting study (1959) in which he has demonstrated the changes in sperm heat production with variations in environmental factors, including pH, tonicity, and centrifugation, attention is drawn to the narrow margin between the freeenergy change of anaeorbic glycolysis which is associated with ATP synthesis and the energy expenditure involved in flagellation. The data suggest that in bull sperm under anaerobic conditions the rate of ATP synthesis does not keep pace with that of ATP hydrolysis.

Although adequate data are available for the ATP-splitting activity of sperm fragments and siierni extracts (see Nelson, 1954; Burnasheva, 1958), the rate of ATP hydrolysis in whole sperm is difficult to assess, inasmuch as the value of inorganic phosphate liberated is the net result of hydrolysis over synthesis or the phosphorylation of ADP. This is clearly indicated in Table 13.12, in which the energy from ATP-splitting is seen to be insufficient for the energy requirements of movement. This procedural quandary was noted by Lardy, Hansen and Phillips (1945) who demonstrated in aerobic suspensions of bull sperm an increase in nucleotide-phosphate release in the presence of cyanide, an inhibitor of phosphorylation processes.

G. BIOSYNTHETIC ACTIVITY

Although spermatozoa are generally regarded as fully differentiated by the time they reach the epididymis, some questions have arisen with respect to their biosyn


BIOLOGY OF SPERMATOZOA


765


thetic ability even after ejaculation. Such metabolic cofactors as ATP, for example, are most certainly synthesized (at least from ADP), at the expense of organic substrates, throughout the motile life span. More complex substances may also be synthesized. Hakim (1959) has reported that polynucleotide phosphorylases can be extracted from human sperm which, when incubated with nucleotide phosphates, cause the formation of dinucleotides, as determined chromatographically. Thus, for example, a mixture of ADP and guanosine diphosphate (GDP), in the presence of suitable enzyme, forms some ADP-GDP. In another type of study employing intact bull sperm, Bishop and Lovelock indicated that C^*-labeled acetate is incorporated into fatty acid (see Austin and Bishop, 1957).

The possibility of jirotein synthesis by sperm was suggested by Bhargava (1957), who reported the incorporation of labeled amino acids into the protein fraction of bull spermatozoa as assayed by radioactivity counting. These conclusions have since been contradicted by Martin and Brachet (1959) who suggest, on a basis of autoradiographic data, that the uptake and synthesis can be attributed to cellular components other than to the sperm in the sample. This finding falls more nearly in line with the general conclusion that RNA, essential for protein synthesis, is absent from mature sperm or is present in only very small amounts (Brachet, 1933; Friedlaender and Frasei-, 1952; Leuchtenberger, Leuchtenberger, Vendrely and Vendrely, 1952; Mauritzen, Roy and Stedman, 1952). In this connection it is of interest to recall the observations of Wu, McKenzie, Fang and Butts (1959) on the contrasting metabolic capacities of testicular and seminal bull sperm. Relatively clean preparations of spermatozoa expressed from incised testis, but not sperm from the ejaculate, can oxidize glucose by way of the hexose monophosphate shunt, thereby supplying a source of ribosc which is available for RNA in the cai'lici' stages of sperm differentiation.

IX. Sperm Flagellation

The characteristics and mechanics of s]ierm movement are discussed in con


siderable detail in several recent reviews dealing with both invertebrate and vertebrate material (Gray, 1953, 1955, 1958; Gray and Hancock, 1955; Bishop, 1961). Sperm motility, closely related to muscular contraction, on the one hand, and to general flagellar and ciliary activity, on the other, represents an important physiologic process with implications l)eyond the specific behavior of the gametes. For the present context, however, only certain more general aspects of the problem are pertinent.

By the tiirn of the century the significance of the flagellum for sperm motility was well established (see Wilson, 1925). As early as 1898, Engelmann had succeeded in cutting off the tails of frog spermatozoa to find that the flagella continued to move if the separations were made close to the heads. Ciaccio (1899) and particularly Koltzoff (1903) discussed the elementary mechanisms of flagellation and went so far as to compare the process with contraction of muscle. In 1911, Heidenhain postulated that the chemical energy rec}uired for motility must be distributed throughout the flagellum, a concept generally conceded today (Gray, 1958). Ballowitz (1888, 1908) emphasized the significance of the longitudinal fibrils of the axial bundle for motility. In the history of sperm biology these two decades, immediately before and after 1900, constitute the "Age of Flagellation."

A. WAVE PATTERNS

Largely through the efforts of Sir James Gray (1953-1958) many details of the process of flagellation have been recorded, most attention having been focused on the sperm of the sea urchin and bull. Although there exists much natural variation among species in the overt characteristics of the phenomenon, basically the same fundamental mechanism is involved. Propagated waves originate at the base of the flagellum and progress distally toward the tip. The major bending-couple is two-dimensional, but as it sweeps distally it is accompanied by, or is converted into, a three-dimensional wave which gives the sperm a helical spin about the axis of forward progression (Gray, 1955, 1958). In squid sperm under experimental conditions the two components of movement, lateral vibration and rotation, can


SPERM, OVA, AND PREGNANCY


be separated and analyzed individually (Bishop, 1958f). Wave co-ordination involves not only the initiation of the beat, which may be a function of the basal granule, but also the propagation of the conduction wave along the flagellum. The velocity of wave propagation has been calculated for bull sperm to be 600 to 700 /x per sec. (Bishop, 1961).

The frequency of beat, stroboscopically determined, is on the order of 20 per sec. for the bull and 15 per sec. for man (Ritchie, 1950; Rothschild, 1953; Rikmenspoel, 1957; Zorgniotti, Hotchkiss and Wall, 1958). Wave amplitude in bull sperm is 8 to 10 fi, about 20 times the diameter of the tail. These values are at best only first approximations, because wave characteristics change not only with progression along the length of the flagellum, but also with environmental conditions such as temperature and viscosity of the medium.

B. SPERM VELOCITY

Many attempts have been made to determine the speed of sperm travel (see Bishop, 1961). As a general rule, the methods used give data for translatory rather than absolute velocities (Table 13.13). Speeds up to 350 /* per sec. have been recorded for bull sperm. Rikmenspoel (1957) has presented an extensive correlation of the variations in bull sperm velocity with changes in frequency and amplitude of wave formation and with alterations in viscosity and temperature of the environment. The effect of current flow on stallion sperm velocity was demonstrated by Yam TABLE 13.13

Translatory velocities of mammalian

spermatozoa, in vitro

(Buffered saline or saline-plasma, 37°C.)


Species


Average Velocity


Reference



H per sec.



Man


23


Adolphi, 1905



14


Botella Llusia et al., 1957


Horse


87


Yamane and Ito, 1932


Ram


80


Phillips and Andrews, 1937


Bull


123


Rothschild, 1953



114


Moeller and VanDemark, 1955



105


Rikmenspoel, 1957



94


Gray, 1958


ane and Ito ( 1932 ) . They found that sperm orient themselves by rheotaxis, or are oriented physically, against a current, and that up to a limit, as the opposing flow is increased, the speed of movement also increases. When the opposing current flow was varied from to 20 ju. per sec, sperm velocity increased from 87 to 107 yu, per sec. Under the conditions of the experiment, the results might be attributable merely to the direction given the sperm, thereby reducing the randomness of movement. Nevertheless, these findings may have some bearing on the problem of active sperm transport in vivo, where ciliary or other currents play a role. From a comparison of the data on sperm velocities (Table 13.13) and those previously cited on sperm transport, the conclusion is inescapable that in most mammals, migration is not dependent on active swinnning movements alone.

C. HYDRODYNAMICS

Initiated by the theoretical speculations and mathematical derivations of Sir Geoffrey Taylor (1952), a considerable body of information has accrued which permits an evaluation of the mechanics and forces involved in si)erm movement (Gray and Hancock, 1953; Hancock, 1953; Rothschild, 1953; Machin, 1958; Xelson, 1958b; Carlson, 1959). From these considerations it is clear that a spiral or three-dimensional pattern of flagellation is more eflficient than a two-dimensional wave motion; Taylor calculates that the resulting sperm velocity in the former case may be up to twice as great, depending on the configuration of the sperm cell, for a given amount of energy expended. Employing these mathematical derivations and experimental data for wave characteristics such as frequency and amplitude, Gray and Hancock (1953) found good agreement in calculated and observed values for the velocity of sea-urchin sperm of about 190 fx per sec. The power output required to effect this activity has also been calculated. For sea urchin sperm, Carlson (1959) obtained a value of about 3 X 10~" erg per sec. per sperm. Comparable figures for bull sperm have been estimated as ranging from 2xl0~^to3x 10~^ erg })er sec. per sperm, depending on


BIOLOGY OF SPERMATOZOA


767


certain theoretical assumptions underlying the analysis (Rothschild, 1953, 1959; Nelson, 1958b).

At the present time, such information may seem limited in its application to problems of sperm physiology in relation to the reproductive process as a whole. From a broad point of view, however, it obviously affords a biophysical measure of what the sperm can accomplish, and constitutes a link between the metabolic energy produced, on the one hand, and the work performed during activity, on the other (Table 13.12).

D. MECHANISM OF MOTILITY

Speculation concerning the physical basis for activity of cilia and, by implication, flagella has a long tradition (Grant, 1833; Ankermann, 1857; Schafer, 1904). Of the various theories proposed, the only one to persist is that which conceives of the flagellum as a diminutive contractile system (Ciaccio, 1899; Koltzoff, 1903; Ballowitz, 1908; Heidenhain, 1911). Other types of biochemical systems can be imagined to account for sperm movement, but the evidence, particularly of the past few years, favors the concept of a contractile protein mechanism, generally associated with the fibrillar system of the tail (see Bishop, 1961).

Brief mention has been made of certain salient features of the motility process. It is clear that ATP is essential for sperm activity, as it is for many other physiologic processes reciuiring energy. A constant sujiply of ATP is maintained by the glycolytic and/or oxidative processes of metabolism (Engelhardt, 1958 j. Certain experiments have indicated that extractable ATP is not significantly depleted during sperm activity (Hultin, 1958), thus further supporting the view that resynthesis of the nucleotide accompanies its dephosphorylation. The presence and general localization of ATPase in the flagellum have been noted; by its specific action on ATP as substrate, chemical energy associated with high-energy" phosphate bonds is liberated.

The ATP-ATPase type of enzyme system is widely distributed throughout the animal and plant kingdoms; it has been extensively studied and closely identified Avith the contractile svstem of muscle. It


was of major significance that the contractile protein itself, myosin, was found to possess the ATP-splitting activity which leads to contraction (Engelhardt and Lyubimova, 1939). ATPases thus represent the essential link between the biochemical and mechanical events (Engelhardt, 1958). Myosin alone is incapable of shortening, but when combined with actin, the complex undergoes contraction in the presence of ATP. This can be readily demonstrated in simplified muscle systems such as glycerinatetl fiber models (Szent-Gyorgyi, 1949; Varga, 1950) or actomyosin thread preparations (Portzehl and Weber, 1950).

As a result of their previous studies of the biochemistry of muscle, and the overt similarities of muscle contraction and sperm flagellation, Engelhardt and his associates undertook a detailed study of motility of bull sjierm. They extracted from sperm cell homogenates a partially purified l)rotein which showed ATPase activity and was tentatively called "spermosin" (Engelhardt, 1946). Refinements in extraction and luirification procedures since that time have resulted in tlie jireparation of a product with many of the properties of myosin, isolated by similar techniques from muscle. Meanwhile, work was being reported from several other laboratories confirming the occurrence of ATPase in sperm and sperm tail preparations of a variety of species (Felix, Fischer, Krekels and Mohr, 1951 ; Nelson, 1954, 1955b; Utida, Maruyama and Nanao, 1956; Bishop, 1958a; Tibbs, 1959). Although not all of these preparations are unequivocally associated with contractile protein or contractile protein alone, the evidence seems clear that the sperm tail possesses high ATPase activity.

More recent publications from Engelhardt's institute indicate that material of a high degree of purity can be extracted from bull sperm tails which probably is the contractile protein, "spermosin," responsible for movement (Engelhardt and Burnasheva, 1957; Burnasheva, 1958; Engelhardt, 1958). Ai^iiroximately 80 per cent of the ATPase activity of the whole sperm is concentrated in the tail fraction, isolated by centrifugation. Substrate specificity and cationic requirements of the enzyme have led to the conclusion that it is very similar to muscle


•68


SPERM, OVA, AND PREGNANCY


I On 9


ACTOMYOSIN


ACTOSPERMOSIN



O (D (D ®


© (5) ©0


© F-Actin

(g) Spermosin

@ Spermosin + F-Actin (ratio I2:i)

@ Spermosin + F-Actin + AT P ( 4 23 x 10"" M)

© Myosin

© Myosin + F-Actin (ratio 2:1)

@ Myosin + F-Actin + AT P (4 23 x 10"" M)

Fk;. 13.18. Complex-formation and viscosity change upon addition of adenosine triphosphate (ATP) in system composed of contractile protein extracted from bull sperm and actin from rabbit muscle. The response of muscle actomyosin is shown at the right for comparison. (From 8. A. Burnasheva, Biokhimiia, 23, 558-563. 1958.)


myosin. Further similarity is indicated by the chiim that "spermosin" can combine with actin, extracted from muscle, to form an "actospermosin" complex (Burnasheva, 1958). This complex undergoes viscosity changes similar to those shown by actomyosin, upon the addition of ATP (Fig. 13.18). It is to be noted that, thus far, physical methods have not been applied to the study of the protein isolated from sperm by these investigators. Attempts to extract an actinlike protein from bull sperm have thus far proved unsuccessful. Whether the contractile system of sperm is eventually resolved as a single component system, as suggested by Burnasheva, or a double component system as in muscle, remains for further investigation to demonstrate.

Although these extraction experiments give strong evidence in favor of a myosin


like protein in sperm flagella, the picture is far from complete. Rather striking differences have been shown, for example, in the response of fish sperm ATPase to cation concentration when compared with the behavior of muscle ATPase (Tibbs, 1959). Moreover, a comparison of structural details of the sperm flagellum before and after KCl-extraction procedures fails to indicate the source of the extractable protein; indeed, very little change can be detected in electron micrographs of mammalian sperm subjected to such treatment (Bishop, 1961). The motile mechanism of spermatozoa has been investigated also by the preparation and reactivation of cell models, comparable to the glycerinated models of muscle. Hoffmann-Berling (1954, 1955, 1959) first accomplished this with sperm of the locust, Tachijcines; as in the case of muscle models, glycerol-extracted sperm were reactivated by treatment with ATP at suitable concentration. This phenomenon has since been demonstrated with sjierm of the squid, Loligo, and of several species of mammals (Bishop, 1958b, e; Bishop and Hoffmann-Berling, 1959). The methods of extraction, ATP concentrations, ionic requirements, and response to sulfhydryl inhibitors are roughly similar to those applicable to muscle models. The general nature of the response to ATP, however, is strikingly different in that the addition of the nucleotide initiates flagellation which may continue, in bull sperm for example, for as long as 2 hours (Bishop and HoffmannBerling, 1959). Apparently, contraction-relaxation cycles are induced in the models which in frequency and amplitude are similar to those of normal fresh sperm. However, as a result of the complete loss of permeability and co-ordination properties of the flagellar models, wave propagation along the flagellum fails to occur and forward movement is insignificant. Among other interesting features of these virtually dead but ATP-reactivated sperm models is the fact that they can be reversibly immobilized by treatment with the MarshBendall (relaxing) factor, prepared from rabbit muscle according to the method of Portzehl (Bishop, 1958c). Moreover, the models are capable of flagellation against a force inijiosed by increasing the viscosity


BIOLOGY OF SPERMATOZOA


7(>9


of the surrounding medium (Bishop, 1958f ; Bishop and Hoffmann-Berling, 1959).

Such biochemical approaches as these suggest that the molecular basis of sperm motility is very similar to that of the contractile protein system of muscle. The identification of this system in the sperm is less securely established, but it is assumed to be localized in the longitudinal fibrils of the flagellum. The universality of the 2 X 9 + 2 pattern of filaments seems to demand that considerable significance be attached to them. The filaments appear on chemical grounds to resemble a fibrous protein which could be contractile in nature. Both solubility data (Schmitt, 1944; Bradfield, 1955) and the results of proteolytic digestion of sperm flagella (Hodge, 1949; Grigg and Hodge, 1949) support this view. The positive form birefringence of sperm tails further indicates an orderly arrangement of highly asymmetric structural units which may indeed be the components of the longitudinal fibrils themselves (Schmitt, 1944). X-ray diffraction measurements also suggest a high degree of organization with regular spacing of the structural elements (Lowman and Jensen, 1955). These reports on sperm flagella do not prove that the longitudinal fibrils are contractile protein, but they lend credence to that assumption. Excellent supporting evidence, moreover, is that obtained by Astbury and Weibull ( 1949) in their study of an entirely different type of flagellar system, the isolated flagella of bacteria. These investigators concluded that the x-ray diffraction pattern of flagellar preparations is characteristic of the k-m-e-f group of fibrous proteins and, further, that both the a- and ^-configurations can be demonstrated in unstretched and stretched fibrillar preparations. Astbury and Saha (1953) refer to these bacterial flagella as "monomolecular muscles."

It is to be stressed that the longitudinal filaments of spermatozoa show no consistent cross-striation or periodicity which might be compared with that of the striated muscle fibril (Bishop, 1961). In human sperm prepared for electron micrography, SchultzLarsen (1958) found an indication of periodicity with intervals of about 20 A, but this phenomenon is irregular and remains to l)e confirmed. Cross-striations at inter


vals of 500 to 700 A were found in Arbacia sperm by Harvey and Anderson (1943), but these have been interpreted as aggregation artifacts rather than as true components of structural periodicity.

Whereas the physical basis for sperm motility is thus fairly well established in a contractile protein system possibly associated with the flagellar filaments, no fully satisfactory theory of the operation of the mechanism has been advanced. The suggestion of Bradfield (1955) that the cylindrically arranged i^eripheral fibrils fire off progressive contraction waves in successive order was put forth hypothetically to describe a plausible but untested description of flagellation. Afzelius (1959) proposes, on the basis of ultrastructural differences in members of the pairs of peripheral fibrils, that the mechanism may function along the lines of the interdigitating-fibril scheme described for striated muscle by Huxley and Hanson (1954, 1957). Other more conservative speculations have been suggested (c/. Bishop, 1958f; Gray, 1958; Nelson, 1959), and final analysis of the precise nature of the contraction-relaxation waves and their synchronous operation in the sperm flagellum must await further experimental innovation and investigation. A striking gap currently persists between the ultrastructural interpretations of spermatozoa and the molecular characteristics associated with motility.


X. Fertilizing Capacity of Treated Spermatozoa

A wide range of environmental factors has been employed in the study of mammalian sperm responses, dating from the very earliest investigations of the gametes (van Leeuwenhoek, 1678). Three principal criteria have served as end points in the investigations of sperm physiology — motility, metabolism, and fertilizing capacity. Interrelated and interdependent in vivo, any of these properties alone or in combination can be assessed following experimental manipulation of the sperm in vitro. The chemical factors known to modify motility and metabolic behavior of sperm may be arbitrarily grouped roughly as follows: electrolytes including the hydrogen ion, enzymatic inhibitors, chelating compounds, and a variety


770


SPERM. OVA, AND PREGNANCY


of uncoupling agents which include sulfliyclryl-blocking agents, hormones {e.g., thyroxine), antibiotics, and surface-active substances (Mann, 1954, 1958bj. Other types of environmental factors which induce profound effects on sperm behavior involve dilution of the cell suspension, temperature changes, ionizing radiation, and certain biologic fluids and cell extracts.

The action of such agents on sperm motility and metabolic activity, but not necessarily on fertilizing capacity, is reviewed in detail elsewhere (Hartman, 1939; Mann. 1954; Bishop, 1961) ; the effect on fertilizhig capacity per se will be briefly presented here. Alteration of the fertility rate by pretreatment of spermatozoa is, of course, an established procedure. In an extreme sense, this is accomplished by the extension of the life span of sperm for purposes of artificial insemination (Anderson, 1945; Emmens and Blackshaw, 1956; Salisbury, 1957), or, conversely, the curtailment of survival by spermicidal agents (Mann, 1958b; Jackson, 1959).

A. DILUTION OF THE SPERM SUSPENSION

Chang (1946a) drew attention to the dilution effect on mammalian sperm by demonstrating that artificial insemination of rabbits was successful with a given number of sperm suspended in a small amount (0.1 ml.) of saline medium, whereas the same number of sperm in a larger volume (1.0 ml.) failed to bring about fertilization. Mann (1954) suggested that the dilution effect in mammalian sperm might in part be the same general type of response as that occurring in invertebrate sperm, in which the phenomenon has been extensively investigated (Gray, 1928; Hayashi, 1945; Rothschild, 1948, 1956a, b; Rothschild and Tuft, 1950; Mohri, 1956a, b). The studies of mamn:ialian sperm by Emmens and Swyer (1948), Blackshaw (1953a), and White (1953) indicate that some essential substance, or substances, is lost during dilution of the sperm suspension. Such loss can be partially counteracted by the addition to the diluent of K+ (Blackshaw, 1953b; White, 1953) or of seminal plasma or certain large molecular compounds (Chang, 1959). The nature of the loss, the protective


effect of colloidal substances, and the intracellular changes involved in the dilution eft'ect in mammalian sperm are still obscure. The alterations in the sperm are probably not mere physical changes but rather chemical alterations wdiich involve the metabolic state. The dilution phenomenon in invertebrate spermatozoa, for example, seems to involve an activation of the cytochrome system or other changes in respiratory pattern induced by such factors as pH or copper ions of sea water (Rothschild, 1950, 1956b). Rothschild (1959) has shown an increase in both the initial heat production and [prolonged heat production of bull sperm diluted 1:3 with balanced saline solution, compared with the heat output of sperm in seminal plasma.

B. TEMPERATURE EFFECTS

Numerous studies have indicated a direct effect of temperature change on the overt behavior and survival of spermatozoa, but little attention has been directed toward the possible effect on fertilizing capacity of pretreatment of the gametes. One earlier investigation (Young, 1929c) indicated that exposure of guinea pig sperm in the epididymis to 45°C. for 30 minutes reduced the fertility rate, and treatment at 47°C. seriously impaired motility; nevertheless, those embryos which were produced by females inseminated with sperm treated at 45 to 46°C. were apparently normal. Hagstrom and Hagstrom (1959) recently demonstrated that the fertilization rate of sea urchins is enhanced by exposure of sperm to either slight increases or decreases in temperature before union of the gametes. The pronounced temperature changes to which sperm are exposed during vitrification are of a far different order of magnitude and are surprisingly well tolerated when properly controlled (see Artificial Insemination) .

C. IONIZING RADIATION

When very severe, irradiation can lead to impairment of motility and metabolism in animal spermatozoa; lower doses induce change in nuclear components with conseciuent abnormalities in development. Hertwig (1911) first demonstrated the paradoxical effect of fertilizing frog eggs with sperm


BIOLOGY OF SPERMATOZOA


771


exposed to radium emanations. At lower levels of treatment, abnormal young were produced, increasing in percentage and severity with increase in dosage. At high levels of radiation, normal young developed. The latter effect was attributed to the parthcnogenetic development of eggs stimulated by sperm incapable of participating in fertilization. This was confirmed by Rugh (1939) who found an increase in embryonic al)normality and death following fertilization by sperm x-irradiated with doses from 15 to 10,000 r; sperm treated with 50,000 r, however, failed to enter the eggs and a high proportion (91 per cent) of the parthenogenetic young were viable.

Since parthenogenesis is not readily induced in mammals, no such paradoxical effect is to be expected. Impairment of fertilization and induction of embryonic abnormalities have, however, been caused by x-irradiation of sperm in vitro. Irradiation of rabbit and mouse sperm induced changes as manifested by embryonic abnormalities and chromosomal aberrations after fertilization of normal eggs (Amoroso and Parkes, 1947; Bruce and Austin, 1956). y-Radiation when administered at doses of 32,000 to 65,000 r from a radiocobalt source depressed the motility of rabbit sperm (Chang, Hunt and Romanoff, 1957). After treatment with these high exposures, the sperm that were able to reach the ova showed little if any impairment in fertilizing cai^acity. However, even at a dosage of 800 r, blastocyst formation was retarded, and at 6500 r it was prevented altogether. Johansson (1946) had reported similar findings in fowl; high levels of x-irradiation (3000 to 12,000 r) reduced motility of sperm, whereas relatively low levels (600 to 1200 r) impaired development. The w^ork of Edwards (19541957) on the mouse indicates that irradiation, either x-ray or ultraviolet (nonionizing), while permitting fertilization, can render the male gamete incapable of taking part in development. Comparable radiomimetic effects were obtained by treatment of mouse sperm with either trypaflavine or toluidine blue (Edwards, 1958).

The effect of irradiation in mammalian sperm may be similar to that suggested for invertebrate sperm. At certain levels of x


irradiation the fertilizing capacity of sea urchin sperm is reduced, and the cause has been attributed to the formation in the medium of hydrogen peroxide, produced by splitting of water molecules and recombination of free radicals (Evans, 1947). It has also been suggested that stable organic peroxides, rather than hydrogen peroxide, are formed and that these are toxic to sperm, possibly acting by the oxidation of enzymatic sulfhydryl groups (Barron, Nelson and Ardao, 1948; Barron and Dickman, 1949; Barron, Flood and Gasvoda, 1949).

D. IONIC AXD OSMOTIC EFFECTS

Despite a mass of data concerning the action of electrolytes and pH changes on sperm motility, and recently on sperm heat production (Rothschild, 1959), relatively little has been done to assess the fertilizing capacity of pretreated sperm. Although certain ions in excess seem to have unusually detrimental effects on sperm survival, in vitro — for example, calcium, manganese, lithium, and chloride (Lardy and Phillips, 1943; MacLeod, Swan and Aitken, 1949) — of more surprising interest is the general resistance of sperm to nonbalanced saline media (see Bishop, 1961 ). Rabbit sperm, for instance, can tolerate 2.0 per cent NaCl for many hours if brought gradually into the hypertonic medium (Anderson, 1945), and bull sperm retain motility for several hours in isotonic KCl. Determinations of the degree to which fertilizing capacity is affected by such treatment might yield very significant results.

Chang and Thorsteinsson (19581)) have made an important beginning with this aim in view. They found that the fertilizing capacity of rabbit sperm is unimpaired by exposure for brief periods (10 to 20 minutes) before insemination in Krebs-Ringer solutions of one-half or twice isotonic concentration. Of jiarticular interest was the finding that motility, but not fertility, was dein-essed by treatment with the hypertonic medium ; one can assume that some recovery occui'red in the female tract. Beyond the limits of this range of tonicity, fertilizing capacity was reduced, as judged by observation of recovered tubal eggs; yet even witli solutions 0.1 oi' 4 times isotonic


772


SPERM. OVA, AND PREGNANCY


strength (which completely inhibited motility, in vitro) fertilization occurred, although at a low rate. Chang and Thorsteinsson also studied the tolerance of sperm to osmotic variation in relation to simultaneous changes in pH. In isotonic KrebsRinger medium, rabbit sperm withstood short exposure to acid or alkaline conditions over a pH range of about 5.6 to 10.0, based on observations of motility and conception rate. Under hypo- or hypertonic conditions, however, the upper limit of the pH range tolerated was significantly depressed. This work emphasizes once again the unusual resistance or adaptation of the mammalian germ cell to changes in ionic environment.

E. EFFECTS OF BIOLOGIC FLUIDS

Some effects of certain biologic fluids with which sperm come in contact have been discussed in previous sections. It is clear, for example, that seminal fructose serves the gametes as glycolytic substrate at the time of ejaculation; uterine fluid, or certain of its components, aids in the capacitation phenomenon of sperm during transport through the genital tract. In studies of the effect on fertilizing capacity, sperm have been treated, in vitro, with seminal plasma, urine, normal blood serum, and antisperm serum, as well as with isolated products of the female tract itself.

The beneficial effect of seminal plasma as sperm diluent, for example, was demonstrated in the rabbit by Chang (1947b). Tests on 33 rabbits showed the advantage (percentage of fertilized eggs I of homologous plasma over saline when the does were inseminated with a minimal number of sperm. It was subsequently indicated that heterologous plasma from human semen was equally effective when used as a diluent for rabbit sperm (Chang, 1949). Bull seminal plasma, however, was injurious to rabbit sperm and caused a significant reduction in fertilizing capacity. It is not clear whether the favorable action of plasma, when it occurs, is due to a specific factor or set of factors, or whether it is caused by a nonspecific action such as chelation by the amino acid or polypeptide components present. The role of chelating substances in


extending the motility, metabolism, and fertilizing capacity of sperm has been demonstrated in several invertebrate and vertebrate species (Lorenz and Tyler, 1951; Tyler and Rothschild, 1951; Tyler and Tanabe, 1952; Tyler, 1953; Rothschild and Tyler, 1954). Such an effect was indeed suggested by the work of Chang, since preparations of dead heterologous sperm were as effective as seminal plasma in augmenting fertility in the rabbit (Chang, 1949). These findings may have some bearing on those cases in which, it has been claimed, resuspension of human sperm in foreign plasma improves motility and fertilizing capacity (see Rozin, 1958). A possible detrimental effect of seminal plasma on the fertilizing capacity of sperm was indicated by the demonstration of Chang (1957) that plasma destroys or counteracts the capacitation response of sperm within the rabbit genital tract (see above).

Although it is generally believed that urine is harmful to spermatozoa, Chang and Thorsteinsson (1958a) have shown that rabbit sperm tolerate exposure to 50 per cent urine for 10 to 15 minutes with no disturbance in conception rate. A urine concentration of 75 per cent can seriously impair sperm motility, in vitro, but even this treatment does not prevent fertilization when these same sperm are artificially inseminated into receptive does.

As has long been known, normal blood serum sometimes agglutinates spermatozoa, usually in a head-to-head type of aggregation. This is regarded as a nonspecific agglutination response, and the serum factor which brings it about can be destroyed or effectively reduced by heating to approximately 60°C. In an investigation of the effects of sera on homologous and heterologous sperm, Chang (1947a) demonstrated a complement-like agglutinating component which generally was toxic to the sperm of both its own and of other species; the one exception was the factor in human serum which was ineffective on human sperm. The substance in rabbit serum was found chemically unstable, thermolabile, and nondialyzable. Such an agent was detectable in the sera of man, bull, rabbit, guinea pig, and rat; very little is known, however, concern


BIOLOGY OF SPERMATOZOA


773


ing its origin or possible role, if any, during normal reproductive processes.

The effects of antiserum on the gametes have been discussed in a previous section. It will be recalled that the tyi)ical cellspecific agglutination and inniiobilization responses can render the sperm incapable of fertilization. Further, the experiments of Kiddy, Stone and Casida (1959) suggest a differential effect of treatment with high and low concentrations of antisperm serum. The former impairs fertilizing capacity; the latter results in abnormal development after fertilization (see section on Lnmunologic Problems) .

The impendency of fertilization provokes consideration of several types of sperm responses which can profitably be introduced here and further elaborated on in the chap


ter by Blandau. These responses involve interrreactions of the sperm and egg, or egg exudates, and concern such processes as chemotaxis, sperm activation, sperm agglutination, and the acrosome reaction.

With respect to the occurrence of chemotaxis, that is, the directed movement toward the egg in response to a chemical gradient from the egg, the evidence relating to animal gametes is essentially negative (Rothschild, 1956b). Many earlier claims for sperm chemotaxis among lower animals (see Hcilbrunn, 1943), and certain recent reports on mammalian species (Hiibner, 1955; Schuster, 1955; Schwartz, Brooks and Zinsser, 1958), can be readily ascribed to "trapaction," that is, the accumulation of sperm in the vicinity of the egg or egg substance, and not to a nonrandom movement of the


k


% - '^


?^B




Fig. 13.19. Fertilizin reaction in nianinial. Agglutination of rabbit .sperm in immediate vicinity of egg collected from rabbit oviduct. Sperm agglutinates form predominantly in head-to-head patterns. (From D. W. Bishop and A. Tvler, J. Exper. Zool., 132, 575-601, 1956.)



Fig. 13.20. Electron micrographs of sea iirchiu spermatozoa (llonicentrotus pulclicrriniu.s) : A, control, formalin-fixed in sea water; B, formalin-fixed two seconds after addition of egg water showing breakdown in acrosomal region and extrusion of protoplasmic mass; C, formalin-fixed 20 seconds after addition of egg water; D, formalin-fixed three minutes after addition of egg water. The agglutination which results from the addition of egg water is reversed at 2.5 minutes. (Photographs courtesy of J. C. Dan.)

774


BIOLOGY OF SPERMATOZOA


spenn toward it. The iihenomenon of chemotaxis has, however, been established as occurring in some primitive ph^nts, such as certain ferns, mosses, and brown algae, and the various attempts to determine the nature of the chemical stimulus and the mechanism of the response have been attended by some success (Pfeffer, 1884; Shibata, 1911; Cook, Elvidge and Heilbron, 1948; Cook and Elvidge, 1951; Rothschild, 1951b, 1956b; Wilkie, 1954; Brokaw, 1957, 1958a, b).

Activation of sperm by homologous eggs and egg exudates has been described in some invertebrate species, and the stimulating activity has been attributed to the fertilizin (gvnogamone I) present in the egg jelly coat (Lillie, 1919; Tyler, 1948; Rothschild, 1956b). The source of the activator and the specificity of the reaction are, however, somewhat controversial. The increase in motility, when observed, may or may not be accompanied by a substantial enhancement in respiratory activity (Rothschild, 1956b).

The species-specific agglutination of invertebrate spermatozoa by fertilizin of homologous eggs constituted the keystone of the fertilizin theory advanced by Lillie (1919) to account for the specificity and "cell recognition" inherent in the process of fertilization. The nature of the serologiclike gametic substances — egg fertilizin and sperm antifertilizin — and the role these substances may play in the fertilization process have been extensively studied by Tyler and coworkers (1948-1959). Sperm agglutination by egg exudates has been demonstrated in many species of animals in both the invertebrate and lower vertebrate groups (see Tyler, 1948). The phenomenon is also exhibited by mammalian gametes (Fig. 13.19), among which some degree of species specificity is displayed (Bishop and Tyler, 1956 ) . A current view of the possible significance of these gametic substances to fertilization may be found in the recent review by Tyler (1959).

Spermatozoa not only seem to interreact serologically with egg exudates resulting in agglutination and/or loss of fertilizing capacity; they also can be stimulated under some circumstances to undergo morphologic change, most spectacularly characterized by


the acrosome reaction (Dan, 1952, 1956; Colwin and Colwin, 1955, 1957). The forcible release of material from the sperm head (Fig. 13.20), apparently induced by the presence of egg fertilizin, involves the protrusion of a filamentous projection which seems to play a vital, if, as yet obscure, role in the initial stage of the fertilization process.

XI. Conclusion

The notation of a conclusion to the Biology of Spermatozoa seems singularly inappropriate. Both the intensity and the expanse of current research indicate that one is merely taking stock of accumulating data and transient concepts — that in the future lies the answer to most of the ciuestions raised in the pages above. On the one hand, the properties of spermatozoa can be expected to become increasingly clear by our delving more deeply into the nature and activity of the cell, a fruitful approach in its own right and beneficial to the more practical concerns of fertility, sterility, and animal breeding. On the other hand, the recognition of the general characteristics of sperm behavior, movement, metabolism, and survival seems likely to shed brighter light on comparable processes and systems in other cells and tissues, including the nature of cell regulation and adaptation, energy utilization, aging, and movement inherent in ciliary activity, flagellation, and muscular contraction.

If much of the foregoing seems more fragmentary than complete, more provocative and speculative than dogmatic or resolute, this survey may then serve some purpose. The accomplishments have been many, but even more fascinating developments lie ahead.

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14



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Pages where the terms "Historic" (textbooks, papers, people, recommendations) appear on this site, and sections within pages where this disclaimer appears, indicate that the content and scientific understanding are specific to the time of publication. This means that while some scientific descriptions are still accurate, the terminology and interpretation of the developmental mechanisms reflect the understanding at the time of original publication and those of the preceding periods, these terms, interpretations and recommendations may not reflect our current scientific understanding.     (More? Embryology History | Historic Embryology Papers)
Young WC. Sex and internal secretions. (1961) 3rd Eda. Williams and Wilkins. Baltimore.
Section A Biologic Basis of Sex Cytologic and Genetic Basis of Sex | Role of Hormones in the Differentiation of Sex
Section B The Hypophysis and the Gonadotrophic Hormones in Relation to Reproduction Morphology of the Hypophysis Related to Its Function | Physiology of the Anterior Hypophysis in Relation to Reproduction
The Mammalian Testis | The Accessory Reproductive Glands of Mammals | The Mammalian Ovary | The Mammalian Female Reproductive Cycle and Its Controlling Mechanisms | Action of Estrogen and Progesterone on the Reproductive Tract of Lower Primates | The Mammary Gland and Lactation | Some Problems of the Metabolism and Mechanism of Action of Steroid Sex Hormones | Nutritional Effects on Endocrine Secretions
Section D Biology of Sperm and Ova, Fertilization, Implantation, the Placenta, and Pregnancy Biology of Spermatozoa | Biology of Eggs and Implantation | Histochemistry and Electron Microscopy of the Placenta | Gestation
Section E Physiology of Reproduction in Submammalian Vertebrates Endocrinology of Reproduction in Cold-blooded Vertebrates | Endocrinology of Reproduction in Birds
Section F Hormonal Regulation of Reproductive Behavior The Hormones and Mating Behavior | Gonadal Hormones and Social Behavior in Infrahuman Vertebrates | Gonadal Hormones and Parental Behavior in Birds and Infrahuman Mammals | Sex Hormones and Other Variables in Human Eroticism | The Ontogenesis of Sexual Behavior in Man | Cultural Determinants of Sexual Behavior


Reference: Young WC. Sex and internal secretions. (1961) 3rd Eda. Williams and Wilkins. Baltimore.


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