Book - Sex and internal secretions (1961) 6

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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 C Physiology of the Gonads and Accessory Organs

The Accessory Reproductive Glands of Mammals

Dorothy Price, Ph.D.

Professor Of Zoology, The University Of Chicago

and

H. Guy Williams- Ashmari, Ph.D.

Associate Professor, Ben May Laboratory, The University Of Chicago


I. Introduction

embryonically from the mesonephric or Wolffian duct (ductus deferens) i.e., the ampullary glands (glandula vasis deferentis) and seminal vesicles or vesicular glands, and those deriving from the urogenital sinus or urethra, namely the prostate and bulbo-urethral or Cowper's glands (see chapter by Burns). The anatomic relationships established in the fetus are retained to a considerable degree postnatally so that the ampullary glands and seminal vesicles are associated with the ducti deferentes. However, in some mammals the seminal vesicles empty into the pelvic urethra close to the openings of the deferent ducts but separate from them; no ejaculatory ducts are present. The prostatic and bulbourethral glands are associated with the proximal and distal urethra, respectively. The secretion of the prostate is discharged, in most cases, through multiple ducts that join the prostatic urethra at the level of the colliculus seminalis. The ducts of the bulbo-urethral glands drain into the urethra in the region of the urethral bulb.

In addition to these accessory reproductive glands, there are small mucus-secreting glands (of Littre) opening into the urethra along its length, and preputial glands (which are modified sebaceous glands) emi)tying their secretion on the prepuce.

In many female mammals, homologues of the male prostate and bulbo-urethral glands develop in the fetus. These glands may retrogress prenatally, remain vestigial, or develop postnatally and become functionally active. These homologues are the female prostate glands (para-urethral glands of Skene) and the bulbovestibular (major vestibular or Bartholin's glands). In addition, there are urethral glands (minor vestibular) which are homologous with the male urethral glands of Littre, and female preputial or clitoridal glands corresponding to the male preputials. The major vestibular, when present, and the minor vestibular and clitoridal glands are functional in many mature females. In a few cases, well developed prostate glands which are actively secretory have been found in females of four mammalian orders.


B. General Characteristics

The male accessory reproductive glands of higher mammals have many characteristics in common. Typically, all possess (1) a secretory epithelium which is enormously increased in effective secretory area by villous infoldings, or by a compound tubuloalveolar structure, (2) an underlying layer of connective tissue (the lamina propria) and (3) smooth muscle fibers. It is now well established that the secretory activity of the epithelial cells is normally under the control of testicular hormones. The secretions pass from the cells into the lumina of the glandular alveoli where they are usually stored until ejaculation.

The sensory innervation includes various types of sensory nerve endings in the connective tissue, and free nerve endings in the epithelium. The autonomic innervation is parasympathetic (nervi erigentes) and sympathetic (hypogastric nerve) from the pelvic plexus. If the plexus is resected or the sympathetic chain above is interrupted, there is no reflex ejection of the glandular secretions. When the hypogastric nerve is stimulated, peristaltic waves of contraction occur in the ductus deferens, and there is contraction in the seminal vesicles and prostate which partially empties the stored secretion from the lumina of these glands. Stimulation of the parasympathetic system or the administration of pilocarpine results in an increased output of prostatic secretion.

There are marked dissimilarities in gross structure, character of the epithelia, and the chemical nature of the secretions in the various glands — prostates, seminal vesicles, bulbo-urethral, and ampullary (Mann, 19o4a). There are also differences in structure and function between homologous glands in related forms. The nomenclature that was applied to the glands in early descriptive studies was often based on anatomic relationships and gross morphologic structure in adults. This resulted in some confusion in classification, but most of the disputed points have been clarified and some of the homologies have been established by embryologic study. The extensive studies of INIann (1954a) show clearly that


TABLE 6.1 Occurrence of male accessory reproductive glands and their homologues in females"

Male*

Female<^


Order


Bu


Pr


Sv


Am


Bv


Pr


Genera and species with functioning female prostates


Monotremata . . .


+


_?


_


_


+


_



Marsupiala


+


+




+




Insectivora


+


+


±


±


+


+


Erinaceoii.s europeus (Deanesly, 193-4) Hemicentetes (Lehmann, 1938) Talpa europea (Godet, 1949)


Chiropt era


+


+


±


±


+


=t


Coelura afra (Mathews, 1941) Taphozous sp. (Mathews, 1941) Nycteris luteola (Mathews, 1941) Carioderma cor (Mathews, 1941)


Primates


+


+


±


=b


+




Carnivora


±


+



zb


±




Perissodactyla...


+


+


+


+


+


_



Artiodactyla ....


+


+


+


±


+




Hyracoidea


+


+


+






Proboscidea


+


+


+


+





Sirenia



+


+






Cetacea



+







Edentata


±


+


+






Pholidota



+


+






Rodentia


+


+


+


=t


±


±


Arvicanthus cinereus (Rant her, 1909)









Rattus norvegicus (Marx, 1931, 1932; Korenchevsky



and Dennison, 1936; Korenchevsky, 1937; Witschi,


Mahoney and Riley, 1938; Price, 1939; Mahoney,


1940, 1942; Mahoney and Witschi, 1947)


Mastomys erythroleucus (Brambell and Davis, 1940)


Apodemus sylvaticus (Raynaud, 1942, 1945)


Microtus arvalis (Delost, i953a, 1953b)


Lagomorpha. . . .


+


+


±


+


+


±


Sylvilagus floridanus (Elschlepp, 1952)


"Compiled from Oudemans, 1892; Engle, 1926a; Retief, 1949; Eckstein and Zuckerman, 1956 and others. + indicates the presence of a well developed functioning gland. — indicates either a small vestigial gland or the absence of any rudiment.

^ Bulbo-urethral (Cowper's), prostate, seminal vesicle and ampuUary glands.

"^ Bulbovestibular (Bartholin's) and prostate glands (para-urethral glands of Skene); genera and species refer only to those in which functioning female prostates have been reported in the listed references.


homologous organs do not necessarily have the same chemical functions.

Finally, there is variability among orders of mannnals and families within orders, with respect to the accessory glands which are present (Table 6.1). The prostate is the only u;land that is found almost universally.


C. Survey Of The Glands

1. Bulbo-urethral Glands and Bulbovestibular

Bulbo-urethral (Cowper's). The bulbourethral glands are compound tubulo-alveolar glands resembling mucous glands in some respects. Their secretion is a viscid lubricant which is em])tied into the bulbar region of the pelvic urethra. There may be a single pair of glands as in the monotremes, primates, and rodents, or as many as three pairs (Fig. 6.1), as in some marsupials (Chase, 1939; Rubin, 1944). Their relative size, gross structure, and complexity vary widely. For example, they are small, compact, bean-shaped glands in man, relatively enormous, complicated glands in squirrels, and large, cylindrical glands in the boar.

Bulbo-urethral glands are notably lacking in Cetacea, Sirenia, and certain carnivores such as seals, walruses, sea lions, all raustelids, and the bear and dog (Oudemans, 1892; Engle, 1926a; Eckstein and Zuckerman, 1956). (Oudemans made a point of the fact that they are not present in aciuatic mammals, but it is rather doubtful if their absence is related to an aquatic environment. BuLBOVESTiBULAR (Bartholin's) . The bulbovestibular or major vestibular glands are also compound tubulo-alveolar glands which resemble their male homologues in structure and secrete a mucus-like substance. Their secretory function is under control of ovarian hormones and they involute when the ovaries are removed. They are widely distributed in the various orders of mammals although the information is fragmentary with respect to some groups. A single pair of glands is the general rule and they are usually much smaller than the bulbo-urethral. In the female opossum, the single pair of glands is homologous with the smallest of the three pairs of Cowper's glands. In the adult, they are well developed and filled with colloid (Rubin, 1944). In monotremes the ducts open at the base of the clitoris, in opossums into the urogenital sinus canal, and in hyenas (where they are well developed) into the urogenital canal close to the base of the clitoris (Eckstein and Zuckerman, 1956). In many other females the ducts open into the vestibule. In the adult human female, Bartholin's glands resem])le Cowper's glands closely in histologic structure.

2. Male and Female Prostate Glands

Male prostate. The prostate is a compound tubulo-alveolar gland in which the gross structure is variable and may be (1) disseminate or diffuse, in which the glandular acini remain within the lamina propria around the urethra and do not penetrate the voluntary muscle of the urethra, (2) a type in which the gland forms a '"body," sometimes lobed, outside the urethral muscle, or (3) a combination of both types. A disseminate prostate is found in some marsupials (Fig. 6.1) and edentates, and in sheep, goats, the hippopotamus, and the whale. The bull and boar prostates have a disseminate region as well as a discrete body of the gland. In mammals in which there is a glandular body, there may be a solid, compact prostate as in the dog and man, or several lobes as in rodents (Figs. 6.2 and 6.3), lagomorphs (Fig. 6.4), and insectivores.



Fig. 6.L Male opossum reproductive tract. B, bladder; C, Cowper's glands; D, ductus deferens; E, epididymis; P, penis; Pr, prostate I, II, III surrounding the urethra; T, Testis; U, ureter. (Redrawn from C. R. Moore, Phj'siol. ZooL, 14, 1-45, 194L)

A prostate gland has been found in all mammals that have been studied except monotremes, and is the only accessory gland in carnivores such as the ferret, weasel, dog, and bear, and in cetaceans — whales, dolphins, and porpoises. Oudemans (1892) considered that monotremes and marsupials lack prostate glands but possess well developed urethral glands. His classification of glands as "urethral" (glands of Littrei or "prostatic" depended on whether the glandular acini remained in the urethral stroma or penetrated the muscle to form a



Fig. 6.2. Mule hamster accessory repidiluri i aspect.

body outside. It is now recognized that marsupials such as the opossum have a disseminate prostate, and in Didelphys Virginian a there are three regions which differ


5EMINAL VESICLE .- RIGHT


DORSAL PROSTATE


^VENTRAL PROSTATE


gl.-inds. Above, ventral aspect; below, dor.sal


clearly in histologic structure (Chase, 1939). It may be considered that there are three prostatic "lobes" probably differing in function as well as in structure. Although Oude



Fig. 6.3. Male guinea pig accessory reproductive glands. (From E. Ortiz, D. Price, H. G. Williams-Ashman and J. Banks, Endocrinology, 59, 479-492, 1956.)


mans concluded that monotremes lack prostate glands, he described a concentration of urethral glands at the neck of the bladder in the duckbill platypus. Ornithorhyncus poradoxicus. The diagrams in his monograph suggest that this concentration of complicated glands is a disseminate type of prostate.

There has been confusion in the nomenclature of the lobes of the prostate in the rat and in the descriptions of the structure of the lobes. In early studies the application of human anatomic terminology to rodents resulted in designation of the lobes as anterior (ventral), middle, and posterior (dorsal). Later terminology, more suitable for


cfuadrupedal animals, led to anterior (cranial), middle, and posterior (caudal). Unfortunately, combinations of these two systems of nomenclature still occur in the literature, and there is uncertainty as to the number of histologically distinguishable regions or lobes. In view of the current interest in the chemical composition of the glands and their secretions the subject will be reviewed.

For many years the prostate was usually described as being composed of three pairs of lobes: cranial or anterior (coagulating glands) bound to the seminal vesicles; middle or dorsolateral nearly encircling the urethra dorsolaterally, and the ^-entral or


Fig. 6.4. Rabbit accessory reproductive glands; lateral aspect. Left, domestic male; center, cottontail male; right, cottontail female. B, bulbo-urethral gland; C, coagulating gland (vesicular gland); D, ductus deferens; P, paraprostate ; Pr, prostate; V , urethra; VC, urogenital canal (vestibulum) ; V, vagina. (Redrawn from J. G. Elschlepp, J. Morphol., 91, 169-198, 1952.)


liosterior (Moore, Price and Gallagher, 1930; Callow and Deanesly, 1935; Price, 1936). Korenchevsky and Dennison (1935) noted that the histologic structure of the dorsal lobe (or region) is quite similar to that of the coagulating glands whereas the lateral lobes more nearly resemble the ventral. This has been confirmed in histologic and functional studies (Price, Mann and Lutwak-Mann, 1955). Gunn and Gould (1957a) reported differences in histologic structure and functional activity in the two lobes.

The lateral lobes can be distinguished grossly from the dorsal by anatomic relationships and color, but the glandular lobules form a continuous mass and can be separated into distinct lateral and dorsal lobes only by dissection. This can be accomplished with considerable accuracy in immature males and young adults; in large rats it is more difficult because of distention of the alveoli and overlap of lobules in contiguous regions. The ventral tips of the lateral lobes extend down and partially underlie the ventral lobes to which they are loosely bovmd. The dorsal prostate is somewhat butterfly-shaped with a single cranial region and wings extending caudally along the urethra much as in the hamster (Fig. 6.2). By dissection in the midline, it can be divided into right and left lobes. The dorsal and lateral prostates are drained by 50 or more ducts opening into the roof of the prostatic urethra (Witschi, Mahoney and Riley, 1938). Those from the dorsal region open more dorsally; those from the lateral lobes, laterodorsally.

Some of the confusion in prostatic terminology arises from the general application of the word "lobe" to (1) organs that are grossly anatomically distinct, (2) regions that do not form entirely discrete structures but can be distinguished histologically, (3) ])arts of the gland which contain two histologically different portions, and (4) regions that differ, not in histologic structure but in response to hormones and in the tendency to ])athologic growths (human and dog).

The lobation of the human prostate has been the subject of controversy for some time. It is of especial interest because one region, the posterior or dorsal lobe, is commonly the site for prostatic carcinoma and another, the more anterior or ventral region, for benign prostatic hypertrophy. The lobes have been described as posterior, anterior, middle, and two lateral, or as posterior and anterior, or outer and inner (medullary). The component parts of these regions have been discussed extensively (see Moore, 1936; Huggins and Webster, 1948; Retief, 1949; Franks, 1954). Lowsley (1912) studied the embryologic development of the human prostate and concluded that the gland derives from five independent groups of tubules. A cranial posterior or dorsal group (lobe) arises from the dorsal wall of the prostatic urethra or urogenital sinus; right and left lateral lobes originate from the prostatic furrows and grow back to form the main part of the base of the gland; a middle lobe derives dorsally from the urethra between the bladder and ejaculatory ducts; a ventral or anterior lobe forms but regresses and becomes insignificant.

Although these prostatic buds or tubules form independent groups in their embryonic origin there is no clear separation into such groups in the human prostate postnatally. However, Huggins and Webster (1948) were able to distinguish clearly two different regions, a posterior and an anterior lobe, by differential response to estrogen administration. The extent of the anterior or ventral lobe, as delimited by them, apparently includes the tubules of the middle and lateral lobes as described by Lowsley.

The pioneer studies of Walker (1910a) on the coagulating function of discrete glands of the prostatic complex in rats and guinea pigs (Fig. 6.3) were followed by specific identification of coagulating glands in several rodents including mice and hamsters (Fig. 6.2) and in the rhesus monkey (van Wagenen, 1936). However, a copulation plug in the vagina of females has been reported in some marsupials, insectivores, chiropterans, the chimpanzee among the primates (Tinklepaugh, 1930), and several genera of rodents in which coagulating glands have not been identified. Eadie (1948a) found that in an insectivore, Condylura cristata, there is a peculiar prostatic secretion from paired ventral lobes. It contains an enormous number of amyloid bodies resembling the corpora amylacea present in the prostate gland of man and some other mammals. These prostatic concretions are generally considered abnormal, but Eadie suggested that this unusual secretion, which was found in all breeding males, might be instrumental in the formation of a unique type of copulation plug. A large urethral" gland which lies between the prostate and bulbo-urethral glands and surrounds the urethra is peculiar to certain species of bats. Mathews (1941) considered it probable that the presence of this gland is correlated with the formation of a large copulation plug, but he did not ascribe a specific coagulating function to the gland (which bears a histologic resemblance to the bulbo-urethral glands in some bats).

The difficulties of homology and classification can be illustrated by the case of the rabbit. Differences of opinion have existed concerning the nomenclature and homologies of the seminal vesicles (or prostatic utricle), vesicular glands (seminal vesicle or prostate), and paraprostate glands (or superior Cowper's glands). In studies on embryologic development and histologic structure, Bern and Krichesky (1943) clarified the problem. They established that the domestic rabbit has true seminal vesicles, vesicular glands (which are considered as probably homologous with the coagulating glands of rats), prostates, paraprostates (usually similar to the bulbo-urethrals in histologic structure but in about one-third of the cases, one or more of the paraprostates resembled the prostate histologically), bulbo-urethral glands, and glandular ampullae. Elschlepp (1952) compared the accessory glands of the cottontail, Sylvilagus floridamis, with those of the domestic rabbit, and concluded that coagulating glands (avoiding the usage of "vesicular glands" which has often been used synonymously with seminal vesicles) , dorsal prostates, and bulbo-urethral glands are homologous in the two species. The adult cottontail has neither paraprostates nor seminal vesicles (Fig. 6.4) . Classification of the glands in the hedgehog and shrew has also presented problems (see discussion in Eckstein and Zuckermann, 1956; Eadie, 1947). Among the Sciuridae, many possess a bulbar gland which differs from their true Cowper's glands (Mossman, Lawlah and Bradley, 1932). It is evident that among mammals there are many potentialities for forming accessory glands with varied anatomic structure, histologic characteristics, and functional activities.

Female prostate. In fetuses of many female mammals, small cords of cells which represent the homologues of the male prostate bud off from the epithelial lining of the urethra. These primordia normally retrogress or remain vestigial and only rarely continue to develop after birth. In the human female, these rudimentary structures are known as para-urethral glands of Skene. They have also been referred to as periurethral glands. However, it seems advisable, as Witschi, Mahoney and Riley (1938) suggested, to restrict the usage para-urethral and peri-urethral to the aggregations of mucus-secreting glands that have short ducts opening into the urethra. These clearly differ from the true female prostate glands.

In contrast to the rudimentary prostate glands which are retained postnatally by some female mammals, relatively large, well developed female prostates have been reported postnatally in some insectivores, chiropterans, rodents, and lagomorphs. The male accessory glands of many species in these orders are exceptionally well developed and the prostates are usually lobed. Female prostates are tubulo-alveolar glands, as are their male homologues, and they too form lobes, but the glands are never as large as those of the male. Their secretory activity is apparently dependent mainly on ovarian androgens, but the function, if any, of the secretion is obscure. Extensive research has shown that the administration of androgens to rodents, either to pregnant females or fetuses, to fetal lagomorphs, and to pouch-young oppossums, results in the formation and retention of prostates in females which normally do not have such glands (see chapter by Burns).

Deanesly (1934) described vaginal glands in the female hedgehog and suggested that one pair is homologous with the external prostates of the male. The female glands extend dorsolaterally on either side of the urethra and a single duct from each lobe opens into the vagina. They seem to be active during the breeding season and to retrogress in the anestrum. In another insectivore,


Hemicentetes, there is a pair of large "paravaginal glands" which are functionally active in the mature female and have large acini filled with secretion. They resemble the male prostate in histologic structure and anatomic position, but have no ducts (Lehmann, 1938). In adult European moles, most females have bilobed ventral prostate glands which undergo cyclic changes in the epithelium. The prostate of the male is also bilobed and ventral in position and the homology in the two sexes is clear (Godet, 1949).

Mathews (1941) studied the anatomy and histophysiology of the male and female genital tracts of nine species of African bats. Female prostates are well developed in four species, less conspicuous in a fifth, and absent from the remaining four. There is a marked tendency for greater development of the glands in pregnant and lactating females. In three species, the female prostates surround the urethra (as do their male homologues), but in Nycteris luteola the female prostate appears ventrally, whereas the male prostate in this species is limited to the dorsal aspect of the urethra. Mathews considered that the female prostates represent greatly enlarged female urethral glands which are homologous with the male ])yostate.

The occurrence of female prostates and their relation to hormones have been most extensively studied in rodents. The first description of a well developed female prostate gland seems to be that of Rauther (1909), who found such a gland ventral to the neck of the bladder in the African field rat, Arvicanthis cinereus. In Rattus norvegicus, Marx (1931, 1932) reported the sporadic occurrence of female prostates. Korenchevsky and Dennison (1936) and Korenchevsky (1937) found prostates in 9 of 56 females and stated that the glands wei'c atrophic, but when androgens were administered, these glands resembled the male ventral prostate. On this basis, the homology of the glands of the female with the \-entral prostate of the male was suggested. Further studies (Witschi, Mahoney and Riley, 1938; Mahoney, 1940, 1942; Malioney and Witschi, 1947) showed that the female prostate of the rat is homologous with only the most medioventral part of the male prostate ; the lobes are bilateral or unilateral, with the right the preferred side; each lobe has a single duct which opens into the urethra. The incidence of female prostates varies markedly in different strains, and can be increased by selective inbreeding, w^iich also increases the occurrence of bilateral compared with unilateral lobes. The frequency of female prostates was increased in the Wistar stock from 28 to 99 per cent, but when selective inbreeding was stopped, the frequency declined.

In young untreated female rats, the prostate, when present, develops a histologic structure identical with that of the male homologue, but at about 6 weeks of age the epithelium undergoes regression (Price, 1939; Mahoney, 1940) and becomes histologically well developed again only during pregnancy and lactation (Burrill and Greene, 1942; Price, 1942). Thus, the female prostate of the rat is not only homologous with a part of the ventral prostate of the male on the basis of embryologic development, but during early postnatal development and in periods of pregnancy and lactation it resembles its male homologue histologically (Fig. 6.46). In addition, it is functionally equivalent (see Section II) to the male ventral prostate in the secretion of citric acid (Price, Mann and LutwakMann, 1949).

Brambell and Davis (1940) found large, well developed prostate glands in every one of 104 female African mice, Mastonujs erythroleucus Temm. These glands consist of paired lobes, each draining into the urethra by a single duct. They resemble the ventral prostate of the male in position, shape, and histologic structure. On the basis of this evidence it was concluded that the female glands are the homologues of the male ventral prostate. In some cases, the female prostates are nearly as large as their male homologues and are actively secretory. Brambell and Davis correlated hypertrophy and secretory activity with the luteal phase of the cycle and gestation.

Female prostate glands have also been described in the field mouse, Apodemns sylvaenms sylvaticus. Raynaud (1942, 1945) found bilobed prostates in 51 immature and adult females collected in the vicinity of Vabre (Tarn) and in 3 females from three other regions of France. However, the lobes were macroscopically visible in only 10 females; in all others, the glands were identified in histologic preparations. There w^as great variability in histologic structure, but a well developed epithelium showing secretory activity was found during pregnancy and lactation. Raynaud established that the female prostate is homologous with a part of the male ventral prostate. He concluded that there is a probability that bilobed female prostates exist normally in all females of Apodemus sylvaticus.

The prostate glands in adult female field voles, Microtiis arvalis P., are considered homologous with the ventral lobes and part of the lateral lobes of the male prostate (Delost, 1953a, b). The lobes in the female are lateral in position in part of the gland, but in other regions they completely surround the urethra. The structure is identical with that of the ventral prostate of the male. The epithelium appears secretory in normal adult females, and during gestation this activity is intense.

Bilobed female prostates were found in the 37 adult cottontail rabbits examined by Elschlepp (1952). They lie on the dorsal wall of the vagina (Fig. 6.4) and are similar histologically to the prostate of the male. The glands are larger in pregnant than in nonpregnant females and contain more secretion.

In summary, well developed female prostate glands are present in immature and adult females of many species. They may occur as ventral, lateral, or dorsal lobes; the lobes may be unilateral or consistently bilateral; their occurrence may be sporadic or reach an incidence of 100 per cent; they are found both in laboratory strains and in wild populations. The genetic studies of Witschi and his collaborators show that the incidence in rodents can be increased by selective inbreeding. In certain populations of wild rodents the character has become established. A striking example of this is the presence of large prostates in all female Masto)7iys erythroleucus. The secretory activity of the glands seems to be controlled mainly by ovarian androgens (sec Section III) . No function can be ascribed to the secretion.

3. Seminal Vesicles

The seminal vesicles are paired, usually elongated glands which may appear relatively simple externally (Figs. 6.2 and 6.3) but are subdivided internally by complicated villous projections. The name refers to an old misconception that they are sperm reservoirs. The seminal vesicles are relatively enormous and distended with secretion in some mammals, for example, the rat, guinea pig, and hamster; they are large in others such as the boar, and in still others, as in man, they are small and compact.

Seminal vesicles are absent from the monotremes, marsupials, carnivores, and cetaceans that have been studied, and from some insectivores, chiropterans, primates, and lagomorphs. Variability exists among the edentates ; the sloths and the armadillo have seminal vesicles which are well developed in the two-toed sloth and armadillo, but are very small and rudimentary in the three-toed sloth. Among the lagomorphs, the seminal vesicle of the domestic male rabbit is a large unpaired gland whereas the seminal vesicles in the adult cottontail rabbit are vestigial or absent although they develop for a time in the fetus (Elschlepp, 1952).

4. Ampullary Glands

These organs are glandular enlargements arising from the ampullae of the ducti cleferentes or the posterior region of the ductus if a distinct ampullary enlargement is not present. They may be only slight glandular enlargements of the wall, or discrete glands which nearly encircle the ductus deferens as in rats, some mice, and hamsters (Fig. 6.2). They are vestigial in certain pure line strains of mice (Horning, 1947) and lacking in guinea pigs (Fig. 6.3). In some bats, they attain very large size. In general, they are absent from many mammalian orders and variable in others (Table 6.1).

D. Evolutionary History of Accessory Reproductive Glands of Mammals

The well developed male accessory glands which characterize the niamnialiaii class as a whole, and form such a conspicuous part of the reproductive tract in most mammals, are not found in nonmammalian vertebrates. These glands appear as anatomically distinct organs in the primitive prototherian mammals, the monotremes, which are definitely mammalian but which also retain certain anatomic characteristics of their reptilian ancestors and still lay shelled eggs. However, it has been suggested that in the evolution of the three groups of living mammals from mammal-like reptiles, the line of descent of monotremes is entirely separate from that of marsupials and placentals. Furthermore, the last two groups are probably parallel branches of the mannnalian stock.

The accessory glands of modern mammals represent, then, the parallel evolution of discrete glands that probably began their development very early in the evolutionary history of mammals. In gross structure, size, and internal complexity they are unique accessory organs among vertebrates. Modern reptiles have no such glands; the seminal plasma is composed mainly of secretions from the epididymis and the renal tubules of the sexual segment of the long, lobulated kidney. Both these regions become highly secretory during the breeding season (see chapter by Forbes) . Parenthetically, the semen of birds (a later offshoot from the reptilian line than mammals) contains only a small amount of seminal plasma (Mann, 1954a) which is secreted in the cock almost entirely by the seminiferous tubules and vasa efferentia (Lake, 1957). In modern mammals, the epididymal epithelium is still an important accessory secretory area (see chapter by Bishop) , but the bulk of the seminal plasma comes from glandular elaborations of quite different regions, the urogenital sinus (a derivative of the primitive cloaca) and the posterior part of the Wolffian ducts, the ducti deferentes.

Modern monotremes are specialized forms but in certain characteristics they are primitive. They show almost diagramatically some of the first steps in the evolution of accessory glands. The bulbo-urethrals are already well developed but the concentration of complicated urethral glands at the neck of the bladder in the duckbill platypus almost certainlv illustrates the derivation of a specialized gland (the prostate) from simpler glands which are nmnerous along the urethra. The probable evolution of prostates and bulbo-urethral glands from smaller, simpler urethral glands has been suggested in the past. Observations of Bruner and Witschi (1946) support this concept. In experiments on fetal hamsters, it was found that masculinized females developed prostate glands but the ducts joined the collecting ducts of the urethral glands and did not open directly into the urogenital sinus. According to these workers, this may represent an intermediate stage in the development of specialized glands.

The history of the cloaca may well be important in relation to development of accessory glands (Retief, 1949). The cloaca is retained in modern reptiles; in monotremes it is subdivided cranially into ventral urodeum or urogenital sinus and a dorsal coprodeum; it is represented by a pocket in marsupials but is lost as a discrete structure in all higher mammals. The first development of a separate urogenital duct or urethra as it occurs in monotremes may be correlated with the first appearance of discrete accessory glands from this specific region in mammals.

The marsupials illustrate a more advanced type of glandular development with three histologically distinguishable regions in the disseminate prostate and three pairs of bulbo-urethral glands. Seminal vesicles and ampullary glands are found only among higher mammals.

The size and structural complexity of these unique glands in mammals raises the question of the adaptive value of relatively large accessory glands associated with the mammalian reproductive tract. This is a matter only for speculation. The evolution of such glands with increased surface for secretion and enlarged storage space may, perhaps, have been correlated with a tendency for an increase in volume of seminal plasma in the ejaculate of mammals. Mann (1954a) pointed out the variability in the volume of ejaculated semen and in the sperm density in various species. With regard to the volume of seminal plasma, he stated, "In lower animals it may be so scarce that the emitted semen takes the form of a very thick lump of spermatozoa, closely packed together. There is little seminal plasma in bird semen and even among some of the mammals, but on the whole, the higher mammals including man, produce a relatively dilute semen with a considerable l)roportion of seminal plasma." A second suggestion, more speculative, is that the evolution of large mammalian glands may also have compensated for loss of accessory reproductive function in the kidney. The kidney of mammal-like reptiles and ancestral mammals may have contributed to the formation of seminal plasma (as is true in modern reptiles, amphibians, and fishes), but the compact kidney of warm-blooded, metabolically active mammals may be ill adapted for such a purpose.

II. Function of the Male Accessory Glands

A. Introduction

The only known function of the male accessory glands is to secrete the seminal plasma. The proportion of this fluid which originates from the various secretory organs, or even from different lobes of the same gland, varies greatly from one species to another. There is also remarkable species variation in the volume and composition of the individual secretions. The functional activity of the accessory glands is governed primarily by hormones of testicular origin. The output of androgens is subject to the control of the anterior hypophysis, and many factors (e.g., age, light, season, temperature, and diet) affect the secretory activity of the hypophysis and testis. Thus, it is not surprising that in a given individual, there may be marked fluctuations in the cjuantity and chemistry of the secretions of the accessory glands, and hence of the seminal plasma.

The development by Charles Huggins of ingenious surgical procedures enabled the secretory activity of the canine prostate to be measured by simple volumetric methods. Such studies of prostatic secretion in the dog established the quantitative relationships between the function of prostatic epithelium and the androgenic status of the host. In other species, serial collection of the individual secretions in the same animal has not been achieved for purely technical reasons. The use of "split ejaculates" has given some insight into the glandular origin of various conii)oncnts of the seminal plasma, but this techniciue does not provide uncontaminated secretions from any one gland. However, in the last two decades extensive analyses of the chemical and enzymatic constituents of the individual secretions stored in the accessory glands, and of the whole seminal plasma, have been performed. The levels of many of these substances and enzymes are dependent on androgenic hormones. These findings have provided a basis for sensitive chemical methods for the bioassay of androgens. Moreover, knowledge of the biosynthesis of these substances by the accessory glands may point to the primary biochemical locus of action of androgenic steroids. This chemical approach to the study of the accessory glands has received great impetus from the pioneer studies of Thaddeus Mann.

The secretions of the accessory glands of many species are a repository for huge Cjuantities of substances which are present only in trace amounts in other tissues and body fluids. It is obvious that the seminal plasma must provide an ionically balanced and nutritive milieu suitable for the survival of sperm in the vagina and uterus. Certain substances secreted by one or more of the accessory glands, e.g., fructose, undoubtedly serve as a source of energy for the sperm. However, there is no evidence that any component of mammalian seminal plasma, or any one of the accessory glands, is absolutely indispensable for fertility. Artificial insemination is successful in some mammals if sperm from the epididymis are diluted in a suital)ly prepared medium, placed in a female in the correct stage of the estrous cycle, and deposited in a region of th(; female tract where there is maximal opiiortunity for their successful ascent. Removal of the coagulating glands in guinea l)igs (Engle, 1926b) , or dorsolateral prostate (Gunn and Gould, 1958) in rats does not prevent insemination and fertilization. Blandau (1945) extirpated the seminal vesicles and coagulating glands of rats and found that when these males were mated there was no copulation plug and, evidently as a result, the spermatozoa did not penetrate the vaginal canals of the females. Thus the secretions of the coagulating gland and seminal vesicles in the rat assist the transport of sperm in the female.

The following section will consider the output and composition of the secretions, and their hormonal regulation, primarily from a chemical standpoint, rather than in relation to the anatomy and embryology of the structures from which they originate.

B. Volumetric Studies of Secretion

1. Prostatic Isolation Operation

Volumetric studies of the secretion of canine prostatic fluid have yielded great insight into the factors which determine the functional activity of male accessory glands. The dog is devoid of both seminal vesicles and bulbo-urethral glands, and if the urine is suitably deviated, practically pure prostatic secretion can be collected from the urethra. Eckhard (1863) ligated the neck of the bladder of dogs and obtained prostatic fluid by urethral catheterization. This technique was used by a number of investigators to study the secretory activity of the i:)rostate gland (Mislawsky and Bormann, 1899; Sergijewsky and Bachromejew, 1932; Winkler, 19311. A superior modification of the operation was introduced by Farrell (1931, 1938; Farrell and Lyman, 1937). The output of prostatic fluid was increased greatly either by electrical stimulation of the nervus erigens or by the injection of cholinergic drugs such as pilocarpine. These early prostatic isolation operations suffered from the signal disadvantage that, for technical reasons, they permitted only brief experiments.

In 1939, Huggins, Masina, Eichelberger and Wharton developed a simple surgical procedure which enabled frequent collection of canine prostatic secretion over long l)eriods of time. The original technique was modified slightly by Huggins and Sommer (1953) and is depicted in Figure 6.5. The bladdei' is separated from the prostate gland, the urine voided through a supral)ubic canula, and the animals circumcised. Healing was complete within one week after surgery, and the animals were maintained in good health. Prostatic fluid could be collected at fre(iucnt intervals for as long as two years. Normal adult dogs were found to secrete 0.1 to 0.2 ml. of prostatic fluid per hour without external stimulation. Following the administration of pilocarpine, the canine prostate secreted as much as four times its weight of fluid (60 ml.) in one hour. The amount of secretion obtained in response to a standard dose of pilocarpine remained relatively constant for three months or more, and bore no direct relationship to the weight of the gland. The volume and composition of the fluid varied with the time and intensity of the cholinergic stimulus. Huggins (1947c) found that, after a single intravenous injection of pilocarpine, the volume and the content of total protein, certain enzymes (acid phosphatase, fSglucuronidase and fibrinogenase ) , and citrate were maximal in the first 15 minutes, then declined progressively in three succeeding quarter-hour periods. But the chloride content always rose initially from the low values of the resting secretion and reached maximal levels after the first 15-minute period. If the drug was administered intramuscularly, maximal values for total protein and citrate were found in the first period, whereas those for the volume and enzyme content were higher in the second and third periods. It was concluded from experiments involving the repeated intravenous injection of pilocarpine that acid phosphatase and fibrinogenase were definitely secreted and not simply washed out of the gland. However, a "washing out" process does occur after an initial stimulus with respect to total protein and citrate levels.

It was observed by Huggins, Masina, Eichelberger and Wharton (1939) that infectious diseases {e.g., pyelonephritis, distemper) often decreased the volume of stimulated fluid. This effect seemed to be due to inhibition of the hypophysis, because it could be overcome by injection of gonadotrophin. Soon after castration (7 to 23 days) the secretion ceased and was restored by the administration of testosterone propionate. Androgens also initiated secretion in immature animals. In castrate dogs maintained on testosterone, neither adrenalectomy nor removal of the thyroid and parathyroid glands affected the rate of prostatic secretion. Huggins (1947c) observed that in normal



Fig. 6.5. The canine prostatic isolation operation. The connection of the prostatic urethra with the bhulder has been severed and the prostatic secretion is collected by way of the penis. (From C. Huggins and J. L. Sommer, J. Exper. Med., 97, 663680, 1953.)

animals, secretion was unaffected by injection of either progesterone or desoxycorticosterone.

Cystic hyperplasia of the prostate occurs in many senile dogs. The volume of fluid secreted by such hypertrophied glands in response to pilocarpine was smaller than that obtained from young adult animals (Huggins and Clark, 1940).

Injection of diethylstilbestrol into normal adult dogs abolishes prostatic secretion. Administration of gonadotrophin restores secretion in such estrogen treated animals, which suggests that the primary effect of estrogens under these conditions is on the hypophysis (Huggins, 1947c). Estrogens also antagonize the stimulatory effects of injected androgens. In castrate dogs receiving testosterone, injection of large doses of diethylstilbestrol decreases the output of prostatic fluid to very low levels (Huggins and Clark, 1940). The neutralization of androgen action by estrogens in this situation is pronounced but not complete. Thus the acid phosphatase activity of prostatic fluid collected from animals treated with both testosterone propionate and diethylstilbestrol is of the same order of magnitude as that of normal secretion, despite the fact that the volume of the secretion is extremely low (Huggins, 1947c). The ratio of diethylstilbestrol recjuired to antagonize maximally the action of testosterone was found to be about 1 : 25. In dogs with either normal or cystic prostate glands, injection of amounts of estrogen sufficient to decrease prostatic secretion leads to shrinkage of the prostate. Large doses of estrogen cause the canine prostate gland to enlarge ; the dorsal segment undergoes squamous metaplasia and the ventral lobe becomes atrophic (Huggins and Clark, 1940; Huggins, 1947c). If both estrogen and androgen are administered simultaneously, the dorsal region becomes squamous and the ventral portion of the gland retains its columnar epithelium, although the volume of the prostatic secretion may be drastically reduced.



Fig. 6.6. The c-anine prostatic translocation operation. (From C. Huggins and J. L. Sommer, J. Exper. Med., 97, 663-680, 1953.)


2. Prostatic Translocation Operation

Huggins and Sommer (1953) transposed the prostate gland of the dog from its natural position to the perineum, as depicted in Figure 6.6. This procedure permitted the size of the prostate to be measured in the living animal, and provided prostatic fluid quite uncontaminated with other material. Pilocarpine was used as a secretory stimulus. Using this technique, Huggins and Sommer found that the effects of androgens and estrogens on prostatic size and secretion were similar to those obtained with dogs that had undergone the prostatic isolation operation.

C. Chemical Composition of the Glandular Secretions=

Electrolytes. Water is the main constituent of prostatic and seminal vesicle secretions and of seminal plasma, all of which are approximately iso-osmotic with respect to blood serum. The vesicular secretion is usually more alkaline than the prostatic secretion and has a higher dry weight, mainly because it contains more protein. The electrolyte content of the secretions varies widely between different species (Huggins, 1945; Mann, 1954a). In general, sodium is the main cation, although this is not true of boar vesicular secretion which is very rich in potassium. Chloride tends to be the main anion in those species whose accessory gland secretions do not contain large amounts of citrate. In this connection it is instructive to compare the resting prostatic fluid of man, and the pilocarpine-stimulated prostatic fluid of the dog (Huggins, 1945, 1947c). The human secretion has a much greater citrate and calcium content, and a much smaller chloride level than the corresponding canine fluid, although the total concentration of osmotically active substances is of the same order of magnitude in both secretions.

Zinc. Berti'aiid and X'hidesco (1921 » found large amounts of zinc in human semen. The highest concentration of zinc is present in the first fraction of the ejaculate, which is largely prostatic secretion (Mawson and Fischer, 1953). In the rat, the zinc content of the dorsolateral prostate is especially lii<i;li (.Mawson and Fisclicr, 1951 ). After intracardiac injection, Zn*'^ is concentrated by this tissue 15 to 25 times more than any other organ, including the ventral prostate (Gunn, Gould, Ginori and Morse, 1955). The dorsolateral prostate of the rat consists of two parts which are functionally and anatomically distinct, and only the lateral portion concentrates Zn*'^ (Gunn and Gould, 1956a, 1957a). A rapid uptake of Zn^'s by slices of the rat dorsolateral prostate in vitro has been noted by Taylor (1957) .

The zinc content of the rat dorsolateral prostate, and its uptake of Zn*'^, are under hormonal control. The concentration of zinc in this gland increases 6- to 10-fold between the 35th and 100th day of life (Fischer, Tikkala and IMawson, 1955). In the adult rat, Gunn and Gould (1956b) observed a marked decrease in Zn^^ uptake after castration, which could be prevented by androgen treatment. In immature (Alihar, Elcoate and JNIawson, 1957) and hypophysectomized (Gunn and Gould, 1957b) animals, the administration of testosterone or gonadotrophin increased the zinc levels and the rate of Zn*'^ uptake, whereas estradiol was ineffective.

The physiologic function of the zinc in seminal plasma is problematical. This metal is an integral component of the enzyme carbonic anhydrase. The distribution of carbonic anhydrase among the various lobes of the prostate gland was studied by Mawson and Fischer (1952). In the rat, the posterior prostate contains about the same amount of carbonic anhydrase as the erythrocytes, whereas the ventral prostate contains very little of this enzyme. The lateral portion of the posterior prostate contains about 6 times as much zinc as the median part. But only a small portion of the zinc in the rat dorsolateral prostate, and in human semen, can be accounted for as carbonic anhydrase (Mawson and Fischer, 1953). According to Gunn and Gould (1958), dorsolateral prostatectomy, which removes most of the zinc from semen, is without influence upon either fecundity or fertility in the rat.

Fructose. The presence of a reducing and yeast-fermentable sugar in mammalian semen has been known for some time (McCarthy, Stepita, Johnston and Killian, 1928; Huggins and Johnson, 1933). It was assumed by early workers that this sugar was glucose. Although Yamada (1933) reported that human semen contained a sugar which reacted as a ketose, it was not until 1945 that the nature of the main reducing sugar in most mammalian semens was elucidated by Thaddeus Mann. Using a highly specific enzymatic method of estimation, Mann (1946) showed that the semen of men and many other mammals contains little or no glucose. The reducing and yeast-fermentable sugar of bull seminal plasma was isolated in the pure state and identified as d( — ) -fructose on the basis of its optical rotation and formation of methylphenyl fructosazone.

Mann (1949) found fructose in the semen of the bull, ram, boar, goat, opposum, rabbit, guinea pig, mouse, hamster, and man. It is also present in the European mole iTalpa) and hedgehog {Erinaceus) (Mann, 1956). Table 6.2 shows that the fructose content of semen varies greatly from one species to another. In the five species indicated, the total fructose accounts for all the yeast-fermentable carbohydrate present, but these species also contain nonfermentable sugars. The semen of some animals contains glucose. This is true of the rabbit, in which both glucose and fructose are detectable (]\lann and Parsons, 1950), and the cock (Mann, 1954a), the semen of which is devoid of fructose. Ketoses other than fructose are found in certain semens. For example, the vesicular and ampullar secretions of the stallion possess carbohydrates which react in colorimetric tests as

TABLE 6.2 Reducing sugars of semen Values obtained from Mann (1946) and expressed in terms of milligrams of fructose per 100 ml. of semen.


Species


Total

Reducing

Sugar


Fructose


Yeast Fermentable Sugar


Nonfer mentable

Sugar


Bull


937 443 528 295 31


910 340

417

141

9


917

370

411

144

10


25


Ram


73


Rabbit"


117

149


Boar


21


" Small amounts of glucose were occasionallj' fovmd in rabbit semen by Mann and Par.-ons (1950).


ketoses, but cannot be fructose as they are not fermented by yeast (Mann, Leone and Polge, 1956) .

In most mammals, fructose is formed mainly in the seminal vesicles (Huggins and Johnson, 1933; Davies and Mann, 1947; Mann, 1949; Ortiz, Price, Williams-Ashman and Banks, 1956). But in the rat, the seminal vesicles secrete little fructose, most of which originates from the dorsal prostate and coagulating glands (Humphrey and Mann, 1949).

Metabolic pathways for the biosynthesis of seminal fructose by the accessory glands have been studied extensively. From the results of experiments on diabetic animals, Mann and Parsons (1950) concluded that blood glucose was the precursor of seminal fructose. The hyperglycemia resulting from the administration of alloxan to rabbits was accompanied by a parallel increase in the fructose content of semen. Injection of insulin into such diabetic animals led to a fall in the levels of both blood glucose and seminal fructose. Similarly, the concentration of fructose in human semen was found to be abnormally high in diabetic patients. Mann and Lutwak-Mann (1951b) incubated minced accessory gland tissue with glucose and observed the formation of small amounts of fructose. Cell-free extracts of bull seminal vesicle showed marked phosphoglucomutase and phosphohexoisomerase activity. The same preparations hydrolyzed both glucose 6-phosphate and fructose 6-phosphate to the corresponding free sugars. Slices of seminal vesicle glycolyzed glucose at much greater rates than fructose. On the basis of these facts, Mann and Lutwak-Mann (1951a, b) postulated that the conversion of glucose to fructose involved an initial phosphorylation of glucose to glucose 6-phosphate by hexokinase, with adenosine triphosphate as the ])hosphate donor. After enzymatic isomerization of glucose 6-phosphate to fructose 6-phosphate, the latter was dephosphorylated to free fructose. It was assumed that any glucose formed by the dephosphorylation of glucose 6-phosphate was rcutilized, whereas fructose was not, and hence accumulated in the secretion. This formulation is consonant with the properties of the hexokinase of seminal vesicle (Kellerman, 19551 wliicli, at low sugar concentrations, phosphorylates glucose at much faster rates than fructose.

It was suggested by Mann and LutwakMann (1951a, b) that the dephosphorylation of hexosemonophosphates by accessory glands was catalyzed by an alkaline phosphatase which attacked the 6-phosphate esters of both glucose and fructose. Kuhlman (1954) claimed, on histochemical evidence, that rat seminal vesicle contains a phosphatase specific for fructose 6-phosphate which is most active in the vicinity of pH 7. Kellerman (1955) stated that, although the microsome-bound alkaline phosphatase of guinea pig seminal vesicle hydrolyzes the 6-phosphate esters of glucose and fructose at approximately the same rate, the mitochondria of this tissue contain a phosphatase which, at pH 5.8, hydrolyzes fructose 6-phosphate ten times as rapidly as glucose 6-phosphate. However, Hers (1957a) was unable to confirm these observations.

An alternative pathway for the conversion of glucose to fructose was suggested by Williams-Ashman and Banks (1954a), who found that certain fructose-secreting accessory glands of rodents contain an enzyme, ketose reductase, which catalyzes the reversible oxidation of sorbitol to fructose. This enzyme attacks a number of higher polyols and uses diphosphopyridine nucleotide (DPN) as a specific hydrogen acceptor (Williams-Ashman, Banks and Wolf son, 1957). The presence of an active ketose reductase in male accessory sexual tissues was confirmed by Hers (1956, 1957a) who discovered another enzyme, aldose reductase, which catalyzes the reduction of glucose to sorbitol with dihydrotriphosphopyridine nucleotide (TPNH) as the hydrogen donor. Hers suggested that seminal fructose was formed from glucose by the combined action of ketose and aldose reductases, as follows:

Glucose + TPNH + H+ ;^± Sorbitol + TPN+

Sorbitol + DPN+ <=^ Fructose + DPNH + H+

Glucose + TPNH + DPN+

-^ Fructose + TPN^ + DPNH

this mechanism for fructose biosynthesis accounts for the following observations ( Hers. 1957a) : (1) extracts of sheep seminal vesicle convert C'^-labelcd glucose to fructose without rupture of the carbon chain; (2) TPNH and DPN are required for this transformation, during which sorbitol is produced, and becomes radioactive; (3) inhibitors of aldose reductase {e.g., glucosonej inhibit the conversion of glucose to fructose and sorbitol; and (4j sorbitol is present, alongside fructose, in the secretions of sheep seminal vesicle and of certain accessory glands of other species [vide injra) .

The relative importance of these phosphorylative and nonphosphorylative pathways for the biosynthesis of seminal fructose remains to be determined.^

The fructose content of semen and of the accessory glands is strictly controlled by testicular hormones. The experiments of Mann and Parsons (1950), depicted in Figure 6.7, show that in the sexually mature rabbit, seminal fructose levels fell dramatically soon after castration. This decrease in seminal fructose was prevented by implantation of a pellet of testosterone. Later administration of androgen to the orchidectomized animals restored seminal fructose to normal levels. Measurement of the fructose content of ejaculated semen may be a sensitive index of androgenic activity, and has the signal advantage that the timesequence of changes which result from alterations in the level of circulating androgen can be determined without sacrifice of the animal. This "fructose test" has been used to assess the production of testicular androgen, or the hormonal activity of exogenous substances, in man (Harvey, 1948; Landau and Longhead, 1951; Tyler, 1955; Nowakowski and Schirren, 1956; Nowakowski, 1957) and in other animals (Gassner, Hill and Svdzberger, 1952; Branton, D'Arensbourg and Johnston, 1952; ]\Iann and Walton, 1953; Glover, 1956; Davies, Mann and Rowson, 1957) .

The amounts of fructose in semen and in the accessory glands are not determined solely by androgenic hormones, as Mann (1954a, 1956) has emphasized. The relative size and storage capacity of the accessory

^Samuels, Harding and Mann (1960) measured the aldose and ketose reductase levels in the various accessory glands of the rat, and in the seminal vesicles of the sheep, bull, boar, and horse. They found that the level of fructose production could be correlated with the activity of the least of the two enzymes.



Fig. 6.7. Postcastrate fall and testosterone-induced rise of seminal fructose in the rabbit. The pellet contained 100 mg. of testosterone. (Redrawn from T. Mann, The Biochemistry of Semen, Methuen & Co., 1954.)

glands play an important role. Another factor which complicates the fructose test is frequency of ejaculation. In man and the stallion, a single ejaculation largely depletes the seminal vesicles (Mann, 1956). In the bull, however, the seminal vesicles have a remarkable storage capacity such that the fructose content of eight consecutive ejaculations obtained within one hour is practically the same (Mann, 1954a) . Blood sugar levels can influence seminal fructose, but there is no evidence that the abnormally large quantities of fructose in diabetic semen (Alann and Parsons, 1950) result from increased output of androgenic hormones. Interruption of the blood supply to an accessory gland is another factor which affects the amount of fructose it secretes (Clegg, 1953). In immature animals, there seems to be a short time after birth when the accessory glands will not produce fructose in response to testosterone (Ortiz, Price, Williams-Ashman and Banks. 1956). Obstruction of the ejaculatory ducts will, of course, prevent the appearance of fructose in semen, as Young (1949) found in a patient with congenital bilateral aplasia of the vas deferens.


The anterior hypophysis may determine indirectly the secretory activity of accessory glands, and the amounts of fructose and other chemical substances which accumlate therein. Mann and Parsons (1950) showed that hypophysectomy in the rabbit results in a decline in the fructose content of semen, and of the prostate gland and glandula vesicularis. These changes were similar to those induced by castration, and could be reversed by treatment with androgens or with gonadotrophin. The deleterious effect of inanition, or a deficiency of certain B vitamins, on fructose formation in the accessory glands is almost certainly related to a concomitant depression of gonadotrophin secretion by the anterior pituitary gland (Lutwak-Mann and Mann, 1950 ) . In the bull (Davies, Mann and Rowson, 1957j, underfeeding leads to a greater depression of fructose levels in semen than of sperm formation.

Analysis of fructose in excised ]irostate gland or seminal vesicle has been used widely as an indicator of androgenic activity. This procedure has yielded much information concerning the relationship between the time of onset of androgen secretion by the testes and the initiation of spermatogenesis. In the rabbit (Davies and Mann, 1949), rat (Mann, Lutwak-Mann and Price, 1948), bull (Mann, Davies and Humphrey, 1949), boar (Mann, 1954b), and guinea pig (Ortiz, Price, Williams-Ashman and Banks, 1956) , fructose can be detected in the accessory glands before spermatozoa are produced. The androgenic potency of exogenous substances can be determined by application of the fructose test to the accessory glands of animals castrated before or after puberty. The increase in fructose content of the coagulating gland of castrated rats in response to testosterone is greater than the corresponding change in organ weight (Mann and Parsons, 1950) . The prostate gland and seminal vesicle of the rat (Rudolph and Samuels, 1949; Rudolph and Starnes, 1955; Rauscher and Schneider, 1954) and the seminal vesicle of the guinea pig (Levey and Szego, 1955b; Ortiz, Price, Williams-Ashman and Banks, 1956) behave in a similar fashion. This technique has provided evidence for the slight androgenic activity of progesterone (Price, Mann and Lutwak-Mann, 1955), and for the antagonistic (Parsons, 1950) or synergistic (Gassner, Hill and Sulzberger, 1952) influence of estrogens on the action of androgens.

Intact vascular and neural links are not necessary for the male accessory glands to accumulate fructose after androgenic stimulation. Subcutaneous transplants of these tissues into male rats will grow and produce fructose. After castration, the fructose content falls and can be restored by testosterone therapy (Mann, Lutwak-Mann and Price, 1948). Fructose secretion was also observed in accessory tissues transplanted into female hosts which had received androgens (Lutwak-Mann, Mann and Price, 1949), or were injected with gonadotrophins. which probably stimulate the secretion of ovarian androgens (Price, Mann and Lutwak-]Mann, 1955).

The fructose in seminal i^lasma serves as a source of energy for spermatozoa under both anaerobic and aerobic conditions (Mann, 1954a).

Sorbitol. Sorbitol has been detected in the seminal vesicles of the sheep ( Hers, 1957a, b) and the coagulating gland of the rat (Wolfson and Williams-Ashman, 1958), as well as in the semen of many species ( King, Isherwood and Mann, 1958; King and Alann, 1958, 1959). The sorbitol content of semen tends to be high in those animals which exhibit high levels of seminal fructose, although it is also present in the semens of the stallion and cock, which are virtually devoid of fructose (Table 6.3). Sorbitol can be synthesized in the accessory glands by the action of either ketose reductase (WilliamsAshman and Banks, 1954a; Williams-Ashman, Banks and Wolfson, 1957; Hers, 1956, 1957a) or aldose reductase (Hers, 1956, 1957a). Under anaerobic conditions, spermatozoa will not glycolyze sorbitol (unlike fructose) to lactic acid, but will reduce both fructose and glucose to sorbitol. In oxygen, spermatozoa readily oxidize sorbitol (Mann and White, 1956), and also form sorbitol from glucose and fructose. The interconversion of fructose and sorbitol by spermatozoa is catalyzed by a DPN-specific ketose re(Uictas(> (sorbitol dehydrogenase) which is similar to that of the accessory glands (King and ^Nlann, 1959). Although the spermatozoa can affect the ratio of the levels of soi'bitol and fi-uctose in seminal plasma, most of the seminal sorbitol is probably derived from the accessory glands.

Inositol. During his studies on the vesicular secretion of the boar, Mann (1954b) isolated large amounts of a crystalline, nonreducing carbohydrate which he identified rigorously as ?weso-inositol. This cyclic polyol was found only in the seminal vesicle, being absent from the epididymis and Cowper's gland. The concentration of inositol in boar vesicular secretion was as high as 2.6 gm. per 100 ml., and constituted as much as 70 per cent of the total dialyzable material therein. Using a specific microbiologic method of estimation, Hartree (1957) found that in the boar, the inositol content of seminal plasma was usually greater than 600 mg. per 100 ml., although much smaller quantities (less than 60 mg. per 100 ml.) were present in the bull, ram, stallion, and man. In all of the species examined, the bulk of the inositol in seminal plasma was in the free state, and in amounts much greater than those in blood or cerebrospinal fluid. In most animals, seminal inositol originates from the seminal vesicles, but it has been detected in the prostate gland of the hedgehog, and in the ampullar secretion of the stallion.

The levels of inositol in human semen, together with those of fructose, are increased after the administration of testosterone according to Kimmig and Schirren (1956).

The physiologic function, if any, of the inositol in seminal plasma is unknown. Since boar vesicular secretion, unlike other body fluids of the pig, contains immense amounts of inositol and very little sodium chloride, j\Iann (1954b) suggested that inositol is concerned with the maintenance of the osmotic ecjuilibrium of boar seminal plasma.

Ascorbic acid. Deproteinized extracts of the seminal plasma of many species reduce 2,6-dichlorophenol indophenol in the cold. This property has been attributed to the presence of ascorbic acid in the semen of the bull (Phillips, Lardy, Heiser and Ruppel, 1940), guinea pig (Zimmet, 1939), and man (Nespor, 1939; Berg, Huggins and Hodges, 1941; Huggins, Scott and Heinen, 1942). However, it is now established that ascorbic acid does not always account for the total reducing power of semen. In some animals, e.g., the boar, ergothioneine is responsible in large part for the reduction of indophenol [vide infra), and bull semen contains sulfite and another, unidentified, reducing substance (Larson and Salisbury, 1953) . Nevertheless, ascorbic acid is undoubtedly present in seminal plasma. Employing a specific analytical method based on the formation of its dinitrophenylhydrazone, Mann (1954a) found that the seminal vesicle secretion of the rat, bull, guinea pig, and man contains ascorbic acid in amounts varying from 5 to 12 mg. per 100 ml. Mann's values for the ascorbic acid content of human semen (10 to 12 mg. per 100 ml.) agree well with those reported by Berg, Huggins and Hodges (1941), which were based on indophenol reduction.



TABLE 6.3 Sorbitol and fructose content of fresh seminal

plasma In some oases the .samples represented semen which had lieen pooled; the number of individuals is given in brackets. (From T. E. King and T. Mann, Proc. Roy. Soc. London, ser B, 151, 2262-13, 1959.)


Number of Species \ Samples Sorbitol ; Fructose

Analyzed



Ram. . . Rabbit. Bull . . . Boar. . . Stallion Dog .. . Cock... Man . . .


mg./lOO ml.

150-600 (12)

40-150 (4)

120-540 (14)

20-40 (4)

<1 (4)

<1 (5)

<1 (14)

154 (3)



Amino sugars. After hydrolysis with acid, boar semen contains considerable amounts of amino sugars (Mann, 1954a). The epididymal "semen" contains more amino sugar than the vesicular secretion.

Ergothioneine. The vesicular secretion of the boar (Leone and Mann, 1951 ; Mann and Leone, 1953) is a rich source of ergothioneine. This sulfur-containing base is also found in the accessory glands of the European hedgehog and mole (Mann, 1956) , and in the ampullar secretion of the stallion (Mann, Leone and Polge, 1956). Little or no ergothioneine is present in the semen of the bull, ram, and man.

Experiments with S^^-labeled precursors suggest strongly that seminal ergothioneine is not synthesized in the animal V)ody (Alelville, dtken and Kovalenko, 1955; Heath, Rimington and Mann, 1957). Because orally ingested S'^'"'-labeled ergothioneine l)asses into the seminal plasma of the boar (Heath, Rimington, Glover, ]Mann and Leone, 1953) , it is possible that those accessory glands which secrete ergothioneine concentrate this substance from the blood.

Mann and Leone (1953) are of the opinion that the function of ergothioneine in seminal plasma is to protect the spermatozoa from the poisonous action of oxidizing agents. It is remarkable that the seminal fluids of the boar and the stallion, both of which contain ergothioneine, have common characteristics which would render their spermatozoa especially sensitive to oxidizing agents, viz., large volume, low sperm density, and small content of glycolyzable sugars.

PoLYAMiNES. Large amounts of spermine and spermidine are present in the prostate gland of many species (Harrison, 1931 ; Rosenthal and Tabor, 1956). The chemical structure of these polyamines was elucidated by Dudley, Rosenheim and Starling (1926, 1927). Human seminal plasma contains as much as 300 mg. spermine per 100 ml., most of which is derived from the prostate gland. If human semen is allowed to stand for a few hours at room temperature, the spermine present crystallizes in the form of spermine phosphate ("Boettcher's crystals").- Both spermine and spermidine are oxidized by the diamine oxidase of human seminal plasma (Zellcr, 1941; Zeller and Joel, 1941). These polyamines via their degradation products are highly toxic to spermatozoa (Tabor and Rosenthal, 1956) , and it seems unlikely that

"In a letter written to llie Royal Society of London in November 1677, Antoni van Leeuwenhoek described for the fir.'^t time the presence and movement of spermatozoa in human semen. In the same letter, he also mentioned that "threesided bodies," which were "as bright and clear as if they had been crystals," were deposited in the aged semen of man. These crystals were undoubtedly composed of spermine phosphate. The liistory of the discovery of spermine in semen is admirably summarized by Mann (1954a), with special reference to the contributions of Louis Vauquelin (see footnote 3), and also of Alexander von Pcihl, whose claims for the therapeutic proiinities' of spermine aroused much interest and controversy at the end of the 19th century.


their presence in seminal plasma is of functional value.

Choline DERIVATIVES. Florence (1895) described the formation of brown crystals upon the addition of a solution of iodine in potassium iodide to semen. This reaction was used as the basis of a medico-legal test for semen stains. Bocarius (1902) showed that choline was responsible for the formation of this material. In the rat, the seminal fluid is by far the richest source of choline of any tissue or body fluid (Fletscher, Best and Solandt, 1935). A series of careful studies by Kahane (Kahane and Levy, 1936; Kahane, 1937) revealed that human semen contains very little free choline immediately after ejaculation, but that large amounts of the free base are formed if the semen is allowed to stand at room temperature. Lundquist (1946, 1947a, b, 1949) isolated phosphorylcholine from human seminal plasma and showed that it was converted to choline and inorganic phosphate by seminal acid phosphatase. However, the French investigators (Diament, Kahane and Levy, 1952. 1953; Diament, 1954) isolated a-glycerophosphorylcholine from the vesicular secretion of rats, and suggested that this substance, rather than phosphorylcholine, was the precursor of free choline in aged semen. Lundquist (1953) also found glycerophosl^horylcholine in the vc'sicular secretion of the rabbit, rat, and guinea pig. WilliamsAshman and Banks (1956) showed that the amount of glycerophosphorylcholine in rat vesicular secretion falls rapidly after castration, and can be restored to normal levels by administration of testosterone. Rezek and Sir (1956) found both ]')hosi)liorylcholinf and glyceroi)hospliofylclu)line in lunnan ejaculates.

A thorough study of the wat('i'-s()lul)lf choline (lerivati\-cs in seminal plasma was made by Dawson, Maiui and White (1957). They foimd (Tabh'().4i that in most species, gh^cerophosphoryh-holine is the only derivati\'e preseiu, but ill man there are considerable (|uantities of phosphoiyh'lioline as well. The lattei- substance is rai)idly dephospholylated after ejaculation, but glycerophosphoryh'holine is not degradeil by enzymes in seminal phisnia or \'esiculai' secretion. In



TABLE 6.4

Phosphor ylchoUne and a-glycerophosphorylcholine in semen and in secretions of accessory reproductive glands


Species


Concentration (mg. per 100 gm.) of:


Phosphorylcholine a-Glycerophosphorylcholine


Ram

Ram

Bull

Bull

Bull

Bull

Bull

Cioat

Boar

Boar

Boar

Stallion. . . Stallion. . .

Man

Rat

Rat

Rabbit... Hedgehog. Hedgehog. Monkey. . . Cock..'....


Semen

Seminal pla.sma

Semen

Seminal plasma

Vesicular secretion

Epididymal secretion

Ampullar secretion

Semen

Seminal plasma

Vesicular secretion

Epididymal secretion

Semen

Ampidlar secretion

Semen

Vesicular secretion

Seminal vesicle

Semen

Secretion of "Prostate I and 11"

Secretion of "Prostate III"

Vesicular secretion

Semen






Present




256-380



Present Present Present Present


1185-1942

1601-2040

237-460

110-496


1490

94

1382-1550

108-235

190

3060

38-113

120

59-90

654; 530-765"

190-515"

215-370

Present



° Results from Williams-Ashman and Banks (1956); all other values from Dawson, Mann and White

lf57).


the bull and boar, the epidiclymi.^ is the ])rincii)al source of the glyceroiihosi)horylcholine of the seminal plasma.

Williams-Ashman and Banks (1956) in-ovided evidence that the choline moiety of the glycerophosphorylcholine in vesicular secretion is not derived from a direct reaction between glycerol and cytidine diphosphate choline. The latter nucleotide was shown to be a precursor of lecithin in rat seminal vesicle tissue. The glycerophosphorylcholine of seminal plasma may originate from the enzymatic degradation of the choline-containing lipids of the seminal vesicle epithelium.

Choline and glycerophosjihorylcholine are not metabolized by spermatozoa, and do not affect their respiration (Dawson, Mann and White, 1957). There is no evidence that the water-soluble choline derivatives of seminal plasma serve any useful function.

Lipids. That lipid-containing granules are l^resent in human seminal plasma has been known for more than a century. They are found in prostatic secretion (Thompson, 1861 1, and were termed "lecithin-kornchen" by Fuerbringer (1881). However, Scott ( 1945 ) showed that lecithin is absent from both of these fluids, and that the majority of the phospholipid therein is phosphatidyl ethanolamine. Neutral fat is virtually absent from human seminal plasma and prostatic secretion, one-third of the total lipid of which can be accounted for as cholesterol.

According to Boguth (1952), about onethird of the total plasmalogen in bull semen (30 to 90 mg. per 100 ml.) is in the seminal plasma. In the ram, only 10 per cent of the seminal plasmalogen is found outside the spermatozoa (Hartree and ]Mann. 1959).

Large amounts of 7-dehydrocholesterol were found in the preputial gland and epididymis of the rat (Ward and ]\Ioore, 1953) . One gram of the hydrocarbon heptacosane (CH3(CHo)o5CH3) was isolated from an alcoholic extract of 18 liters of human semen by Wagner-Jauregg (1941). The partition of heptacosane, and of steroidal estrogens (Diczfalusy, 1954) and androgens (Dirscherl and Kniicliel, 1950) between the sperm and plasma of human semen remains to be determined.

Citric acid. Citric acid was first detected in human semen by Schersten (1929). The distribution of citric acid in the semen and in the secretions of the accessory glands of various species is summarized in Table 6.5. In some animals, {e.g., the rat and man), citric acid is produced mainly by the prostate gland, and in others {e.g., the bull, boar, and guinea pig), most of it originates from the seminal vesicles.

The citric acid content of the seminal plasma and of the secretions of accessory glands depends on androgenic hormones. Citric acid disappears from these fluids after castration, and is formed again after treatment with testosterone. This citric acid test" has been used to determine the time of onset of secretory function in accessory glands (Mann, Davies and Humphrey, 1949; Ortiz, Price, Williams- Ashman and Banks, 1956), hormonal influences on secretion in subcutaneous transplants (Mann, LutwakMann and Price, 1948; Lutwak-jNIann. Mann and Price, 1949 L the androgenic ac


TABLE 6.5

Citric acid in semen and in the secretions of accessory reproductive glands


Species


Material


Citric Acid


Reference




(tng./lOO gm.)




Man


Semen


140-637


Huggins and Xeal (1942)



Man


Prostatic secretion


480-2()88


Huggins and Neal (1942)



Man ....


Seminal vesicle secretion Hypertrophic adenoma of the


15-22 201-1533


Huggins and Neal (1942) Barron and Huggins (1946a)



Man




prostate gland





Man


Carcinoma of the prostate gland


12-137


Barron and Huggins (194()a)



Bull


Semen


510-1100


Humphrey and Mann (1949)



Bull


Seminal gland secretion


670


Humphrey and Mann (1949)



Bull


Ampullar semen


550


Humphrey and Mann (1949)



Bull


Epididvmal semen



Humphrey and IVIann (1949)



Bull


Epididymis


18


Humphrey and Mann (1949)



Boar


Semen


130


Humphrey and Mann (1949)



Boar


Cowper's gland secretion Epididymal semen Seminal yesicle secretion



Humphrey and Mann (1949)



Boar



Humphrey and Mann (1949)



Boar


580


Humphrey and Mann (1949)



Ram


Semen Semen


110-260 110-550


Humphrey and Mann (1949) Himiphrey and Mann (1949)



Rabbit



Rabbit


Epididymis


54


Humphrey and Mann (1949)



Rabbit


Prostate (I, II and III)


62


Himiphrey and Mann (1949)



Rabbit


Cowper's Gland


42


Humphrey and Mann (1949)



Rabbit


Ampulla


273


Himiphrey and Mann (1949)



Rat


Seminal vesicle Coagulating gland Ampulla Dorsolateral prostate


39


Humi)hrey and :\Iann (1949) Hunii)hrc\ and Mann (1949)



Rat



Rat



Humplucv and Mann (1949)



Rat


20


Humphrey and Mann (1949)



Rat


Ventral prostate Semen


122


Humphrey and Mann (1949) Mann, Leone and Polge (1956)



Stallion


8-53



Stallion


Seminal vesicle


77


Mann, Leone and Polge (1956)



Guinea pig. . . .


Seminal vesicle


153-216


Ortiz, Price, Williams-Ashman Banks (195())



Guinea pig. . . .


Semiiuil vesicle sec'retion


320-357


Ortiz, Price, Williams-Ashman Baidvs (1956)



Guinea pig. . . .


Coagulating gland


26-40


Ortiz, Price, Williams- Ashman Banks (1956)



Guinea pig. . . .


Lateral i)rostate


16-20


Ortiz, Price, Williams- Ashman Banks (1956)



Guinea i)ig. . . .


Doi'sal ])i(is1at(>


47-75


Ortiz, Price, Williams-Ashman Banks (195(i)



Dog


Prostatic secretion


0-30


Barron and Huggins (1946a) Barron and Huggins (1946a) Mann, Leone and Polge (1956)



Dog


Prostate gland Vesicular secretion


8



Jackass


22-82

tivity of various hormones (Mann and Parsons, 1950; Price, Mann and Lutwak-JMann, 1955; Ortiz, Price, Williams-Ashman and Banks, 1956), and the effect of nutrition on the onset of androgen secretion and sperm formation in bull calves (Davies, Mann and Rowson, 1957).

The androgen-induced changes in the citrate levels in semen and various accessory glands are reminiscent of similar alterations in the fructose content of these tissues. However, the concentrations of these substances do not necessarily parallel one another in response to hormonal stimulation. In the postcastrate animal, the fall in citric acid and its reappearance after androgen treatment is usually more sluggish than that of fructose. Also, the seminal fructose of some species may not be secreted by the same accessory organ (or lobes of the gland ) which produces citric acid. Thus in the rat, fructose is secreted by the anterior and dorsolateral prostate, whereas citric acid is derived from the seminal vesicles and dorsolateral and ventral prostates, but is totally absent from the anterior prostate (Humphrey and Mann, 1949). In the guinea pig, however, the seminal vesicles are the principal source of both fructose and citric acid (Ortiz, Price, Williams-Ashman and Banks, 1956).

Using a strain of rats in which the incidence of the female prostate is very high, Price, ]Mann and Lutwak-JVIann (1949) showed that the growth of this gland which follows the injection of testosterone is accompanied by a tremendous increase in its content of citric acid. In this way the female prostate resembles the ventral prostate gland of the male rat.

Citric acid is synthesized in the i)rostate gland by the usual reactions of the tricarboxylic acid cycle (Williams-Ashman, 1954; Williams-Ashman and Banks, 1954b). No other organic acids are present in more than trace amounts in the secretions of those accessory glands that accumulate citrate. The enzymatic machinery for the degradation of citric acid via the tricarboxylic acid cycle is jH-esent in the rat ventral prostate gland OVilliams-Ashman, 1954; Williams-Ashman and Banks, 1954b; Williams-Ashman, 1955) and there is no evidence, despite suggestions to the contrary (Awapara, 1952a), that citric acid accumulates because it cannot be oxidized. It has been suggested that a common denominator affecting the androgendependent accumulation of citric acid and fructose in the accessory glands is the intracellular balance between the oxidized and reduced forms of DPN and TPN (Talalay and Williams-Ashman, 1958).

Mann (1954a) has summarized the ideas of various authors concerning the possible functional role of citric acid in seminal plasma. All of these suggestions are based more upon conjecture than experimental fact.

Catecholamine^. There is evidence for the presence of both epinephrine and norepinephrine in seminal pla.sraa (Brochart, 1948; Beauvallet and Brochart, 1949). Extracts of human prostate and seminal vesicle contain a monoamine oxidase which oxidizes catecholamines (Zcller and Joel, 1941). Katsh (1959) detected serotonin and histamine in human ejaculates.

Amino acids. Chromatographic studies have revealed the presence of many free amino acids in human semen (Jacobbson, 1950; Lundquist, 1952), from which crystalline tyrosine was isolated by WagnerJauregg (1941). According to Barron and Huggins (1946b), human prostatic adenoma is very rich in free glutamic acid, and the nonprotein amino-nitrogen of this and dog prostatic tissue is high. Bovine seminal plasma contains free serine, alanine, glycine, and aspartic and glutamic acids (Gassner and Hopwood, 1952). A similar distribution of amino acids is found in the vesicular and ampullary secretions of the bull. The free amino acid levels of bull seminal plasma fall greatly after castration. In the rat, Marvin and Awapara (1949) found that the concentration of free amino acids in the whole prostate decreased markedly following orchidectomy, and could be restored to normal levels in the castrate animal by treatment with androgen. In this species, Awapara (1952a) observed that the content of free amino acids in the ventral lobe of the prostate was much higher than in the dorsal lobe. After castration, there was a marked drop in the content of most amino acids with the exception of aspartic and glutamic acids, which seemed to remain at almost normal levels (Awapara, 1952b j.

Seminal plasma and the secretions of the male accessory glands contain a battery of proteolytic enzymes [vide infra). For this reason, changes in the levels of free amino acids in these fluids resulting from hormonal treatments should be interpreted with caution. Jacobbson (1950), for example, has shown that in human semen, the nonprotein nitrogen and amino-nitrogen content increases many fold within 60 minutes after ejaculation.

Prostaglandin. A vasodepressor substance, designated j^rostaglandin, was found by von Euler (1934, 1936) in the prostatic and vesicular secretions of man, and also in the accessory glands of sheep (von Euler, 1939). The prostaglandin of ram prostate was i^urified by Bergstrom ( 1949), who suggested that it was an unsaturated fatty acid devoid of nitrogen. According to Eliasson (1957), the prostaglandin of human semen and of the prostate gland of sheep are identical.

The pharmacologic effects which result from the injection of seminal plasma icf. Kurzrok and Lieb, 1931; von Euler, 1934, 1936, 1939; Goldblatt, 1935; Cockrill, Miller and Kurzrok, 1935; Asplund, 1947) are complex, and are probably due to the combined action of many constituents of this fluid. The hypotensive action of protein fractions of the secretions of some accessory organs (Freund, Miles, Mill and Wilhelm, 19581 is discussed below.

Uric acid. Bull seminal vesicles may contain as much as 70 mg. per cent of uric acid (Leone, 1953). The uric acid content of the semen of other animals is much lowci' (Mann, 1954a).

Urea. The urea content of human and ram semen is much higlici' than that found in the bull, boar, and stallion (Mann. 1954a).

Major protein constitiiknts. Human seminal plasma contains from 3.5 to 5.5 gm. of protein-like material per 100 niL (Huggins, Scott and Heinen, 1942). Less than 18 per cent of this material is coagulable by heat, and as much as 68 per cent of it is dialyzable. Thus the majority of the seminal proteins of man can be classified as proteoses. Electrophoretic analyses of the nondialyzable proteins of human seminal plasma have been performed by Gray and Huggins ( 1942 1 and by Ross, Moore and Aliller (1942). The major components bore some correspondence to those of blood serum, although the amount of albumin was small. The proteins of bovine seminal plasma are less dialyzable, and more coagulable by heat, than those of man (Larson and Salisbury, 1954). Electrophoretic studies showed the presence of three major and eight minor constituents, which seemed to be distinct from the proteins of bovine blood serum. In this species giycoor lipoproteins were present only in very low concentrations. Larson, Gray and Salisbury (1954) found that the bovine seminal plasma proteins are highly antigenic. They obtained immunologic evidence that the major protein constituents of this fluid are distinct from any of the main ])rot(>ins of either blood or milk.

Enzymes, (i) Acid phosphatase. Kutscher and Wolbergs (1935) discovered that human semen and prostate contain a very active phosphatase which is optimally active at pH 5 to 6. This enzyme is resjjonsible for the greater phosphatase activity of male as compared with female urine. Its secretion by the prostate accounts for the fact that male urine collected from the renal pelvis exhibits very little enzyme activity (Scott and Huggins, 1942). Human prostatic acid phosjihatase hydrolyzes a number of phosphate monoesters (Kutscher and Worner, 1936; Kutscher and Pany, 1938). The enzyme has been purified extensively (London and Hudson, 1953; Boman, 1954; London, Sommer and Hudson, 1955). In addition to hydrolyzing phosphate esters, human prostatic acid phosphatase catalyzes the transfer of phosphate from various donors to alcohols such as glucose, fructose, and methanol (London and Hudson, 1955; Jeffree, 1957). L-Tartrate inliibits the enzyme competitively (AbulKadl and King, ^1948).

'I'hc activity of acid phosphatase in the human jjrostate is low in childhood and increases about 20 times at i)uberty (Gutman and Gutman, 1938a). In adult men, the acid phosphatase content of semen seems to reflect the circulating levels of androgenic hoi-niones (Gutman and Gutman, 1940). High levels of acid })hosphatase are also present in osteoplastic metastases of prostatic carcinoma (Gutman, Sproul and Gutman, 1936). Acid phosphatase does not seem to enter the circulation from the prostate gland in healthy individuals unless they are subject to prostatic massage. But in about 65 per cent of men with metastatic carcinoma of the prostate, the serum levels of this enzyme are abnormally high (Gutman and Gutman, 19381); Robinson, Gutman and Gutman, 1939; Huggins and Hodges, 1941). The diagnosis and prognostic evaluation of carcinoma of the prostate in men has been aided greatly by measurements of the acid phosphatase levels of serum. Inhibition of the acid phosphatase activity of blood serum by L-tartrate has been used as an index for the outflow of prostatic acid phosphatase into the serum in neoplastic diseases of the prostate gland (Abul-Fadl and King, 1948; Fishman and Lerner, 1953).

The prostate gland of the monkey (Gutman and Gutman, 1938a) and dog (Huggins and Russell, 1946), and the seminal vesicle of the guinea i)ig (Bern and Le\y, 1952) exhibit powerful acid phosphatase activity, whereas the levels of this enzyme in the prostate of the rabbit (Bern and Levy, 1952) and rat (Huggins and Webster, 1948) are relatively low. The properties of these enzymes from different species are strikingly similar (Novales and Bern, 1953) . In the monkey and dog, the prostatic acid jihosphatase activities are controlled by androgenic hormones. This is also true in the rat (Stafford, Rubinstein and Meyer, 1949), and guinea pig (Ortiz, Brown and Wiley, 1957).

{2} Alkaline phosphatase. An enzyme, activated by magnesium ions, which hydrolyzes a variety of phosphate monoesters at pH 9, is present in the seminal fluid and accessory glands. In some species, e.g., the l)ull (Reid, Ward and Salisbury, 1948 ) , the levels of seminal alkaline phosphatase are much greater than those of the acid phosphatase. In the rat, alkaline phosphatase activity in the prostate and seminal vesicles decreases markedly after castration (Stafford, Rubinstein and Meyer, 1949).

(3) 5'-Nucleotidase. Reis (1937, 1938) noticed that human seminal plasma dcphosphorylated adenosine 5'-phosphate and inosine 5'-phosphate very rapidly. He proposed the term 5'-nucleotidase" for enzymes which specifically hydrolyze the 5'-monophosphates of ribose and its nucleosides. Mann (1945) reported that bull seminal plasma is exceedingly rich in 5'-nucleotidase. The enzyme was purified from this source by Heppel and Hilmoe (1951a). It was inactive towards adenosine 2'- and 3'phosphates, but catalyzed the hydrolysis of the 5'-monophosphate esters of adenosine, inosine, cytidine, uridine, and ribosyl nicotinamide. The 5'-nucleotidase of bull semen is optimally active at pH 8.5, and requires magnesium ions for maximal activity.

(4) Inorganic pyrophosphatase. Heppel and Hilmoe (1951b) reported the presence of an inorganic pyrophosphatase in bull seminal plasma. The enzyme was not purified extensively, and it is not clear whether it is different from other in'rophosphatases in semen.

(5) Nucleotide pyrophosphatases. The enzymatic hydrolysis of adenosine triphosphate (ATP) by seminal plasma was observed by Mann (1945) and by MacLeod and Summerson (1946). Three distinct ATPases were isolated from bull seminal plasma by Heppel and Hilmoe (1953). The first of these enzymes catalyzed the hydrolysis of ATP to inorganic pyrophosphate and adenosine 5'-phosphate. The other two catalyzed the liberation of inorganic orthophosphate from ATP, and were active at pH 5 and pH 8.5 respectively. The possible identity of any of these proteins with other enzymes which hydrolyze the pyrophosphate linkage of ]iyridine nucleotides (Williams-Ashman, Liao and Gotterer, 1958) and cytidine diphosphate choline (Williams-Ashman and Banks, 1956) remains to be established.

The physiologic function of any of the phosphatases in seminal plasma is unknown.

{6) Proteolytic enzymes. The proteolytic activity of human semen was first noted by Huggins and Neal (1942), and has been studied extensively by Lundquist and his collaborators. An enzyme similar to pepsinogen, and probably secreted by the seminal vesicles, was discovered in human seminal plasma by Lundquist and Seedorf (1952). Three other proteolytic enzymes were partially purified from human semen by Lundquist, Thorsteinsson and Buus (1955). The first enzyme resembled chymotrypsin, and the second was an aminopeptidase. The third enzyme hydrolyzed benzoylarginine ethyl ester, and seems to be identical with the arginine ester hydrolyzing enzyme described in male accessory reproductive glands by Gotterer, Banks and Williams-Ashman (1956). The relationship of these enzymes to the hydrolysis of fibrin or fibrinogen by prostatic secretion is discussed below with reference to the coagulation and liquefaction of semen.

(7) Glycosidases. Using phenolphthalein glucuronide as a substrate, Talalay, Fishman and Huggins (1946) determined the /?-glucuronidase activity of the male accessory glands of the rat. The levels of this enzyme in the epididymis fall about 50 per cent after castration, and can be restored to normal levels by the administration of testosterone (Conchie and Findlay, 1959). When the corresponding phenol- or p-nitrophenol-glycosides were employed as substrates, Conchie, Findlay and Levvy (1956) showed that the epididymis of the rat is particularly rich in y3-iV-acetylglucosaminidase. The levels of this enzyme were found by Conchie and Mann (1957) to be very much greater than those of seven other glycosidases in male accessory secretions. The levels of various glycosidases in the epididymis of rodents increases enormously at puberty. In adult animals the activity of some of these enzymes {e.g., a-mannosidase and /?-iV-acetylglucosaminidase) fell to negligible values after castration, and were restored only partially by treatment with testosterone.

(8) Miscellaneous enzipnes. The kneels of a number of oxidizing enzymes in human seminal plasma were studied by Rhodes and Williams-Asluiuiii (1960», who noted the presence of a x'cry active TPN-linked isocitric dehydrogenase. The ability of luiinan semen to hydrolyze acetylclioline is rather fe(>!)le, and the bulk of the activity resides in the seminal plasma (Zeller and Joel, 1941). According to Sekine (1951), boar semen exhibits powerful choline esterase activity, wliicli is confined mainly to the s])ermatozoa. The activity of phosphohexoisomerase (Wiist, 1957) and lactic dehydrogenase (MacLeod and Wroblewski, 1958) in human seminal plasma has been documented.

The levels of the following soluble enzymes have been determined in the accessory glands of male rodents: phenol sulfatase (Huggins and Smith, 1947). nonsi)ecific esterase (Huggins and ]\Ioulton, 1948), enolase, and dehydrogenases for lactate, malate, glucose 6-phosphate, 6-phosphogluconate and isocitrate (Williams- Ashman, 1954; Rudolph, 1956), aldolase and a-glycero])hosphate dehydrogenase (Butler and Schade, 1958). The nucleoside phosphorylase and adenosine deaminase activities of bull seminal vesicle were measured by Leone and Santoianni (1957). The vesicular secretion of the bull is rich in flavins, and exhibits strong xanthine oxidase activity (Leone, 1953). Leone and Bonaduce (1959) described a very active diphospho]5yridine nucleotidase in the vesicular secretion of the bull.

Conclusions. The foregoing survey indicates that, just as the size and morphology of the accessory glands differ profoundly, so there are wide species variations in the chemistry of their secretions, which comprise the seminal plasma. Some seminal constituents {e.g., fructose) are found in many mammals. Other substances, such as ergothioneine, are present in appreciable amounts in the seminal plasma of only a few species. The biochemistry of the accessory glands is still in its infancy, and it may be expected that future research will disclose other species-restricted comjionents of seminal plasma. Mann (1954a, 1956) I'ightly emphasizes that the finding of substantial concentrations of certain substances in the semen of only relatively few species does not necessarily detract from their physiologic value. The high levels of ci-gotliioneine in the seminal plasma of the boar and stallion is a case in point. The cjacuhitcs of these species have peculiarities which may render their spermatozoa particularly susceptible to the immobilizing action of oxidizing agents, and the suggestion (Mann and Leone, 1953; IMann, Leone and Polge, 1956) that ergothioneine, in virtue of its reducing properties, serves a protective function in boar and stallion semen seems an eminently reasonable one. However, the accessory glands of many animals secrete certain substances {e.g., glycerophosphorylcholine, spermine, citric acid) that do not appear to be of any particular value for the survival of spermatozoa in the male or female genital tracts. Perhaps these substances are simply by-products of the secretory mechanisms of the glands from which they originate, or represent biochemical vestiges.

The widespread occurrence of fructose in accessory gland secretions deserves further comment. The only other situation where large amounts of fructose are present in mammalian extracellular fluids under normal physiologic conditions is in the fetal blood of ungulates (Bernard, 1855; Bacon and Bell, 1948; Alexander, Huggett, Nixon and Widdas, 1955). Mammalian spermatozoa metabolize glucose just as well as fructose as a source of energy under anaerobic and aerobic conditions. Indeed, glucose has been used widely as the sole glycolyzable sugar in artifi^l diluents employed in the storage of semen for artificial insemination (Mann, 1954a). Thus fructose does not seem to be more beneficial than glucose to the well being of spermatozoa. There is evidence that the utilization of fructose, in contrast to glucose, is not impaired in the diabetic state (Chernick, Chaikoff and Abraham, 1951 ; Renold, Hastings and Nesbett, 1954) . It is conceivable that the presence of fructose in semen w^ould render the spermatozoa relatively insensitive to insulin. But it would seem more probable that the physiologic value of seminal fructose is related to factors other than the maturation or survival of spermatozoa. Mann (1954a) has pointed out that if glucose were the only glycolyzable sugar in semen, its concentration would not be expected to exceed that of blood. The transformation of blood glucose into seminal fructose by the accessory glands permits the establishment of very high levels of fructose in semen. Furthermore, the formation of seminal fructose is strictly controlled by androgenic hormones, and it would be hard to conceive of a similar hormonal dependence of glucose levels in semen.

Although the volume and chemical composition of seminal plasma are influenced by many factors, androgenic hormones are undoubtedly the principal determinants of the secretory activity of the accessory glands. Chemical and enzymatic constituents of accessory gland secretions such as fructose, citric acid, and acid phosphatase have proved to be exquisitely sensitive indicators of androgenic activity. The application of such "chemical tests" for androgen action has provided important corroborative evidence for previous conclusions, based on purely morphologic studies, that the initiation of mature secretory function of the accessory glands precedes the appearance of sperm in the seminiferous tubules, and also that the adverse effects of malnutrition on the functional activity of the prostate gland and seminal vesicle are mediated via the hypophysis. Chemical investigations have established that the major portion of certain components (glycerojihosphorylcholine, glycosidases ) of the seminal plasma of some species originates from the epididymis. The way to the successful treatment of metastatic carcinoma of the prostate in man by antiandrogenic measures was paved by the availability of a chemical systemic index of the hormonal dependence of many of these neoplasias, viz., the acid phosphatase of blood serum. Changes in the chemistry of some accessory organs (e.g., the fructose content of the rat coagulating glandj seem to l)e more sensitive indicators of the action of exogenous androgens in castrated animals than the weights or histologic structure of these organs. The application of such chemical methods to the bioassay of androgens holds much promise for the future. Finally, it may be mentioned that chemical studies of the secretions of the accessory glands have given insight into the homology of these organs. The finding of high concentrations of citric acid, but not of fructose, in the rat female prostate after stimulation with androgens shows that the secretion of this tissue resembles that of the ventral prostate gland of the male rat. On the other hand, structures which are usually considered to be anatomically and functionally homologous may secrete quite different substances. Thus in the guinea pig and bull, both citric acid and fructose are secreted by the seminal vesicles, whereas in the rat, citric acid is produced by the seminal vesicles and fructose is formed only in the dorsolateral prostate and coagulating glands.

D. Metabolism of the Prostate and Seminal Vesicle

The metabolism of the male accessory reproductive glands, and the activity of many enzymes therein, are influenced profoundly by steroid hormones. In adult animals, excision of the testes results in a rapid decline in the respiration, but not of the anaerobic glycolysis, of slices of the prostate gland of the dog (Barron and Huggins, 1944), and of the rat prostate (Homma, 1952; Nyden and Williams-Ashman, 1953; Bern, 1953; Rudolph and Starnes, 1954; Butler and Schade, 1958) and seminal vesicle (Rudolph and Samuels, 1949; Porter and Melampy, 1952; Rudolph and Starnes, 1954j. The post-castrate fall in oxygen consumption by these tissues can be reversed by the administration of testosterone. The respiration of the epithelium (but not of the muscle) of the guinea pig seminal vesicle responds in a similar way to androgen deprivation (Levey and Szego, 1955b). The stimulatory effect of testosterone on the respiration of the prostate gland and seminal vesicle of castrated rats is not prevented by the simultaneous administration of hydrocortisone (Rudolph and Starnes, 1954).

The activity of a number of respiratory enzymes in the rat prostate gland is decreased by castration to about the same extent as the respiration of slices of this tissue. This is true for the succinic and cytochrome oxidase systems (Davis, Meyer and McShan, 1949), and for fumarase, aconitase, and malic dehydrogenase (Williams-Ashman, 1954). But the succinic oxidase levels in two other androgen-sensitive tissues are uninfluenced by castration, viz., the epithelium of the guinea pig seminal vesicle (Levey and Szego, 1955b), and the levator ani muscle of the rat (Leonard, 1950). In the rat prostate, androgens have little influence on the activity of the glycolytic enzymes enolase and lactic (l(>hydrogenase, and of the TPN-specific enzymes which oxidize isocitrate, glucose 6-phosphate and 6-phosphogluconate (WilliamsAshman, 1954; Rudolph, 1956). The enzymatic machinery responsible for the respiration of the male accessory glands seems to be similar to that of other mammalian tissues (Barron and Huggins, 1946a, b; Nyden and Williams-Ashman, 1953; WilliamsAshman, and Banks, 1954b; Williams-Ashman, 1954, 1955; Levey and Szego, 1955a). Glock and McLean (1955) have shown that, as in most other mammalian tissues, the levels of DPN in rodent prostate and seminal vesicle are higher than those of DPNH, whereas the content of TPNH is much greater than that of TPN.

Nyden and Williams-Ashman (1953) found that the respiration-coupled synthesis of long-chain fatty acids from acetate by ventral prostate slices m viti'o was depressed by castration to a greater extent than the respiration, and could be restored to normal levels by testosterone therapy. Certain other synthetic reactions (the incorporation of P^--labeled inorganic phosphate into phospholipids, total nucleic acids, and phosphoproteins) were less sensitive to androgens under these conditions. However, in experiments involving the injection of P-^--labeled inorganic phosphate into animals, the administration of androgen increased the turnover of various acidinsoluble phosphorus containing fractions. Thus Levin, Albert and Johnson (1955) observed that testosterone increases the turnover of various phospholipids in the lirostate gland and seminal vesicle. In the seminal vesicle, Fleischmann and Fleischmann (1952) found that the entry of P-'into the desoxyribonucleic acid fraction was increased 100-fold by androgen administered to castrate rats, whereas the sjiecific radioactivity of the ribonucleic acid was increased only 2-fold. Cytoplasmic basophilia in the rat seminal vesicle (Melampy and Cavazos, 1953), and the endoplasmic reticulum of the ventral prostate gland (Harkin, 1957a), which are intimately associated with cytoi)lasmic ribonucleic acid, are influenced profoundly by androgenic hormones.

Transamination between glutamate and cither pyruvate or a-ketoglutarate was shown by Barron and Huggins (1946b) to proceed rapidly in canine and human prostate tissues. Awapara (1952a. bl reported that the ahmine (but not aspartic) transaminase activities of the ventral prostate gland of the rat were decreased by castration, and increased by testosterone therapy.

Rudolph and Starnes (1954) studied the water distribution in the rat accessory glands. The extracellular water in normal seminal vesicles and prostates was 13.8 per cent and 8.5 per cent, respectively. The corresj^onding values in castrate animals were 37.0 per cent and 31.8 per cent. The growth of the glands which resulted from treatment with testosterone was accompanied by a greater increase in the intracellular water than in extracellular water. Rudolph and Samuels (1949) provided evidence that changes in the water content of seminal vesicles induced by treatment of castrate rats with testosterone did not \)recede metabolic changes (e.g., fructose synthesis) in this tissue.

The pronounced effects of androgen administration in vivo on the metabolism and enzymatic activity of the accessary glands cannot be mimicked by the addition of androgens in vitro. Dirscherl, Breuer and Scheller (1955) reported that low levels of testosterone stimulated the respiration of mouse seminal vesicles if the control respiration was low. But others have found that the respiration and glycolysis of male accessory glands are uninfluenced by the direct addition of androgens /// I'itro except at high concentrations (>5 X 10~^ m), at which testosterone is inhibitory (Bern, 1953; McDonald and Latta, 1954, 1956; Andrewes and Taylor, 19551. According to Farnsworth (1958), the direct addition of testosterone to prostate tissue impedes citrate synthesis to a greater extent than oxygen consumption. Williams-Ashman (1954) found that the in vitro addition of testosterone did not affect the activity of a number of respiratory and glycolytic enzymes in the rat A-entral prostate gland.

The mechanism of action of androgenic hormones at a molecular level is not known. There is no evidence that androgens are directly involved in the large changes in the activity of some enzyme systems in accessory glands which follow the administration or deprivation of these hormones. Recent studies wliich indicate that minute concentrations of certain steroid hormones can stimulate the transfer of hydrogen between pyridine nucleotides by isolated enzyme systems deserve further comment. A soluble enzyme in human placenta catalyzes an estradiol- 17y8-dependent exchange of hydrogen between TPNH and DPN (Talalay and Williams-Ashman, 1958). There is evidence in favor of the hypothesis (Talalay, Hurlock and Williams-Ashman, 1958; Talalay and Williams-Ashman, 1960) that estradiol- 17^ transports hydrogen in this reaction by undergoing reversible oxidation to estrone:

Estrone + TPNH + H+ ^ Estradiol-17/3 + TPN EstradioI-17/3 + DPN ^

Estrone + DPNH + H+ TPNH + DPN ^ TPN + DPNH

Hagerman and Villee (1959), however, believe that estradiol- 17/;^ and estrone mediate transhydrogenation between TPXH and DPN by a mechanism which does not involve oxido-reduction of the steroids. Hurlock and Talalay (1958) showed that a soluble 3a-hydroxysteroid dehydrogenase isolated from rat liver catalyzes hydrogen transfer between pyridine nucleotides in the presence of catalytic levels of androsterone and some other 3a-hydroxysteroids. In this instance also, it seems that the steroids act in a coenzyme-like manner by undergoing alternate oxidation and reduction. However, biologically inactive steroids such as etiocholan-3a-ol-17-one are even more active than androgenic substances such as anch'osterone in this isolated enzyme system. Hurlock and Talalay (1959) reported that the particle-bound 3a- and 11^-hydroxysteroid dehydrogenases of rat liver react at comparable rates with both TPX and DPN, and they suggest that these dehydrogenases might function as transhydrogenases in the presence of their appropriate steroid substrates. The hydroxy steroid dehydrogenases for which there is direct or circumstantial evidence for their ability to function as transhydrogenases are localized either in the microsomes (endoplasmic reticulum) or in the soluble cell sap. Other enzymes that catalyze the transfer of hydrogen between pyridine nucleotides are bound to the mitochondria of many animal tissues (Stein,


Kaplan and Ciotti, 1959; c/. Talalay, Hurlock and Williams-Ashman, 1958). These mitochondrial transhydrogenases do not require steroid hormones as cof actors. According to Hmiiphrey (1957), the large cytoplasmic particles of rat prostate gland and seminal vesicle are devoid of transhydrogenase activity. Slices of human prostate gland convert testosterone to androst-4ene-3,17-dione (and other metabolites) (Wotiz and Lemon, 1954; Wotiz, Lemon and Voulgaropoulos, 1954). This suggests that the human prostate contains a 17/3hydroxysteroid dehydrogenase which could conceivably function as a transhydrogenase in the presence of low levels of testosterone. Baron, Gore and Williams (1960) reported the presence of androsterone-stimulated transhydrogenase reactions in the prostate gland of rodents and man. On the contrary, Williams-Ashman, Liao and Gotterer (1958), and Samuels, Harding and Mann (1960) were unable to demonstrate any activation by testosterone of hydrogen transfer between TPNH and DPN in rat prostatic tissue. DPNH and TPNH serve rather different metabolic functions (c/. Talalay and Williams-Ashman, 1958), and it is possible that steroid-mediated transhydrogenations might exert a controlling influence over the balance between the oxidized and reduced forms of pyridine nucleotides in the extramitochondrial regions of certain cells. However, at present there is no direct evidence in support of this hypothesis (cf. Talalay and Williams-Ashman, 1960).

E. Coagulation of Semen

Mammalian semen is emitted from the urethra as a liquid. In some species, e.g., the bull and the dog, the semen remains permanently in the liquid state. But the seminal fluid of many other mammals may undergo remarkable changes in its physical IM^operties on standing. Rodent semen clots rapidly and, if ejaculated into the vagina, forms a solid vaginal plug. This structure assists fertilization by preventing an outflow of semen from the vagina after copulation (Blandau, 1945). The subsequent dissolution of the vaginal plug, probably as the result of the action of leukocytic enzymes, was studied by Stockard and Pajianicolaou (1919). A copulatory plug lias also been described in certain Insectivora, Chiroptera, and Marsupiala (Camus and Gley, 1899; Engle, 1926a; Courrier, 1925; Eaclie, 1948a, bj.

It has been stated that in the opposum (Hartman, 1924) and in the bat (Courrier, 1925), the vaginal plug results from the coagulation of the female secretions by seminal plasma. However, the semen of many other species clots on its own accord. Camus and Gley (1896, 1899) were the first to recognize that in the rat and guinea pig, the clotting process involves the solidification of the vesicular secretion by an enzyme of prostatic origin, which they termed vesiculase. The classical experiments of Walker (1910a, b) showed that this enzyme is secreted solely by the anterior prostate or "coagulating" gland. In the rhesus monkey, the secretion of the cranial lobe (but not of the caudal lobe) of the prostate gland coagulates the vesicular secretion (van Wagenen, 1936) . The "soft calculus" frequently present in the urinary bladder of male but not female rats is l^robably formed by clotting of the seminal vesicle secretion by the action of enzymes from the coagulating gland (Vulpe, Usher and Leblond, 1956) .

More recently, the mechanism of action of vesiculase has been studied in considerable detail. A crude preparation of the proteins of the vesicular secretion that are clotted by this enzyme can be obtained in a stable form, and the clotting process may l)e measured quantitatively by simple spectrophotometric procedures (Gotterer, Ginsburg, Schulman, Banks and Williams-Ashman, 1955; Gotterer and Williams-Ashman, 1957; Zorgniotti and Brendler, 1958). The over-all coagulation process is extremely sensitive to the ionic strength of the solution in which it takes place, and is abolished by the addition of metal chelating agents such as Versene (ethylcnediaminetetraacetic acid), o-i)henanthroline, and a,adipyridyl, and also by heavy metals such as mercuric ions. The inhibitory action of Versene can be overcome by manganous ions, or by somewhat higher concentrations of calcium ions. Experiments involving the delayed addition of either heavy metal ions or of metal chelating agents established that till' coagulation process can be separated into two distinct phases (Gotterer and AVilliams-Ashman, 1957). The first of these requires a metal ion such as Mn++, is inhibited by Versene, and does not necessarily involve the precipitation of insoluble material. The second phase, which is insensitive to the action of metal chelating agents, is inhibited by mercuric ions and leads to the formation of a coagulum. The coagulated material is protein in nature.

Further fractionation of the vesicular secretion by Speyer (1959) led to the isolation of a heat-stable protein, coagulinogen, which is the precursor of the insoluble material of the vaginal plug, but is not clotted by vesiculase. Speyer (1959) isolated another, heat-labile protein from vesicular secretion which he designated procoagulase, and which is converted into a clotting enzyme coagulase by the action of vesiculase. The coagulation of the seminal vesicle secretion by the prostatic enzyme vesiculase thus seems to take place by the following reactions:

„ , Vesiculase „ ,

rrocoagulase > Coagulase

Coagulinogen °^^" '^^^ — ^ Coagulated protein

Only the first reaction is inhibited by Versene.

Partial purification of vesiculase has l>een achieved (Gotterer, Ginsburg, Schulman. Banks and Williams-Ashman, 1955). Vesiculase is quite distinct from another enzyme in the secretion of the coagulating gland of guinea pigs which hydrolyzes, inter alia, tosyl-L-arginine methyl ester (TAMe) (Gotterer, Banks and WilliamsAshman, 1956). Unlike thrombin, vesiculase does not hydrolyze TAMe and does not clot fibrinogen. The dissimilarity between the coagulation of blood and of semen is further borne out by the failure of thrombin to clot the proteins of the vesicular secretion, and by the inability of TA]\Ie (which depresses the action of thrombin) to inhibit vesiculase action.

Electrical stimulation of the head of the guinea pig induces ejaculation without voiding of either urine or feces (Batelli, 1922). Ejaculates obtained in this manner from normal, sexually mature guinea pigs coagulate rapidly. After castration, the semen is no longer coagulable, but becomes so a few days after treatment with androgens (Moore and Gallagher, 1930). This "electric ejaculation test" can be used as an indicator for androgenic activity (c/. Sayles, 1939, 1942).

It is generally believed that human semen is ejaculated as a fluid, and then coagulates (Lane-Roberts, Sharman, Walker and Wiesner, 1939; Joel, 1942; Huggins and Neal, 1942; Lundquist, 1949), although some authors state that it is emitted in a gelatinous form (Pollak, 1943; Hammen, 1944; Oettle, 1954). But there is no doubt that the semen from normal men subsequently liquifies if kept at room temperature.^ Human semen possesses strong fibrinolytic activity (Huggins and Neal, 1942; Harvey, 1949; Ying, Day, Whitmore and Tagnon, 1956). The prostate gland of men secretes a proteolytic enzyme, fibrinolysin, which is probably responsible for the phenomenon of liquefaction. Prostatic fibrinolysin is produced in large amounts by certain cancers of the prostate in man, and seems to enter the circulation since there is a pronounced bleeding tendency in such patients (Tagnon, Schulman, Whitmore and Leone, 1953; Scott, Matthews, Butterworth and Frommeyer, 1954; Swan, Wood and Owen, 1957). Canine semen, which does not clot, contains little fibrinolysin, but is rich in another proteolytic enzyme, fibrinogenase, which hydrolyzes fibrinogen. Little fibrinogenase is present in human semen (Huggins and Neal, 1942). The presence of related proteolytic enzymes in the secretions of the male accessory glands is described above.

^ Louis Nicolas Vauquelin published the first paper on the chemistry of seminal fluid (Vauquelin, 1791). This remarkable study includes a detailed and accurate account of the liquefaction of human semen which is quite unexcelled by later writings. It also describes the formation, in ejaculates which had stood for three or four days, of "cristaux transparens, d'environ une hgne de long, tres-minces, et qui se croisent souvent de maniere a representer les rayons d'une roue. Ces cristaux isoles nous ont offer, a I'aide d'un verre grossissant, la forme d'un solide a quatre pans, termines par des pyramides tres-allongees, a quatre faces." Although Vauquelin believed that these crystals were composed of calcium phosphate, Mann (1954a) has pointed out that he had, in reality, obser\ed the deposition of spermine phosphate in aged semen.


Freund and Thompson (1957) reported that intravenous injection of crude guinea pig coagulating gland secretion into rabbits or guinea pigs induces hypotensive shock. Edema results if the secretion is injected locally. The secretion of the coagulating gland of the rat does not possess these properties. Further studies by Freund, Miles, Mill and Wilhelm (1958) showed that two main protein fractions can be separated from the secretion of the guinea pig coagulating gland by preparative starch electrophoresis. Fraction I was hypotensive and a potent permeability factor in rabbits and guinea pigs. It hydrolyzed TAMe rapidly and may be identical with the TAMehydrolyzing enzyme described in guinea pig coagulating gland l)y Gotterer, Banks and Williams-Ashman (1956). The latter enzyme is not present in the coagulating gland of the rat. Fraction II isolated by Freund and his associates is ]n"obably vesiculase.

III. Structure and Function in Relation to Hormones

A. INTRODUCTION

Some of the effects of removal of the testes in males have been recognized ever since castration was first practiced on man and domestic animals. Aristotle's writings include accurate descriptions of the effects of castration on secondary sex characters in birds and in man. The classical studies of John Hunter (1792) laid the basis for an understanding of the relation between the presence of the testes and the size and functional state of the accessory reproductive glands of mammals, although he did not postulate the existence of testicular hormones.

Hunter demonstrated experimentally that the seminal vesicles of guinea pigs are not reservoirs for semen and concluded that this a])plies to the seminal vesicles in man and in other mammals. He not only described the gross anatomy of the seminal vesicles in many species (hedgehog, iiiole. man, boar, bull, horse, buck, mouse, rat, beaver, guinea pig) and their absence fi'om others, but he observed tliat they arc smaller in the gelding than in the stallion. In refei'ence to other glands, he generalized


that "the prostate gland, Cowper's glands and the glands along the urethra . . . are in the perfect male large and pulpy, secreting a considerable quantity of slimy mucus which is salt to the taste . . . while in the castrated animal these are small, flabby, tough and ligamentous, and have little secretion." In addition, he made the equally important discovery that the testes of mammals (and birds as well) are very small in winter in animals which have their seasons of copulation" and the seminal vesicles and prostates are "hardly discernable." He concluded that "from these observations it is reasonable to infer that the use of the vesiculae in the animal oeconomy must, in common with many other parts, be dependent upon the testicles."

Over 100 years later, many of his observations were rediscovered, extended, and interpreted in the light of the first demonstration that the testis is an endocrine organ (Berthold, 1849). In the early part of the 20th century the interest in attempting to isolate and characterize androgens from testis tissue and urine led to a search for rapid and dependable bioassay methods. The cock's comb provided a sensitive and convenient test object (Pezard, 1911). In addition, some of the accessory reproductive glands of mammals were found to atrophy rapidly after castration and proved also to be sensitive indicators for the presence of androgenic hormones. Cytologic tests using the rat prostate, seminal vesicles, and Cowper's glands were developed and an electric ejaculation test in the guinea l)ig was devised (Moore, 1932, 1939). Weights, sizes, cytologic structure, and mitotic activity in mouse seminal vesicles were suggested as bioassay methods for androgenic hormones (Deaneslv and Parkes. 1933).

After th(> successful isolation and chemical characterization of androgens and estrogens from various sources, interest centered on the fundamental relationships of androgens to normal develojjment, histologic structure, and secretory activity of the accessory glands in many species of inainmals. The effects of estrogens and gestagens and the competitive and synergistic i'elationshii)s of steroid hormones were examined. The results of this early work contributed extensively to the fields of biochemistry, biology, and medicine. More recently there have been studies on the relation of hormones to the ultrastructure, histochemistry, and metabolism of the glands, and to the chemical composition of their secretions ( Section II I .

In the following section, the hormonal control of structure and function will be discussed with particular reference to the luniierous studies on the prostate glands and seminal vesicles of rats and mice.

B. EFFECTS OF ANDROGENS

The term androgen will be used in the collective sense for substances that are capable of stimulating accessory reproductive glands in castrated animals and maintaining normal histologic structure and secretory activity in the epithelium. Androgenic substances are formed by the testes, ovaries, and adrenal cortex. All androgens which have been characterized are steroids. The urine contains many androgen metabolites, mainly in the form of their conjugates with either glucuronic or sulfuric acids. Testosterone is the principal androgen secreted by the testis and this substance, or tiie longer acting testosterone propionate, is most commonly used as a replacement for testicular androgen. In the last two decades a number of unnatural androgens [e.g., 17a-methyl testosterone) have been synthesized and found to possess strong biologic activity. The relationship between chemical structure of steroids and andro


genic activity in a variety of bioassay procedures is discussed by Dorfman and Shipley (1956).

1. Testicular Androgens

The effects of endogenous and exogenous androgen on weight, histologic structure, and secretory activity of the accessory glands have been reviewed by Moore (1939), Price (1947), Burrows (1949), Dorfman (1950), Dorfman and Shipley (1956) and many others. Aspects of metabolic activity have been treated by Roberts and Szego (1953) and Mann (1954a).

The first detailed cytologic studies of male accessory glands and the changes following castration and hormone administration were made on the prostates, coagulating glands, and seminal vesicles of adult rats (Moore, Price and Gallagher, 1930; Moore, Hughes and Gallagher, 1930). Extensive research on structure and function of these and other accessory glands in many species followed this early work, but the cytologic structure of prostates and seminal vesicles of rats and mice remains one of the most sensitive indicators for androgenic hormones.

Rat PROSTATE AND SEMINAL VESICLES. Ventral prostate. In the normal adult gland, the columnar secretory epithelium has basal nuclei with conspicuous nucleoli and chromatin particles, and a supranuclear clear zone or light area in the cytoplasm corresponding to the position of the Golgi zone (Figs. 6.8, 6.9, and 6.14). In osmium preparations, the Golgi apparatus appears as



Figs. (3.8 Axn 6.9. Rat ventral prostate from a normal adult male. X 5UU and lOUU. Boinnhematoxylin preparations. (From C. R. Moore, D. Price and T. F. Gallagher, Am. J. Anat., 45, 71-107, 1930.)


heavy strands or networks (Figs. 6.19 and 6.22) which do not conform precisely to the shape or area of the cytoplasmic clear zone. Mitochondria are distributed as rods or granules in all parts of the cell. The secretion in the lumina of the alveoli is eosinophilic and mainly granular. A basement membrane rests on a stroma of connective tissue containing smooth muscle strands and blood vessels. Occasional small basal cells are wedged between the tall secretory cells. These observations were made by light microscopy of tissues fixed and stained by routine methods (Moore, Price and Gallagher, 1930).


Electron microscopy (Harkin, 1957a) shows that the epithelial cells have an endoplasmic reticulum or ergastoplasm composed of membrane-lined sacs with a finely granular component in the spaces between them (Figs. 6.27 to 6.29) ; the outside of the thin membrane is studded with Palade's granules (Palade, 1955). The arrangement of the sacs tends to parallel the long axis of the cells, but in cross section the pattern ai)pears concentric or lamellar, particularly in the supranuclear region (Fig. 6.29). The ergastoplasmic sacs occupy more space than the matrix apically, but basally the two are equally prominent (Fig. 6.28). The mem


TABLE 6.6

Summary of the effects of testicular androgen, on the rat prostate and coagulating glands


Normal Males


Castrated Males


General Characteristics


All lobes alveoli witli folded inueosa; secretion in the lumina.. columnar epithelial cells: cytoplasm granular or foamy. supranuclear clear zone in cytoplasm (ventral lobe)

Golgi supranuclear networks

mitochondria as rods or granules

nuclei basal or central

stroma of connective tissue and smooth muscle


Size reduced; villi lost; secretion reduced

Size reduced; pseudostratified; cytoplasm less dense

Clear zone lost

Reduced in amount; fragmented

Still numerous but reduced in relative numbers

Shrunken and pyknotic

Increased fibromuscular tissue


Specific Characteristics


Ventral lobes

Histochemical observations:

secretion in lumina strongly PAS- and alkaline phosphatase-positive . . .

cytoplasm weak PAS, strong alkaline phosphatase activity;

basophilic reaction except in clear zone

Golyi accumulations of PAS-positive granules

stroma some alkaline phosphatase activity

Electron microscopic observations:

cytoplasm moderately distended ergastoplasmic sacs

Golgi supranuclear microvesicular complex

mitochondria numerous, prominent apically

Lateral lobes

Histochemical observations:

cyioplasm luminal border organelle with high concentrations of zinc and basophilic material; osmiophilic, argentophilic

nucleoli high concentrations of zinc and marked basophilia

stroma high concentrations of zinc; basojihilic material (jresent Dorsal lobes Histochemical observations:

cytoplasm in apical region strongly basophilic; basally, some zinc

nucleoli high concentrations of zinc and marked basophilia

stroma basophilic material; strong alkaline i)hosphatase reaction

Electron microscopic observations:

cytoplasm distended ergastoplasmic cisternae;

Coagulating glands (anterior prostate) Histocheinical observations:

secretion strongly PAS-positive

cytoplasm weak P.\S reaction

stroma some alkaline phosphatase activity

Electron microscopic observations:

cyioplasm extremely dilated ergastoplasmic cisternae


Some phosphatase activity retained Phosphatase activity low


Sacs collapsed; granvilar component reduced

Reduced in size

Reduced in relative numbers


Cisternae collapse


granules reduced


pears unalterc


Cisternae collapsed; granules reduced



hrane is continuous with the outer nuclear membrane. The Golgi complex is conspicuous as microvesicles midway between nucleus and lumen. Mitochondria lie in the matrix between the sacs and are very prominent in the most apical region. Microvilli project into the lumina of the alveoli with no interruption of the cytoplasmic, or plasma, membrane. The membrane at the base of the cell is double (see Fig. 6.28) ; one component, the basement membrane, continues unbroken under adjacent cells; the second forms a part of the double plasma membrane between cells. Brandes and Groth (1961) have confirmed Harkin's findings and added further observations. Nuclei contain patches of granules which are frequently along the inner nuclear membrane; the Golgi complex consists of vesicles, vacuoles, and parallel meml)ranes; vesicles and granules surrounded by smooth-surfaced membranes are disposed in the cytoplasmic matrix and are more numerous apically; the dilated sacs or cisternae of the supranuclear region seem to intercommunicate.

Histochemical studies of basophilia, alkaline phosphatase activity, and the localization of i^eriodic acid-reactive carbohydrates (Periodic acid-Schiff or PAS reaction) add further information (Table 6.6). Davey and Foster (1950) found basophilia (which was abolished by ribonuclease) distributed through the cytoplasm except in the clear area described by Moore, Price and Gallagher (1930) as corresponding to the position of the Golgi zone. Stroma of the ventral prostate shows some degree of alkaline phosphatase activity but luminal secretion and epithelial cells are strongly positive (Bern, 1949a), especially at the luminal and basal borders (Stafford, Rubenstein and Meyer, 1949). The secretion also gives a fairly intense PAS reaction whereas the epithelial cells are only slightly reactive; occasionally the Golgi apparatus is visible as PAS-positive granules (Leblond, 1950).

After castration, there is reduction in cell height and loss of the cytoplasmic clear zone (Fig. 6.15) within 4 days. On subsecjuent days, cell size continues to decrease and nuclei become small and pyknotic (Figs. 6.10. 6.16 to 6.18). The Golgi apparatus begins to fragment by 10 days; by 20 days it consists of granules much reduced in amount (Fig. 6.20) and the basement membrane of the cells disappears (Moore, Price and Gallagher, 1930).

Harkin (1957a) reported changes observable by electron microscopy within 24 hours after castration; distention of apical ergastoplasmic sacs and reduction in size and number of microvilli. By 2 days, there is dilation of Golgi microvesicles, collapse of the apical ergastoplasmic sacs, and reduction in mass of apical cytoplasm; at 4 days, massive collapse of sacs, reduction in mitochondrial number, and increase in electron-dense bodies (Fig. 6.30). The granular component is not reduced until 8 days after castration or longer. Brandes and Portela (1960a) noted, briefly, collapse in the cisternae of the ergastoplasm, loss of the ribonucleic acid- (RNA) rich granules from the membranes of the endoplasmic reticulum, and apparent increase in mitochondria but with a reduction in their size (Table 6.6).

The distribution of alkaline phosphatase in the stroma, epithelium, and secretion is unchanged 32 days after castration; the stroma is still reactive at 120 days but the epithelium is completely atrophic (Bern and Levy, 1952). (Quantitative determinations of alkaline and acid phosphatases showed, however, that activities of both enzymes are reduced markedly by 8 days (Stafford, Rubenstein and Meyer, 1949). The epithelium loses the ability to secrete citric acid (see Section IT).

Changes after gonadectomy are prevented or reversed by administration of androgenic substances. Extracts of bull testes (:\Ioore, Price and Gallagher, 1930) prevented involution of the epithelium in castrates (Fig. 6.11 and 6.21) and androsterone, testosterone, and testosterone propionate prevented or repaired castration changes CMoore and Price, 1937, 1938). The response of the castrate to androgen is rapid; cell hypertrophy begins within 23 hours after a single injection of testosterone propionate into males castrated for 40 days ; at 35 hours mitotic activity begins and reaches a maximum at 43 hours (Burkhart, 1942).

Ergastoplasmic sacs in the epithelial cells




I I'- <; II) 0.13. Rat ventr;il lu-o-tatc (Figs. 6 10. H.U) and coagulal iim aland ( Fm- C) 12, (i.lo). All pliotomicrographs , lOUU. Fig. 6.10. 20-day cabtiatc. Fig. 6.11. 20-day ca.^tratf injected with testis extract. Fig. 6.12. 20-day castrate. Fig. 6.13. 20-day castrate injected with testis extract. (From C. R. Moore, D. Price and T. F. Gallagher, Am. J. Anat., 45, 71-107, 1930.)


are prevented from collapsing by treatment of castrates with testosterone and the process is reversed if the androgen is given after castration changes have developed (J. C. Harkin, personal communication). Alkaline and acid phosphatase levels are essentially normal in castrates injected with testost(M'one propionate (Stafford, Rubinstein and Meyer, 1949).

Lateral prostate. The epithelial cells in normal adult glands are columnar and the nuclei are basal, but cell size and nuclear position arc more variable than in the ventral prostate ( Korenchevsky and Dcnnison. 193.51. The Golgi apparatus appears as


l^rominent supranuclear networks in osmium stainecl preparations (Rixon and Whitfield, 1959).

Histochemical studies employing a dithizone zinc stain demonstrated high concentrations of zinc in the apical jiart of the cells (Gunn and Gould, 19o6a). Fleischhauer (1957) observed (macroscopically) heavy staining that was visible in this lobe after intravenous or subcutaneous injections of dithizone. In mifix(Hl frozen sections, he found in tlic basal regions of all epithelial cells numerous stained granules which lie interpreted as zinc-positive material. 'Hie nature of a rather wide diffusely



m


m


IB




IB


m


0'^




IE


m



m


m


m


Figs. 6.14-6.26



Figs. 6.14-6.22. Rat ventral prostate. Figs. 6.14-6.21. Camera lucida drawings X 3000. Figs. 6.19-6.22. Mann-Kopsch preparations for Golgi apparatus. Fig. 6.14. Normal male. Figs. 6.156.18. From males castrated for 4, 10, 20 and 90 days. Fig. 6.19. Normal male. Fig. 6.20. 20-day castrate. Fig. 6.21. 20-day castrate injected with testis extract. Fig. 6.22. Normal male ; photomicrograph X 1000. (From C. R. Moore, D. Price and T. F. Gallaglier, Am. J. Anat., 45, 71107, 1930.)

Figs. 6.23-6.26. Rat coagulating gland. Figs. 6.23-6.25. Camera lucida drawings X 3000. Fig. 6.23. Normal male. Fig. 6.24. 20-day castrate. Fig. 6.25. 20-day castrate injected with testis extract. Fig. 6.26. Normal male; photomicrogaph X 1000; Mann-Kopsch preparation for Golgi apparatus. (From C. R. Moore, D. Price and T. F. Gallagher, Am. J. Anat.. 45, 71-107, 1930.)




Fig. 6.27. R:it xcniinl |.in~i,ii( ikhihiI mil. I ,li . 1 1 uiiiuicrograpli X 8500; LuftV ])prmanganate fixative. Aliciuvilli lxIlikI a^ i)iul()n^atioii.-. ol the cytoplasm into the lumen; a major part of the cytoplasm is a labyrinth of ergastoplasmic sacs with scattered mitochondria ; nuclei are basal ; half-way between nucleus and cell apex is a zone of small vesicles and canals, the Golgi complex (From J. C. Harkin, un])u})lishe(l.)



stained area in the ai)ical cytoi)lasin was not clear. Ki.xoii and Whitfield 11959) reported high concentrations of zinc in the apical cytoplasm, nucleoli, and stroma in fixed tissues stained with dithizone. Tn the apical cytoplasm, the zinc is conccnti atcd at the tip of the cells in a "luminal l)order organelle" which is osmiophilic (distinct from the (Jo].o;i apparatus), argent()i)hilic,


and basophilic. Nucleoli and subepithelial sti'oma are basophilic.

Castration results in a typical pattern of involution in the epithelial cells: size is !•(■( bleed, nuclei become small and jwknotic, and changes occur in the density of the cytoplasm (Korenchevsky and Dennison, 1935; Price, Mann and Lutwak-Mann, 1955). The zinc content of the gland (dorsolatcral or lateral prostate) and the rate of Zn*^^ uptake decrease after gonadectomy as does the secretion of citric acid and fructose (see Section II).

Dorsal prostate. The epithelium in the dorsal prostate of normal rats is columnar or cuboidal depending on distention of the alveoli; nuclei are basal and stain heavily; there is cytoplasmic vacuolization which is usually limited to the basal region (Korenchevsky and Dennison, 1935) .

Brandes and Groth (1961) described the ultrastructure of two different cell types in the dorsolateral (or dorsal) lobe. These types differ in the relation of cytoplasmic matrix to endoplasmic reticulum. In both, the matrix is moderatelv homogeneous and


contains small particles, but in cell type 1, the matrix appears as separate profiles and the reticulum as membrane-bounded individual cavities. In cell type 2, the reticulum forms dilated membrane-bounded cisternae which are intercommunicating and the cytoplasmic matrix is reduced mainly to thin strands appearing isolated within the cisternae.

Gunn and Gould (1956a) reported a zinc-negative histochemical reaction in the epithelium of the dorsal prostate. Fleischhauer (1957) observed a slight dithizonestain macroscopically, and in unfixed frozen sections, individual groups of cells contain the distinctive basal zinc-positive granules that are characteristic of all epithelial cells



Fl(.i. li..'N. l;;il v,l,ii;,l |Mm-;;:h, n,uM,:,l in.ih. 1 ,1. r i ,, ,i i li i in . ,-i:i [ .1, . 2(;,()()(); 1 ),-| ll ( )ll's chrome o^niic and hxatixi . Il;i>:il pari ot epithelial cell to show the character of the granular component and ergastopla>iiiic -acs which are essentially equal in amount in this region. Double basement membrane indicated by arrow. (From J. C. Harkin, Endocrinology, 60, 185-199, 1957.)


l'"i(..G.2U. Rat \(;iilial lUo.-Uilu, iiuinial iiiak;. I'^lcctionnu' acid fixative with sucrose. Supranuclear region of epithelia plasmic sacs. (From J. C. Harkin, unpublished.)


.-laph Is.iiOO; Paladps osmic •ell sliowing laniellatetl ergasto


in the lateral lolje. Tiiere is no diffuse staining of the apical cytoplasm. Nucleoli arc intensely zinc-positive after fixation and staining with dithizone; nucleoli, apical cytoplasm and stroma are basophilic fRixon and Whitfield, 1959). The stroma is also strongly alkaline phosphatase - positive (Bern, 1949a).

Epithelial cells respond to castration l»y reduction in cell and nuclear size, and loss of granulation in the cytoplasm (Korenchevsky and Dennison, 1935). Brandes and Portela (1960a) observed in electron micrographs the V)eginning of collapse of the


cisternae of the endoi)lasmic reticulum, reduction in RNA-rich particles, and changes in mitochondria. Histochemical studies ( Iicni and Levy, 1952) indicate that distribution of alkaline phosphatase activity remains unchanged. Fructose content is reduced in the gland after castration (Price, Mann and Lut\vak-]\Iann, 1955).

CocKjulating gland {anterior prostdte) . In normal males, the ei)ithelium is columnar and rests on a well marked basement membrane; nuclei stain heavily and homogeneously and ai'e situated midway between the basement meml)i';iiie and lumen. The cvtoplasm is not as granular as in the ventral prostate and appears vacuolated, particularly in the basal region and around the nuclei; the apical cytoplasm is condensed and granular (Fig. 6.13, a gland from a castrated male injected with testicular extract, illustrates essentially the characteristics of the normal epithelium). Golgi bodies ( Fig. 6.26) form large networks close to the luminal end of the cells (Moore, Price and Gallagher, 1930).

The striking characteristic of these cells in electron microscopy (Brandes, Belt and Bourne, 1959; Brandes and Groth, 1961) is the great dilation of the cisternae of the endoplasmic reticulum (Fig. 6.31) which fill the greatest part of the cell and are particularly distended in the basal region. The





Fig. 6.30. Rut \entral prostate, 4-day castrate. Electronmicrograph X 26,000; Dalton's chrome osmic acid fi.xative. Portion of nucleus and di.stal region of epithelial cell. An electron dense body lies above the nucleus and below dilatated Golgi microvesicles. Arrow points to collapsed ergastoplasmic sacs. (From J. C. Harkin, Endocrinology, 60, 185-199, 1957.)


Fig. 6.31. Rat coagulating gland, normal male. Electronmiciogiaplis, lefl X 7200; upper and lowei- right X 39,000. Caulfield's modification of Palade's osmic acid fixative. Left, ba.sal portions of two epithelial cells; right, details of basal region: bni, basement membrane; ci, dilated cisternae; cm, plasma membrane; cy, cytoplasmic matrix; G, Golgi complex; bn, limiting membrane of endoplasmic reticulum; w, mitochondria; n, nucleus. (From D. Brandes, unpublished.)


cytoplasmic matrix appears as strands within the cisternae. The Golgi complex is represented by parallel rows of membranes, vacuoles, and smaller vesicles.

Histochemically (Table 6.6), the secretion is intensely PAS-positive and the cytoplasm is slightly reactive (Leblond, 19501. The stroma is strongly alkaline phosphatase-positive (Bern, 1949a).

The effects of castration arc not ai)i)arent by light microscopy as early as in the ventral prostate and seminal vesicles. At 10 days after castration the cells are slightly smaller and the cytoplasm less dense; by 20 days, the cells are markedly reduced in size, nuclei smaller, cytoplasm clear, basement membrane absent or less well defined (Figs. 6.12 and 6.24). The Golgi apparatus is reduced in amount but not fragmontcnl. It still retains the shape of strands or threads which cap around the nucleus at 90 days of castration but the mass is reduced (Moore, Price and Gallagher, 1930).

Brandes and Portela (1960a) state that castration produces gradual and slow collapse of cisternae in the endoplasmic reticulum, changes in mitochondria, and reduction and loss of RNA-rich particles from the membranes. Studies of functional activity show that the ability to secrete fructose and vesiculase is lost (see Section II).

Depending on the length of the interval between the operation and administration of the hormone, treatment of castrates with testis extracts (Figs. 6.13 and 6.25) or testosterone prevents or repairs histologic and functional changes.

Seminal vc.'iicles. The secretory epithelium is colunuiar in normal males; nuclei are basal and contain one or two conspicuous nucleoli and smaller chromatin masses (Table 6.7). Seci-etion granules, surrounded


ACCESSORY MAMMALIAN REPRODUCTIVE GLANDS


409


TABLE 6.7 Summary of the effects of testicular androgen on rat and mouse seminal vesicles


Normal Males


Castrated Males


General Characteristics


Rat and mouse

mucosa folded; acidophilic secretion in lumen Villous folding reduced; secretion greatly reduced

columnar epithelial cells: secretion granules supranuclear Cell size reduced; granules lost

Golgi supranuclear networks \ Reduced in volume; fragmented

mitochondria as rods or granules ' Apparently reduced in relative numbers (rat)

nuclei basal; nucleoli prominent I Nuclei shrunken and pyknotic; nucleoli disappear

stroma of connective tissue and smooth muscle Amount appears increased


Specific Characteristics


Rat Histochemical observations : secretion in lumen PAS-positive, intensity variable; acidophilic

ci//opZasm slight PAS reaction; strongly acid phosphatase-positive;

strongly basophilic

secretion granules in epithelium strongly acid phosphatase-positive

nuclei strong acid phosphatase reaction

stroma slight PAS reaction; acid phosphatase-positive; strong alkaline

phosphatase reaction

Mouse Histochemical observations :

secretion in lumen moderately PAS-positive and acidophilic

cytoplasm moderately basophilic at base and lateral margins of cells;. . .

apical granules acid phosphatase-positive secretion granules in epithelium weakly PAS-positive and acidophilic... Golgi region; granules PAS-positive and acidophilic stroma intensely PAS- and alkaline phosphatase-positive Electron microscopic observations: cytoplasm complex pattern of basal and lateral ergastoplasmic membranes;


Phosphatase activity reduced

Slight basophilia

Activity lost

Remained weakly acid phosphatase-positive

Acid and alkaline phosphatase activity reduced


Secretory granules less acidopliiliWeakly basophilic


abundant RNA-rich granules

Golgi region; parallel arrays of smooth-surfaced membranes and vesicles


Reduced in number; less acidophilii Phosphatase activity reduced


Ergastoplasmic channels less distended and contorted Relative number reduced


by vesicular zones are present in the supranuclear region (Fig. 6.36) and resemble the secretion in the lumen in staining reactions. The Golgi complex appears in osmium preparations as irregular networks or a vesicular structure (Fig. 6.32); the basement membrane is poorly defined or absent (Moore, Hughes and Gallagher, 1930).

The extracellular secretion is only slightly PAS-positive but varies in the intensity of reaction; there is little reaction in the epithelial cells except in some cells with stained granules; fibers of the lamina propria, smooth muscles, and walls of arterioles are weakly reactive (Leblond, 1950; ]\Ielampy and Cavazos, 1953). Stroma and capillaries are strongly alkaline phosphatase-positive (Bern, 1949a; Dempsey, Greep and Deane, 1949; :\lelarapy and Cavazos, 1953). The cytoplasm, secretion granules, nuclei, and stroma give an intense acid phosphatase reaction; the cytojilasm is strongly baso


philic and the reaction is abolished by ribonuclease (]\lelampy and Cavazos, 1953).

The response to castration is rapid. In 2 days the cells are reduced in height mainly by reduction in apical mass ; secretion granules are few, small, and indistinct. By 10 days, cells are small, secretion granules are gone, nuclei are small with heavily staining chromatin (Fig. 6.35), and Golgi bodies have begun to fragment; at 20 days, these changes are more advanced and the remnant of the Golgi bodies (Fig. 6.33) occupies almost the entire supranuclear region (Moore, Hughes and Gallagher, 1930). In a cytometric study, Cavazos and ]\Ielampy (1954) found a statistically significant reduction in cell height by 6 hours after castration; by 48 hours many nucleoli are smaller than normal and by 60 hours most nucleoli are small; nuclear diameters are reduced but change more slowly.


410


PHYSIOLOGY OF GONADS



'0 (?rCf


f-\


m


m



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^


Figs. 6.32-6.36. Rat seminal vesicle; camera lucida drawing.s ,: 3000. Figs. 6.32-6.34. ManuKopsch preparations for Golgi apparatus. Fig. 6.32. Normal male. Fig. 6.33. 20-day castrate. Fig. 6.34. 20-day castrate injected with testis extract. Fig. 6.35. 10-day castrate. Fig. 6.36. 20day castrate injected with testis extract. (From C. R. Moore, W. Hughes and T. F. Gallagher, Am. J. Anat., 45, 71-107, 1930.)


Gonadectomy causes gradual reduction and disappearance of alkaline phosphatase activity (Dempsey, Greep and Deane, 1949; Melampy and Cavazos, 1953) and hypophysectomy, with consequent diminution of testicular hormones, gives similar results (Dempsey, Greep and Deane, 1949). Bern and Levy (1952) reported some retention of alkaline phosphatase activity in the fibromuscular tissue of castrates. Acid phosphatase activity decreased within 10 days following castration (Melampy and Cavazos, 1953).

In early experiments (Moore, Hughes and Gallagher, 1930) , administration of bull testis extracts to castrated rats maintained normal histologic structure or repaired involutional changes (Figs. 6.34 and 6.36),


and androsterone, testosterone, and testosterone propionate gave similar resvdts (Moore and Price, 1937, 1938). Androgen treatment in castrates produced detectable changes within 2 days. Burkhart (1942 1 observed cell hypertrophy ahd enlargement of nuclei 23 hours after a single injection of testosterone propionate into 40-day castrates; mitotic activity began at 35 hours and reached a maximum at 43 hours. Cavazos and Melampy (1954) treated castrates with testosterone propionate and found increases in nuclear diameter within 12 hours, cell height within 24 hours, and nucleolar size l)y 36 hours; mitotic activity was evident at 48 hours. The same hormone restoi'cd normal alkaline and acid phosphatase activity in castrates within 10 days


ACCESSORY MAMMALIAN REPRODUCTIVE GLANDS


411


(Dempsey, Grecp and Deane, 1949; Melampy and Cavazos, 1953).

Mouse prostate and seminal vesicles. Ventral prostate. The epithelial cells in the adult gland are low to moderately tall columnar; acini are surrounded by a thin fibromuscular layer; lumina contain finely granular, acidophilic secretion. The cytoplasm appears somewhat foamy with a clear zone in the supranuclear Golgi region and rather dense basophilia near the lumen (Franks, 1959). In approj)riate histologic preparations, the Golgi apparatus is visible as a network in the apical cytoplasm close to the nucleus and the twisted strands are oriented parallel to the long axis of the cell (Horning, 1947).

Brandes and Portela (1960c) observed by electron microscopy an endoplasmic reticulum of cisternae or vesicles that are usually flattened but have dilations. RNA-rich granules are attached to the outer surface of the thin membranes bounding the vesicles and occur also in the cytoplasmic matrix; the arrangement of cisternae may be parallel or in a random pattern. The luminal margin of cells exhibits small cytoplasmic projections covered by the cell membrane. There are also extensions of the margin which ai)pear similar to fragments of cytoplasm that seem to lie free in the lumen, and are presumably detached from the apical tips of cells. The lumina also contain structures that resemble profiles of the endoplasmic reticulum and mitochondria. The supranuclear Golgi complex consists of vacuoles of various sizes and flattened vesicles; endoplasmic reticulum and mitochondria are present in the Golgi zone.

Histochemical findings (Table 6.8) indicate alkaline phosphatase activity in the stroma with a positive reaction in the basement membrane, endothelium of blood vessels, and sheaths of smooth muscle fibers (Brandes and Bourne, 1954). With longer incubation periods of the tissue (Bern, 1949a), the epithelium and secretion in the lumina are strongly reactive and the stroma shows some activity. Brandes and Bourne (1954) reported acid phosphatase activity in the epithelium; the Golgi region gave the strongest reaction and the nuclei were moderately positive. Luminal secretion, ag


gregations of granules in the Golgi region and apical cytoplasm, basement membranes, and capillary endothelium were PA8-positive. Sulfhydryl and disulfide reactions were moderately strong in epithelial cells and basement membranes.

Gonadectomy results in typical cell retrogression with reduction in cell height and nuclear size. Brandes and Bourne (1954) summarized their results as follows: after gonad removal, the Golgi apparatus showed some fragmentation and was not so dense by 12 to 14 days; alkaline phosphatase activity was slightly less intense by 4 days; changes in acid pliosphatase activity in the Golgi region were evident by 4 days and marked by 21 to 22 days; the PAS reaction was reduced by 8 days and almost lost in the epithelium by 21 to 22 days. Subcutaneous implantation of pellets of testosterone propionate 13 to 32 days after castration produced a rapid return to normal of Golgi apparatus and phosphatase activity and a gradual recovery of normal PAS reactions. Allen (1958) reported a significant increase in mitotic activity in the epithelium of 30-day castrates within 30 to 36 hours following a single injection of 16 /xg. of testosterone propionate; peak activity was reached in 42 to 48 hours.

Dorsal prostate. The epithelial cells resemble rather closely those of the coagulating gland but the cytoplasm is more granular, the centrally placed nuclei darker, and the Golgi apparatus in the apical cytoplasm (in close contact with the nucleus) is less dense than the Golgi networks in the coagulating gland (Horning, 1947). Histochemically, the distribution of phosphatase activities and PAS reaction in normal males, castrates, and castrates treated with testosterone propionate are similar to the findings in the coagulating gland (Bern, 1949a, 1951; Brandes and Bourne, 1954).

Coagulating gland {anterior prostate). The secretory cells are columnar and the nuclei are approximately midway between basement membrane and lumen. The cytoplasm is granular and the condensed Golgi apparatus is a flattened network oriented transversely in the most apical region of the cytoplasm (Horning, 1947).

Electron microscopic studies by Brandes


412


PHYSIOLOGY OF GONADS


TABLE 6.8

Summary of the effects of testicular androgen on the mouse prostate and coagulating glands


Normal Males


Castrated Males


General Characteristics


All lobes alveoli with folded


lucosa; secretion in luniina.


columnar epithelial cells

cytoplasm of epithelial cells granular or foamy

nuclei basal .

stroma of connective tissue and smooth muscle

Histochemical observations:

secretion in lumina PAS-positive

cytoplasm acid phosphatase-positive; sulfhydryl reaction

intracytoplasmic granules PAS-positive; near luminal border

Golgi region PAS-positive granules; strong acid phosphatase activity.

basement membrane PAS- and alkaline phosphatase-positive

Stroma PAS- and alkaline phosphatase-positive

Electron microscopic observations:

epithelial cells with microvilli

Golgi complex smooth surfaced membranes and vesicles


Iveolar size; loss of villi and bulk of


Reduction i

secretion

Reduction in cell size; pseudostratification .\ppears less dense Shrunken and pyknotic Fibromuscular increase

Almost completely negative

Phosphatase activity reduced

Almost completely negative

Almost completely negative

Activity retained; less intense

Activity retained in sheaths of smooth muscles

Cell size reduced


Specific Characteristics


Ventral lobes

Histochemical observations : secretion in lumina strong alkaline phosphatase activity cytoplasm alkaline pliosphatase-positive

Golgi loose networks in apical cytoplasm

Electron microscopic observations: cytoplasm; ergastoplasm with generally flattened cisternae.. Dorsal lobes Histochemical observations :

Golgi compact networks in apical cytoplasm

Coagulating glands (anterior prostate) Histochemical observations : Secretion in lumina intense protein reaction; PAS-positive;.

sulfhydryl reaction

cytoplasm high concentrations of RNA basally and apically

protein reactions, intense apically

sulfhydryl reaction, especially strong apically

Golgi condensed apical networks

Electron microscopic observations: cytoplasm extremely dilated ergastoplasmic cisternae


Reduced in amount; fragmented Cisternae collapsed; reduced granules

Retluced in amount; fragmented


Some PAS reaction retained

Sulfhydryl reaction lost

Markedly decreased

Greatly reduced

Reaction lost

Reduced in amount; fragmented

Cisternae collapsed; granules reduced


and Portela (1960b) show that these epithelial cells are characterized by an endoplasmic reticulum with greatly dilated cisternae. This dilation is more marked in the middle of the cell and in the basal region (Fig. 6.37) where the dilated cisternae appear as intercommunicating channels in which the cytoplasmic matrix forms isolated profiles or strands containing mitochondria and other organelles. The matrix is more abundant in the Golgi region and protrudes from the luminal margin of the cells as microprojections covered by tlu; cell membrane.

Alkaline phosphatase activity is localized in the stroma (Bern, 1949a, 1951 ; Brandes


and Bourne, 1954; Bern, Alfert and Blair, 1957) ; acid phosphatase activity (Brandes and Bourne, 1954) is found in the epithelium and is particularly strong in the Golgi zone. Brandes and Bourne (1954) and Bern, Alfert and Blair (1957) reported PAS-positive reactions in the epithelial cells in the Golgi region and apical cytoplasm, and intense reactions in luminal secretion, basement membrane, and stroma. Sulfhydryl and disulfide reactions are evident in luminal secretion, epithelium (especially in the a]ucal region), basement membrane, and fibromuscular tissue. The reactions are stronger than in the ventral prostate. Bern, Alfert and Blair (1957) found high con


ACCESSORY MAMMALIAN REPRODTTCTIVE GLANDS


4i;


cy


/Jf-!^


pc


N.pc

y



. .... ^


cy



\


m


..^cy


^"^^


^m


V*'


^


cy




IS


Fig. 6.37. Mouse coagulating gland, normal male. Electronmicrograph X 39,000. Caulfield's modification of Palade's osmic acid fixative. Basal portion of an epithelial cell; insert, details of basement membrane region: bm, basement membrane: ci, dilated cisternae: cm, plasma or cell membrane; cy, cytoplasmic matrix; ?n, mitochondrion: /;, nucleus. (From D. Brandes, unpublished.)


centrations of RNA basally and apically in the epithelial cells. Strong protein reactions are present in luminal secretion and apical regions of the cells.

The response to gonadectomy (Brandes and Bourne, 1954) includes reduction of alkaline and acid phosphatase activity within 4 days, PAS reactions by 8 days, and slight


fragmentation and loss of density of the Golgi apparatus by 12 to 14 days. Changes are more marked after longer periods of castration (Table 6.8), although Bern (1951) observed retention of stromal alkaline phosphatase for long periods. RNA concentrations in the cell (Bern, Alfert and Blair, 1957) are greatly decreased but some


414


PHYSIOLOGY OF GONADS




Fig. 6.38. Mouse seininul \esick\ norniMl male. Pliotomicrograpli preparation. (From E. Howard, Am. J. Anat., 65, 105-149, 1939.)


50. Houm-liematoxvlin


accunuilation remains in the apical region. The sulfhydryl reaction is partially lost luit the cells retain apical reactivity.

Testosterone propionate implanted subcutaneously 13 to 32 days after testis removal (Brandes and Bourne, 1954) rapidly restored the Golgi apparatus and enzyme activity to normal, and the PAS reaction returned gradually. Allen (1958) showed that the epithelium of 30-day castrates responds to a single injection of 16 fig. of testosterone propionate by an increase in mitotic activity within 30 to 36 hours.

Se7ninal vesicles. The epithelial cells in adult glands are colunniai- with basal nuclei and secretory granules surrounded by halos in the supranuclear cytoplasm (Fig. 6.35) ; the epithelium rests on a layer of smooth muscle and connective tissue stroma (Howard, 1939).

In electron micrographs (Deane and Porter, 1959), it can be seen that the surface membranes of secretory cells Ivdvv microvilli which extend into the lumen. The supranuclear region contains secretory granules enclosed in vesicles or cisternae, smaller membrane-bound vesicles, and parallel arrays of smooth Golgi membranes. Moderately distended ergastoplasmic cliannels with membranes studded with pi'csumed libonucleoprotein ])articles lorm comi)lex convolutions along the latci'al luai'gins and at the base of cells. The nucleus possesses clumped chromatin along the meml)rane (Figs. 6.39, 6.42, and 6.43). Fu


jita (1959) described essentially the same type of endoplasmic reticulum and the presence of microvilli and secretion granules. In addition, small granules in the Golgi region were interpreted as precursors of secretory granules.

Histochemical preparations (Table 6.7) show strong alkaline phosphatase activity in stromal elements (Atkinson, 1948; Bern. 1951 ) ; acid phosphatase activity is present in the apical or Golgi region of the epithelial cells (Deane and Dempsey, 1945). Secretory material and secretory granules in the lumen and cytoplasm are acidophilic and weakly PAS-positive, whereas the reticulum in the lamina propria is intensely PAS reactive (Fig. 6.44). Lateral margins and basal regions of the cells (Fig. 6.45) are moderately basophilic (Deane and Porter, 1959).

Following castration of adult mice, the secretory epithelium retrogresses with loss of secretion granules and reduction in cell heiglit and nuclear size. Effects of gonadectomy may be retarded and not uniform in all cells (Howard, 1939), but changes within 5 days have been rei:)orted for cell and nucleai- size (Martins and Rocha, 1 929 ) .

{electron mici'osco])ic and histochemical studies (Table ().7l reveal marked changes within a week aftei' gonad removal (Deane and Porter, 1959). Cell size is reduced, there are fewer secretory granules, ergastoplasmic channels are less distended and


ACCESSORY MAMMALIAN REPRODUCTIVE GLANDS


415



Fig. 6.39. Mouse seminal vesicle, normal male. Electronmierograph X 4200; osmic acid fixation with sucrose. Epithelial cells showing basal and lateral ergastoplasmic channels and membranes, and supranuclear \-psicles containing .secretory granules. (From H. W. Deane and K. R. Porter, unpubli.shed.)


coin-oliitcd (Fig. 6.40). The relative number of riboniicleoprotein particles is somewhat leduced, secretory granules are less acidophilic, and the cytoplasm is only weakly basophilic (Fig. 6.45). Secretion granules were still visible by electron microscopy 10 days after castration but they were not visible at 25 days (Fujita, 1959) .

Atkinson (1948) found that alkaline phosphatase activity disappears almost completely from the stroma within 10 days, but Bern, Alfert and Blair (1957) observed retention in the fibromuscular tissue.

Martins and Rocha (1929) reported complete prevention of castration effects by injection of extracts of bull or goat testes. The epithelium of castrates responds readily to androgens. A single dose of 16 /x,g. of testosterone propionate in 30-day castrates resulted in increased mitotic activity beginning 30 to 36 hours after treatment and


reached a peak at 42 to 48 hours (Allen, 1958). Administration of testosterone to castrates completely restored the fine structure to normal (Fujita, 1959). Alkaline phosphatase activity in the stroma returned to normal within 10 days with testosterone propionate administration (Atkinson, 1948). The same hormone given to normal males for one week resulted in increased cell height, more abundant and acidophilic secretion and secretory granules, increased basophilia (Fig. 6.45), more distended and convoluted ergastoplasmic channels (Fig. 6.41), and a relative increase in ribonucleoprotein particles (Deane and Porter, 1959).

Discussion. The secretory cells in the epithelia of rat and mouse prostatic lobes and seminal vesicles have many histologic characteristics in common and some marked dissimilarities. In light microscopy with


416


PHYSIOLOGY OF GONADS




V\v, 6 40 Mouse seminal vesicle, 7-(lay castrate. Electronmicrograph X 4200; osmic acid fixation wjtli sucrose. Note the reduction in cell height, number of secretory granules, and contortion of the ergastoplasmic membranes. Arrow indicates microvilli. (From H. W. Deane and K. R. Porter, unpublished.)


routine fixation and stains, the most obvious differences are in cell height, position and staining intensity of the nuclei, presence or absence of secretory granules, and in such cytoplasmic characteristics as the supranuclear clear zone in the rat ventral prostate and the basal vesicular region in the coagulating gland. The Golgi apparatus varies in density, structure, and position in the apical cytoplasm. Studies on ultrastructure reveal striking differences in the degree of dilation and the disposition of the endoplasmic reticulum. In the rat ventral prostate the most dilated cisternae are in the supranuclear region; in the dorsal lobe, generally distended vesicles are disposed throughout the cytoplasm in both cell types; in the coagulating gland, there is extreme dilation of the sacs, particularly in the basal region. The flattened vesicles of the mouse ventral prostate are disposed at random; the coagulating gland, like that of the rat, shows greatly dilated cisternae, especially basally. Moderately distended ergastoplasmic channels in the basal and lateral regions of cells are characteristic of the mouse seminal vesicles.

Changes following castration are detect


able by light microscopy within 2 days in the rat seminal vesicle; 4 days in the ventral prostate; 10 days in the coagulating gland.

Harkin (1957a) suggested a correlation between distention of the sacs and secretory activity of the cells in the rat ventral prostate. Within 24 hours after gonadectomy, he observed dilation of the sacs, but within 2 days, collapse of the apical sacs was marked and by 4 days, there was general collapse of the vesicles in other regions of the endoplasmic reticulum. At this stage secretory activity of the cells was apparently reduced.

Brandes and Portela (1960a, b, c) discussed the relation of the cisternae to secretion in the mouse glands. They proposed that the extremely dilated cisternae of the coagulating glands contain secretory products which are released into the lumina of acini by some undetermined mechanism. They found no evidence that the Golgi complex is involved in the elaboration of secretory material in the cisternae, but histochemical findings suggest that it might take part in formation of secretory products that are not intracisternal. The ventral prostate


ACCESSORY MAMMALIAN REPRODUCTIVE GLANDS


417




Fig. 6.41. Mouse seminal vesicle, intact male treated with testosterone propionate for 7 days. Electron micrograph X 4200; osmic acid fixation with sucrose. Note increase in cell height, abundance of secretory granules and contortion of the ergastoplasmic membranes. (From H. W. Deane and K. R. Porter, unpublished.)


is characterized by flattened vesicles. Brandes and Portela doubted that there is transport of secretory material to these cisternae and release from the intracisternal spaces into the acinar Imnen. They suggested an apocrine type of release involving extrusion of portions of the apical cytoplasm from the


free margin of cells. The possibility of implication of the Golgi apparatus in the production of secretory material was considered.

From a study of the rat, Brandes and Groth (1961) concluded that the dilated cisternae of coagulating glands, dorsolat


418


PHYSIOLOGY OF GONADS



Fig. 6.42. Moil. M - :,,:ii il , i -id, , nui n, il n, ,i , I i_ (,;(,) l-.l, ,ii,m micrograpli X 36,000: omiik- ;ici(l tixation; section liiat((l with iii.myl cicclatc to enhance the density of nucleoi)ro1(Mn.-^. Infianuclear region of an epithehal cell; nucleus at the top ; ergastopla.sniic channel.s with inomhianes studded with particles; arrow indicates a mitochondrion. (From H. W. Deane and K. R. Porter, unpublished.)


eral and ventral prostates contain secretory products, and that it is probable that the membranes of the endoplasmic reticulum (or the granules associated with them) play an active role in the syntheses of the proteins present in the glandular secretions. Attemi)ts have been made to correlate


structure of cells as observed by light and electron microscopy with histochemical localizations of mucoproteins (PAS reaction), alkaline and acid phosphatase activity, and basophilic material. Various interpretations have been offered for the functional significance of these substances, all of which


ACCESSORY MAMMALIAN REPRODUCTIVK GLANDS


419


are under control of andi'ogcnic hormones of testicular origin. Leblond (1950) stated that the presence of PAS-positive granules in the Golgi region supjiorts the concept of participation of the Golgi apparatus in the secretory process. Brandes and Portela (1960b) suggested that the vesicles and vacuoles in the Golgi zone might represent presecretory or secretory material. The possibility that collapse of ergastoplasmic sacs after gonadectomy might be correlated with reduction in PAS-positive secretory material was proposed by Harkin (1957a).

Alkaline and acid phosphatase activity is also found in the Golgi region, but the significance of this localization is not clear. Harkin (1957a) suggested that reduction in acid phosphatase activity following castration might be correlated with the decrease in numbers of mitochondria.

The histochemical pattern of enzyme activity has been discussed by Brandes and Bourne (1954), and the functional significance of distribution of epithelial and stromal alkaline phosphatase activity has been treated by Bern (1949a).

Cytoplasmic basophilia that was abolished by ribonuclease was demonstrated in the epithelial cells of the rat seminal vesicle (Melampy and Cavazos, 1953). In the mouse seminal vesicle, Deane and Porter (1959) found cytoplasmic basophilia (all of which was attributable to ribonucleic acid) localized in regions which corresponded to the distribution of ergastoplasmic membranes with their associated particles of presumed ribonucleoprotein. The relative number of particles was apparentlj^ reduced after one week of castration, and increased with testosterone propionate administration to normal males. These changes were not considered marked enough to account for the pronounced reduction in basophilia following gonadectomy, and the increase with androgenic hormone treatment of normal males.

Rixon and Whitfield (1959) found high concentrations of zinc, lipid, and basophilic material in a luminal border organelle in the lateral prostate of rats. Silver staining demonstrated fibrils, and it was suggested that zinc may be involved in the ergastoplasmic reticulum, possibly with lipopro


tein, and would be associated with the microsome fraction in homogenatcs.

In the discussion of changes in structure and histochemical localizations of substances after gonadectomy and with hormone administration, no specific mention was made of differences in response among the glands. There arc, however, pronounced differences in rate of regression following withdrawal of testicular hormone, and in rate and degree of response to administered androgen. These differences in hormone sensitivity or threshold have been established by such end points as changes in histologic structure, weight (which includes increase in mass of cells and accumulation and storage of secretion), and secretion of specific substances such as fructose and citric acid (Mann, 1954a). In order of sensitivity they are first, secretory function, second, histologic structure, and finally, weight, whichis frecjuently used as an end point (Dorfman and Shipley, 1956).

Responsiveness of the epithelial cells depends on many factors and varies with specific glands, age of the animal, genetic strain, and species. A few examples will illustrate these points. Following castration of adult rats the seminal vesicles retrogress more rapidly than the ventral prostate and recjuire higher doses of testosterone propionate to restore normal histological structure (Price, 1944a). The ability of the seminal vesicles and the ventral ])rostate in young rats to respond to testosterone propionate increases with age to a peak which is specific for the organ (Price and Ortiz, 1944; Price, 1947). This is true also for the female prostate (Price, 1944b) and the accessory glands in young male hamsters (Ortiz,' 1947). The effect of age on responsiveness in the mouse ventral prostate was studied by Lasnitzki ( 1955a ) who cultured glands from mice 4 to 6 weeks of age and 6 months old in normal control medium and in the presence of testosterone propionate. Young prostates regressed on the control medium but retained normal histologic structure when the hormone was added. In the control medium, older glands maintained normal structure and became hyperplastic with addition of the androgen. Franks (1959) also found differences re


420


PHYSIOLOGY OF GONADS



. (i 1.; .Mnii-i -(iiiiiiit Ill III ill - iiiir ~|icciiiii'ii. Ill, muilii Ml Kill, and preparation as Fig. 6.39). Suprunuclcur icgiuu of au epilht'lial c-ell ; nucleus at the hottoui; numerous membrane-bound vesicles with secretory granules in the larger vesicles; arrows indicate some of the parallel arrays of smooth-surfaced Golgi membranes. (From H. W. Deane and K. R. Porter, unjiuhlished.)


lated to age in the response of cultured mouse ventral prostate to testosterone propionate. The Long-Evans and SpragueDawlcy strains of rats differ in responsiveness of the ventral prostate of adult hypojihysectomized castrates to testosterone


propionate (Lostroh and Li, 1956). Species differences in rate of retrogression of accessory glands in adult castrates are marked. As reported above, changes in histologic structure occur rapidly in rats, more slowly and less uniformly in mice (Howard, 1939),


ACCESSORY MAMMALIAN REPRODUCTIVE GLANDS


421



Fig. 6.44. Mouse seminal vesicle, normal male. Photomicrofirapli - 250. Carnoy's fixative, stained by the periodic acid-Schiff method, counterstained with hematoxylin. Note the intense PAS-reaction in the reticulum in the lamina propria and surrounding the smooth muscle fibers; secretory material in the lumen is moderately reactive. (From H. W. Deane and K. R. Porter, unpublished.)


ctncl slowly and somewhat incompletely in guinea pigs (Sayles, 1939, 1942) and hamsters (Ortiz, 1953). It should be noted that in adult castrated guinea pigs the ability to secrete fructose and citric acid is apparently lost in cells which show only partial retrogression histologically (Ortiz, Price, Williams-Ashman and Banks, 1956).

Another type of variation in responsiveness is demonstrated when weights of seminal vesicles and ventral prostrates in immature castrated rats are used as end points for the potency of various C19 steroids (Dorfman and Shipley, 1956 L When lower dosages of testosterone and 17aniethyl-A^-androstene-3^ , 17/?-diol are given the ventral prostate is more responsive than the seminal vesicles, but at high dosage levels the percentage increase in seminal vesi


cle weight equals or exceeds that of the prostate. Three other C19 steroids are far more effective on the ventral prostate than on seminal vesicles.

The female prostate in adult rats responds histologically and gravimetrically to a number of C19 steroids (Korenchevsky, 1937). These findings have been confirmed and extended by Huggins and Jensen

(1954) and Huggins, Parsons and Jensen

(1955) in hypophysectomized female rats. These workers examined the relation of molecular structure to the growth-promoting ability of the steroids.

Atrophy in male accessory glands of rats and mice has been reported under conditions of inanition and vitamin deficiency. These results are not usually attributable to reduction in responsiveness of the glands


422


PHYSIOLOGY OF GONADS



Fig. 6.45. Moil-^c .^einiiial mmcIi- l'li(iiniiiicinmn|ilis x 7UU. C'ain(>\-'- ti\ati\ c-nictliylcnie blue. Top, normal mali' ; in k Idle 7-ila\ ca-tiaN , iMJitoin, intact male ticatcil wil li ic.-tostcionp proprionate for 7 days. Hasoi)lidic inatciial ( ciga-tDplasm) occurs at the hasc and along lateral margins of cells; the Golgi zone appears clear; secretory granules are unstained. Basophilic material and Golgi zone are less evident after castration and more highly developed after testosterone treatment than normal. (From H. W. Deane and K. R. Porter, unjniblished.)


themselves Init to (liiuiniition in <2;onadotrophin titer by way of pituitary inhibition. Moore and Samuels (1931) showed that gonadotrophin or androgen treatment repaired the atrophied accessory glands in vitamin B-deficient rats and in those on limited food intake. Further, Lutwak-Mann and Mann (1950) demonstrated reduction of fructose and citric acid in accessory glands of rats on a vitamin B-deficient diet, but treatment with chorionic gonadotrophin not only restored the levels of these substances to normal but produced hypersecretion. Grayhack and Scott ( 1952 ) reported that the growth response to testosterone propionate of the ventral and posterior prostate in castrated rats on reduced food intake, vitaminfree casein, or glucose was little different from normally fed rats at lower dosages, but at higher levels there was less resj^onse in rats on limited dietarv intake. Testosterone


propionate did not produce normal stimulation of the accesisory glands in castrated mice on limited food intake (Goldsmith and Nigrelli, 1950) . In adult rats, a folic acid antagonist (Aminopterin) partially prevented the reduction in jirostatic weight produced by estradiol but did not interfere with testosterone stimulation of the prostate in castrated adults or intact immature animals (Brendler, 1949).

The senile changes which occur in adx'anced age in the prostate glands of the rat, mouse and of man have been describetl by Moore (1936) and interjireted on tht. basis of decrease in testicular androgen. Presenile variations in histologic structui'e are pr()l)al)ly ivhited to changes in responsiveness to andi'ogens. In a brief report on electron microscopy (Harkin, 1957b), involutional changes in the rat ventral prostate were described. With increasing age.


ACCESSORY MAMMALIAN REPRODUCTIVE GLANDS


423


eloctron-denye material is deposited in the Golgi region of epithelial cells and these bodies are said to resemble structures found in hyperplastic prostates in man.

2. Adrenal Androgens

A large Ixxly of evidence jioints to effects of hormones from the adrenal cortex on the accessory reproductive glands of male rats and mice and on prostate glands of female rats. The significance of this relationshi}) is unknown and the effects are slight in many cases. Reviews by Parkes (1945), Ponse (1950), Courrier, Baclesse and Marois (1953), Moore (1953) and Delost (1956) deal extensively with the subject. In man, a relationship between pathologies of the adrenal cortex and virilism is well recognized (Dorfman and Shipley, 1956).

The marked development of the ventral prostate in young castrated rats (Price, 1936) was attributed by Howard (1938) to the action of androgen from the adrenal cortex. The same explanation was suggested for the extensive development of the seminal vesicles and prostate in young castrated mice (Howard, 1939). The ventral prostate does not develop in immature castrated-adrenalectoraized rats according to Burrill and Greene (1939a) and Howard (1941), but Gersh and Grollman (1939) did not confirm these findings. The impairment of prostate and seminal vesicle development in young castrated-adrenalectomized mice (Howard, 1946) was considered to be the result of poor physical condition rather than loss of adrenal androgen. Gonadectomy in young male mice of an inbred strain (Woolley and Little, 1945a. b) produced adrenal cortical carcinoma correlated w^ith strong stimulation of the prostate and seminal vesicles. Spiegel (1939) castrated young guinea pigs and found the development of adrenal-cortical tumors and evidence of stimulation of prostates and seminal vesicles.

In the field vole {Microtus arvalis P.), Delost (1956) observed extensive development of the ventral prostate in young castrated males. Gonadectomy of adult males during the breeding season results in atrophy of seminal vesicles, and dorsal and lateral prostate, whereas the ventral prostate


shows an intense secretory activity by one month after testis removal. Adrenalectomy of castrates produces complete involution of the ventral prostate. Outside the breeding period, there is atrophy of all accessory glands except the ventral prostate which exhibits strong activation that can be prevented by adrenalectomy.

The prostate gland of young female rats undergoes development and differentiation, and resembles the male ventral prostate with which it is homologous (Price, 1939; Mahoney, 1940). Development still occurs following ovariectomy (Burrill and Greene, 1939b; Price, 1942) or adrenalectomy (Burrill and Greene, 1941), but not in ovariectomized-adrenalectomized females. A comparison of the responsiveness of female and male prostates indicated that the male gland is more sensiti\'e to adrenal androgens (Price, 1942).

Autotransplants of adrenals into one seminal vesicle of adult castrated rats produced slight local stimulation of the gland and also androgenic effects on the other seminal vesicle and on the ventral prostate (Katsh, Gordon and Charipper, 1948). But androgenic action was local and barely discernible in somewhat similar experiments (.lost and Geloso, 1954). Price and Ingle (1957) autotransplanted adrenals into seminal vesicles and ventral prostates of adult castrates and observed definite but local stimulation of seminal vesicles, coagulating glands, and ventral prostates. Negative results of adrenal transplants in seminal vesicles of nonadrenalectomized rats were reported by Moore (1953). Takewaki (1954) failed to detect any androgenic effect of autotransplants of adrenals placed subcutaneously in contact with seminal vesicle grafts in castrated males.

The finding that treatment of young castrated male rats with adrenocorticotrophin caused stimulation of the ventral prostate (Davidson and Moon, 1936) has been confirmed by Deanesly (1960) who observed, in addition, a slight stimulation of the seminal vesicles. Nelson (1941) also found androgenic effects on accessory glands following ACTH treatment but Moore (1953), van der Laan (1953), and Takewaki (1954) obtained negative results. In hypophysec


424


PHYSIOLOGY OF GONADS


tomized-castrated rats, ACTH was reported ineffective in increasing ventral prostate weight (van der Laan, 1953; Grayhack, Bunce, Kearns and Scott, 1955) , but Lostroh and Li (1957) obtained some growth of ventral pi'ostates and seminal vesicles at certain dosage levels. They emphasized that dosage is a critical factor in demonstrating the androgen-secreting ability of the adrenal cortex under ACTH stimulation. Administration of ACTH to hypophysectomized-castrated-adrenalectomized rats does not affect the accessory glands.

There has been no general agreement on the androgenicity of desoxycorticosterone on the accessory glands (see reviews by Parkes, 1945 and by Courrier, Baclesse and Marois, 1953). Lostroh and Li (1957) reported that ll-desoxy-17-hydroxy-corticosterone and 11-dehydro-corticosterone displayed an androgenic activity equivalent to 4 fxg. of testosterone propionate on the ventral prostates and seminal vesicles of hypophysectomized-castrated adult rats. Corticosterone, cortisone, and hydrocortisone were ineffective. Grayhack, Bunce, Kearns and Scott (1955) found cortisone ineffective on the weight of ventral prostates in hypophysectomized castrates. In the field vole (Microtus arvalis P.), Delost (1956) produced effects on the ventral prostate by cortisone administration.

3. Ovarian Androgens

It has long been known that mammalian ovaries can secrete androgenic hormones which have virilizing effects in females. Discussion of the evidence for androgenic activity of the ovary has been presented by Ponse (1948) and Parkes (1950). More recently the subject has been extensively reviewed (Ponse, 19541), 1955). Much of the interest in ovarian androgens in the rodent has centered on the question of their site of origin in the ovary and the effects of temperature and gonadotrophin administration on androgen production (see reviews, and Chapter 7 by Young) . However, the use of male accessory glands as bio-indicators for ovarian androgen has contributed to our knowledge of the responsiveness of the glands.

Methods for approaching this problem include transplantation of ovaries into vai'i


ous sites in castrated male rats and mice; ])lacing ovarian autotransplants into the ears of females ; transplantation of male accessory glands into females; and observations of prostate glands in females of the so-called female prostate strains of rats. The use of such females as hosts for grafts of male prostatic tissue has permitted a direct comparison of responsiveness in these homologous glands.

The first observations that ovarian grafts maintain normal prostates and seminal vesicles in castrated males w^ere made in guinea pigs (Lipschiitz, 1932) and mice (de Jongh and Korteweg, 1935). Hill (1937) transplanted ovaries into the ears of castrated male mice and obtained stimulation of the prostate and seminal vesicles. Deanesly ( 1938) reported similar findings in rats. Local effects from ovaries grafted into seminal vesicles of castrated rats w^ere shown by Katsh (1950). Takewaki (1953) also found local stimulating effects on seminal vesicles when rat ovaries and seminal vesicles were transplanted close together into the spleens of gonadectomized males and females.

In experiments in which ventral prostates and seminal vesicles were transplanted subcutaneously into adult female rats (Price, 1941, 1942) it was sliown that the ventral prostate is well maintained in virgin females (Fig. 6.46) and highly stimulated during jiregnancy and lactation in the host. It is comi)letely retrogressed in spayed females. Seminal vesicle grafts, however, are stimulated only rarely. This occurs only in females that have littered repeatedly and have been lactating for long periods. This indicates that the threshold of response of the ventral prostate to ovarian androgens is lower than that of the seminal vesicles. Evidence of functional stimulation with production of fructose and/or citric acid was obtained in coagulating glands, and ventral, lateral, and dorsal prostates trans])lanted into female rats in which the ovaries were stimulated by gonadotrophin treatment (Price, Mann and LutwakMann, 1955). It may be assumed that the effects were attributable to ovarian androgens since ventral prostate grafts in spayed females (Greene and Burrill, 1939) are not stimulated histologicallv when gonado


ACCESSORY MAMMALIAN REPRODUCTIVE GLANDS


425



Fig. 6.46. Rat male and female prostates. Photomicrographs X 650. Bouin-hematoxylin preparation. Top, host prostate from an adult virgin female; middle, male prostate graft from the above female host; bottom, prostate from a pregnant female. Note the semi-regressed epithelium in the female prostate of the virgin female host compared with the columnar epithelium and light areas in the male prostate graft and the prostate from a pregnant female. (From D. Price, Anat. Rec, 82, 93-113, 1942.)


trophin is administered. Great stimulation of ventral prostate grafts in the ovarian bursa of females was obtained by Ponse (1954a).

The female prostate gland is normally jiartially retrogressed in adult females except during pregnancy and lactation (Fig. 6.46) when it appears stimulated (Burrill and Greene, 1942; Price, 1942); in spayed females it is atrophic (Price, 1942). The striking development of the female prostate in pregnancy and lactation in a number of species of mammals is discussed in Section I. Hernandez (1942) obtained stimulation of female prostates by autotransplants of oA-aries into ears, hind legs, or tails of rats.

Transplantation of rat ventral prostates into virgin females shows that the male


prostate has a lower threshold to ovarian androgens than the female gland and maintains high epithelium and cellular light areas whereas the epithelium of the host prostate (Fig. 6.46) is low and retrogressed (Price, 1942).

4- Progesterone

The administration of progesterone in relatively enormous doses has stimulating effects as determined by weight, histologic structure, and function of some of the accessory glands in castrated male rats, mice, and guinea pigs. The literature has been reviewed by Greene, Burrill and Thomson (1940), Parkes (1950), and Price, Mann and Lutwak-Alann (1955).

Burkhart (1942) treated adult 40-day


426


PHYSIOLOGY OF GONADS


castrated rats with one or two 20-mg. doses of progesterone and observed a slight stimulation of mitotic activity in the ventral prostate and seminal vesicles after 55 hours but a pronounced hypertrophy of epithelium and connective tissue in both glands. The ventral prostate is more sensitive to progesterone than the seminal vesicles.

In castrated rats (Price, IMann and Lutwak-Mann, 1955), treatment with 25 mg. of progesterone daily stimulated the secretion of fructose or citric acid in seminal vesicles, coagulating glands, and ventral, lateral and dorsal prostates, and produced histologic changes in the last three glands. The effect, however, was only equivalent to that of about 5 /xg. of testosterone propionate. The lowest threshold to the hormone is in the ventral prostate and the highest in the seminal vesicles. Lostroh and Li (1957) found no effects of 0.5 mg. of progesterone daily on the ventral prostate and seminal vesicles of hypophysectomizedcastrated adult rats, but the same dose of 17a-hydroxy progesterone was the androgenic equivalent of 4 /xg. of testosterone propionate. It may be noted that the following transformations are involved in the biosynthesis of testicular androgens: cholesterol —> pregnenolone — > progesterone — > 17a-hyclroxy progesterone — > androst-4-ene3,17-dione -^ testosterone (Dorfman, 1957). The slight androgenic actions of progesterone and 17a-hydroxy progesterone may result from their conversion to androstane derivatives by extragonadal tissues. The findings of Katsh (1950) that progesterone crystals implanted directly into the seminal vesicles of castrated rats have no stimulating influence may be significant in this regard.

C. EFFECTS OF ESTROGENS

Administration of estrogenic hormones to normal males affects the accessory glands both indirectly and directly. The effects fall into three categories: inhibition as evidenced by weight changes, involution of the epithelium, and loss of secretory activity (attributable to inhibition of pituitary gonadotro])hin and reduction in endogenous androgen) ; direct stimulation of fibromuscular tissue; and stimulation of hyi)erplasia and stratified squamous metaplasia of the


ei)ithelium with possible keratinization. In no case does estrogen induce secretory activity of epithelial cells. The reduction in seci'etion as determined cjuantitatively (see Section II) may result from castration atrophy of secretory cells, or from hyperplastic and metaplastic transformations of the e])ithelium witli resultant loss of normal secretory function.

The observed responses to estrogen treatment in glands of intact and castrated males, and in organ cultures of prostatic tissue, represent the dual effects of androgen withdrawal and estrogen addition. The extensive literature on the effects of estrogen and the evidence for so-called antagonistic, cooperative and synergistic effects of simultaneous administration of androgen and estrogen have been discussed extensivelv (Zuckerman, 1940; Emmens and Parkes, 1947; Ponse, 1948; Bern, 1949b; Burrows, 1949).

The observation that administration of estrogen to intact male rats causes atrophjof the accessory glands which is mediated by way of reduction of pituitary gonadotrophin and failure of secretion of testicular hormones was made by Moore and Price (1932). Estrogen-induced atrophy was prevented by simultaneous treatment with gonadotrophin or androgen. Direct stimulating effects were reported by Freud

(1933) and David, Freud and de Jongh

(1934) who observed fibromuscular growth in seminal vesicles of estrogen-treated castrated rats and stratification in the duct epithelium of the lateral prostate. Simultaneous treatment with androgen enhanced the hypertrophic effect of estrogen on the fibromuscular wall of the seminal vesicle but prevented epithelial change in the lateral prostate ducts. Korenchevsky and Dennison (1935) found estrogen stimulation of the muscular layer of the rat seminal vesicle with no effect on the epithelium, but in coagulating glands (and to a lesser degree in the dorsal prostate ) there was not only fibromuscular hypertrophy but also metaplastic transformation of the epithelium with stratification; changes in the ventral and lateral lobes were slight and the epitiieHum was unaffected. Androgen treatment prevented the jxithologic changes induced by estrogen. Harsh, Overholser and Wells


ACCESSORY MAMMALIAN REPRODUCTIVE GLANDS


427


(1939) noted stratified, sqiuinious epithelium in the ducts of the seminal vesicles, and ducts and acini of coagulating glands following estrogen administration. But a slight delay in castration atrophy and a weak stimulating effect of estrogen on seminal vesicle epithelium were observed by Overholser and Nelson ( 1935 ) and Lacassagne and Raynaud (1937).

Ovaries transplanted into castrated male rats (Pfeiffer, 1936) induce fibromuscular hyi^ertrophy in host seminal vesicles and coagulating glands; stratified sciuamous cornified epithelium appears also in coagulating glands, and hyperplasia and metal^lasia are present in lateral prostates. Estrogenic stimulation of fibromuscular tissue occurs (Price, 1941) in seminal vesicle grafts in normal female hosts but no such effects are evident in ventral prostate grafts.

Burkhart (1942) injected a single dose of estradiol benzoate into 40-day-castrated rats and observed no effect on the ventral prostate. But in the seminal vesicles, hypertrophy of epithelial cells occurred by 27 liours after treatment and by 55 hours, mitotic activity was evident in the epithelium and to some extent in the connective tissue.

In histochemical studies (Bern and Levy, 1952) , metaplastic changes were observed in the seminal vesicle epithelium after estrogen treatment but no cornification occurred; the replacing epithelium was alkaline phosphatase-positive in contrast to the negative reaction in the original epithelium (Table 6.7). Fibromuscular hypertrophy was found but no definite alteration in enzyme concentrations except an absence of activity in edema of the subepithelial stroma. No metaplastic changes appeared in the coagulating gland epithelium, but the ducts of the dorsal prostate underwent metaplasia; alkaline phosphatase activity of the stroma in both glands was retained as in the castrate. The ventral prostate ejnthelium was atrophic but still enzyme-active after 120 days of treatment and the stroma reacted positively.

The effects of estrogen on the accessory glands of mice are far more marked than in rats. Long continued and strong doses of ■estrogen cause hyperplasia, metaplasia and


keratinization in the epithelium of mouse coagulating glands (Lacassagne, 1933 ) . The same effects, with fibromuscular hypertrophy, were described in coagulating glands and prostates by Burrows and Kennaway (1934), Burrows (1935a), and de Jongh (1935) who prevented epithelial metaplasia in prostates by simultaneous treatment with androgen. Burrows (1935b) studied the localization of responses to estrogenic compounds and found that in order of time of response, the coagulating gland is first, seminal vesicles next, and finally the prostatic lobes. Changes begin in the urethral ends of ducts and jirogress peripherally into the acini. Li the degree of response, the coagulating glands and seminal vesicles show the most drastic changes with the appearance of stratified, squamous, keratinizing epithelium and ultimate loss of acini. The effects on the lobes of the prostate include stratified, cornifying epithelium but the changes are not so i)ronounced. Some hypertroj^hy of fibromuscular stroma occurs in all the glands and hyperplasia is marked in the fibromuscular wall of the seminal vesicles.

Tislowitz (1939) found stimulation of mitotic activity in muscle and connective tissue of seminal vesicles and ventral prostate glands of immature castrated mice treated with estrogen. Stratification and cornification appear in the ventral prostate epithelium, with mitoses in the basal cell layers and also in seminal vesicle epithelium. Allen (1956) compared the mitogenic activity of a single dose of 16 /xg. of estradiol benzoate on seminal vesicles, coagulating glands, and ventral prostates of 30-daycastrated mice. Significant increases in mitotic activity occur in seminal vesicles and coagulating glands about 24 hours after treatment; the ventral prostate does not respond significantly until 72 hours and gives a low absolute value of mitoses.

Horning (1947) studied some of the initial changes in prostatic epithelium of intact mice receiving estrogen. Slight hypertrophy of epithelial cells and extensive fragmentation and dispersal of hypertrophied portions of the Golgi network occur by 8 days in the coagulating gland. At the same period, hypertrophic changes are less pronounced in the ejuthelium of the dorsal


428


PHYSIOLOGY OF GONADS


prostate and there is only slight fragmentation of the Golgi apparatus. In the ventral prostate no epithelial hypertrophy is found but the Golgi network hypertrophies without fragmentation or dispersal. The ventral prostate is definitely less sensitive to estrogen than the other two glands.

After longer periods of estrogen administration, Bern (1951) observed fibromuscular hypertrophy of the seminal vesicle and intense alkaline phosphatase activity as in untreated intact males (Table 6.7) ; the epithelium, which is normally negative in enzyme activity, becomes positive and the beginning of metaplastic changes is occasionally visible. In the coagulating gland, stratified scjuamous metaplasia with masses of keratin is found and the metaplastic epithelium is strongly alkaline phosphatasepositive; enzyme activity is retained in the stroma but is variable. Bern, Alfert and Blair (1957) reported that the metaplastic coagulating gland epithelium is strongly alkaline phosphatase reactive, virtually PAS-negative, has dense homogeneous RNA concentrations decreasing in amount from base to lumen, and a dense homogeneous cytoplasmic protein reaction with a gradient of increasing intensity from base to lumen. The enlarged vesicular nuclei of the metaplastic epithelium have lower concentrations of deoxyribonucleic acid (DNA) than the nuclei of normal epithelial cells. The cytoplasm of these cells is as reactive as normal cells for sulfhydryl groups, and the newly formed keratin is intensely reactive; the greatest concentrations of disulfide groups are in superficial keratin.

In the dorsal prostate (Bern, 1951), estrogen causes fibromuscular hypertrophy with variable retention of alkaline phosi)hatase activity. Metaplastic changes involving basal cell proliferation and stratification begin and the metaplastic epithelium is intensely alkaline phosphatase active, a rvversal of the normal reaction.

Brandes and Bourne (1954), using diethylstilbestrol, observed an increase in fibromuscular stroma in coagulating glands. dorsal and ventral prostates, and epithelial hyperplasia and stratification in varying degrees. The most pronounced changes occurred in the coagulating gland. The effects of estrogen on Golgi networks, and on PAS


and acid and alkaline phosphatase reactions are in general similar to the results of castration (Table 6.8).

Ventral prostate glands have been grown in culture by the watch glass method, with estrogens added to the medium. Lasnitzki (1954, 1958) reported hyperplasia and squamous metaplasia of the epithelium in young i^rostate tissue from C3H mice. In older glands, stimulation of fibromuscular tissue occurred. Franks (1959) using the C57 strain and a different culture medium observed no epithelial hyperplasia and metaplasia, but obtained increases in stroma and muscle. He attributed atrophic changes in the epithelium, which appear more marked in estrogen-treated than in control cultures, to direct inhibition by the hormone. Ventral prostate tissue from young mice is more sensitive to estrogen than tissue from adult or old males.

In the dog, Huggins and his collaborators demonstrated the effects of estrogen and combinations of estrogen and androgen on histologic structure and secretion in the prostate (see Section II). Estrogen causes decrease or increase in prostatic size depending on the dosage and on the levels of endogenous or exogenous androgen (Huggins and Clark, 1940) . Sciuamous metaplasia of the epithelium of ducts and acini occurs with estrogen treatment, but only in the posterior lobe.

Discussion. The results of estrogen administration to rats and mice vary with species, age of animal, specific gland under consideration, dosage, duration of treatment, and presence or absence of endogenous or exogenous androgen. Interpretation of the findings rests on the understanding that androgen directly stimulates mitotic and secretory activity in the epithelial cells. Estrogen inhibits i)ituitary function and thus i-eduees testicular androgen in intact males. It directly increases mitotic activity in th(> epithelium of the accessory glands, and inckices (>pithelial liy|)erplasia and iiietaphisia, and fibromuscular hyperplasia. Whether the effects of simultaneous presence of androgen with exogenous estrogen are classified as protective, competitive (antagonistic), or cooperative (synergistic) on the acce.ssoiy glands depends on the


ACCESSORY MAMMALIAN REPRODUCTIVE GLANDS


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relative levels of the two hormones. Both affect mitotic activity directly.

In a comparison of the effectiveness of androgen and estrogen on mitotic activity, Allen (1956, 1958) showed that a dose of 16 fjig. of testosterone propionate induces statistically significant increases in mitotic activity of the epithelium of seminal vesicles, coagulating glands and ventral prostates of castrated mice in 30 to 36 hours. The same dose of estradiol benzoate increases mitotic activity in 24 hours in seminal vesicles and coagulating glands, but not until 72 hours in ventral prostates.

Differences in responsiveness to estrogen are evident between rats and mice but in both species there is a gradient of reactivity with coagulating glands showing the most marked changes, seminal vesicles next, and prostatic lobes least. In glands of both species, the duct epithelium is more sensitive than acinar epithelium and the first observable effects are on urethral ends of ducts. Hyperplastic and metaplastic responses to estrogen occur also to varying degrees in accessory glands of other mammals — man, monkey, dog, cat, ground squirrel, and guinea pig. Zuckerman (1940) reviewed the effects of estrogen in male and female rodents and other mammals and suggested from the evidence that "stratified squamous proliferation or metaplasia is usually a primary response of tissue in whose development oestrogen-sensitive entodermal sinus epithelium has played a part."

On the basis of the pathologic effects of estrogen on the mouse coagulating glands and the protective action of androgen, several workers originally suggested that benign prostatic hypertrophy in man might result from a primary imbalance in the normal ratio of estrogenic to androgenic hormones in the male organism (Zuckerman, 1936). Further study has not supported this concept.

D. HORMONAL CONTROL OF SPONTANEOUS PROSTATIC NEOPLASMS

Spontaneous tumors of the prostate occur in rodents rarely if at all, but benign growths are extremely common in aging dogs and men, and prostatic cancer is a major prol^lem in man. It is noteworthy that


neoplasms of the seminal vesicles in man are rare (Dixon and Moore, 1952).

1. Benign Growths

In the dog, prostatic enlargement which is essentially due to cystic hyperplasia of the epithelium occurs in almost all senile males with functioning testes, but is not found in castrates (Huggins, 1947b). In these prostatic growths, which characteristically involve the entire gland, tall columnar secretory epithelium is always present in some acini. Canine prostatic hyperplasia is under control of testicular androgens (Huggins and Clark, 1940) and marked involution of these tumors as evidenced by their size and secretory activity (see Section II) can be induced by gonadectomy or treatment with suitable dosages of estrogen. Estrogen overdosage, however, causes prostatic enlargement and a metaplasia of the posterior lobe which does not resemble cystic hyperplasia. Huggins and Moulder (1945) reported that dogs feminized by estrogen-secreting Sertoli cell tumors of the testis do not have cystic hyperplasia. The important factors in this pathologic growth seem to be age and testicular androgens (Huggins, 1947b), but prolonged administration of testosterone propionate to aged castrate dogs results in normal-appearing prostates and not cystic hyperplasia.

Benign prostatic hypertrophy in man is rarely encountered before the age of 40 (Moore, 1943; Huggins, 1947b) but it is extremely common in old men. It differs markedly from prostatic hyperplasia in dogs; the lesions are limited to the medullary region of the prostate and are spheroidal neoplastic nodules involving, usually, both epithelium and fibromuscular tissue; other nodular types occur but are less frequent (Huggins, 1947b; Franks, 1954). The prostatic epithelium is composed of tall secretory cells (Huggins and Stevens, 1940). Despite the fact that castration may be followed by some shrinkage of hypertrophied human prostate tissue (White, 1893; Cabot, 1896; Huggins and Stevens, 1940), it is generally admitted that this treatment is of little value. Estrogen treatment results in changes in the acini of the inner or medullary (periurethral) part of the prostate, and stratification with squamous metaplasia


430


PHYSIOLOGY OF GONADS


of the duct epithelium, but there is little effect on nodular stroma and acini (Huggins, 1947bj. Since benign prostatic hypertrophy has not been observed in men castrated early in life, testicular androgen is presumably involved in its etiology (Huggins, 1947b). However, it is doubtful whether androgens are causative agents for this disease. Lesser, Vose and Dixey (1955) found that in men over the age of 45 who had received androgen treatment for noncancerous conditions, the incidence of benign enlargement of the prostate was no greater than in untreated controls.

2. Prostatic Cancer

Prostatic cancer is a common disease in elderly men. This carcinoma, which characteristically arises in the posterior (outer) region of the prostate, consists of an abnormal growth of cells resembling adult prostatic epithelium rather than undifferentiated tissue (Huggins, Stevens and Hodges, 1941 ). It was found that these neoplasms are hormone-dependent and usually are influenced by anti-androgenic therapy; those which fail to respond are not adenocarcinomas with acini present in the tumor, but are undifferentiated carcinomas with solid masses of malignant cells (Huggins, 1942). However, the two types intergrade and both contain large amounts of acid phosphatase and are considered cancers of adult prostatic epithelium. The beneficial effects of castration or estrogenic treatment or both simultaneously, on metastatic carcinoma of the prostate in man were first demonstrated by Huggins and his collaborators (Huggins and Hodges, 1941 ; Huggins, Stevens and Hodges, 1941 ; Huggins, Scott and Hodges, 1941 ; Huggins, 1943, 1947a). This discovery was facilitated by the availability of a chemical index of the activity of the neoplasm, namely, the acid phosphatase activity of blood serum.

Although testosterone increases the level of serum acid phosphatase in patients with prostatic cancer (Huggins and Hodges, 1941 ; Sullivan, Gutman and Gutman, 1942 1 , androgen treatment does not always exacerbate the growth of the tumor (Trunnel and Duffy, 1950; Brendler, Chase and Scott, 1950; Brendler, 1956; Franks, 1958) or may even decrease it (Pearson, 1957). It is ciues


tionable if androgens induce prostatic cancer, inasmuch as their prolonged administration neither increases the incidence of this disease in man (Lesser, Vose and Dixey, 1955) nor induces it in dogs (Hertz, 1951).

The response of human metastasizing prostate cancer to anti-androgenic therapy is often very dramatic, but neither castration nor treatment with estrogens cures this disease. The tumor may regress for considerable periods of time, but eventually it recurs and begins to grow again. A small proportion of cases do not benefit at all (Huggins, 1957; Franks, 1958). Nevertheless, castration and/or estrogen therapy remain the best treatment for prostatic carcinoma in man (Nesbit and Baum, 1950; Huggins, 1956; O'Conor, Desautels, Pryor, Munson and Harrison, 1959).

Huggins and Scott (1945) suggested that the failure of some patients with prostatic cancer to obtain long lasting improvement from castration or estrogen treatment, or the two combined, lay in the secretion of androgenic substances by the adrenal glands. Early attempts to study the effect of bilateral adrenalectomy on human prostatic cancer were thwarted by the lack of suitable adrenal cortical steroids for adequate substitution therapy. But with the advent of cortisone, bilateral adrenalectomy could be accomplished with ease (Huggins and Bergenstal, 1951, 1952). It seems, however, that adrenalectomy is of limited value to patients with prostatic cancer in relapse after orchiectomy and/or treatment with estrogens (Whitmore, Randall, Pearson and West, 1954; Huggins, 1956; Fergusson, 1958).

E. EFFECTS OF CERTAIN AROMATIC HYDROCARBONS (CARCINOGENS)

Spontaneous tumors have not been found in the prostate glands of rodents, but tumors can be induced in rats and mice by treatment with carcinogenic chemicals such as benzpyrene and metiiylcholanthrene. There lias been considerable interest in inducing such tvnnois and studying their inception and growth, and the iH'lation of steroid hormones to their de^•elopment. Such investigations have contributed to an understanding of early neoi)lastic changes in the rodent prostate, but have had limited applicability


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to the })rol)lcm of hormonal control of prostatic cancer in man.

The first induction of prostatic cancer in rodents was accomplished by Moore and Melchionna (1937) who injected benzpyrene in lard directly into the rat anterior prostate (it should be noted that Moore and ]\Ielchionna used "anterior" in the sense of ventral as indicated by their histologic descriptions of a characteristic clear zone in the peripheral cytoplasm of the epithelial cells; this is typical only of the ventral lobe). The treatment was followed within 210 days by the development of squamous cell carcinomas in 72 per cent, and sarcomas in 5 per cent, of intact rats. Essentially similar results were obtained in an equivalent number of rats castrated at the time of carcinogen injection. Castration after tumors had developed did not cause atrophy of tumor cells. No metastases were found but there was anaplasia of cells and the tumors were invasive. The squamous metaplasia occurred in columnar secretory epithelium which was close to, or in contact with, benzpyrene cysts. The sequence of changes was reduction in cell height, loss of the clear area in the peripheral cytoplasm, pseudostratification, true stratification, development of intercellular bridges, and formation of keratohyaline. It was concluded that testicular androgen is not an important factor in the development of these squamous cell carcinomas, but on the basis of a small series of experimental animals it was suggested that exogenous androgen treatment in castrates may increase the incidence of sarcomas.

In 1946, Dunning, Curtis and Segaloff implanted compressed methylcholanthrene l)ellets into rat prostates (lobe not specified) and induced metastasizing squamous cell carcinomas. The tumors were transplantable and metastasized equally well in male and female hosts. Bern and Levy (1952) injected methylcholanthrene in lard into ventral prostates of intact Long-Evans rats and induced extensive neoplasms within 7 to 9 months. All but one were squamous cell carcinomas; the exception was a sarcoma. Quantitative determinations of enzyme activity showed a loss of alkaline phosphatase in cancerous prostates but no significant changes in acid phosphatase activity. Histo


chemically, the stroma and capillaries were alkaline phosphatase reactive, but the carcinomas had virtually lost the strong alkaline phosphatase activity of the epithelium of origin (Table 6.2). There was some pseudoreaction or reaction in sloughed keratin and necrotic areas.

Allen (1953) injected a suspension of methylcholanthrene in distilled water into ventral prostates or coagulating glands of intact and castrated rats. All were autopsied 180 days later. A high percentage of squamous cell carcinomas and a few sarcomas developed; metastases occurred in a few cases. There was no statistically significant difference between tumor incidence in the ventral prostate and coagulating gland. Tumors of the ventral prostate were found in 70.6 per cent of the intact rats and in 100 per cent of the castrates; in castrates injected with testosterone propionate there were tumors in 57.7 per cent of the animals, and in castrates treated with estradiol benzoate, 77.8 per cent. It was concluded that tumor incidence was highest in castrates and lowest in intact males or castrates treated with testosterone propionate, and that estrogen did not affect tumor incidence. ]\Iirand and Staubitz (1956) placed methylcholanthrene crystals in ventral prostates of 99 intact Wistar rats and observed the effects for over 300 days. The resulting tumors were classified as 30 squamous cell carcinomas, 3 leiomyosarcomas, and 2 adenocarcinomas; squamous cell carcinomas and adenocarcinomas metastasized. Fragments of squamous cell carcinomas were transplanted and survived and metastasized more successfully in males than in females.

Horning (1946) imjiregnated strips of tissue from mouse dorsal prostates and anterior prostates (coagulating glands) with crystals of methylcholanthrene and inserted them as subcutaneous homografts into intact males. By this method adenocarcinomas were induced in grafts of both dorsal prostate and coagulating gland.

In mice of the RIII and Strong A strains (Horning and Dmochowski, 1947) methylcholanthrene in lard was injected into dorsal and anterior prostates (coagulating glands). Squamous cell carcinomas and sarcomas developed in Strong A mice, but only sarcomas in RIII. Squamous metaplasia of


431


PHYSIOLOGY OF GONADS


the epithelium occurred in the RIII strain but no malignant proliferation of metaplastic cells followed. It was noted that the epithelial changes which occurred with raethylcholanthrene treatment were "almost identical" with the secjuence of changes following prolonged estrogen administration in Strong A mice (Horning, 1947).

Horning (1949, 1952) studied the effects of castration, diethylstilbestrol, and testosterone propionate on growth rates of prostatic tumors transplanted as grafts. Tumors were induced in ventral prostate, dorsal prostate, and coagulating gland tissue of Strong A mice by wrapping pieces of epithelium around crystals of methylcholanthrene and transplanting the grafts su!)cutaneously into 75 intact males. Of the 54 tumors which developed, 42 were adenocarcinomas or secreting glandular carcinomas, 10 were squamous cell carcinomas, and 2, spindle cell sarcomas. Neoplastic development began, apparently, in epithelium in a nonsecretory phase, and hyperplastic changes followed the sequence of mitosis, abnormal cell division, and pyknosis accompanied by an increase in fibromuscular tissue. Three distinct types of epithelial proliferation then occurred; one, with tonguelike groups of early malignant cells, gave rise to secretory glandular carcinomas; the second, from acinar ejMthelium, and the third, from duct epithelium, developed into squamous cell carcinomas with keratinization and formation of keratin pearls. Some grafts had foci of the first and third type and the evidence suggested that the tumors subsequently became squamous cell carcinomas. Both tumor types were transplantable and were cari'icd through many serial transi)lantations without losing their histologic characteristics.

In an effort to study effects of testicular androgen on growth, transplants of an adenocarcinoma were made into intact and castrated males. The tumors grew rapidly and progressively in intact mice but regressed in castrates. Testosterone propionate administration to castrated males bearing regressed tumors resulted in a resumption of tumor growth in some cases. Gonadectomy of the host had little effect on the growth of transplanted s(|uamous cell carcinomas. An


drogen-dependence of secreting glandular carcinomas was suggested.

When stilbestrol pellets were implanted into one flank of intact males and a glandular carcinoma into the opposite flank, the effects varied from slight to pronounced retardation of the tumor but complete regression did not occur. The squamous cell carcinomas were insensitive to stilbestrol.

Additional experiments (Horning, 1952) involved transplanting pieces of prostatic epithelium impregnated with methylcholanthrene alone, or with the carcinogen combined with stilbestrol or testosterone propionate into intact males (groups of 35 for each treatment). The carcinogen alone induced 8 adenocarcinomas and 5 squamous cell carcinomas; carcinogen and stilbestrol, 23 squamous cell carcinomas and 3 sarcomas; carcinogen and testosterone propionate, 2 squamous cell carcinomas and 1 sarcoma. The increased tumor incidence with estradiol was interpreted as an inhibitory action of the estrogen on secretory epithelial cells, making them more susceptible to methylcholanthrenc.

Brandes and Bourne (1954) made homografts of pieces of ventral and anterior prostate (coagulating gland) impregnated with methylcholanthrenc into intact males of the Strong A strain, and studied histochemical changes. The grafts underwent squamous metaplasia and the processes of epithelial proliferation, stratification, and keratinization were completed within 10 days in some cases. Histochemical changes from the normal i)attern (Table 6.8) occurred concurrently. Alkaline phosphatase activity disappeared early; acid phosphatase activity became weak in nuclei and cytoplasm but keratohyalin granules were strongly reactive; PAS-positive reactions were gradually lost in luminal secretion and intracytoplasmic granules but retained in the basement membrane. In some grafts there was transformation into squamous cell carcinomas and when this happened phosjihatase activity was lost but the basement membranes were still PAS-positive.

Lasnitzki (1951, 1954, 1955a. b, 1958) grew ventral jirostate glands from C3H and Strong A mice in culture by the watch-glass t('chnif|ue and added methylcholanthrenc to the medium. Hyperplasia and squamous metaplasia resulted in glands ex]ilanted


ACCESSORY MAMMALIAN REPRODUCTIVE GLANDS


433


from both young and older mice. When estrone and carcinogen were added simultaneously to cultures of young glands, squamous metaplasia was increased; with older glands, hyperplasia was inhibited and stromal increase occurred. The relation of vitamin A to the response of prostates to methylcholanthrene was studied (Lasnitzki. 1955b). Vitamin A added to the medium caused an increase in secretion and deposition of PAS-positive material in the secretory cells but did not influence growth or development; the vitamin added simultaneously with methylcholanthrene did not influence hyperplasia, but did prevent keratin formation and degenerative changes in the secretory epithelium; excess vitamin A following the carcinogen prevented formation of keratin and decreased hyperplasia.

Summary. Treatment of rat and mouse accessory glands with benzpyrene or methylcholanthrene has induced precancerous and cancerous changes which led to the development of adenocarcinomas, squamous cell carcinomas, and sarcomas. The first type of tumor has been induced in large numbers only in mice and by the homograft method.

The evidence suggests that, in mice, growth of adenocarcinomas is androgendependent, but squamous cell carcinomas are little affected by androgen loss or estrogen treatment. The incidence of tumors has been increased by simultaneous administration of estrogen and carcinogen but reduced by the administration of androgen with carcinogen. In rats, it has been affirmed and denied that incidence and growth of squamous cell carcinomas are reduced in intact males ; estrogen has not affected tumor incidence. Species and strain differences in response are marked.

F. EFFECTS OF NONSTEROID HORMONES

There is e-\'idence that hormones from the anterior pituitary may directly affect the weight and histologic structure of accessory glands, or act synergistically with androgen. However, the findings have been somewhat conflicting. Dosage level, age, and strain of rats have varied, and questions have been raised with respect to the purity of the hormone preparations.

Attention was focused on the pituitary in relation to accessory glands when Huggins and Russell (1946) observed that prostatic


atrophy is more marked in the hypophysectomized than in the castrated dog. Van der Laan (1953) found the ventral prostates of hypophysectomized-castrated immature rats less responsive to testosterone propionate than the glands of castrates; a crude extract of beef pituitaries restored responsiveness in hypophysectomized-castrates. Prostates of young adult hypophysectomized-castrated Sprague-Dawley rats were also less responsive (total weight of dorsal and ventral prostates) to testosterone propionate than those of castrates (Grayhack, Bunce, Kearns and Scott, 1955). Paesi, de Jongh and Hoogstra (1956) administered pituitary extracts simultaneously with a low dose of testosterone propionate to hypophysectomized-castrated rats and reported a slightly greater ventral prostate weight than with the androgen alone.

To identify the hormones of the anterior pituitary that are capable of affecting the accessory glands or influencing their responsiveness to androgen, the following hormone preparations have been injected alone and in various combinations into hypophysectomized-castrated rats: prolactin (luteotrophin; LTH), growth hormone (somatotrophin; STH), adrenocorticotrophin ( ACTH ) , interstitial cell-stimulating hormone (luteinizing hormone; ICSH; LH), follicle stimulating hormone (FSHl. In addition, chorionic gonadotrophin and thyroxine have been administered. Of these hormones, only prolactin and growth hormone have been shown to act directly on accessory glands (for comprehensive data on negative and positive results of these hormones see Grayhack, Bunce, Kearns and Scott, 1955; Lostroh and Li, 1956, 1957 ». The degree to which contamination with prolactin or growth hormone might influence the assay of ICSH preparations by the ventral prostate test has been examined by Lostroh, Squire and Li (1958).

1. Prolactin {LTH)

When Pasqualini (1953) treated castrated adult rats with testosterone propionate followed by administration of a lower dose of androgen plus LTH, the amount of secretion in the seminal vesicles was greater than with androgen alone. Prostate weights were increased slightly by LTH with androgen. Van der Laan (1953) reported that in adult


434


PHYSIOLOGY OF GONADS


hypophysectomized-castrated rats LTH had no effect on ventral prostate weiglit. Grayhack, Bunce, Kearns and Scott (1955) made the same observation for prostate weights in young adult Sprague-Dawley rats, but found that LTH augmented the effect of testosterone propionate on prostate weight.

A difference in response between LongEvans and Sprague-Dawley strains of rats was observed by Lostroh and Li (1956). In immature hypophysectomized-castrated Long-Evans rats, LTH alone had no effect on ventral prostate or seminal vesicle weights, and no synergistic effect when administered with a low dose of testosterone propionate; in Sprague-Dawleys, however, the weights of ventral prostates and coagulating glands were increased by LTH but, again, no synergism occurred with exogenous androgen. Chase, Geschwind and Bern (1957) reported that in immature hypophysectomized-castrated Sprague-Dawleys, LTH did not affect weights of ventral prostates or coagulating glands but it did increase seminal vesicle weight. When LTH was administered with testosterone propionate, glandular tissue in the ventral prostate was increased and weights of coagulating glands (in some cases) and seminal vesicles were significantly higher than with androgen alone.

In the immature hypophysectomized Sprague-Dawley rats that were not castrated, LTH alone did not affect ventral prostate weight but when given simultaneously with ICSH it acted synergistically (Segaloff, Steelman and Flores, 1956). These results were confirmed by Lostroh, Squire and Li (1958) for the Sprague-Dawley strain, but in Long-Evans rats, LTH neither increased prostatic weight, nor augmented prostatic response to ICSH.

Antliff, Prasad and Meyer (1960) have shown that in the guinea pig, LTH had no effect on seminal vesicles of castrated or hypophysectomized males, but when it was administered with subminimal doses of testosterone propionate, seminal vesicle weight and epithelial height were increased.

2. Growth Hormone {STH)

Van der Laan (1953) found no effects of STH on ventral prostate weights in young hypophysectomized-castrated rats. Huggins,


Parsons and Jensen ( 1955) observed only slight effects on weights of ventral prostates and seminal vesicles with administration of STH to young hypophysectomized-castrated Sprague-Dawley rats, but a synergistic effect on weight was evident with simultaneous treatment with STH and testosterone propionate.

In hypophysectomized-castrated LongEvans rats (Lostroh and Li, 1956, 1957), STH produced slight histologic changes and significant weight increases in the ventral jirostate; when administered with testosterone propionate, an additive effect on weight was obtained. The changes in the seminal vesicles were less evident. The effects on Sprague-Dawley rats included weight increases in ventral prostates and seminal vesicles and a greatly enhanced weight response when STH and testosterone propionate were administered simultaneously (hypophysectomized-castrates in this strain gave a limited response to the androgen I . Chase, Geschwind and Bern (19571 found no consistent weight increases of ventral prostates, coagulating glands or seminal vesicles in young hypophysectomized-castrated Sprague-Dawleys treated with STH or STH and testosterone propionate. Simultaneous administration of STH, LTH and testosterone, however, induced significant increases in all accessories above the weights produced by the androgen alone.

Lostroh, Squire and Li (19581 determined that STH had no effect on the ventral prostate response to ICSH in hypophysectomized Long-Evans rats, but })roduced an enhanced response in Sprague-Dawleys. It was concluded that the Long-Evans strain is jjreferable for the testing of crude ICSH extracts, inasmuch as neither STH, LTH, nor both simultaneously, affect the response of the ventral prostate to ICSH.

With regard to the action of STH on histologic structure of the prostate in hypopiiysectomized-castrated rats, it should be noted that the effects are slight; nuclei api)ear vesicular, and the connective tissue stroma is increased (Lostroh and Li, 1957). The synergistic action of STH on prostate growth in hypoi)hysectomized-castrated rats when administered simultaneously with testosterone is more striking. In a general discussion of the many biologic effects of


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growtli hormone, Li (1956) wrote, Does this ability to act as a synergist mean that growth hormone plays a permissive or supporting role in the biological action of a hormone or of a biological agent? It is not unreasonable to assume that growth hormone creates the necessary and sufficient environment for other biological agents to exercise the full scope of their functions."

Acknowledgments. We are greatly indebted to Drs. Thaddeiis Mann, James Harkin, Helen Deane, Keith Porter, and David Brandes for generously supplying luipublished data and electronmicrographs. Mrs. Eva Brown provided invaluable assistance in the preparation of the manuscript. We wish to thank our artist. Mr. Kenji Toda, for many of the original figures. The researches of one of us (D. P.) cited in the chapter were supported in part by grants from the Dr. Wallace C. and Clara A. Abbott Fund of the University of Chicago and by Research Grants 2912 and 5335 from the National Institutes of Health, Public Health Service.

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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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