Book - Sex and internal secretions (1961) 4
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SECTION B The Hypophysis and the Gonadotrophic Hormones in Relation to Reproducetion
Physiology of the Anterior Hypophysis in Relation to Reproduction
Roy 0. Greep, Ph.D.
Professor Of Anatomy, Harvard School Of Dental Medicine, Boston
I. Introduction
The elucidation of the major functions of the anterior lobe of the pituitary gland will likely stand as an epic in the scientific achievements of the past half century. The series of discoveries which revealed that this part of the hypophysis exercises direct or indirect control over a wide spectrum of biologic processes opened a new physiologic frontier. In no part of this new territory was understanding to be encompassed more spectacularly than by the revelation that the reproductive processes of the Vertebrata as a whole are mediated by secretions of the pituitary gland. It is intriguing to dwell on the evolutionary emergence of this gland which has so wide a control over the growth and development of both somatic and genital structures of the vertebrate body. That this regulator of so many organs and processes arises embryologically from the lining of the oral cavity and that it bears an ancient, and perchance functional, relationship to the primitive diencephalon is intriguing. The significance of these matters belongs to the future, but of immediate concern are the relationships of the pituitary to sex functions and it is to these that attention is addressed in this chai)ter.
The coverage of literature has mainly
been restricted to the period since the last
edition of this work. The number of papers
published in these years is large in proportion to the concomitant advancement of
the subject. Owing to interruptions of work
in reproductive physiology during World
War II tmd-the postwar emphasis on those
aspects of pituitary physiology which relate
to the adrenal cortex and the thyroid, interest in the study of gonadotrophic hormones waned. The papers cited, by no means
a complete list, are important in one or more
of the following respects: a pertinent contribution to knowledge, an intelligent correlation of significant physiologic data, useful theorizing, or a guide to the literature
in this and related fields. In several instances reference is made to earlier works.
These help to bring into perspective current
information and thought.^
' The following review articles, books and monographs are especially pertinent and valuable : Hisaw, 1947 ; Evans and Simpson, 1950 : Ershoff, 1952 :
II. The Hypophyseal Gonadotrophins
During fetal development and in infancy the gonads normally come under little or no important pituitary hormonal influences; a possible exception has been noted in the rabbit (.lost, 1951). Beyond infancy, however, it is clear that the arousal of gonadal functions, including the slow prepubertal, as well as the more spectacular spurt of pubertal development, is entirely dependent on gonad-stimulating hormones secreted by the anterior lobe of the hypophysis. There has been extensive exploration of the question as to whether the hypophysis secretes one or more than one gonadotrophin. Although there is yet very little, if any, knowledge of what the hypophyseal cells actually secrete, there is substantial evidence for the present widely held belief that in the mammals, at least, the anterior hypophysis secretes three trophic substances which stimulate and govern gonadal activity. These are: the follicle-stimulating hormone (FSH) ; the luteinizing hormone (LH) or interstitial cell-stimulating hormone (IC8H) ; and prolactin or lactogenic hormone, which has luteotrophic properties and has been termed luteotrophin (LTH).
The chemical evidence for the existence of separate gonadotrophins was reviewed by Li and Evans (1948) and Li (1949). Exhaustive expositions of the chemistry of the adenoliypophyseal luteotrophin, the first of the gonadotroi:)hins to be isolated, have been provided by White (1949) and by Li (19571. The work on FSH and LH was brought upto-date in 1955 by Hays and Steelman. The results of 25 years of work in these areas leave no doubt that the various biologic activities which have been demonstrated in whole anterior lobe tissue can be segregated by chemical fractionation procedures. The fact that these activities are identifiable with separate single protein fractions does not provide evidence that they are in fact secreted in the form of proteins with which the biologic activities have been identified. Furthermore, it is important to realize that the pituitary fractions equated with separate gonadotrophins have been regarded as chemical entities on criteria of purity which have been undergoing revision as advances are made in protein chemistry.
Nalbandov, 1953a ; Sayers and Brown, 1954 ; Benoit
and Assenmacher, 1955 ; Cowie and FoUey, 1955 ;
Benson and Cowie, 1957; Burrows, 1949; Parkes,
1952, 1956 : Harris, 1955 ; Chester Jones and Eckstein, 1955: and Pickford and Atz, 1957.
Several attempts have been made to find extractive procedures b}^ which it would be possible to obtain all or nearly all the pituitary trophic hormones, including the gonadotrophins, from a common batch of starting material (Fevold, 1943; Schwenk, Fleischer and Tolksdorf, 1943; Koenig and King, 1950). Although success in this undertaking would, of course, greatly enhance the potential supply of anterior hypophyseal hormones, such procedures have thus far not proved feasible primarily because of the great differences in the solubility characteristics of the several hormones. Ellis (1958), however, has been able to obtain folliclestimulating, luteinizing, and thyroid-stimulating hormone (TSH) in good yield from common batches of frozen whole sheep pituitaries.
Studies have been made of the biologic activity of cell-particulate fractions obtained by ultracentrifugation of homogenized anterior hypophyseal tissue, but it has not been possible to relate gonad-stimulating activity to any specific cellular organelle (McShan and Meyer, 1952; Brown and Hess, 1957).
By the use of histochemical procedures, not in themselves definitive, the secretion of FSH and LH has been ascribed, respectively, to specific cell types in the anterior pituitary (see chapter by Purves).
A. Follicle-Stimulating Hormone
1. Chemical Features
Chow, van Dyke, Clreep, Rothen and Shedlovsky (1942), working with swine pituitary, obtained an FSH i)rei)aration that was free of other contaminating activities but was physiochemically heterogeneous. In 1949 Li, Simpson and Evans ol)tained an FSH fraction (ovine) that was free of otheitrophic factors and that behaved as a single protein by electrophoretic procedures and ultracentrifugation; it was not tested for constant solubility. The hormone FSH behaves as a protein and there is both chemical and histochemical evidence indicating that it is a glycoprotein. FSH from swine and sheep has been variously estimated on the basis of incomplete data to have an isoelectric point of 4.5 and a molecular weight of 70,000 (Li, 1949).
That the carbohydrate residue in FSH may play an important role in physiologic activity is suggested by the finding that the FSH activity of anterior pituitary extracts is destroyed by salivary amylase and TakaDiastase (McShan and Meyer, 1938; Abramovitz and Hisaw, 1939). The resistance of FSH to proteolysis, however, is notable. In fact, McShan and Meyer (1938), Chen and van Dyke (1939), Chow, Greep and van Dyke (1939), and McShan and Meyer (1940) obtained considerable purification of FSH by means of a selective inactivation or destruction of LH with crude trypsin. More recently, Steelman, Lamont, Dittman and Hawrylewicz (1953) and Steelman, Lamont and Baltes (1955, 1956) have used pancreatin digestion to prepare swine FSH having an activity of 2.5 to 9 times the Armour Standard (264-151X).
In 1950 van Dyke, P'an and Shedlovsky advanced the purification of swine FSH to about 80 to 85 per cent "pure." Since then, Li and Pederson (1952), Steelman and his associates (1953, 1955, 1956), and Leonora, McShan and ]\Ieyer (1956) have made additional improvements in the extraction, recovery, and purification of FSH with increased specific activity.
Steelman, Kelly, Segaloff and Weber (1956) described the preparation of an "apl)arently homogenous follicle-stimulating hormone" with 30 to 50 times the activity of Armour Standard. By placing highly purified FSH on diethylaminoethyl cellulose and utilizing gradient elution, they obtained a 4- to 5-fold concentration of activity as dctcrniined by the augmentation test of Steelman and Pohley (1953). Preliminary study of this FSH in the ultracentrifuge and l)y ])aper electrophoresis revealed no evidence of heterogeneity. The molecular weight was calculated to be 29,000 as op])Osed to previous estimates. Total carboliydrate content was 7 to 8 per cent and was comprised of 50 per cent hexosamine and detectable quantities of mannose, galactose, and fucosc. Tests for LH, TSH, adrenocorticotropliic hormone (ACTH), and somatotrophic hormone (STH) were negative at the dosage level of 0.5 mg. of the FSH preparation. Latest among studies of purification of FSH is that of Ellis (1958). Using metaphosphoric acid and ethanol precipitation, he obtained a satisfactory concentrate of FSH which he was able to purify further by chromatography on diethylaminoethyl cellulose and starch electrophoresis at pH 4. This FSH preparation was electrophoretically monodisperse, free of LH and TSH, and had 30 to 40 times the potency of Armour Standard.
The development of satisfactory procedures for the isolation of FSH has been an exceedingly difficult in-ol)lcm and most workers are agreed that the methods are not finalized and that no product thus far obtained has satisfied all the modern criteria of purity. Certainly, also, there are too few studies with the highly imrified preparations to establish the biologic characteristics of this hormone. The difficulty in ascribing to FSH the status of a homogeneous chemical compound is emphasized l)y the work of Steelman, Lamont and Baltcs (1955, 1956) and by Steelman (1958). Using jiancreatin digestion followed by chromatography on hydroxyl apatite, they obtained 3 FSH fractions, all with considerable relative activity, and are in doubt as to whether the ])rechromatographed material can be regarded as homogeneous.
There is no doubt, as we survey the vertebrates as a whole, that there is a pituitary factor immediately concerned in follicular maturatibiTancl to this extent follicle-stimulating hormone is a proper term. Caution should be exercised lest terminologic convenience be confused with the fact. Nevertheless, with modern methods of protein research it is very probable that a folliclestimulating hormone will be isolated soon, and perhaps characterized as a polypei^tide with known amino acid composition and sequence.
Whatever the precise nature of FSH, it is carried to its target organs by the blood. A preliminary study (Mc Arthur, Pennell, Antoniades, Ingersoll, Oncley and Ulfelder, 1956) of postmenopausal plasma fractionated by the cold ethanol method of Cohn has revealed that the gonadotrophic activity, pituitary in origin and presumed to comprise mainly FSH, is contained in Fractions II and III. This means that FSH as such may well be in combination with proteins not only in the pituitary but also in the blood stream.
2. Physiologic Effects in Females
One activity of the anterior lobe of the pituitary brings about the maturation of egg-bearing ovarian follicles. In mammals this property resides in the follicle-stimulating hormone as we know it and, in lower vertebrates, in a follicle-stimulating component of the hypophyseal complex. In mammals FSH acts on the ovary to promote the development of primary follicles into large fluid-filled vesicular structures of the type that engaged the attention of de Graaf in his search for the ovum. Primary follicles* consist of a large oocyte surrounded by a single layer of flattened granulosal cells. As development proceeds, additional layers (6 to 9) of granulosa cells are proliferated to form a spherical mass with the ovum at the center. It is at this stage that the follicle • becomes overtly sensitive to the action of FSH. Its further development, including the secretion of the follicular liquor, the mitotic proliferation of granulosa cells, and the molding of surrounding stroma into an investing layer of thecal cells, seems to be largely controlled by FSH. In all mammals studied, the growth and maturation of the ovum itself seems independent of the action of this hormone. In the monkey (Green and Zuckerman, 1947), rat (Mandl and Zuckerman, 1952), and hamster (Knigge and Leathem, 1956) the ovum reaches its full size well before the appearance of the follicular antrum.
FSH, by promoting follicular enlargement, controls to a large degree the growth of the ovary. In immature animals injected with different amounts of FSH, the weight of the ovary can be taken as a measure of FSH activity, providing the hormone injected is pure. The size to which the ovary can be forced to develop in either immature or adult mammals seems limited only by the number of responsive follicles available. With excessive dosage over periods of 5 to 10 or more days, ovaries of massive size have been developed in many species of birds (Witschi, 1955) and mammals (Casida, Meyer, ^NlcShan and Wisnicky, 1943; Dowling, 1949; ^Vlarden, 1952), including man (Davis and Hellbaum, 1944; Maddock, 1954). In the absence of luteinizing hormone, a situation insured by removal of the recipient animal's own hypophysis, FSHstimulated ovaries present the appearance of being a mass of translucent cystic follicles. During a normal estrous cycle the number of follicles developing to maturity differs among species, but for any given species the number is quite constant (Brambell, 1956). Inasmuch as the size of the ovaries in the continuous (nonseasonal) breeder remains reasonably constant, it follows that the level of FSH stimulation must also be maintained at a similar norm. Follicular enlargement, which is brought about in large measure by the accumulation of antral liquor, occurs at a constant rate for any given species (Hisaw, 1947; Brambell, 1956). Accordingly, when ovarian enlargement is induced with increasingly greater dosages of exogenous FSH, the growth of individual follicles is not accelerated but more and more follicles are brought to maturity; the number, however, is not precisely proportional to the dose. The simultaneous development of an excessive number of Graafian follicles is the basis of the many demonstrations of artificially induced superovulation and superfetation in rats (Evans and Simpson, 1940), rabbits (Pincus, 1940; Parkes, 1943), sheep (Zawadowsky, 1941; Hammond, Jr., Hammond and Parkes, 1942; Casida, Warwick and Meyer, 1944), and cattle (Dowling, 1949; Hammond, Jr., 1949). The administration of FSH to immature animals hastens the maturation of the ovaries and leads to marked sexual precocity in all mammals tested, including the monkey (Simpson and van Wagenen, 1953) and cattle (Casida, Meyer, McShan and Wisnicky, 1943). Inasmuch as FSH activity may be restricted to the growth of follicles, accelerated puberty in the instances noted must have occurred in part through the concomitant secretion of endogenous gonadotrophins, among which LH would be pi'imarily necessary.
That FSH secretion fluctuates rhythmically during reproductive cycles in sexually mature female mammals is suggested bv the evidence of Simpson, van Wagenen and Carter (1956), showing an increase in the level of pituitary FSH content during the follicular phase of the menstrual cycle in monkeys. This is in general agreement with the older literature on other mammals (van Dyke, 1939; Smith, 1939). On the other hand, the possibility that rhythmic fluctuation in the secretion of gonadotrophin may not be confined to FSH is discussed below.
3. Physiologic Effects in Males
In males FSH acts on the spermiogenic cells in the testes with the same selectivity that it exhibits for germinal tissues in the female gonad. The growth of the testes is predominantly due to FSH and is a reflection of an increase in the size of the seminiferous tubules and of spermatogenic activity (Greep and Fevold, 1937; Creep, van Dyke and Chow, 1942; Simpson, Li and Evans, 1951). When FSH is administered to immature male rodents the size of the testes is greatly increased, but an acceleration of the appearance of fully formed spermatozoa has not been observed. True sexual precocity in males has not, therefore, been obtained, even when every other aspect of the reproductive system is fully developed. Nelson (1952) cited evidence for the view that FSH may be responsible only for the proliferation of spermatogonia and primary spermatocytes. He speculates that an androgenic influence may participate in the final stages of spermatogenesis.
Testes developed under the stimulation of exogenous FSH exhibited no change in the number, appearance, or secretory activity of the intertubular cells of Leydig (Greep and Fevold, 1937; Greep, van Dyke and Chow, 1942; Simi)son, Li and Evans, 1951 ) . The unchanged male accessory sexual structures register clearly the fact that no androgen was produced as a result of the treatment.
4. FSH in Relation to Compensatory Gonadal Responses
A difference in the responses of male and female gonads to FSH is that under exogenous FSH stimulation the testes do not develop beyond their maximal normal adult size as do the ovaries. It has been noted that the sexes also differ in the degree of compensatory hypertrophy which is elicited by unilateral gonadectomy, a response that is l)riniarily elicited by FSH. The observation by Carmichael and Marshall (1908) that unilateral ovariectomy leads to compensatory hypertrophy of the remaining ovary is now commonplace. Similar experiments on tiie male were not reported until 1940 (Addis and Lew). Compensatory gonadal hypert rophy is much less pronounced in the male ; the 50 per cent of testis mass remaining after unilateral gonadectomy increases only to 56 per cent of the weight of paired gonads of comparable controls, whereas the ovary increases up to 70 per cent. Clearly, in no instance is all the lost tissue regained through compensatory hypertrophy. It is of incidental interest in this connection that Edwards (1940) showed that unilateral castration in adult male animals leads to a halving of the sperm count. In the unilaterally ovariectomized female it is well established that the opposite ovary sheds at each ovulation the full number of eggs characteristic of the species (Asdell, 1924; Brambell, 1956). It is also of interest that in the unilaterally spayed rat the number of primary oocytes remains at the level normal for one ovary (Mandl and Zuckerman, 1950), indicating that FSH, as suggested by most older studies, does not influence ovogenesis.
5. Assay
In recent years the ability of FSH to synergize with human chorionic gonadotrophin {HCCPr has been utilized in the development of methods for the bioassay of FSH. The end point is weight of ovaries from immature rats (Evans and Simpson, 1950; Steelman and Pohley, 1953) or mice (Brown, 1955) which have also been injected with a constant and substantial amount of HCG. These methods are more sensitive than other, older assay procedures involving ovarian weight increase following injection of FSH alone into immature, intact rats or mice, but they are less specific than the assay based on ovarian weight increase (Evans, Simpson, Tolksdorf and Jensen, 1939) or re-establishment of minimal follicular growth (histologic) in hypophysectomized immature rats (Evans and Simpson, 1950). The methods of Steelman and Pohley and of Brown have clear advantages over the indirect assay based on the weight of the uteri in FSH-injected, intact, immature mice as introduced by Klinefelter, Albright and Griswold (1943).
Testis weight increments in hypophysectomized male rodents serve as a convenient measure of FSH activity, provided, however, that the FSH is biologically pure (Greep, van Dyke and Chow, 1940; Simpson, Evans and Li, 1950).
The activity of FSH in an aqueous medium is known to be augmented by a variety of substances. The explanation of the effectiveness of these cofactors is unknown, but several of them such as heme (McShan and Meyer, 1941) and zinc and copper salts, probably merely delay absorption and provide a more sustained effective blood level of the hormone.
B. Luteinizing Hormone (Interstitial Cell-Stimulating Hormone)
1. Chemical Features
Working independently, Li, Simpson and Evans (1940a, b) and Shedlovsky, Rothen, Greep, van Dyke and Chow (1940) isolated LH from sheep and swine pituitaries, respectively. The two preparations have similar physiologic properties (Greep, van Dyke and Chow, 1942; Simpson, Li and Evans, 1942a, b ) , but they differ chemically (Chow, van Dyke, Greep, Rothen and Shedlovsky, 1942) and are distinguishable by immunologic methods (Chow, 1942). The sheep hormone had an isoelectric point of 4.6 and a molecular weight of 40,000, whereas that of swine had an isoelectric point of 7.45 and a molecular weight of 100,000. Ellis (1958) has recently achieved a considerable concentration of LH activity, using cation exchange resins. Squire and Li (1959), using a modified extractive procedure, chromatographic separation, and zone electrophoresis on starch, obtained tw^o equally active "ICH" proteins; one of these, "^-ICH," has been partially characterized: it has an isoelectric point near 7.3 and a molecular weight (calculated) of approximately 30,000. This fraction had "a high degree of homogeneity," showed no evidence of contamination with other pituitary hormones, and was active by the ventral prostate test at a total dose of 0.5 /xg. LH, like FSH, is a glycoprotein. The biologic activity of LH is far more resistant to glycolytic enzymes than is that of FSH ; its resistance to proteolytic enzymes, however, is much less.
The available preparations of LH seem to be more satisfactory than those of FSH in point of comprising a hormone as a chemical entity. However, LH, as indicated by the recent work of Squire and Li (1959), may in turn reveal its biologic activity in some small part of a protein complex.
2. Physiologic Effects in Females
The many early studies based on the action of LH extracts injected into intact immature rats are uninformative, since the participation of endogenous gonadotrophins cannot be excluded nor safely discounted. Histologically, the action of LH has been detected with certainty in long-term hypophysectomized immature female animals by repair of the atrophic ovarian interstitial cells (Simpson, Li and Evans, 1942a, b».
The ovarian manifestations of the action of LH administered to intact immature female animals are notably inconspicuous. There is no increase in ovarian weight and no evidence that LH promotes the secretion of gonadal hormones. The interstitial cells appear a little more swollen than in untreated animals, and in guinea pigs in particular so-called pseudolutein bodies may appear. Conversely, the effect of LH on ovaries of adult rats are evident macroscopically. There occur an excessive number of ovulated follicles, many hemorrhagic follich's, and widespread luteinization of medium to large unruptured follicles, the ovaries being generally enlarged due to the synergism of the administered LH with endogenous FSH.
In hypophysectomized immature female rats under treatment with FSH, the notable effect of introducing LH is to impose an intensification of the enlargement of the ovaries and to promote a sudden growth of the reproductive tract (Greep, van Dyke and Chow, 1942». That this latter effect is due to the initiation of estrogen secretion is uneciuivocal. The cells of the theca inktcrna which remain atrophic under FSH stimulation swell and ar(|uire the cytologic and histochemical criteria of actively secreting cells only if LH, among the pituitary gonadotrophins, is administered.
The importance of LH in bringing about the secretion of the ovarian hormone estradiol-17/3 has been mentioned. The facts that the thecal cells are maintained in a stimulated condition and that continuous uterine growth occurs during the follicular phase of a reproductive cycle suggest that LH is secreted during this time. Other lines of evidence likewise indicate that LH not only may be secreted throughout each estrous or menstrual cycle, but that it may play a dominant role in sexual periodicity. As early as 1937 Dempsey expressed the view that, as a step in accounting for the estrous cycle, it is necessary to assume that fluctuations occur in the secretion of LH, thus bringing about ovulation and corpus luteum formation. Indeed, McArthur, Ingersoll and Worcester (1958) reported a marked elevation in the urinary excretion of LH by women at the midpoint of the normal ovulatory menstrual cycle. This view is further supported by the fact of persistent estrus in rats and guinea pigs (for reviews of literature on spontaneously occurring or induced persistent estrus, see Noumura, 1958 and chapter by Everett) in which the causative factor appears to involve an inability to elicit a discharge of LH by reflex neurogenic mechanisms.
Although meager study has been made of the ovulating property of pure LH, it is known to have this function (Greep, Chow and van Dyke, 1942; Hisaw, 1947). There seems little reason to suppose that the pure hormone would behave differently in this trespect from preparations having some FSH contamination. From information available, ovulation may, with reasonable assurance, be ascribed to a sudden elevation in the secretion of LH. Once ovulation has taken place, the conversion of the follicle into a luteinized body — a corpus luteum — is unquestionably attributable to LH action.
3. Physiologic Action in Males
The pi'imary cITcct of LH on the male gonad is to promote the maturation, maintenance or i-epair of the interstitial tissue leading to the elaboration of androgenic hoi'mone iC.i'eep. Fevold and Hisaw, 1936; Fraenkel-Conrat, Li, Simpson and Evans, 1940). This function is most clearly demonstrated by the injection of LH into hypophysectomized, immature male rats after liyi)ophyseal deficiency has resulted in marked testicular atrophy. In these animals the shrunken, spindle-shaped, and cytochemically empty cells of Leydig are quickly restored to the swollen, epitheloid, lipid-rich type. The lipoid vacuoles exhibit histochemical reactions that are characteristic of, but not necessarily specific for, steroidal substances. More specifically, the seminal vesicles, prostate and Cowper's glands undergo enlargement and each elaborates its special exocrine secretion in proof of induced testicular endocrine function. The testes also enlarge somewhat, due to swelling of the tubules and some minimal stimulation of their spermatogenic epithelium. The latter response is believed to be brought about by the direct spermatogenic action of testosterone per se and can, in fact, be duplicated by the injection of this hormone alone. In hypophysectomized adult male rats given LH from the time of operation, the entire reproductive system, including all components of the testes, is maintained in a normal condition (Greep and Fevold, 1937). Here it is presumed that the action of LH is confined to stimulation of the interstitial cells, because all other aspects of the response can be dujilicated by exogenous androgens.
4. An Extragonadal Activity
The possibility that gonadotrophins have an effect on the adrenal cortex has always been attractive. However, only in the mouse has any clear-cut action been demonstrated. LH has a trophic influence on the so-called X-zone in the adrenal glands of mice (Chester Jones, 1949) . This zone of highly eosinophilic cells is present at birth and disappears at puberty in the males and during the first gestation in females. Chester Jones (1950) found that although the zone persists after hypophysectomy in prepubertal mice, the cells therein revert to an atrophic state. It seemed likely, then, that the zone might be responsive to a factor from the pituitary. A check of available preparations revealed that extracts rich in LH were markedly trophic to the X-zone. Neither FSH nor ACTH had any beneficial action in this regard. These findings fit nicely with the fact that the X-zone reappears after castration of postpubertal male mice (Howard, 1939) , i.e., at a time when the secretion of LH is greatly increased (for a review see Chester Jones, 1955, 1957).
5. FSH and LH in Relation to Estrogen Secretion
The relation of FSH to estrogen secretion by the ovary has not been fully elucidated. Present knowledge in this area is deficient mainly because there has been little opportunity to study the activity of FSH as an entity free of LH with which it exhibits synergism. Acting with but minute amounts of LH, the quantitative responses to FSH are considerably enhanced as determined by the growth response of the female gonad (reviewed by Fevold, 1939, and by Evans and Simpson, 1950), yet the qualitative nature of the response may not be altered (Fraenkel-Conrat, Li, Simpson and Evans, 1941). To the extent of present knowledge, biologically pure FSH does not elicit the secretion of estrogen by the ovary of the hypophysectomized rat, and there is convincing evidence that it also does not evoke the secretion of androgen by the testes of similarly operated animals (Fevold, 1939; Greep, van Dyke and Chow, 1942; Evans and Simpson, 1950; Simpson, Evans and Li, 1950; Simpson, Li and Evans, 1951). However, FSH does play an essential and possibly even a primary role in the promotion of estrogen secretion by the ovary, inasmuch as it prepares the follicular apparatus on which LH can act to promote estrogen production. That the injection of highly purified FSH preparations into intact, immature rats and mice (Moon and Li, 1952; Thomopoulou and Li, 1954) has evoked the secretion of estrogen is explicable, perhaps, on the basis of endogenous LH release.
6. FSH and LH, Interactions
LH is believed to interact in some manner with FSH at the ovarian level to produce greater stimulation than could be expected on the basis of the sum of the separate actions of these hormones. This effect has also been termed synergism or ]'»otentiation. It is a real and striking phenomenon, for which no satisfactory explanation is available. The principle of potentiation has been employed extensively to intensify artificially induced gonadal stimulation in sheep and cattle (Hammond, Jr. and Parkes, 1942; Casida, Meyer, McShan and Wisnicky, 1943).
An interplay between FSH and LH seems to operate in the accomplishment of ovulation. The ovulatory response to LH is well known to be intensified when acting in conjunction with FSH (Foster, Foster and Hisaw, 1937; Pincus, 1940; Chang, 1947a, b; Hisaw, 1947; ISlarden, 1952). The reader is referred to Hisaw (1947) for a thoughtful analysis of the literature. FSH acting alone in hypophysectomized animals will bring follicles to maturity, but seldom does it lead to their rupture (Knobil, Kostyo and Greep, 1959). LH, on the contrary, given as a quick acting stimulus in the presence of ripe follicles, is an efficient ovulator. Inasmuch as the exact nature of the ovulatory stimulus has not been elucidated, the term "ovulatory hormone" has been widely used. It has the advantage of being less committal as to the nature of the factors operating. In reality, it would be a very difficult matter to test the ovulating capacity of LH in the complete absence of FSH, since only nearmature follicles can be ovulated, and their existence is dependent on the action of FSH.
Rapid involution and disappearance of persisting corpora lutea in hypophysectomized adult rats (Bunde and Greep, 1936; Greep, 1938) was induced by injecting impure LH extracts and combinations of FSH and LH. Although pure LH did not produce the histolytic response, it is worth noting that it also did not elicit any maintenance of hiteal function (Greep, van Dyke and Chow, 1942 ».
When FSH and LI I were adniinistered concurrently to intact or hypophysectomized, immature male rats a synergism was noted in respect to the increase in weight of the testes (Greep, Fevold and Hisaw, 193(31.
7. Assay
Several means are available for detecting the activity of LH, but no one has proved fully satisfactory as a means of quantifying this hormone. Oldest of these methods is the augmentation and luteinization test, as developed in the laboratory of Fevold and Hisaw. It involved a comparison of the qualitative ovarian response of intact, immature female rats to FSH alone as opposed to that of animals receiving FSH and LH simultaneously. A positive test for LH was manifest by augmentation of the ovarian weight and induction of extensive luteinization, resulting in what has often been termed "mulberry ovaries." When hypophysectomized test animals were employed, the results were less striking both quantitatively and qualitatively, but interference by endogenous LH was precluded.
The possibility of using the weight increase of the accessory sexual structures in immature male rats as a test for LH suggested itself from studies of the effects of the separate gonadotrophins in male animals. The ventral lobe of the prostate was the most sensitive. Employing hypophysectomized animals and pure LH, Greep, van Dyke and Chow (1941) demonstrated a satisfactory dose-response relationship. The test is simple, ofi'ers an objective measurement, and is moderately sensitive. It has the disadvantage of being indirect — the prostate response is androgen-mediated and reflects the action of LH on the testicular Leydig cells. The specificity of the prostate test for LH has been questioned by Segaloff, Steelman and Flores (1956). Pursuant to a report by Grayhack, Bunce, Kearns and Scott (1955) that jirolactin enhanced the ventral j^rostate response to testosterone in castrated rats, Segaloft" and his colleagues reported that prolactin sensitized the ventral prostate to the action of androgen secreted in response to LH. Lostroh and Li (1956) were not able to confirm that prolactin synergizes with testosterone, and Lostroh, Squire and Li (1958) found a strain variation with respect to the specificity of the ventral prostate as a test for ICSH. Wlien the Long-Evans strain of rats was used, neither prolactin nor growth horiiionc. or any combination of these, altered the prostatic response to exogenous ICSH; with the Sj)raguc-Dawley strain both growth hormone and lactogenic hormone effected significant enhancement of the response to ICSH. It seems that hypophysectomized animals of any strain can be used for the assay of purified ICSH, but only those of the Long-Evans strain are suitable of the assay of impure preparations. Loraine and Diczfalusy (1958) concluded that the response of the ventral prostate of the hypophysectomized immature male rat to human menopausal gonadotrophin and HCG is not affected by the simultaneous administration of prolactin. Most important, perhaps, is the fact that FSH does not affect the prostatic assay for LH (Greep, van Dyke and Chow, 1941 ; Lostroh, Squire and Li^, 1958 j.
According to Lostroh, Squire and Li (1958) , "the hypophysectomized male rat of the Long-Evans strain has been a satisfactory animal for assaying preparations of ICSH when the weight of the ventral prostate is used as a measure of the activity and the response is represented as a logarithmic function of the dose. . . . The sensitivity of the assay is such that a total dose level of from 0.010 to 0.060 mg. is sufficient for assay purposes when the subcutaneous route is employed."
The Weaver finch test for LH, developed by Witschi (1940, 1955), is gaining acceptance as a valuable means of identifying this gonadotrophin (Segal, 1957). The test is carried out on African finches of the genera Euplectes and Steganura. Regenerating white feathers of the ventral pterylae in females or in males in eclipse plumage react to a single injection of LH by the formation of a black bar formed as a result of melanm-dep6sition. The test is held to be specific for pituitary LH, but the evidence for this view is not yet conclusive. The reaction is independent of the sex glands. The observation that the Weaver finch test does not detect LH in menopausal urine (Witschi, 1955) as does the ventral prostate method (Greep and Chester Jones, 1950b; Loraine and Diczfalusy, 1958) needs further exploration.
Evidence submitted within the past year (Parlow, 1958) suggests that LH produces a fall in ascorbic acid content of the ovaries of pseudopregnant rats and promises to form the basis of a further and much more sensitive means of assaying the luteinizing hormone.
C. Luteotrophin (Prolactin)
1. Chemical Features
Prolactin has been isolated in a form satisfying all the available physicochemical criteria of protein homogeneity and exhibiting no biologic activity other than that believed to be attributable to the hormone (for latest evidence see Li, 1957). Prolactin was the first in this category of protein hormones to be obtained in crystalline form (White, Catchpole and Long, 1937; White, 1943) — a notable achievement even though crystallization is no necessary indication of purity. Prolactin has mainly been obtained from the hypophyses of beef and sheep and although there may be minor species differences in the composition of the hormone as indicated by a slight difference in solubility, the preparations otherwise behave identically. They have an isoelectric point of 5.7 and a molecular weight of about 3200 (White. 1949).
2. Physiology of Luteotrophic Hormone (Prolactin)
Riddle, Bates and Dykshorn (1932) proposed the term prolactin to describe a pituitary activity which stimulated the crop sac of pigeons. In mammals prolactin was shown to possess also lactogenic and luteotrophic activity. It has not been unequivocally demonstrated that prolactin, lactogenic hormone, and luteotrophin are identical nor that luteotrophic manifestations are essential for luteal function except in the rat. For the purposes of this chapter, however, it is regarded as semantically legitimate to discuss LTH as a gonadotrophin, although scientifically neither its activity nor its identity with prolactin has been rigorously proved.
As noted, the rat is the only animal in which a luteotrophic action of prolactin has been conclusively demonstrated (Astwood, 1941 ; Cutuly, 1941 ; Evans, Simpson and Lyons, 1941 ; Evans, Simpson, Lyons and Turpeinen, 1941 ) . Confirmatory observations have been reported by Lyons (1942), Tobin (1942), Fluhmann and Laqueur (1943), Everett (1944), Sydnor (1945). Greep and Chester Jones (1950a). Tests of the luteotrophic action of prolactin in other species have not been encouraging. Intensive treatment with prolactin has singularly failed to prolong the functional life of the corpus luteum in monkeys (Hisaw, 1944; Bryans, 1951) and women (Holmstrom and Jones, 1949, Bradbury, Brown and Gray, 1950). By administering prolactin along with HCG during the postovulatory phase of the cycle in regularly menstruating women, Fried and Rakoff (1952) prolonged the functional life of the corpus luteum up to 13 days but neither hormone given alone was effective. These findings suggest that LTH may only act in synergism with a luteinizing hormone in man.
By recent demonstration, the lactogenic hormone appears to have a luteotrophic action in sheep (Moore and Nalbandov, 1955j. The corpora lutea of anestrous ewes, ovulated with pregnant mare serum gonadotrophin, were maintained in a functional state for 20 to 30 days by daily injections of 200 I.U. of prolactin. Without such treatment the newly ovulated corpora lutea regressed rapidly. However, since the prolactin-treated animals were intact and their ovaries were heavily luteinized, the authors could not be certain that the prolongation of the functional life of the corpora lutea was attributable solely to the injected hormone. The testing of LTH in the recently ovulated and hypophysectomized rabbit (which seems not to have been done) would furnish valuable basic information.
3. Detection and Assay of Prolactin Activity
Luteotrophic hormone produces no conspicuous morphologic changes in the mammalian ovary; the response can in fact only be detected by demonstrating that preformed corpora lutea are functional. Luteotrophic activity per se, therefore, cannot be estimated directly in mammals, but rests on the evocation of progesterone secretion so that tests for LTH (reviewed by Astwood, 1953) are, in effect, tests for progesterone. Thus in the appropriately prepared rat I^TH activity can be demonstrated qualitatively and a rough estimate of potency made by the appearance of deciduomas (Astwood, 1939, Lyons, Li, Johnson and Cole, 1953), maintenance of early gestation (Cutuly, 1941, and 1942). interruption of estrous cycles (Desclin, 1949; ]\Iayer and Canivenc, 1951 j, and appearance of diestrual smears in HCG-treated hypophysectomized rats (Astwood, 1941; van der Kuy, van Soest and van Prooye-Belle, 1953) . In other mammals tests for LTH likewise hinge on the demonstration of some progesterone-like effect; these vary with the species (Cowie and Folley, 1955).
In practice the assumption is made that prolactin and luteotrophin are identical, and luteotrophic preparations are assayed by measurement of their capacity to stimulate the pigeon crop sac as determined by either the "systemic crop weight" method introduced by Riddle, Bates and Dykshorn (1933) or the "local micromethod" of Lyons and Page (1935). Modifications have been introduced in the "weight" method by Clarke and Folley (1953), and in the "local" method by Lyons (1937) and Reece and Turner (1937). (For detailed review of assay literature see White, 1949, or Cowie and Folley, 1955.)
III. Pituitary Gonadotrophic Hormone Content and Related Evidence of Secretory Activity
A valuable method of studying the physiology of the pituitary gland is to remove it at autopsy or by surgery and ascertain the nature and quantity of gonad-stimulating hormone activity residual within the gland as shown by the gonadal response elicited in some appropriate recipient test animal, i.e., immature rat, mouse, chick, pigeon. Early investigators relied mainly on imjilanting the fresh gland or injecting minced tissue, but this procedure is quantitatively unreliable inasmuch as the tissue tends to remain viable for an indeterminate time (Hellbaum and Greep, 1940). This ^difficulty was obviated by freezing (Fraenjkel-Conrat, Simpson and Evans, 1940a) or iby desiccating the glands in acetone followed by trituration in water or saline. As an alternative procedure, the pituitary glands of small animals can be crushed between glass slides, then speedily air-dried and recovered as a powder, without loss of potency (Kupperman, Elder and Meyer, 1941). Others preferred to test fresh ground homogenate of the pituitary tissue.
Pituitary gonadotrophic jiotency, a representation of the aiiiount of gonadotrophins
present at the moment of death, is a resultant of the rate of synthesis and the rate of
liberation. Whether such potency measurements provide any reasonable clue to the
actual rate of secretion has been debated.
The significance of such measurements is
enhanced by the preponderance of cases
in which the potency has correlated well
with function, as revealed by the gonads and
reproductive tracts of the donors (Meyer,
Biddulph and Finerty, 1946; Robinson and
Nalbandov, 1951; Kammlade, Welch, Nalbandov and Norton, 1952; Bahn, Lorenz,
Bennett and Albert, 1953a; Greeley and
Meyer, 1953; Nalbandov, 1953b, a review;
Simpson, van Wagenen and Carter, 1956 j.
These correlations, however, were not always close and in a few instances, as noted
elsewhere (page 261) , pituitary potency and
function diverged sharply. As long ago as
1939 Smith surmised that the usefulness of
whole gland assays in the study of pituitary
physiology had been nearly fulfilled. The
prediction seemed reasonable but the technique, with some refinements, is still widely
used and is one of the important means
of gaining insight concerning the probable
level of gonadotrophic function. It would be
exceedingly helpful to be able to determine
the blood level of the gonadotrophins, but
even this would not reflect the rate of entry
of the gonadotrophins into the blood stream
unless the rate of destruction and excretion
were also evaluated.
For the most part the levels of circulating
gonadotrophins in normal animals have
been found/to be very low and are generally
below the /sensitivity of the available criteria for defecting them. Significant gonadotrophic activity has not been found in the
blood or urine of ruminants (reviewed by
Benson and Cowie, 1957) and the fact has
been re-emphasized by the circumstance
that no trace of such activity was found in
volumes of sheep blood as large as 500 ml.
(Bassett, Sew^ell and White, 1955). Normal
rat plasma has also yielded consistently
negative results except for one unconfirmed
report of a trace of LH activity (Hellbaum
and Greep, 1943) . The plasma of gonadectomized rats, however, like the plasmas of
menopausal women or gonadectomized men
and women, was early found to contain demonstrable gonadotrophic activity (Emery, 1932). Later, Hellbaum and Greep
(1943) reported finding FSH, but no LH,
in 10 ml. plasma from castrated male rats.
Recently this result has been confirmed in
substance by Cozens and Nelson (1958).
Using closely spaced injections, they administered to hypophysectomized, immature
female rats up to 24 or 32 ml. plasma from
spayed adult female rats over a 4-day period ; although they found a clear indication
of FSH activity, ICSH was not apparent.
A. Phylogenetic Considerations
The end points of cross-species testing of pituitary glands for gonadotrophic potency have comprised mainly gonad stimulation in immature male and female rats and mice and in 1-day-old male chicks, ovulation in estrous rabbits or pregnant mice (Ladman and Runner, 1951), ovulation or spermiation in frogs, and coloration of sparrow bill or Weaver finch feathers. The conditions of the assay have varied greatly; the test animals have been of different ages at the start of treatment; they have been hypophysectomized in one study and intact in the next; the dosage has been expressed in integers or fractions of whole glands or as milligrams, wet weight or dry weight, of pituitary tissue. The data often do not permit comparisons of assays of pituitaries from a given species, leaving aside the difficulties of making interspecies comparisons. The extensive literature on this topic has been reviewed repeatedly (Smith, 1939; van Dyke, 1939; Burrows, 1949; Chester Jones and Eckstein, 1955).
1. Ascidians
On the basis of tests in three intact 26day-old mice, Carlisle (1950) claimed to have detected gonadotrophic activity in the neural gland of ascidians. Dodd's (1955) recent evidence of more substantial nature casts doubt on the validity of this claim — yet leaves the question open, since 1 of Dodd's 10 intact 19-day-old mice receiving as much as 230 neural glands each gave a positive response. The further statement by Carlisle (1954) that mammalian pituitary and urinary gonadotrophins promote ovulation and sperm discharge in the ascidians has likewise been challenged (Dodd, 19")oi.
2. Fish
Fish have been reported to respond to gonadotrophins of piscine origin (Hasler, Meyer and Field, 1939; Rasquin, 1951; Hisaw and Albert, 1955; Hoar, 1955; Ramaswami and Simdararaj, 1956, 1957) by exhibiting out-of-season oocyte growth and spawning. It is to be noted, however, that satisfactory control experiments are often lacking. Fish respond less readily to ami:)liibian pituitary. Induced spawning in rainbow trout (Migita, Matsomoto, Kinochita, Sasaki and Ashikawa, 1952) and catfish (Ramaswami and Sundararaj, 1957) has been claimed to follow the injection of frog pituitary. Fish appear to be almost totally insensitive to gonadotrophins of avian and mammalian origin (de Azevedo and Canale, 1938; Hasler, Meyer and Field, 1939; Hoar, 1955); however, Dodd (1955) calls attention to some instances of doubtful or limited effectiveness of mammalian gonadotrophins, especially human urinary gonadotrophins, in fish. Pickford and Atz (1957) brought together the varied and conflicting results of the multitudinous studies of reproductive physiology in fish.
3. Amphibians
Amphibians are notably responsive to gonadotrophins from widely differing phylogenetic sources, yet even among some of the anura the gonadotrophins show a high degree of species specificity (Greaser and Gorbman, 1939; Houssay, 1954). The effectiveness of homoplastic anuran pituitaries in inducing spermiation, ovulation, and oviposition has become a part of classical biology. By contrast, the induction of ovulation in Rana temporaria requires 150 times as much beef as Rana pituitary (Gallien, 1955a), and Houssay has shown that the South American toad, Bufo arenarum, is less sensitive to rat and human hypophysis than to pituitaries of anuran origin. Strangely, the anura, although generally responsive to mammalian gonadotrophins of pituitary or placental origin, have reacted poorly— if at all — to injected brei of elasmobranch pituitaries (Greaser and Gorbman, 1939) or to those of telcosts (Greaser and Gorbman, 1939; Atz and Pickford, 1954).
Pursuing the matter of phyletic considerations further, Atz and Pickford (1954) report that the Russian workers, Stroganov and Alpatov (1951) found frogs more sensitive than fish to sturgeon pituitary; and interestingly, although their data are meager, Wills, 'Riley and Stubbs (1933) obtained ovulation in Bujo americanus and Rana pipiens with gar pike hypophyses. The sturgeon and gar pike, as Greaser and Gorbman (1939) point out, are closer in the phyletic scale to the amphibia than they are to the fish whose pituitaries were ineffective in amphibia.
Some exploratory studies have been made of the action of mammalian FSH and LH in amphibia. The male toad, Bnfo melanostictus, responded to mammalian FSH and LH (Bhaduri, 1951), whereas Bujo bufo responded negatively to FSH (Thorborg and Hansen, 1950). Greze (1949) reported that LH produced a positive spermiation test in Rana esculanta, and Atz and Pickford (1954) , after testing a number of mammalian pituitary preparations, concluded that LH was 40 to 100 times more active than FSH in causing sperm release in Rana pipiens; the positive responses obtained with FSH were easily attributable to an LH contaminant. Wright and Hisaw (1946) studied the response of frog ovaries to purified mammalian gonadotrophins and found that, although FSH ovulated the intact frog, it was not effective in hypophysectomized females. Using the technique of Heilbrunn, Dougherty and Wilbur (1939), Wright and Hisaw applied sheep FSH to frog ovaries in vitro and failed to induce ovulation; but by combining FSH and LH in an appropriate ratio, ovulation was obtained. They believe that the principal function of FSH in the frog is to render the ovary sensitive to an ovulating stimulus. OA'iposition was induced in the salamander, Triturus viridescens, by LH but not by FSH (Mayo, 1937) and Witschi (1937) elicited ovulation in the newt, Taricha torosa, with beef and turkey hypophyses but not with pregnant mare's serum (PMS) gonadotrophin.
The sensitivity of ami)hibians to human pituitary and chorionic gonadotrophins has rendered them extremely useful in clinical assays for diagnostic purposes, especially for the early diagnosis of pregnancy. A number of species of frogs and toads are in use, mainly: (1) the male leopard frog, Ra7m pi-piens (Wiltberger and Miller, 1948; Robbins and Parker, 1949; Haskins and Sherman, 1952) ; positive tests are also obtained with hypophysectomized frogs (Kissen, 1954), (2) the female Xenopus laevis (Thorborg and Hansen, 1951 ; Hobson, 1952) , and (3) the male toad, Bufo arenanim (Hansel) (Galli Mainini, 1948; Allison, 1954; for review, see Houssay, 1954). The Galli Mainini test is held to be relatively more specific for the chorionic gonadotrophin in that positive responses are not obtained with urine from normal men or nonpregnant women, regardless of the presence or absence of functioning gonads. This is in line with Atz and Pickford's finding that spermiation is mainly a function of LH, the urinary output of which in normal men is very low.
4. Reptiles
Few observations have been made on the interrelations of the pituitary and gonads in reptiles (Kehl and Combescot, 1955) and fewer still on the responsiveness of reptiles to heterozoic gonadotrophins. In viviparous snakes hypophysectomy led to regression of gonads and death of the embryos and premature parturition (Clausen, 1940; Bragdon, 1951). Testis impairment following hyl)ophysectomy in male garter snakes was corrected by daily implantation of pituitary glands froni the same species (Schaefer, 1933). Crude extracts of sheep pituitary promoted marked gonad stimulation in the lizard, Uromastix (Kehl, 1944; Kehl and Combescot, 1955), immature alligators, Alligator jnississippiensis (Forbes, 1937), and terrapins, Malaclemmys centrata (Risley, 1941 1 . In extended studies of the chameleon, Anolis carolinensis, Evans (1948) oljtained positive results with mammalian gonadotrophins unspecified.
5. Birds
Among birds males of all ages and mature females are responsive to gonadotrophins of both avian and mammalian sources (Breneman, 1936; Byerly and Burrows, 1938; Nalbandov and Card, 1946; Das and Nalbandov, 1955). These observations are based mainly on the domestic breeds, such as chickens, ducks, and turkeys (reviewed by Nalbandov, 1953a). Evidence suggesting qualitative differences between avian and mammalian gonadotrophins has been obtained by Nalbandov, Meyer and McShan (1951), Das and Nalbandov (1955), and Taber, Clay tor, Knight, Gambrell, Flowers and Ayers (1958). Thus, the ovarian cortex of the sexually immature female chicken, although notably insensitive to mammalian gonadotrophins, has responded to avian pituitary extract. Taber and her associates noted significant follicle stimulation but no precocious ovulation in immature female chickens receiving long-term treatment with acetone-dried avian anterior lobe substance. In contradistinction, the medullary portion of the immature ovary, like the testes of the male bird, responded with increase in weight and androgen production at any age and to hormones of either mammalian or avian source. Following hypophysectomy of prepubertal female birds, the medullary area remained responsive to avian, but not to mammalian, gonadotrophin. Moreover, the effectiveness of mammalian gonadotrophins in maintaining the ovaries and comb of hypophysectomized laying hens was of short duration (Biswal and Nalbandov, 1952; Nalbandov, 1953a); egg-laying continued 5 to 7 days postoperatively, and comb size was sustained for 10 to 15 days. Avian hormone, alone or in combination with mammalian gonadotrophin, was effective over treatment periods lasting up to 35 days. The possibility that these results might have been influenced by the development of immune bodies was not eliminated.
Observations of interest have been made on the hypophyseal control of the rudimentary right gonad in hens (Kornfeld and Nalbandov, 1954). Compensatory hypertrophy of this structure, seen regularly in poulards, has been completely prevented by injections of estrogen, and to lesser extent by androgen. Hypophysectomy, moreover, causes regression of the enlarged right rudiment which cannot be forestalled by injections of hog or sheep gonadotrophins, nor by PMS. Whether chicken gonadotrophins would have the sustaining action they could be presumed to have in this situation, has apparently not been investigated.
Inasmuch as the pituitary-gonad interrelationshii)s are complex and little understood, it is not to be expected that the administration of either isogeneric or heterozoic gonadotrophic complexes would elicit the normal pattern of sexual functions. As a demonstration in point, Witschi (1955) injected sparrows with the suspended powders of human, beef, or turkey hypophyses and found that human hypophyses produced good follicle growth but the oviducts were only incompletely developed; turkey and beef glands, on the contrary, promoted intense stimulation of the reproductive tract and only minor ovarian enlargement. None of these preparations produced a balanced development of the gonads and accessory sexual organs, nor did ovulation and egglaying occur.
Prolactin is present in extracts of the hypophyses of fish, amphibians, and reptiles (Leblond and Noble, 1937) and its presence in avian pituitary was amply demonstrated by Riddle's group in the middle 'thirties. Their classic studies showed that prolactin exerts full control over the proliferative development of the crop sac in pigeons, but it also appeared to have a suppressive action on the gonads in these as well as other birds. Prolactin of mammalian origin administered to laying hens resulted in regression of ovaries, cessation of laying, and appearance of broodiness (Breneman, 1942; Nalbandov, Hochhauser and Dugas, 1945; Nalbandov, 1953a). Nalbandov expressed the opinion that broodiness is secondary to ovarian failure, with attendant reduction in estrogen secretion — a view nicely supported by the earlier finding of Godfrey and Jaap (1950) that broodiness can be quickly terminated by estrogen injections (also see chapter by Lehrman). Prolactin has also been noted to induce broodiness in cocks (Nalbandov and Card, 1946) and atrophy of the developing right gonad in poulards (Kornfeld and Nalbandov, 1954). The mechanism for the suppressive influence of prolactin on the gonads of birds is unknown, but it can be presumed to involve central nervous system centers concerned in some manner with regulation of the anterior pituitary. Prolactin has also been noted to have a calorigenic action (Riddle, Smith, Bates, Moran and Lahr, 1936) , but it has not been possible to correlate this with the induction of broodiness. Likewise, its ability to increase the body temperature of roosters 2 to 4°F. is not contributory, since broody fowl have subnormal temperatures.
6. Mammals
Information on the effectiveness in mammals of pituitary gonadotrophins from lower orders is scanty. As would be anticipated, avian gonadotrophins are by far the most potent. Unfractionated gonadotrophic extracts of avian pituitaries exhibited both follicle-stimulating and luteinizing activity (Leonard, 1937; Witschi, 1937; Gorbman, 1941; Traps, Fevold and Neher, 1947; Nalbandov, Meyer and McShan, 1951). The nature of the gonadal reactions induced in mammals by avian gonadotrojjhins are ciualitatively similar to those produced by mammalian gonadotrophins (Riley and Fraps, 1942a, b; Breneman, 1945; Nalbandov and Card, 1946; Breneman and Mason, 1951).
Moderate folheuhir development and luteinization in rats and mice has been induced by chicken and turkey pituitaries, but full estrous development of the reproductive tracts is generally not attained. Riley and Fraps (1942a, b), using the mouse ovary assay, estimated the ratio of FSH to LH potency in bird hypophyses, and they along with Chance, Rowlands and Young (1939), Witschi (1940), and others, in fact, attempted quantitative estimations of the F8H-LH ratio in hypophyses of a variety of birds and mammals. Such precise numerical values are unfortunately of little significance, owing to the limitations of the bioassays. Nonetheless, two indications seem to emerge from these studies: the FSH-LH ratio varies greatly from one species to another, and within a given species rhythmic fluctuations have been noted. Pituitaries from such cold-blooded forms as fish, frogs, and tui'tles have at l)cst shown liarely positi\-e ti'aces of gonadotrophic activity in rats and mice (for litei'aturc, see Witschi, 1955). In tact, A(hinis and Granger (1941) had to inject 100 times as much frog as mouse hypophysis to obtain minimal stimulation of the mouse ovary.
7. General Considerations
There are probably no generalizations that can safely be drawn from the variety of taxonomic relationships noted in regard to the gonadotrophins ; but, as Zuckerman aptly contends, "knowledge can advance little if we do not generalize." Wide distribution throughout the Vertebrata is suggested for each of the gonadotrophins. The information concerning their ciualitative variation that is revealed by cross-species testing is of fundamental importance to both biology and medicine. Peculiarly, there is little and perhaps no qualitative variation in the gonadotrophins from various species of teleosts (Ihering, 1937; Kazanskii, 1940; Dodd, 1955), but the number of species studied is not a fair sample of the number existent. The situation is different in the amphibia. Here qualitative variation in the gonadotrophins from different s])ecies is much in evidence. Seemingly, some anurans are capable of responding to gonadotrophins from any class of vertebrate, whereas others fail to respond even to gonadotrophins from closely related amphibian species ; albeit the amphibia appear to be far more sensitive to amphibian gonadotrophins than to those from any other class of vertebrates. The reptiles, although little studied, appear to respond sluggishly to nonreptilian gonadotrophins. Birds, too, respond best to bird gonadotrophins, yet in some respects are exceedingly! sensitive to those from a wide variety of mammals. Among the mammals great variations in responsiveness are revealed by every exchange of gonadotrophins between their species. The variations, however, are mainly of quantitative rather than of qualitative nature so far as is known.
The differences in species responsiveness pose difficult problems in the bioassay of the gonadotrophins. It is obvious that the potency units assigned to a gonadotrophin preparation assayed in a foreign species have no significance as to equivalent potency when the same preparation is tested in other species, even when adjustment is made so that the dose per unit of body weight is the same. Moreover, an apparent ineffectiveness in one species may not necessarily mean that the gonadotrophin is biologically inactive; Creaser and Gorbman (1939) state that the effectiveness of a gonadotrophic hormone in a foreign species tends to vary directly with the phylogenetic proximity of the donor and recipient species."
Recently, a marked qualitative species
variation was demonstrated in connection
with another pituitary hormone, somatotrophin. Preparations of swine, ovine, or
bovine somatotrophin were markedly effective in rats and dogs but not in monkeys or
man (for literature, see Smith, Gaebler and
Long, 1955). IVIonkey somatotrophin, however, was effective in monkeys and also in
rats (Knobil and Greep, 1956). Similarly,
fish somatotrophin, which was active in fish,
was inactive in mammals, whereas ox preparations were active in fish as well as in some
mammals (Pickford, 1954; Wilhelmi, 1955).
We have seen that the physiologic properties of a gonadotrophin are as much an expression of the sensitivity of the substrate on which the hormone acts as of the excitations of the hormone itself. This being the situation, the difficulties to be faced in retracing the evolutionary history of the gonadotropins are indeed formidable. However, by extending lines of study that have been followed over the past 20 years, pieces of this fascinating problem may in time be fitted together. At this early departure in the quest, the dictum credited to Medawar to the effect that, "It is not the hormones which evolve but the uses to which they are put" seems grossly premature; but its value in addressing thoughtful inquiry to the matter may be considerable. Another laboratory aphorism to the effect that hormones can be tested down the ])hyletic scale but not in the reverse order undoubtedly contains an element of truth, but as a generalization it would seem to be fraught with much hazard. Along these same lines Witschi (1955), noting the spread in effectiveness of mammalian LH — at least to the amphibia — remarked, "One definite conclusion derives clearly ^ from the now available evidence; namely, that induction of ovulation is a much more general and more ancient function of LH than is its role in the formation of corpora lutea."
B. Age, Sex, Gonadectomy, and Reproductive Rhythms in Relation to Pituitary Gnadotrophins
1. Fetal Gonadotrophins
Experiments cited by Smith (1939j revealed no demonstrable gonadotrophic activity in the pituitary of fetuses except in the hog and horse. The amount of pooled tissue available for such tests with other species was often so small as to make the negative findings inconclusive. The original work of Smith and Dortzbach (1929) on the l)ig fetus is still the most complete. Gonadotrophin was detected in the late fetal stages.
Much experimental evidence bearing on the relation of the hypophysis to the development of the fetal gonads has been obtained over the past several years, princi})ally from the laboratories of Wells in Minnesota and Jost in Paris. These studies are admirably summarized by Jost (1953, 1955, 1956a) and Wells (1956). Each group developed techniques for intra-uterine ablation of fetal endocrine glands, including the hypophysis; the latter being accom])lished, in effect, by decapitation. Decapitation of rat fetuses on day 18 did not lead to abnormalities in the differentiation and development of either the gonads or the accessory sexual structures. By contrast, removal of the testes on day 18 led to deficiencies in the development of the prostate and coagulation glands, and a failure of the Mullerian ducts to regress normally. This, according to Wells, is a deficit in maleness correctable by testosterone and is not to be construed as a feminizing influence. Substitution of testosterone from the time of fetal castration prevented all the impairments of development except the regression of the Miillerian ducts. It would seem that the fetal testis exerts an influence over development of the male accessory structures and that this action is independent of any stiiiuihis fi-oni the hy])()physis.
The effects of fetal decapitation in I'abbits as studied by Jost differ from those in the lat in one important respect. Male fetuses decapitated on the 19th. 20th, or 21st day of gestation were born at term in a semifeminized state as regards the accessory ducts and external genitalia. These changes did not occur when decapitation was performed after the 23rd day nor when the headless fetuses were injected with gonadotrophin. By way of explanation, days 22 and 23 were held to be a determinative phase during which the prospective development of the sexual ducts is established by the fetal testis acting in response to the fetal pituitary. Cytologic examination of the fetal hypophyses during this period revealed the transient presence of McManus-positive cells on the 22nd and 23rd days, which Jost believes may signal a transitory production of gonadotrophins. That the fetal rabbit pituitary actually secretes gonadotrophin on days 22 to 24 would require more positive documentation than is presently available. There are considerations that urge caution. The female rabbit ovary is known to be refractory, at least to exogenous gonadotrophin, throughout the entire infantile period (Hertz and Hisaw, 1934), a finding common to many infant mammals. The fact that McManus-positive material appeared in the pituitary cells is suggestive, but by no means conclusive, evidence of secretion of gonadotrophins. There is also the possibility that maternal gonadotrophins are available to the fetus, but the evidence is controversial (Knobil and Briggs, 1955; Jost, 1956b). It seems clear that any function served the fetus by maternal gonadotrophins may be of minor significance, inasmuch as the maternal pituitary gland has been removed quite early in gestation in several mammals, including the rhesus monkey (Smith, 1954, 1955), without impairment to the development of the fetal male or female reproductive systems. Moreover, in man and horse the available evidence suggests that the pituitary gland is extremely weak in gonadotrophic activity throughout most of the gestation pciiod, including the l)uerperiuin.
=i. Age and Sex
Brencman and ISIason (1951 ) and Breneinan (1945, 1955) made detailed studies of the gonadotrophic i)otency of cockerel and pullet ]5ituitaries (using the chick testis assav) (lnrin^ the initial 3 to 4 months of life and collated these data with growth of the gonads. For 3 weeks postnatally the total glandular potency remained very low in both sexes. Thereafter the cockerel pituitary gained steadily in both total and unit potency, reaching a plateau at about 90 days. The unit potency increased approximately five times and was accompanied by an approximate parallel increase in the weight of the testes of the donor birds. Pullets over 20 days of age showed a similar rise in potency plus a sharp upturn in both unit and total potency coincident with the burst of ovarian enlargement between the 115th and 125th day of life. It is precisely at this point that female chickens were found to become overtly responsive to mammalian gonadotrophins (Das and Nalbandov, 1955; Breneman, 1955). Among adult fowl the pituitaries of nonlaying hens, as tested by the mouse uterine weight method, are about twice as potent as those of laying hens (Riley and Fraps, 1942b). The pituitaries of mature male chickens are markedly more potent than those of females of comparable age; although the male glands are heavier, the difference in potency cannot be accounted for on this basis alone (Riley and Fraps, 1942a; Phillips, 1943; Breneman and Mason, 1951). The pituitary of the male pheasant is likewise more potent than that of the hen pheasant (Greeley and Meyer, 1953).
Among the most systematic studies of gonadotrophic potency in relation to postnatal age and sex are the early ones in rats by Clark /(1935a), McQueen-Williams (1935), and Stein (1935). Gonadotrophin appears in assayable quantity around the end of the 2nd week of life, when the gonads are first becoming responsive to gonadotrophic stimulation. They found a sjjike in the age-potency curve for the female gland at the 3rd postnatal week, which was not exceeded even during sexual maturity.
Pituitaries of adult male rats are markedly more potent in gonadotrophic complex than those of females of com})arable age. Clark's data show that the relative superior potency of the male gland is not attained until some months after puberty. Hoogstra and Paesi (1955) examined the FSH and LH content of the pituitaries of intact immature and adult male and female rats and in agreement with Clark, found the total FSH content of the immature female pituitary greater than that of the adult female or immature male, but not as great as that of the adult male. In terms of FSH per unit of glandular tissue, adult male pituitary appeared to be about five times as potent as the pituitary from adult females of comparable age. The LH content of the pituitaries from immature males and females was low and approximately eciual; in adults, the amount had increased considerably and was greater in the males.
Hollandbeck, Baker, Norton and Nalbandov (1956) estimated the potency of sow pituitaries from birth through 1330 days of age, using the chick testis assay. The results were expressed in terms of the weight of the anterior lobe and correlated with age and body weight. The unit potency decreased linearly with age, whereas total potency showed a linear increase, because, of course, the weight of the pituitary increased with age. The amount of hormone available per unit of body weight was very high at birth and declined through the prepubertal period to a low level which remained nearly constant from puberty onward. This steady drop in available hormone per unit of body weight through the jjrepubertal period is not in accord with the general assumption that an increase in titer of circulating gonadotrophin precedes puberty. Such data do not rule out a likely increase in secretion of gonadotrophin nor, as the authors have suggested, the possibility that an imbalance may have occurred in the ratio at which the gonadotrophic factors, FSH and LH, were secreted. The high potency during infancy was thought to be due to the presence of mainly FSH. The initiation of cyclic ovarian activity at puberty was thought to signify augmented secretion of LH, yielding a more functionally balanced FSH-LH ratio. Similarly, from evidence based on the urinary excretion of FSH and LH in the human female, Brown (1958) showed that the prepubertal period may be characterized by a rising level of LH.
Among the primates informative data relating to the ratio and content of FSH and LH in the hypophysis in relation to sex and age have been obtained only for man. Bahn and associates (1953a-d) studied the FSH and LH content of the human hypophysis in infancy, childhood, maturity, and old age. The glands were obtained from persons dying without prolonged terminal illness and were obtained within 1 to 4 hours after death. The anterior lobes were dissected free, weighed, and kept frozen until time of use. The glands from a given age group were homogenized, pooled, and administered to hypophysectomized immature male and female rats in total doses of homogenate corresponding to 1, 3, 10, and 30 mg. wet weight of anterior lobe tissue; FSH was indicated by follicle growth or stimulation of seminiferous tubules, LH by repair of the Leydig cells. In essential confirmation of earlier studies, they found that the hypophysis of the human infant has no detectable FSH or LH activity. By childhood (4 years) both hormones were present in minimal ciuantity. The hypophyses from women of reproductive age and mature men were notably potent in both FSH and LH: they were essentially equipotent per unit of tissue for each hormone, but the female gland being heavier had a total content greater than that of the male. Using different end points and methods of collection, Witschi (1940), Witschi and Riley (1940), and Witschi (1952) reported a sex difference — they list in round figures the total FSH unitage in hypophyses of humans dying after prolonged illness as 50 R.U. for mature males and 10 R.U. for nonpregnant mature females. In keeping with the observations of Henderson and Rowlands (1938), Witschi (1940) and Witschi and Riley (1940) found the human hypophysis to contain not more than traces of LH as determined by the Weaver finch or rat test. However, the validity of this claim of the Iowa group has been challenged by the more meaningful positive findings of Bahn, Lorenz, Bennett and Albert (1953a, c) that the adult human hypophysis is moderately rich in LH.
Burt and Velardo (1954) studied individual hypophyses from 18 i)atients, 9 males and 9 females, ranging in age from 19 to 82 years. Most of these patients had died after prolonged illness and some had received hormone therapy. The glands were assayed by injecting 7.5 mg. of fresh gland homogenate into hypophysectomized male and female rats. Of these human liypoi)hyses, 6 produced reasonably good follicle growth and formation of corpora lutea, 6 produced only minimal follicle stimulation, and 6 exhibited no gonadotrophic activity. In this small series the authors were unable to detect any correlation of pituitary potency with sex or age. It is interesting, too, that they were unable to predict the gonadotrophic potency in individual glands on the basis of a sampling of the relative percentage of cell types present.
There is general agreement that the concentration of gonadotrophin in the hypophysis tends to increase in old age in many animals, but it is only in women that increased release is thoroughly documented, this being demonstrated by the increase in the blood and urinary titer of gonadotrophin at the time of the menopause. Witschi and Riley (1940) and Witschi (1956) reported that the total FSH content of the hypophyses of postmenopausal women is 4- to 5-fold greater than that of women of reproductive age. The hypophyses of old men show great variability, with some glands reaching the high i)otency of those of postmenopausal women and castrated men. Older men show also a fairly consistent small increase in urinary gonadotrophin beginning at about 60 years of age (Pedersen-Bjergaard and T0nnesen, 1948).
3. Gonadectomy
An increase in the gonad-stimulating potency of the anterior hypophysis following gonadectomy or total loss of gonadal secretory functions through any means other than primary pituitary failure has been found to be an almost invariable occurrence especially in the warm-blooded vertebrates. An early report of a negative finding in castrated pigeons (Schooley, 1937) needs confirmation, inasmuch as Breneman (1945) found that the capon pituitary is distinctly more potent than that of cockerels of the same age. Although the gonadotrophin content in sheep pituitary is high, it is at its l)eak during the time of reproductive quiescence (Kammlade, Welsh, Nalbandov and Norton, 1952) and increases in the ewe following sjiaying (Warwick, 1946).
An objective of recent studies has been to elucidate the effects of gonadectomy on the separate follicle-stimulating and luteinizing activities of the pars clistalis.
Bahn, Lorenz, Bennett and Albert (1953b) found a sharp increase in the FSH content of the human hypophysis at the time of the menopause, with no coincident increase in LH. The ratio of FSH to LH was found to be 1:1 in the pituitaries of women of reproductive age and 3:1 in those from women past the menopause. The failure of LH to increase under these conditions in man is at variance with the observation by jVIcArthur, Ingersoll and Worcester (1958) that appreciably more LH is excreted in the urine after the menopause than during the reproductive years.
In recent years the Dutch workers, Paesi, de Jongh, Gaarenstroom, and Hoogstra introduced modifications in their procedure for studying the gonadotrophic activity of rat pituitaries, which provide in their views an estimate of the relative amounts of FSH and LH (for methods see Hoogstra and Paesi, 1955, 1957). Paesi, de Jongh, Hoogstra and Engelbregt (1955) reported that gonadectomy produced within 3 months a 5-fold increase in the FSH content of the pituitaries of females, and no conspicuous increase in males; LH increased following gonadectomy in each sex, but more so in males than in females. Their finding that gonadectomy tended to diminish the sex difference in pituitary gonadotrophic content in rats is in keeping with the experience of others.
4. Reproductive Rhythms
a. Cijclic Vhanges. In those species in which the reproductive activity is closely tied-in with seasonal alterations in the environment, the expectation is that the peaks in i)ituitary potency would correspond with periods of heightened breeding activity. The few observations available suggest that with certain exceptions this is true.
Valuable preliminary data based on a year-round collection of adult pheasant pituitary has been summarized by Greeley and Meyer (1953). The dried glands were pooled by months or pairs of months and tested in day-old chicks for testis-stimulating potency. The potency of the glands was at a minimum in July when the testes of the donor birds were undergoing rapid regres
sion. Before settling to winter level pituitary potency showed a small increase, while
the testes were in what several workers
have termed a "refractory state" (see Marshall, 1955). The major upsurge in pituitary gonadotrophin, correlating well wdth
weight of donor testes, occurred in January,
February, and March, and reached its maximum in April. The close parallel of hypophyseal potency and testis development
throughout the annual cycle is noteworthy.
It indicates that pituitary gonadotrophin
content reflected accurately the rate of
gonadotrophin release. Benoit's early studies
showed that the duck pituitary is weakest
in the off-breeding season and gathers gonadotrophic potency as added illumination
brings the animals into sexual competence;
also Riley and Fraps (1942b) found a lesser
potency in laying than in nonlaying hens.
Some information has been obtained from mammals. The pituitary of the ground sciuirrel has a much reduced gonadotrophic potency during hibernation (Wells, 1935). A peculiar situation was found to exist with respect to the cottontail rabbit at the time of the breeding season. The pituitary of the male showed an increase in gonadotrophic content, whereas that of the female did not (Elder and Finerty, 1943). Assay of mule deer pituitaries (Grieser and Browman, 1956) has indicated that potency in yearling does is lowest in winter months, gradually increases as spring progresses, and reaches a peak by late fall.
In recent years pituitary gonadotrophic potency has been studied throughout the estrous cycle in the cow, sow, and ewe. The gonadotrophic potency of the cow pituitary is at its lowest point during estrus (Paredis, 1950). In like manner, Robinson and Nalbandov (1951) reported that the gonadotrophin content of sow pituitaries collected during estrus was approximately half that of pituitaries taken at midcycle. The potency remained low in glands taken through the first 8 days of the cycle and then climbed sharply.
Reports on cyclic variations in the gonadotrophic potency of the ewe pituitary are at variance. According to Robinson (1951), potency is at its peak during estrus; Warwick (1946), however, found no difference between glands collected during the breed
2G0
HYPOPHYSIS AND GONADOTROPHIC HORMONES
ing season and those obtained in the nonbreeding season. Kammhide, Welsh, Nalbandov and Norton ( 1952 ) , using the same assay as Warwick- — the chick testis — found the potency highest during anestrum and lowest during estrus. Although the reproductive tract of the anestrous ewe was found to be atrophic, the ovaries contained a few sizeable follicles (Hammond, 1945; Nalbandov, 1953a, b) and ovulation was induced by exogenous gonadotroi)hins. Granting that LH is a requisite for the secretion of estrogen as well as for ovulation, Kammlade, Welsh, Nalbandov and Norton (1952) and Nalbandov ( 1953b j speculated that anestrum in the ewe may perhaps be attributed to an imbalance of the gonadotrophins characterized by increased pituitary FSH potency and a deficiency in circulating LH. They surmised that the follicular development was brought about by the action of virtually "pure" endogenous FSH. In accord with this view Dutt ( 1953 1 and Robinson (1952, 1954) reported that ovulation, followed in some instances by pregnancy (Robinson, 1950), can be induced in the anestrous ewe by the administration of progesterone, thereby suggesting that LH is "stored." Progesterone, as noted elsewhere, can be used to induce or hasten ovulation in hens, estrous rabbits, the persistent-estrous rat, and in monkeys during the anovulatory summer months. According to current opinion, these effects are attributable to excitation of neurohumoral mechanisms which promote the release of LH, Missing for the purpose of the present consideration is knowledge of whether or not the pituitary of the anestrous ewe contains LH. Unfortunately the methods used in studying the ewe pituitary have not been informative in regard to the separate gonudotrophic activities.
Especially noteworthy are the data of Simpson, van Wagenen and Carter (1956) concerning the fluctuation in pituitary gonadotrophic potency in adult female monkeys killed at different stages in the menstrual cycle. The low titer of FSH (in unit and total potency) at the beginning of the cycle quadrupled near the end of the follicular phase and decreased during the luteal phase. The unit and total potency of LH was highest on (lavs 9 to 11. TheFSH to LH ratio
was roughly 1:3 at the beginning and end of the cycle and 1 : 10 through the preovulatory and ovulatory stages. This is mainly because the LH increased relatively much more during that time than did FSH. At the time of greatest concentration, the minimal effective dose for both FSH and LH effects was 1/10 that of pooled sheep pituitary powder. The two hormones were, however, present in the same relative proportions as in sheep pituitaries.
b. Pregnancy. Marked differences exist in the gonadotrophic functions of the hypophysis during pregnancy. In a number of animals, of which the monkey, guinea pig, rat, and mouse are examples, the gonadotrophic functions of the pituitary are assumed by the placenta to the extent that the hypophysis can be ablated without interrupting pregnancy (for review see Smith, 1954). There are authenticated instances in which the ovaries involute especially during the later stages of gestation. Contrariwise, in many animals growth of ovarian follicles continues throughout gestation and, in some, heat ensues immediately following parturition (for review see Williams, Garrigus, Norton and Nalbandov, 1956, and chapter by Young on the mammalian ovary). There are many reports of mating and of spontaneous or artificially induced ovulation during pregnancy. It is, therefore, not unexi)ected that marked variation between species has been noted with respect to the gonadotrophic activity of the hypophysis during pregnancy; it has been reported to increase, decrease, or to show little or no change (reviewed by Cowie and Folley, 1955). Within species, the results have not always been consistent; such discrepancies are probably related to the different procedures that have been used for collecting, storing, and assaying the glands. In a notable example, the pituitaries of pregnant cattle were observed to show a steady increase in gonadotrophic content over those of nonl)rcgnant animals (Bates, Riddle and Lahr, 1935), but in a later study (Nalbandov and Casida, 1940) the potency was found to decline steadily throughout ])regnancy. Simihiily. Robinson and Nalbandov (1951) found that the gonadotrojihic potency of the pregnant sow decreases throughout gesta
PHYSIOLOGY OF ANTERIOR HYPOPHYSIS
261
tion. There is agreement that in man the pituitary gonadotrophic jiotency falls during the second month of gestation and remains barely detectable until a few days postpartum (Bruner, 1951). It has been assumed that in man placental gonadotrophin (HCG) is an important sustaining factor. The pregnant mare, however, also has a rich extrapituitary supply of gonadotrophin, yet shows no decline in pituitary gonadotrophic potency (Hellbaum, 1935). Some have tried to relate the instances of reduced pituitary potency during gestation to high titers of circulating estrogens, but the inconsistencies are again extreme. (For reviews of this controversial literature see Burrows, 1949; Ladman and Runner, 1953; Cowie and Folley, 1955). Recent reports have been few. Ladman and Runner (1953) in a detailed study of the pituitaries of l)regnant mice found changes suggestive of a cyclic fluctuation in potency during gestation. In breeding mule deer the peak potency occurred in the fall and was followed by a sharp decline which persisted during most of gestation with a slow rise occurring near term (May or June).
C. EFFECT OF DIETARY RESTRICTIONS ON
PITUITARY GONADOTROPHIC POTENCY
AND FUNCTION
1. Underfeeding
There is a large bodj' of experimental data showing that poor nutritional status whether caused by reduced food intake or impaired assii^iilation has a deleterious influence on reproductive functions (for comIH-ehengive review of nutrition-endocrine relationships, see Ershoff, 1952 and chapter l)y Leathem). It has long been known that in the common laboratory animals controlled inanition and starvation produce atrophy of the gonads and accessory sex structures and varying degrees of infertility. Similarly, loss of reproductive functions have been observed in human populations during periods of restricted food intake. That the level of circulating gonadotrophins is reduced under these conditions is indicated by involution of the gonads and also, as shown in rats, by the fact that the gonads remain sensitive to administered gonadotrophins (Werner, 1939), the excep
tion being that stimulation of the testis
requires an adequate replacement of vitamin E. Since it has been found that the
pituitary gonadotrophin content remains
at normal (Maddock and Heller, 1947) or
somewhat greater than normal levels (^leites and Reed, 1949; Rinaldini, 1949) during
chronic caloric restriction, it would seem
that the primary defect in pituitary function is failure, not so much in the synthesis,
as the release of hypophyseal gonadotrophins. Aladdock and Heller (1947) also emphasized the dichotomy that is evoked
through inanition between pituitary gonadotrophic content (normal) and gonadotrophic function ( low ) . This involves the
important assumption that the gonadal tissues have not lost their sensitivity to gonadotrophic stimuli as a result of undernourishment. That exogenous gonadotrophins are
effective under these circumstances does not
exclude the possibility of a relative reduction in substrate sensitivity. It is of interest
that castration type cells do not develop in
the pars distalis during inanition despite the
severe reduction in gonadal functions. That
pituitary potency does not increase pari
passu with inhibition of release of gonadotrophin is an indication that production of
gonadotrophins is also impaired during these
periods of restricted food intake.
a. Related observations on inanition and sex function in man. Many times in the history of man he has been exposed to malnutrition of considerable duration. Currently, two-thirds of the world's population is undernourished and many authors incline to the view that actual starvation will become more widespread in the future. Although the associated endocrine disturbances have been given little attention in these conditions, there is convincing evidence that they constitute an extremely important part of the syndrome. Among the clinical manifestations of chronic underfeeding, there are generally symptoms attributable to dysfunction of the thyroid, adrenals, gonads, and pituitary. The few laboratory experiments in human starvation have not made full use of the various indices of endocrine function. Moreover, there is much doubt that studies of the nature of the Minnesota Experiment (Keys, Brozec, Henschel, Michelson and Tavlor, 1950) conducted under situations
262
HYPOPHYSIS AND GONADOTROPHIC HORMONES
of personal security and absence of other than hunger-induced anxieties are to be compared with either mass starvation or the exigencies of war deprivations.
That many of the findings in experimental animals are applicable to man is suggested by the high incidence of impaired reproductive functions in women during states of chronic malnutrition (Keys, 1946; Keys, Brozcc, Henschel, Michelson and Taylor, 1950; Samuels, 1948; Gillman and Gillman, 1951; Zubiran and Gomez-Mont, 1953).
One of the better documented studies of the endocrine aspects of chronic malnutrition in man is being made in Mexico by Zubiran and Gomez-Mont. In 1953 they reported on 529 adult subjects, all with a long history of undernourishment, and 195 autopsies of subjects suffering the effects of starvation. Estrogenic activity as measured by estrogen excretion and by vaginal smears was found to be absent in a high percentage of cases in women of menstrual age. The number in which menstruation had ceased was ecjually high. These evidences of the severity of the disturbances of ovarian function were fully borne out by examination of ovaries at autopsy after prolonged starvation. The ovaries were extremely small and atrophic and not infrequently absent.
It is pertinent also that Zubiran and Gomez-Mont, contrary to the work of Biskind (1946) and of Lloyd and Williams ( 1948) , found no evidence of hyperestrogenism in patients with impaired liver function. Their data show clearly that the excretion of urinary estrogen is low in a high percentage of cases during chronic malnutrition, irrespective of the presence of cirrhosis or the extent of liver impairment. During recovery, however, these workers often found a transitory increase in estrogen excretion sometimes of great magnitude. In males, such increases usually preceded the appearance of gynecomastia which generally outlasted the period of heightened estrogen titers. Zubiran and Gomez-Mont believe that unawareness of the effect of refeeding on estrogen production may furnish an explanation for most of the reported cases of hyperestrogenism which have been attributed to liver damage and the failure to inactivate estrogen.
2. Vitamin Deficiencies
Gonadal dysfunctions of varying severity are also noted in animals fed diets deficient in one or another of the various B vitamins (thiamine, Drill and Burrill, 1944; imntothenic acid, Figge and Allen, 1942; riboflavin, Warkany and Schraffenberger, 1944; pyridoxine, Emerson and Evans, 1940, Nelson and Evans, 1951; biotin, Okey, Pencharz and Lepkovsky, 1950; and B12 , Hartman, Dryden and Gary, 1949; the references cited are inter alia). In each instance the data suggest an impairment of the secretion of gonadotrophins and there is considerable agreement that this, in turn, is attributable to the accompanying inanition rather than to any specific vitamin deficiency per se. With respect to pituitary hormone content, Wooten, Nelson, Simpson and Evans, (1955) reported finding a striking increase in gonadotrophic potency in vitamin Bedeficient rats. In terms of the separate gonadotrophins they found a 3- to 4-fold increase in FSH and little or no change in LH or LTH. With respect to vitamin E, it has been established that in rats and guinea pigs a deficiency of this factor injures the seminiferous tubules and causes resorption of embryos. Inconsistent findings have been reported with respect to the pituitary gonadotrophic potency in vitamin Edeficient rats: an increase was noted by P'an, van Dyke, Kaunitz and Slanetz (1949), and no change by Biddulph and Meyer (1941). The defects produced by absence of vitamin E are in the gonads and are not correctable by administration of gonadotrophin (Mason, 1933; Drummond, Noble and Wright, 1939; Ershoff, 1943). Despite extensive study, the effect of vitamin A deficiency on pituitary gonadotrophin content has not yet been clearly defined (for a review of literature see Ershoff, 1952).
3. Deficiencji in Intake of Protein or of Spe cific Amino Acids
The effect of protein deprivation on hypoi)hyseal functions has been lately re-examined by Leathern (1958) and is discussed in his chapter. He has emphasized the importance of a lal>ile body proteni i-eser\-e and the biologic value of
PHYSIOLOGY OF ANTERIOR HYPOPHYSIS
263
dietary protein for the maintenance of normal reproductive performance and for recovery from the impairments induced by severe protein restriction. Female rats fed a diet containing less than 6 per cent protein showed marked atrophy of the reproductive organs, whereas male rats fed protein-free diets showed little impairment of spermatogenesis or loss of testis weight although the accessory sex organs were reduced by 50 per cent. Considerable data are available indicating that protein restriction impairs the secretion of gonadotrophin in female rats (Samuels, 1950). Less attention has been paid to pituitary hormone content. Leathem (1958) noted a reduction in total gonadotrophic potency in male rats fed a protein-free diet, whereas Srebnik and Nelson (1957) obtained an approximate 2fold increase in FSH content with no change in LH. They found also that protein restriction did not interfere with increases in production and release of FSH and LH following ovariotomy.
Rabbits are surprisingly resistant to protein depletion. Friedman and Friedman (1940) found that female rabbits maintained on protein- free diets continued to manifest estrus and their capacity for restitution of the gonadotrophic hormone potency of the anterior pituitary following ovulation was unimpaired. The relationship of deficiencies in specific amino acids to pituitary gonadotrophic functions have not Ijeen satisfactority elucidated. The conflicting eviderite4s^ reviewed by Ershoff (1952).
IV. Gonadal-Hypophyseal Interrelationships
The dependence of the gonads on the secretions of the anterior lobe of the pituitary has been clearly demonstrated in the mammals and, although less well documented, exists for all vertebrates, to a greater or lesser degree. This dependence has brought about the consideration of the adenohypophysis as a "master gland," but this designation connotes a degree of independent control over the endocrine function of the gonads and the other so-called target organs which is not strictly in accord with the facts. There is strong evidence that each of the target organs is an important factor
in the determination of the functional level of the hypophysis. The gonads and the pituitary play upon each other toward the achievement of a balance of function, harmonious with the fulfillment of the procreative functions of the organism. This reciprocal relationship, long known to endocrinologists, has been referred to as "negative feed-back" or "push-pull." Variations in these interrelationships coincide with the changing epochs of the life of the individual animal. In the mammals the main phases are immaturity, puberty, maturity, and senescence. During each there are subtleties of interplay between hypophyseal and gonadal influences. Mention should be made of the fact that these interactions of the hypophysis and gonads are in large measure dependent on the permissive conditioning of the internal environment by other glands, but especially b}^ the thyroid and adrenals (see chapters by Albert and by Young on the ovary).
A. IMMATURITY
Following birth there is a period during which the ovaries are quite unresponsive to administered gonadotrophins. During this period, extending to about 15 days of age in rats, follicles are present which are indistinguishable morphologically from others which respond readily at an older age. Although the reason for this ovarian insensitiveness to extraneous gonadotrophins during infancy is unknown, Hisaw and Astwood (1942) suggested that there must be ]ihysiologic differences between follicles that are morphologically similar. Zuckerman (1952), on the contrary, believes that the follicles become responsive only after a theca interna has been fully differentiated. Along this same line Hisaw (1947) speculated that the secretion of estrogen by the theca is a necessary preliminary to the attainment of follicular sensitivity to gonadotrophins. He suggested that estrogen acts on the immature follicle in the manner of an organizer, thus rendering the granulosa competent to respond to FSH. Although there is considerable evidence for a direct action of estrogen on ovarian follicles (discussed more fully on page 268 and in the chapter by Young on the ovary) the assump
264
HYPOPHYSIS AND GONADOTROPHIC HORMONES
tion that initiation of ovarian secretion precedes the capacity to respond morphologically to gonadotrophins has not yet been substantiated by experimental evidence.
Under normal circmiistances, it is possible that gonadal maturation is brought about by the elaboration of gradually increasing amounts of pituitary gonadotrophins, primarily FSH, or by a gradual increase in competence of the gonads to respond to gonadotrophins already present even in immaturity. It is tempting to surmise that the gonads of the immature animal elaborate sufficient steroid to suppress the development of gonadotrophic functions. There is some evidence that this may be true in females but the situation is less clear in males. Gonadectomy at birth, as Clark (1935b I has shown, leads quickly to an increase in pituitary gonadotrophic potency in females but not in males. The mechanism which sustains immaturity may therefore not be the same in the two sexes.
Despite the apparent refractoriness of infantile ovaries to gonadotrophins of exogenous origin, they respond readily to excessive endogenous gonadotrophins. Thus, ovaries of newborn rats transplanted to the anterior ocular chambers of spayed adult rats respond quickly as shown by an increase in size, follicle maturation, and ovulation, and by the re-establishment of cyclic estrus in the host (Dunham, Watts and Adair, 1941). Ovaries which would not normally mature until 45 to 70 days of age were functional as grafts at 10 to 20 days of age. This hastening of maturity was not influenced by the period between spaying and grafting, i.e., grafting at the time of spaying was just as effective as grafting at a later time.
It is pertinent that considerable advancement of sexual maturation was seen by Greep and Chester Jones (1950b) in rats in which the ovaries were removed on the 26th day of life and grafted into the neck. These young animals exhibited opening of the vaginal membrane and vaginal estrus within 5 to 10 days. In this strain of rats vaginal patency normally appears at about 55 days of age. It was assumed that in the interval before circulation was re-established in the graft the i)ituitary had increased its secretion of gonadotrophins as it is known to do when "lonadal secretions are (>liniinate(l or
rendered ineffective. The revascularized ovaries responded precociously to this heightened pituitary stimulation. Although ]Mandl and Zuckerman (1951 1 were not able to confirm these findings, it is to be noted that the vaginas in their control rats opened at a mean age of 37 days, which allows small latitude for demonstrating a hastening of vaginal opening by this procedure. Many workers have seen precocious vaginal estrus in immature female parabionts following gonadectomy of their partners (for review see Finerty, 1952» and an increase in blood level of FSH has been detected in rats 7 days after spaying (Cozens and Nelson, 1958). It is evident, therefore, that the pitiiitaries and gonads are in delicate hormonal balance through the period of immaturity, any disruption of which leads quickly to alteration of the endocrine state.
B. PUBERTY AND MATURITY
Puberty is characterized by comj^lex interactions between the gonadotrophins and the sex steroids. These become particularly evident in the cyclical episodes in the life of the mature female (Young and Yerkes, 1943). Experiments which have attempted to unravel these basic pituitary-gonadal interrelationships and the mechanisms responsible for cyclic gonadal functions have involved, as one method of attack, the injection of steroid hormones into intact ])ubertal or adult female mammals.
The influence of the steroid hormones, especially that of the gonadal hormones and their congeners, on the pituitary has been extensively studied. The great variety of experimental procedures employed in these studies has not negated their importance, but it has made comparison of results for a given steroid or between different steroids more difficult and of less validity than might otherwise have been the case. Van Dyke (1939) and Burrows (1949) have provided comprehensive reviews of these data. It will serve the purpose here to examine recent studies wherein a more discriminatory methodology has been employed. These will illustrate the major ai^proaches and recapitulate earlier findings.
When any steroid, natural or synthetic, endogenous or exogenous, acts on the pituitarv. the alternatives ai'c that its function
PHYSIOLOGY OF ANTERIOR HYPOPHYSIS
265
will be increased, decreased, or left unaffected. For practical reasons alone it has been disappointing that the results achieved have been predominantly those of inhibition. Consequently the physician is today fairly well armed with ways of slowing, but not of arousing, pituitary gonadotrophic activities.
C. EFFECTS OF ESTROGENS ON FOLLICLESTIMULATING HORMONE SECRETION
The result of the administration of steroids to animals depends on not only the state of the animal, but also the dose of the hormone and duration of the treatment. Among the ^nown inhibitors of pituitary gonadotrophin secretion, the estrogens are the most effective. There is complete agreement that estrogen in moderate to high dosage inhibits FSH synthesis and liberation. No clear qualitative differences have been encountered in the effect of a variety of natural and synthetic estrogenic steroids in this regard. Quantitative differences in the capacity of the many available estrogens to suppress FSH secretion, however, are readily demonstrable and are directh" referable to the estrogenicity of the steroid. Present evidence suggests that, although l)hysiologically equivalent dosages of a number of estrogenic substances do not produce precisely comparable effects on the pituitary, a close correspondence exists between the ability of an estrogen to induce vaginal estrus and its inhibitory action on the pituitary. "^^^- The influence of relatively large amounts of estrogens on the secretion of gonadotro])hins is of interest, but particularly helpful in resolving the problems of normal physiology are those experiments in w^iich low dosages of estrogens, at or near the naturally occurring levels, have been given. Here, however, there is not agreement with respect to the meaning of the results. It has often l)een claimed that maturity may be* achieved by pituitary function independent of estrogen secretion. In the development of a broader concept. Heller and his co-workers were the first to take the position that amounts of estrogen falling within physiologic limits have no suppressive action on pituitary potency in rats nor on the secretion of gonadotrophins as measured by urinary
excretion (of F8H> in women. Thus Lauson, Heller and Sevringhaus (1938) reported that ovarian development and sexual maturation in rats were not altered by chronic treatment with what they considered to be physiologic quantities of estrogen, nor did the pituitaries exhibit any significant decrease in gonadotrophin content. Heller and Heller (1939) and Heller, Heller and Sevringhaus (1942) found that amounts of estrogen which inhibited ovarian compensatory hypertrophy in rats following unilateral castration did not decrease pituitary potency; and in women doses of estrogen, adequate to control clinical symptoms at the menopause, did not diminish the excretion of gonadotrophin. Heller, Chandler and jMyers (1944) also reported that whereas physiologic doses of estradiol failed to prevent the typical rise in the titer of urinary gonadotrophin following ovariectomy in women, larger doses were completely effective. This series of papers by Heller and his associates has long been puzzling in the interpretation of pituitary-gonadal relationships. It is a weak point in their evidence that they used uterine stimulation in recipient immature intact rats as the end point for the assay of pituitary gonadotrophin potency. It is doubtful if this procedure is sufficiently reliable for the establishment of this important concept. It is to be noted that in the domestic fowl Breneman (1955) likewise has observed no suppressive effects on gonadal maturation using doses of estrogen that are wathin the upper limits of normal blood estrogen levels. Heller's observations have been contradicted by later observations based on more critical assay procedures. Biddulph, Meyer and Gumbreck (1940) suggested that gonadotrophic functions in rats are inhibited by doses of estrogen well below the threshold dose for the estrous reaction of the vagina. Of greatest interest and pertinence, Byrnes and Meyer (1951a) found that the minimal amount of estradiol or estrone required to stimulate the uterus in the spayed member of spayed-intact parabionts was 3 to 4 times that needed to inhibit the pituitary of the same animal as judged by the ovaries of the adjoined twin. In an extension of this study Byrnes and Meyer (1951b) administered estradiol in closely graded doses to single immature (30-dav-old) and
2G6
HYPOPHYSIS AND GOXADOTROPHIC HORMONES
adolescent (55-day-old) rats, intact and spayed, for 10 days. The amounts given were estimated to be within physiologic limits. In the immature animals more estrogen was required to produce positive stimulation of the uterus than to inhibit the pituitary; the evidence for a similar differential in the adolescent group was equivocal. Greep and Chester Jones (1950b), using adolescent rats, observed that a dose of
estradiol benzoate which would inhibit ovarian development in intact rats was adecjuate for uterine maintenance in coetaneous castrates. Byrnes and Meyer (1951b) observed further that the quantity of estrogen required to inhibit the pituitary increased from the immature to the adolescent rats by a factor of 2.77. Although dilution of hormone is involved because of the greater size of the older animals, this did not seem to invalidate the conclusion that the anterior pituitary becomes increasingly less sensitive to the suppressing action of estrogen as sexual maturation progresses.
These data indicate that FSH secretion and estrogen bear a reciprocal relationship and emphasize that FSH secretion is not a pituitary property sui generis but depends
- on the concomitant secretion of steroids.
This idea has an important bearing on how a state of sexual maturity is attained. If we consider that the evidence is in favor of a prepubertal gonad-pituitary interaction, we must assume that the immature gonad has some capacity to secrete estrogen. In an unconfirmed report, Zephiroff, Drosdovsky and Dobrovolskaya-Zavadskaya (1940) have
, claimed that immature rat ovaries have a significant estrogen content. Byrnes and Meyer (1951b) have postulated that refractoriness of the adolescent rat pituitary to estrogen permits higher levels of FSH secretion and thus allows the attainment of l)uberty. It seems clear that future progress in this area will depend on the development of satisfactory methods for measuring the blood level of pituitary and ovarian hormones. Until such information is available it would be helpful merely to have some of the present evidence confirmed. If, for instance, it were firmly established that the pituitary of the immature animal is more sensitive to estrogen than is the uterus, this
would form an excellent starting point for future investigations.
Exceptionally valuable information concerning the effect of administered steroids on pituitary function has been gained from studies of parabiotic rats. The preparation which has been most commonly employed has consisted of immature rats, one of which is castrated at the time of or following the surgical union. Given no treatment, as in control pairs, the castrated animal's pituitary steps up the output of gonadotrophins, thereby stimulating the gonads of the adjoined intact member. The resulting ovarian secretions do not reach the pituitary of the castrated partner and hence exert no inhibitory influence on it. Using substitute steroids administered from the time of spaying, it has thus been possible to ascertain their ability to inhibit the castration-induced hypersecretion of gonadotrophins. The uterus of the castrate provides an index of the estrogenicity of the administered steroid.
Meyer and Hertz (1937) demonstrated convincingly that larger doses of estrogen or androgen are required to inhibit the pituitary of a male than of a female rat. In each the degree of gonadal inhibition in the intact member was roughly proportional to the dose of sex steroid administered to the conjoined castrate. Biddulph, Meyer and Gumbreck (1940) observed that the minimal amounts of sex hormones required for complete inhibition of the postcastration rise in secretion of pituitary gonadotrophins were in females: 0.025 fxg. estradiol, 1.5 /xg. estriol, 1000 /xg. progesterone; and in males: 0.15 fxg. estradiol, 10 ^g. estriol, and 1000 /xg. progesterone. These workers also noted that the order of effectiveness of estrogens in suppressing gonadotrophin secretion paralleled the order of their capacity to i)roduce vaginal cornification in rats, viz., estradiol, estrone, and estriol.
In male animals estrogen damages the spermatogenic epithelium of the testes to the point that spermatogenesis is completely interrupted and the testes shrink to infantile size. At the same time the basophils of the pituitary are severely degranulated and reduced in numbers relative to other cell types, and the gonadotrophic potency of
PHYSIOLOGY OF ANTERIOR HYPOPHYSIS
267
the pituitary nearly vanishes. In the human male the excretion of urinary gonadotrophins is sharply reduced.
D. EFFECT OF ESTROGEN ON LUTEINIZING
HORMONE AND LUTEOTROPHIN
HORMONE SECRETION
An important consideration in assessing the action of steroids on pituitary function is the effect these have on the output of the luteinizing hormone. In most of the prior discussion reference to inhibition of gonadotrophin secretion has been to that of FSH. The assumption that estrogen acts in some manner to elicit a more effective luteinizing stimulus from the pituitary appears in nearly all papers dealing with pituitarygonadal relationships. It constitutes a key point in all theories so far advanced to explain sexual periodicity in the female. The idea that estrogen may act to release LH originated with the "Hohlweg effect." This author (1934) found corpora lutea appearing in the ovaries of immature rats as the result of a single injection of estrogen. This response has not been obtained by other workers (Bradbury, 1947; Greep and Chester Jones, 1950aj using 21-day-old immature rats. It seems that the response is obtained only if the rats are nearing sexual maturity (Burrows, 1949). In adult rats massive daily doses of estrogen induce a type of pseudopregnancy and have a decided effect on the corpora lutea; they increase inxsize to resemble corpora lutea gravidari, and-~ai'e functional as shown by their ability to maintain diestrous vaginal smears for 3 weeks in face of heavy estrogen treatment (Merckel and Nelson, 1940). To what extent such changes are due to LH or LTH, or both, is not clear. It might be expected that the large amount of estrogen would suppress LH secretion. If this were the case, continued LTH secretion would be independent of estrogen titers and, furthermore, would be capable of both maintaining and evoking secretion from the corpus luteum. In the rat, at least, this seems to be consonant with the capacity of pituitary homografts to secrete LTH selectively (Everett, 1956). Hellbaum and Greep (1940, 1943) noted that the pituitaries of castrated rats treated with estro
gen for more than 20 days gradually lost
their LH potency. They presumed that the
LH component was released, but they were
not able to detect it in the bloodstream with
certainty. Greep and Chester Jones (19501)1
tried to demonstrate by several means an
LH-releasing action of estrogen in intact
and gonadectomized rats of both sexes, but
were forced to the conclusion that the fundamental effect of estrogen on the pituitary
appeared to be to reduce the synthesis and
storage of LH. A further point against the
concept that estrogen triggers off an out
l^ouring of LH from the pituitary is the
fact that no sudden upsurge in LH activity
has been observed in the male by an injection of estrogen, as judged by the condition
of the testicular Leydig cells or by the size
and histologic appearance of the ventral
prostate.
There is evidence, however, favoring the concept that estrogen promotes release of LH. Funnell, Keaty and Hellbaum (1951) administered estrogen to a selected group of patients manifesting classical menoj^ausal symptoms and were able to demonstrate LH activity in the urine where previously only FSH activity had been detectable. Confirmatory results ha\-e been reported by Brown (1956, 1959) using patients with secondary amenorrhea.
The relation of estrogen to ovulation bears on the purported LH-releasing action of this substance. Everett, Sawyer and Alarkee (1949) succeeded in hastening ovulation in normal cycling rats by a properly timed injection of estrogen, but the response is not specific inasmuch as other substances, including progesterone, can produce the same effect. The work of Everett (1948) and co-workers (for review see his chapter in this book) suggests that estrogen may act on the anterior hypophysis indirectly through some neural, conceivably hypothalamic, mechanism. Estrogens have been used with variable, but generally poor, success in promoting ovulation. The fact that the estrous rabbit does not ovulate in response to injected estrone (Bachman, 1935) is not considered critical evidence, since ovulation in this species normally involves neural excitations associated with mating. More decisively the persistent
268
HYPOPHYSIS AND GONADOTROPHIC HORMONES
estrous rat, which ovulates with such regularity after the administration of progesterone (Everett, 1940; Hillarp, 1949; Greer, 1953) or testosterone (INIarvin, 1948), does not thus respond to estrogen unless pretreated for several days with progesterone (Everett, 1950). The latter result indicates that progesterone may synergize with or otherwise facilitate the LH-releasing action of estrogen. Hammond, Jr. (1945), on the other hand, succeeded in obtaining ovulation in anestrous sheep with low, but not with high, doses of estrogen. It would seem that the effect of estrogen on LH release varies between species and within species and is greatly influenced by the age of the animal, dosage used, and the time in the sex cycle at which treatment is instituted.
E. DIFFERENTIAL EFFECTS OF GONADAL
STEROIDS ON FOLLICLE-STIMULATING
HORMONE AND LUTEINIZING
HORMONE SECRETION
The root of the matter in the regulation of gonadal function is the interplay of FSH and LH with the sex steroids. With respect to estrogens, the results do not lead to a clear-cut conclusion. Paesi (1952) provided data on a series of immature rats treated for 7 days with doses of estradiol benzoate, ranging from 0.0002 to 100 /xg. daily. The dose-response curve representing the effect of estrogen on ovarian weight was diphasic. Doses of 0.01 to 0.05 /xg. daily decreased the ovarian weight, whereas greater amounts up to 10 fxg. increased the ovarian weight slightly; however, only with 100 /xg. did the gain attain significance. The decrease in ovarian weight with low doses represents, he believes, an impairment of LH release, inasmuch as the ovarian interstitium seemed deficient. The enhanced ovarian weight with excessive dosage was presumed to be due either to increased FSH release or to FSH enhancement by released LH, as suggested by the stimulated interstitium. It is to be noted, however, that even in hypophysectomized immature female rats, excessive doses of estrogen produced significant ovarian enlargement and stimulate the development of an unusual number of medium sized "solid" follicles (Williams, 1940; Pcncharz, 1940; Gaarenstroom and de Jongh, 1946; Payne and Ilellbaum, 1955).
Bradbury (1947) proposed that the effects of estrogen on pituitary gonadotrophic functions are made more accountable when the results are considered in terms of acute and chronic estrogen treatment. The latter is well known to suppress gonadotrophic function and to deplete the gonadotrophin content. His acute experiments lasted only 48 to 120 hours and revealed a significant increase in ovarian weight, which confirms the results reported by Price and Ortiz (1944) and others. Greep and Chester Jones (1950b) also obtained a confirming result and showed further than the reaction was dependent on the presence of the pituitary. In this connection it is pertinent to note that Bradbury found a concurrent reduction in pituitary gonadotrophic potency at 72 to 120 hours after the injection of estrogen and concluded that the initial action of estrogen is to release gonadotrophin (unspecified, but the effect is mainly that of FSH) . The stimulated ovaries showed in addition to accelerated follicular growth a swollen interstitium, suggesting that both FSH and LH had been released. The interstitial cell change was not confirmed in a later study (Greep and Chester Jones, 1950b). Thus, although it seems clear that under a given circumstance the immediate reaction to estrogen may be a slight upsurge in either FSH release or enhancement of FSH action, it is unlikely that FSH production or release is normally dependent on estrogenic action, since both its synthesis and release are greatest when no estrogen is present.
Meyer, Biddulph and Finerty (1946), Gaarenstroom and de Jongh (1948), Greep and Chester Jones (1950a, b), and Byrnes and Meyer (1951b) attempted involved analyses of the effect of gonadal steroids on pituitary function in terms of the separate gonadotrophins, FSH and LH. The wide divergence in their accounts emphasizes how poorly these matters are understood.
F. EFFECTS OF ANDROGENS ON PITUITARY GOXADOTROPHINS
Considerations similar to those enumerated in our discussion of the estrogens apply to the influence of androgens on pituitary gonadotrophins, yet with well defined differences. The effects of androgens have been studied in females quite as much as in males.
PHYSIOLOGY OF ANTERIOR HYPOPHYSIS
269
The early idea that the gonadal hormones were sex specific has been abandoned and the possibility of abnormal functioning is hardly considered when androgens are studied in the female. Perhaps it should be, because in many females among the Mammalia androgens are not known to have a physiologic role. There is much doubt also as to how importantly androgens contribute to the regulation of the pituitary in the female. For the most part, injection of androgen into female mammals produces a deleterious effect on ovarian development, structure, and cyclic functions. Two exceptions may be noted: van Wagenen (1949) injected sexually immature female monkeys with androgen for long periods and obtained a remarkable hastening of the appearance of puberty. The age at first menses was, in fact, reduced by nearly one-half. Androgen injections have also been used successfully to promote ovulation in persistent-estrous rats (Marvin, 1948).
Very soon after the pure androgenic steroids became available, it was established that these substances in adequate dosage (which varied according to the androgenicity of the compound) (1) caused a partial reduction of the pituitary gonadotrophic potency in castrated rats (Heller, Segaloff and Nelson, 1943), and (2) prevented the appearance of castration cells in the pituitary (Nelson, 1935; Nelson and Merckel, 1937a; Wolfe and Hamilton, 1937). The work of Hertz and Meyer (1937) and Nelson (1937i^ placed androgen-pituitary relationships onlTijuantitative basis. The relative efficiency of testosterone propionate and dehydroandrosterone in restricting the secretion of gonadotrophins by gonadectomized parabionts was determined by Hertz and Meyer (1937). Each compound in adequate dosage completely suppressed secretion of pituitary gonadotrophins as shown by absence of ovarian stimulation in the intact partner. Using these compounds in dosages less than that required to produce complete ovarian suppression, the degree of inhibition was found to be roughly proportional to the dose.
The androgens evoke clear-cut alterations in pituitary FSH and LH potency and no doubt are in part responsible for the quantitative sex differences in the pituitary
content and level of secretion of these gonadotrophins. On a comparative basis the pituitaries of adult males are distinctly richer in FSH than those of adult females (Hellbaum and Greep, 1938; Greep and Chester Jones, 1950b; Hoogstra and Paesi, 1955; Paesi, de Jongh, Hoogstra and Engelbregt, 1955), yet in the absence of androgen, as after castration, FSH increases as though an inhibiting influence had been removed. Long-term castrates given large doses of testosterone showed only a partial (Heller, Segaloff and Nelson, 1943) or no reduction in gonadotrophic potency. Examining the effect of androgens on the pituitary gonadotrophins of adult intact female rats, Greep and Chester Jones (1950b) found unexpectedly that within a specified range of dosage (0.1 to 0.5 mg. of testosterone propionate per day) , FSH potency was elevated over that of untreated controls. Similar findings have been reported by Pincus (1950), Hoogstra and Paesi (1957), and Paesi, de Jongh and Willemse (1958) . Hoogstra and Paesi made the additional observation that the increase of pituitary FSH with androgen treatment (2 mg. of testosterone propionate daily) is not limited to intact females, but occurs in intact males and gonadectomized males and females as well. Furthermore, the response was not modified by simultaneous administration of 2 fxg. of estradiol benzoate.
Interest in the androgen-LH relationship is stimulated by the consideration that LH acting independently evokes androgen secretion by the testis, whereas its ability to elicit the secretion of estrogen by the ovary necessarily involves other factors, notably FSH. It would be expected then that the LH-testicular androgen relationship might be subject to somewhat more precise analysis on the basis of experimental data, but this has not been fully realized. There is some evidence that a push-pull mechanism is operative. The injection of testosterone for more than 30 days results in the elimination of LH potency from the pituitary glands of long-term gonadectomized rats (Hellbaum and Greep, 1943; Paesi, de Jongh and Willemse, 1958). The latter authors concluded that testosterone depressed the LH content to lower levels in intact rats than in gonadectomized animals of the same sex.
Intact adult female rats treated with testosterone show cessation of cycles (Nelson and Merckel, 1937b), involution of the corpora lutea, and a marked reduction in ovarian weight (Greep and Chester Jones, 1950b j. The minimal effective inhibitory dose is 10 /tg. daily. A dosage 50 times greater (0.5 mg.j, however, does not produce the same severe degree of ovarian atrophy that is seen with long-term estrogen treatment. The ovaries, although small, have a translucent blistery appearance. This is due to the presence of a small number of semi-atrophic vesicular follicles and to the relatively great reduction in amount of ovarian lipids (Greep and Chester Jones, 1950). From the foregoing evidence it would appear that androgen treatment favors FSH storage over FSH release and that androgen can suppress both the elaboration and I'elease of LH.
The effect of exogenous androgens on the gonads of the intact male rat varies widely with the dose administered. Low to moderate doses of testosterone produced severe testicular injury (Selye and Friedman, 1941 ; Shay, Gershon-Cohen, Paschkis and Fels, 1941; Rubinstein and Kurland, 1941; Ludwig, 1950; Greep and Chester Jones, 1950b) ; maximal reduction in testes weight and complete inhibition of spermatogenesis were obtained with 0.1 mg. of testosterone propionate daily for 30 to 45 days. Large doses of androgen (2 to 20 mg. daily) maintained testis size and sperm production even in the absence of the hypophysis (Walsh, Cuyler and McCullagh, 1934; Cutuly, McCullagh and Cutuly, 1937; Nelson, 1937; Leathem, 1944) . The response is produced by direct action on the tubular epithelium of the testes. A similar effect was produced in hypophyscctomized mice (Nelson and Merckel, 1938), ral)bits (Greep, 1939), and monkeys (P. E. Smith, 1944). Local maintenance of spermatogenesis has also been observed in the tubules in close proximity to intratesticular implants of testosterone pellets in hypopliysectomized rats (Dvoskin. 1947) and monkeys (P. E. Smith, 1944 ». Several androgenic steroids and their derivatives have been tested in regard to their ability to maintain the testes in hypopliy
sectomized rats (Nelson, 1937). It is of interest that the spermatogenic properties of
the various androgens were not found to be
related to their androgenicity as measured
by ability of the compounds to stimulate
the rat prostate or the capon's comb. Pregnenolone, a nonandrogenic steroid, did not
reinitiate spermatogenesis, but it did partly
maintain spermatogenesis, when given immediately after hypophysectomy (Dvoskin,
1949).
In man the testes apparently are not benefited by exogenous androgens at any dose level but are, on the contrary, affected adversely. In 1940 Heckel noted a drop in sperm count during testosterone therapy, a finding that is now a common clinical experience. In the past few years much interest has centered on the recovery phase. Heller, Nelson, Hill, Henderson, Maddock, Jungck, Paulsen and Mortimore ( 1950 », Heller, Nelson, Maddock, Jungck, Paulsen and Mortimore (1951 ), and Ewell, Munson and Salter (1950) reported finding a rebound in spermatogenesis and in sperm counts following the cessation of androgen therapy. They observed that for a year or more following cessation of treatment the sperm counts may be well above the pretreatment levels and that scleroses and hyalinization of the tubular walls may be lessened. The number of cases studied has been extended (Heckel, Rosso and Kestel, 1951; Heckel and ]McDonald, 1952; Swyer, 1956) with poor agreement as to improvement in sperm count. Because the already subnormal testis is further damaged by androgen, there is need for additional study of this interesting phenomenon.
Although rats and man have been extensively investigated, little information is available about other mammals. Wells (1943) injected male ground squirrels with testosterone in daily doses from 0.05 to 20 mg. beginning just before the peak of the annual sexual cycle. He found that the interstitial cells were severely damaged and the testes were somewhat reduced in size, but the spermatogenic capacity of tubular einthelium was unimpaired. His assumption that the release of ICSH was being inhibited has been amply substantiated by other studies. His failure to find tubular ati'()i)hv with the low doses is I'atlu'i' sur
PHYSIOLOGY OF ANTERIOR HYPOPHYSIS
!71
prising and one can only conjecture that the release of FSH was not being interfered with. The higher doses were obviously adequate for direct tubular maintenance irrespective of ICSH or FSH inhibition.
G. OTHER STEROIDS AND OXIDATION PRODUCTS
Several synthetic steroids related to the sex hormones but which exhibit no estrogenic activity have proved to be without effect on pituitary gonadotrophin content (]\Iortimore, Paulsen and Heller, 1951). Lipoadrenal extract and desoxycorticosterone acetate produced some uterine development and some inhibition of hypophyseal secretion of gonadotrophins, whereas cortisone was neither estrogenic nor inhibitory of gonadotrophin secretion (Byrnes and Shipley, 1950). Although exogenous estrogens and androgens inhibited the development of intrasplenic ovarian grafts in guinea pigs (Lipschiitz, Iglesias, Bruzzone, Humerez and Penaranda, 1948), and rats (Takewaki and Maekawa, 1952), progesterone and desoxycorticosterone failed to do so.
O. W. Smith (1944, 1945) maintained on the basis of her investigations that it is not the circulating gonadal hormones per se which influence pituitary function and potency but the oxidation products of these hormones. She used mainly the lactone of estrone prepared by W. W. Westerfeld and by Alan Mather. In her hands the lactone both increased the size of the pituitary and decreased its^gonadotrophic potency. The studies of Bradbury (1947) and of Mortimore, Paulsen and Heller (1951) suggested that any pituitary responses induced by estrololactone may be attributable to its estrogenicity, which is approximately 1/100 that of estrone in regard to both estrogenic activity and pituitary gonadotroiihic inhii)ition.
H. EFFECT OF PROGESTERONE ON PITUITARY GONADOTROPHIC FUNCTIONS
Species vary in the extent to which the secretion of gonadotrophins is influenced by progesterone. In the guinea pig which has a 16- to 17-day cycle, progesterone inhibits the preovulatory swelling (generally attributed to LH) , but growth up to this point is unaffected (Dempsey, 1937). In rodents
with short estrous cycles the influence of progesterone on the secretion of gonadotrophins appears to be minor, in that moderate doses do not alter ovarian maturation or cyclic functions (Greep and Chester Jones, 1950b). Although some inhibition of gonadal functions can be demonstrated after massive doses of progesterone, interpretation is always complicated by the weak estrogenicity and androgenicity of the coml)ound. Similarly, total gonadotrophin content of the pituitary is little altered by progesterone.
Although progesterone is primarily a product of the corpus luteum and exerts a major function during the luteal (and gravid ) phase of the cycle, there are strong indications that in some species the secretion of progesterone is initiated in follicles before ovulation (guinea pig, Dempsey, Hertz, and Young, 1936; rat, Astwood, 1939; Boling and Blandau, 1939; mouse, Ring, 1944). Consistent with this conclusion is the finding that the peak level of progesterone in the blood of cyclic rats coincides with the proestrum as determined by the Hooker-Forbes assay (Constantinides, 1947). By the same means progesterone activity has been found in the blood of laying hens but not in nonlaying hens or roosters (Fraps, Hooker and Forbes, 1948. 1949). The preovulatory appearance of progesterone is believed to be important in the elicitation of behavioral estrus (see chapter by Young ) and as a component of the hypothalamo-pituitary triggering mechanism for ovulation (see chapter by Everett).
Current interest in the progesterone regulation of gonadotrophic functions has been focused on the latter mechanism. Progesterone induces or at least hastens ovulation in cyclic-estrous rats (Everett and Sawyer, 1949a), persistent-estrous rats (Everett, 1940), rabbits (Sawyer, Everett and Markee, 1950), hens (Fraps and Dury, 1943; Rothchild and Fraps, 1949), cattle (Hansel and Trimberger, 1952), anestrous ewes (T. J. Robinson, 1954), anovulatory monkeys (Pfeiffer, 1950) , and women (Rothchild and Koh, 1951 ) . Notably too, both ovulation and luteinization have been observed in ovarian grafts in castrated male rats following administration of progesterone (Kempf, 1949, 1950) . Such grafts normally show only
HYPOPHYSIS AND GONADOTROPHIC HORMONES
follicular development. Since ovulation depends on an acute discharge of LH (discussed elsewhere), the question is raised as to the role of progesterone in this regard. Firstly, it is important to note that progesterone probably is not in itself a releasing agent; rather, it appears to synergize with estrogen to facilitate the triggering mechanism. Moreover, although the possibility exists that progesterone acts directly on the hypophyseal cells to release LH, an impressive body of evidence suggests that progesterone promotes LH release (indirectly) by acting on the central nervous system — probably the hypothalamus (for review see Markee, Everett and Sawyer, 1952 and the chapter by Everett) . Under other conditions, quite the reverse of being a stimulus for ovulation, progesterone is a potent inhibitor of ovulation. There can be little doubt that the suppression of ovulation during the luteal and gravid phases of the reproductive cycle is due to progesterone. Single doses of progesterone prevent ovulation in the mated rabbit, and continued high dosage to cyclic rats (Phillips, 1937), guinea pigs (M0ller-Christensen and Fonss-Bech, 1940), sheep (Dutt and Casida, 1948) , and cattle (Ulberg, Christian and Casida, 1951) postpones ovulation indefinitely.
I. NEUROHYPOPHYSEAL INFLUENCES ON GONADOTROPHIN SECRETION
Recent evidence reviewed by Benson and Cowie (1957) has tended to link the posterior lobe of the hypophysis with the mechanism for release of prolactin. Many years ago Selye (1934) demonstrated that the nervous excitation of suckling served to maintain lactation. A reflex arc was believed to be involved and this has been partially identified by later studies. The stimulus of suckling has been shown to act through the central nervous system (Eayrs and Baddeley, 1956) and the hypothalamo-neurohypophy.seal axis (Andcrsson, 1951a, b) and to effect the release of oxytocin (Cross and Harris, 1950, 1951). The latter causes contraction of the myoejiithelial components of the alveoli, thereby effecting milk ejection (let-down). A number of workers (see Donker, Koshi and Petersen, 1954) have obtained evidence that tlic l)cneficial effect
of oxytocin on lactation is more pronounced than can be accounted for on the basis of change in intra-alveolar pressure. The question then arose as to wdiether oxytocin has an indirect as well as a direct action on the mammary structure. Benson and Folley (1956, 1957) noted that continued administration of synthetic or commercial oxytocin retards mammary involution in lactating rats following the interruption of suckling. Inasmuch as a similar effect was obtained by injecting prolactin, Benson and Folley have speculated that oxytocin may provide the stimulus for release of prolactin, ergo LTH, from the anterior pituitary.
Other lines of evidence have also suggested a relationship between the posterior l)ituitary and gonadotrophic functions of the anterior lobe. Desclin (1956a, b) and Stutinsky (1957) have showai in rats that injections of oxytocin at the time of estrus produce a pseudopregnancy reaction. The results suggested enhancement of LTH release, but the significance of these findings has been questioned because the response is not specific for oxytocin. Pseudopregnancy has been induced by the administration of a variety of nonspecific substances, such as plant juice extract (Dury and Bradbury, 1942) and ovalbumin, etc. (Swingle, Seay, Perlmutt, Collins, Barlow and Fedor, 195i) to female rats at the time of estrus.
V. Anatomic Features Important to
Modern Concepts of Pituitary
Gonadotrophic Function
In terms of general morphology there is little to add to the classical knowledge concerning the hypophysis of mammals. Present considerations turn largely on the details of the vascular supply and the innervation of the gland. In this regard the literature has been notably enriched by Green's (1951a, 1952) comprehensive study of 76 species ranging from amphioxus to man and l)y Wingstrand's monograph (1951 ) on the structure and development of the a\ian jiituitary. The notable advances which have been made in the cytologic and eh'ctron niici'oscopic identification of cell tyi)es in the pars distalis are reviewed in the chapter by Purves.
A distinct regional lobulation of the pars distalis. fii'st described in the eliicken and
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273
duck, was subsequently shown to be widely characteristic of birds (Rahn and Painter, 1941, 18 species; Wingstrand, 1951a, 50 genera). According to these authors the pars distalis in birds comprises two histologically distinct parts, which they designate "cephalic" and "caudal" lobes. The zonation develops during embryonic life and, in stained preparations of the adult gland, is evident to the naked eye. A conspicuous regional distribution of specific cell types has been noted also in the anuran hypophysis (Dawson, 1957) . These observations in lower forms provide an evolutionary background for the numerous instances of regional distribution of cell types described in mammalian pituitaries (Dawson, 1939, 1948; Halmi, 1950, 1952; Purves and Griesbach, 1951a, b, 1955; Ferrer and Danni, 1954). Ferrer (1957) noted a correlation between the arrangement of the vascular supply to the adenohypophysis in rats and the pattern of distribution of basophils. Three zones were recognized, each of which has a particular basophilic picture. Dawson (1957) likewise noted a rather specific relationship of cell types to the vascular pattern in the frog. The observation that a pars intermedia is absent in chickens (Kleinholz and Rahn, 1939) has been confirmed in all species of birds thus far examined (Rahn and Painter, 1941; Wingstrand, 1951; Marshall, 1955).
A. INNERVATION OF THE HYPOPHYSIS
The qu^tion of the innervation of the hypophysislia^HDecome a matter of critical importance in the elucidation of mechanisms regulating the various functions of this organ, especially those of the anterior lobe. Fibers of hypothalamic origin sweep down the infundibulum in great numbers to end mainly in the processus infundibularis and, to a minor and perhajis questionable extent, along the infundibular stem. Early workers generally found some of these neurohypophyseal fibers entering the pars intermedia and terminating in the pars distalis, but never in numbers to inspire confidence in their significance.
Vazquez-Lopez (1949, 1953) advocated the view that there is an adequate anatomic basis for neural control of the adenohypophysis. Using modifications of the classical silver impregnation techniques of Cajal
and Rio del Hortega, he observed presumptive nerve fibers coursing through the pars distalis of the rabbit. These terminated with typical nerve endings in connection with the glandular cells. The fibers were claimed to originate from the tractus hypophysius, to cross over to the pars tuberalis in abundance, and in fewer numbers to follow the general course of the vascular elements to the pars distalis. In 1952 Vazquez-Lopez and Williams, reporting on their examination of these relationships in the rat, described the presence of rather sizeable nerve bundles in the marginal zone of the median eminence, and in the pars tuberalis. They were uncertain of the origin and course of these fibers. In a study of the horse Metuzals (1954), using the Bielchowsky and the Gomori staining methods, traced presumptive nerve fibers from the hypothalamus to their endings in the pars distalis. Of the fibers seen in the pars tuberalis, most are held by Stutinsky (1948), Christ (1951), Nowakowski (1951), and Benoit and Assenmacher (1951a) to be derived from similar fibers which have been observed in the marginal zone of the median eminence and along the inferior aspect of the neural stalk. A dense "secretomotor ground plexus" specifically innervating the gland cells has been described by Metuzals (1956). With respect to Metuzals' preparations, A. J. Marshall (1955) states that they "undoubtedly reveal an extensive and specific innervation of glandular cells in the pars distalis." In the ferret, also, sparse nonmyelinated nerve fibers and end organs of characteristic appearance have been observed (R. N. Smith, 1956) by a modification of Ranson's pyridine-silver method. In all these instances the nuclei of origin of the fibers remain obscure.
Truscott (1944) and Wingstrand (1951a) among others described autonomic fibers entering with and terminating along the sinusoids of the anterior lobe. In the pars distalis of man Hagen (1951) reported the finding of extremely fine fibrillar networks, which were believed to enter the pars distalis from the capsule of the gland; and Westman, Jacobsohn and Hillarp (1943) reported the persistence of an autonomic fiber system in the anterior pituitaries of rabbits after cervical sympathectomy. Metuzals
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HYPOPHYSIS AND GONADOTROPHIC HORMONES
(1956), applying the Bielchowsky-Gros technique to the adenohypophysis of the duck, demonstrated in the capsule and throughout the gland the presence of an autonomic neural formation composed of large strands of nerve fibers replete with ganglion cells and end formations adjacent to parenchymal cells. The presence of even these minor neural contributions to the pars distalis has been vigorously denied by Green (1951b). He was not able to identify such perivascular fibers in the pars distalis, although they were found in the pars tuberalis.
It is significant that all attempts at influencing pituitary secretory function by sympathectomy or by proximal stimulation of the cervical sympathetic trunk have yielded either negative or eciuivocal findings (Friedgood and Pincus, 1935; Friedgood and Cannon, 1940). Although bilateral removal of the superior cervical or stellate ganglia of the sympathetic system in ferrets has been shown to abolish (Abrams, Marshall and Tliomson, 1954) or delay (Donovan and Van der Werff ten Bosch, 1956) the estrous response to added illumination, the latter authors believe this may be accounted for on the basis of an indirect effect ; namely, a diminished amount of light impinging on the retina. All the operated animals showed a marked Horner's syndrome, i.e., globe recession, ptosis, and narrowing of palpebral fissure.
From the foregoing evidence, the conclusion seems clear that the regulation of the pars distalis by secretomotor nerves must be slight, if indeed there is such regulation. All the instances of identification of nerve fibers are open to doubt for the reason that the staining methods do not adequately differentiate nerves from reticular connective tissue fibers. It is to be noted also that by use of phase contrast microscopy in combination with staining i)roccdures. Green (1951b) found no fibers in the rabbit or human pars distalis that could with certainty be identified as nerves.
B. TIIK MKOIAN EMINENCE AND THE I.VFUNDIBULAR STEM
The eminentia has become a focus of interest in attemi)ts to understand neuroendoci'iiic I'clationsliips, because this area is
contiguous with both the hypothalamus and the hypophysis. It is present in vertebrates from amphibians to mammals, and its structure is quite uniform throughout the birds and mammals (Green, 1951a; Wingstrand, 1951b; Nowakowski, 1951). The median eminence is comprised of an inner ependymal zone, a middle coarse-fiber zone made up of the axons of the supraoptichypophyseal tract, and a so-called glandular zone at the surface. The name of the latter was derived from the density of the capillary skein on its surface — it has also been termed the peripheral or marginal zone.
The glandular zone contains the capillary loops of the primary portal plexus (described below) , perpendicularly arranged fibers extending from cells in the ependymal zone and some fibers of nerve cell origin. The latter lie along the base of the glandular zone from which recurrent loops are said to extend toward the surface (Wingstrand, 1951b; Nowakowski, 1952; Assenmacher and Benoit, 1953a, b). Stutinsky (1951) and Vazquez-Lopez and Williams (1952) believed that they have demonstrated fibers from the tractus hypophysius crossing the marginal zone to enter the pars tuberalis, thence to follow the course of the portal veins toward the pars distalis. The existence of such fibers is denied by Rumbaur (1950), Wingstrand (1951a), and Palay (1953a). The innervation of the glandular layer is, as Wingstrand (1951a) states, the key to the postulated control of the pars distalis by a neurovascular mechanism. For birds he states: "No nerve fibers have been seen leaving the eminentia but invariably turn back when they reach the surface. ..."
The glandular zone contains a fiocculent colloid that is demonstrable with the azan trichrome stain, but there is no agreement as to whether this material stains selectively with chronic alum liematoxylin (Gomoril)ositive) like tlic neurosecretory substance in the tractus hypophysius. Benoit finds ahmidant Gomori-positive material in the glandular zone in the duck, whereas Wingstrand's examination of a great variety of birds i'e\-eal('d faintly positive reacting colloid only in a restricted area in the rostral portion of the median eminence. He doubted that it is similar to the heavily staining neuroscci'ctoi'V substance in the supraoptic
PHYSIOLOGY OF ANTERIOR HYPOPHYSIS
275
hypophyseal tract. The central zone exhibits, of course, abundant Goraori-positive material.
In 1957 Dawson presented morphologic evidence of a closer neurovascular relationship in the median eminence of frogs than has yet been observed in mammals. Studying the hypothalamo-hypophyseal relationships at the level of the median eminence in sections stained by modifications of the chrome alum hemotoxylin-phloxine and the aldehyde fuchsin methods, he found that of the fibers from the preoptic tract a surprisingly large number enter and end in the median eminence and have a specific association with the vessels of the portal system. The final simple nerve terminals (dilated with secretion) and the selectively stained secretory substance were arranged in radial patterns about the capillaries. It has not been determined whether the amount of neurosecretory material accumulated about the vessels in the primary plexus is of functional significance, but Dawson noted a specific cell type scattered along the portal vessels as they enter the anterior lobe, and felt that the "rather specific morphologic pattern displayed" suggests a functional interaction of some kind between nervous components of the hypothalamus and glandular units of the anterior pituitary gland, mediated by way of the portal circulation.
C. THE HYPOPHYSEAL PORT.\L CIRCULATORY SYSTEM
A systetH-~o^ hypophyseal portal vessels which drain the tuber cinereum above and supply the adenohypophysis below is present throughout the reptiles, birds, and mammals (Green, 1951). It is indicative of significance that in all these forms the portal vessels provide either the entire or the major vascular supply to the adenohypophysis. Wingstrand (1951a) found no afferent vessels to the pars distalis other than portal vessels in over 50 genera of birds. However, Benoit and Assenmacher (1951b) state that in ducks the pars distalis is inconstantly supplied by a few twigs passing directly from the superior hypophyseal arteries. Crooke (1952) expressed the opinion that the blood supply to the pars distalis in man must be almost entirely by way of the stalk vessels. In examination of over 300
human jntuitaries, he found minute vessels entering from the capsule in only 6 instances. McConnell (1953) reached the conclusion that the pars distalis in man is supplied entirely by the hypophyseal portal vessels, a view which is substantiated by the results from a recent critical study by Xuereb, Prichard and Daniel (1954a). The latter workers (1954b), however, made a further observation wdiich is of cardinal significance. They discovered a vascular link between the neural lobe and the adenohypophysis. Thus, in addition to the "long" portal veins which drain the upper stalk region and supply the sinusoidal network of the anterior and lateral regions of the pars distalis, they found a variety of "short" portal vessels, which arise from an anastomotic link between the superior and inferior hy])oi)hyseal arteries on the lower margin of the infundibular stem. These "short" vessels supply the jjosterior portion of the pars distalis. It will be apparent, therefore, that even though the pars distalis derives all of its blood from the portal vessels, the source of this blood is not limited to that from the superior hypophyseal arteries as was previously thought, but can be supplied by the inferior hypophyseal arteries as well. The matter is of significance in that channels are now known to exist whereby some blood from the systemic circulation can reach the pars distalis without having passed through the primary plexus on the median eminence. Consequently, when the long portal vessels are severed, as in the transection of the pituitary stalk, the possibility remains that in some species collateral channels in the form of these short portal veins continue to sup])ly a portion of the pars distalis.
From a study of the arrangement and structure of the hypophyseal vasculature, Wislocki and King (1936) inferred that the flow of blood in the hypophyseal portal veins was toward the anterior lobe and this has now been confirmed by decisive evidence from work on frogs (Green, 1947), birds (Wingstrand, 1951a), rats (Green and Harris, 1949; Barrnett and Greep, 1951), and man (McConnell, 1953; Xuereb, Prichard and Daniel, 1954a, b). In 1952 Nowakowski, on the basis of his studies of the cat, once more advanced the view that the flow of blood in the portal system is toward
276
HYPOPHYSIS AND GONADOTROPHIC HORMONES
the eminentia. It was his thought that the eminentia has a sensory innervation which registers the content of adenohypophyseal hormones emanating from the pars distalis. The view seems to be completely untenable on anatomic grounds alone.
The capillaries in the primary plexus of the portal vessels show an unusual arrangement in most mammals. From the net which spreads thickly over the surface of the median eminence and the neural stalk, capillary loops and tufts protrude into the wall of the median eminence (Green, 1948; Xuereb, Prichard and Daniel, 1954a). They are not in close proximity with the large nerve tracts of the hypothalamo-hypophyseal neurosecretory system. In birds and mammals they are approached but not closely surrounded by sparse fibrous elements, the nature and origin of which are obscure.
There is a mounting body of circumstantial evidence suggesting that the hypothalamus exerts a large measure of control over the secretory functions of the anterior pituitary. Such control might be effected either through direct nervous connections, the evidence for which (as we have just seen) is scant, or through a relay chain made up of a neural and a vascular link. Anatomically, at least, the latter exists in the form of the hypophyseal portal system.
The neurohumoral concept as outlined by Harris (1947, 1948a) and Green and Harris (1947) holds that pertinent exteroceptive stimuli impinge upon the hypothalamus as the first way-station in the neurovascular reflex arc. Here the sex-related impulses are integrated with existing blood levels of circulating hormones, thence effector impulses travel by nerve conduction to the median eminence and effect the liberation of neurohumors (chemotransmitters). The latter are held to enter the primary plexus and to be distributed to and activate the cells of the pars distalis. A weakness in this theory is at the point of transfer of chemotransmitters from nerve to vessel in the median eminence. The anatomic relationship between the loops or endings of nerves of obscure origin and the capillaries, which lie either on the surface of the median eminence or extend tuft-like into its substance, has not Ix'cn clearlv defined.
D. THE HYPOTHALAMIC AND HYPOPHYSEAL MEDIATED SEXUAL FUNCTIONS
1. Environmental Stimuli
A great number of instances are known in which reproductive processes are conditioned by or are dependent on environmental factors. Although animals breed at a season of the year that is propitious for the survival of their young — warmth and the availability of food being the major factors — this is not through choice on their part. In many birds and mammals the readying of the reproductive system for seasonal breeding and rearing of young is anchored to alterations in the physical environment, especially to changes in length of daylight — a subject which will not again bear expatiation (for a recent review see Amoroso and Matthews, 1955). Darkness, too, may not be an entirely passive stimulus (Burger, Bissonnette and Doolittle, 1942; Jenner and Engels, 1952; Kirkpatrick and Leopold, 1952) , but this concept has not gone unchallenged (Hammond, Jr., 1953). Experiments involving total darkness, moreover, are often subject to the complication of reduced food intake. The ground squirrel (Wells and Zalesky, 1940) and the whitecrowned sparrow (Farner, Mewaldt and Irving, 1953) respond only to changes in the ambient temperature. Propagation in some tropical birds and amphibia is in like manner dependent on the beginning of a rainy season. Food becoming more plentiful at this time seems to be the mediating factor for many tropical birds. In England, certain frogs (Savage, 1935) are lured to the ponds by cheraotactic stimuli arising from ripening algae, but spawning is dependent on another sequence of changes induced in the ionic composition of the water by the algal cycle and perceived through the frog's skin. Animals transported to the opposite hemisphere, generally adapt their breeding activities rather quickly to the same characteristic season irrespective of when it occurs in the calendar year (for review see F. H. A. Maishall, 1942; Burrows, 1949). In many birds, even with gonads in readiness for the mating season, the actual nesting often requires another complex set of extra-organismal stimuli, such as presence of food, presence of mate, density of colony, suitability
PHYSIOLOGY OF ANTERIOR HYPOPHYSIS
277
of breeding site, and avian display and courtship performances (A. J. Marshall, 1955, and chapter by Lehrman).
Thus, for most but not all of the seasonal breeding vertebrates, the primary excitations to breeding activity originate in one way or another in the external environment. Those structures most exposed to such stimuli are the skin and the specialized sense organs, particularly those for seeing, hearing, and smelling. It will be recalled that the sight of his mate incubating is sufficient to initiate in the male pigeon the secretion of crop milk (Patel, 1936). It is also not infrequent, especially among birds, that the presence of a mate is a prerequisite for ovulation. Here again, interesting observations have been made on the pigeon (Matthews, 1939). The female separately caged and denied sight of other birds does not ovulate; but if she is permitted to view, by means of a glass partition in the cage or a mirror, a separately caged male or even a female pigeon, or is allowed to see the mirror image of herself, ovulation and oviposition ensue. The stimulus which causes ovulation is clearly visual and not tactile, olfactory, or auditory. In sheep the presence of the ram is believed to hasten ovarian activity and estrus in ewes (Riches and Watson, 1954; Schinckel, 1954), and it is likely that here both sight and odors play a part.
It is apparent from observation of the behavior of domestic and laboratory mammals that strong nasogenital relationships exist, but the effeet-of'sexual odors and olfactory stimulation on the sexual functions has received only cursory study. Pseudopregnancy has been induced in rats by simply applying a local anesthetic to the nasal mucosa (Shelesnyak and Rosen, 1938) and similar prolongations of luteal function were induced by bilateral excision of the sphenopalatine ganglion, but not by removal of the olfactory bulbs (Rosen, Shelesnyak and Zacharias, 1940). Local irritants applied to the nasal mucosa likewise did not affect the estrous cycle. These authors felt that the nervous factor involved was limited to the nonol factory innervation of the nasal mucosa. Whittcn (1956a) showed that mice rendered anosmic by removal of the olfactory bulbs have significantly smaller ovaries and uteri and fewer corpora lutea. No effect
was observed in anosmic males. In both male and female mice the body weights were reduced.
In a recent study Whitten (1956b) found that external stimuli associated with the male mouse modify the estrous cycle in the female. The incidence of mating after pairing was greatest on the 3rd night and was low on the 1st, 2nd, and 4th nights. The finding suggests that in some females the cycle was delayed, and in others it was advanced, by the presence of the male. In another experiment in which males were caged in a small basket within the females' cage for 2 days before pairing, the incidence of mating on the 1st night was greatly increased. Since presence of the males' excreta had a similar effect on the receptivity of the females, odors were thought to be an important factor in mediating this behavioral response.
The determinate-laying wild birds, i.e., those which stop laying when the number of eggs characteristic of the species has been laid, probably perceive the stimulus for cessation of egg-laying through tactile sensations. Tactile stimuli are also known to come into play strongly during coitus. It is well known that in several mammals (rabbit, cat, mink, ferret, ground squirrel, shorttailed shrew) ovulation is dependent on the coital excitation of receptor areas not all of which are confined to the genitalia. The neural pathways involved are poorly understood.
Knowledge of the effect of sound on reproductive phenomena is exceedingly limited. It would be interesting to ascertain what sequential role, if any, various songs and mating calls play in releasing the manifestations of procreative processes. It is probably of no significance that Benoit (1955) failed to alter the sexual maturation in young drakes with continuous noise, and only indicative that Vaugien (1951) found a hastening of egg-laying in parakeets placed in small containers within hearing distance of an aviary where others of the species were mating. Control females similarly caged beyond hearing range did not lay. Sound, however, was not the only variable in these experiments.
Thus, many extrinsic factors act in some manner to influence the secretion of gonado
HYPOPHYSIS AND GONADOTROPHIC HORMONES
trophins by the anterior hypophysis. Obviously, central nervous pathways are involved, but the anterior pituitary has so little, if any, innervation that it is almost certain that none of the impulses are carried directly to the pituitary by this means. An alternative connecting pathway between brain and hypophysis exists in the form of the portal vessels. One of the most pressing problems in endocrinology is to define the pathways and elucidate the mechanisms by which such exteroceptive stimuli as those mentioned above effect the secretory functions of the anterior hypophysis.
The hypothalamus serves in other respects to receive impulses from higher afferent centers and to instrument these into responses that relate to vegetative functions, for example, breathing, sleeping, and body temperature. It is also known to be the seat of neural mechanisms concerned with sex functions. By location and neural connections the hypothalamus could well be attuned to the reception and integration of sex-related impulses of variable sources. It has often been observed clinically that tumors of the hypothalamus are associated v/ith disturbed sexual functions. Moreover, anxiety states may lead to a cessation of menstrual cycles or impotence without evidence of organic disease. The supposition is that chemotransmitters of some sort, arising in the diencephalon, reach the pituitary by way of the portal veins. Alternatively, no other explanation of the existing evidence is available. Such a chemotransmitter has not yet been identified (Zuckerman, 1954). Extracts of the hypothalamus have thus far ])rovided only inconclusive evidence with respect to release of another trophic factor, corticotrophin, from the adenohypophysis (Guillemin, Hearn, Cheek and Housiiolder, 1957).
2. Electrical Stimulation of the Iljipothalamus A hopeful means of increasing the secretory activity of the anterior pituitary was suggested by the observation that ovulation and pscudoi)regnancy could be produced by application of a strong current to the head or spinal cord in estrous rabbits (Marshall and Verney, 1936) and rats (Harris, 1936). Witli refinement in the technique it has l)c
come possible to apply such stimulation to
precise areas in the pituitary and suprasellar region. Moreover, by use of implanted
electrodes and the remote induction of stimulation it is possible to apply electric excitation of controlled intensity to precise
areas for almost any length of time in unanesthetized animals (de Groot and Harris,
1950, reviewed by Harris, 1955). The
method has been used mainly in connection
with the study of ovulation in rabbits and
pseudopregnancy in rats, but the method
is suitable for much wider application in
the study of mechanisms which regulate the
functions of the pars distalis. By successive
steps an area wherein stimulation leads to
ovulation in estrous rabbits has been fairly
well delimited (Harris, 1937; Haterius and
Derbyshire, 1937; Markee, Sawyer and
Hollinshead, 1946; Harris, 1948b ).^
Stimulation of the various lobes of the hypophysis and of the neurohypophyseal stalk in the unanesthetized rabbit does not promote ovulation, whereas ovulation is quite regularly induced when the stimulating electrodes are in the tuber cinereum just anterior to the median eminence or in areas of the anterior hypothalamus. No part of the supraoptic-hypophyseal tract yielded a positive result. These observations provide strong support for the belief that the hypothalamus exerts a profound effect on the reproductive functions of the pars distalis and show finite conclusively that the connecting link to the pituitary is not neural in character. They otherwise prove nothing with respect to the essentiality of the portal circulation in the mediation of these responses.
Stimulation of the cervical sympathetic fibers leading to the hypophysis neither induces nor interferes with ovulation in rabl)its, according to Haterius (1934) and Markee, Sawyer and Hollinshead (1946). The few successful instances noted by Friedgood and Pincus (1935) might have been due to spread of the stimulus to the hypothalamus, or a response to handling. It is })ertinent to note in this connection that neither sympathetic nor parasympathetic denervation of the hypophysis has led to any detectable change in pituitary functions.
PHYSIOLOGY OF ANTERIOR HYPOPHYSIS
279
3. Lesions in the H ypothahnnus
The puzzling clinical association of lesions in or trauma to the base of the brain in the region of the diencephalon and disorders of the reproductive system set workers to probing this area of the brain in experimental animals as long ago as the turn of the century (for review of the early literature and critical analysis of the evidence, see Anderson and Haymaker, 1948a, b). Clinical or experimental lesions, destroying much of the hypothalamus but leaving the hypophysis intact, ordinarily resulted in genital atrophy accompanied by obesity (Frohlich type syndomej. Diabetes insipidus did or did not ensue, depending on whether the lesion disrupted hypothalamic neural connections with the neurohypophysis. A significant advance was made in 1927 when Smith discovered quite by accident that the chromic acid he was injecting into the sella of rats to destroy pituitary tissues led to extreme obesity through damage to adjacent areas of the brain. It remained for Cahane and Cahane (1935, 1936) to dissociate in rats the sequelae of adiposity and deficiencies in the reproductive system by means of carefully placed lesions. Rats with lesions in the infundibulum showed severe testicular or ovarian atrophy and cessation of estrous cycles with no obesity. Dey (1941, 1943ac), Dey, Fisher, Berry and Ranson (1940), and Dey, Leininger and Ranson (1942) were among the first to use stereotaxic instrumentsJBr a major study of the effect of bilaterally placed hypothalamic lesions on the reproductive organs and sexual behavior. They were able by this means to produce two distinct categories of sexual response in female guinea pigs. (1) Animals with lesions in the median eminence exhibited ovarian atrophy and loss of all reproductive functions. (2) Animals with lesions in the anterior hypothalamus remained in continuous estrus and the ovaries were filled with large Graafian follicles. The lesions in the anterior hypothalamus seemed to interfere with the release of LH. By present thinking it is likely that the secretion of LH continued at a low level but that the reflex arc for acute release at the appropriate time in the cycle was interrupted.
In guinea pigs lesions elsewhere in the hypothalamus produced no impairment of gonadal functions, yet some of the animals did not mate.
Essentially similar findings and somewhat more precise localization of the lesions were reported in rats (Hillarp, 1949). Constant estrus was "consistently" produced by bilateral, relatively large lesions placed immediately anterior and ventral to the paraventricular nucleus, or by smaller, perfectly bilateral, symmetrical lesions within the area between the paraventricular nucleus and the stalk. Lesions in the dorsal and lateral hypothalamus did not affect the reproductive functions. Eighteen other rats with small lesions variously located in the anterior hypothalamus showed cyclic irregularities and impaired reproductive capacity with essentially normal appearing ovaries. Other more recent studies uniformly emphasize the harmful effect of lesions in the ventral hypothalamus and especially in the median eminence on the reproductive functions of the rat (IVIcCann, 1953; Bogdanove and Halmi, 1953) ; man (Anderson, Haymaker and Rappaport, 1950) ; cat (Laqueur, McCann, Schreiner, Rosemberg, Riocii and Anderson, 1955); sheep (Clegg, Santolucito, Smith and Ganong, 1958).
Although lesions in the median eminence often produce some damage to the portal circulation, the infrequency of infarction in the anterior lobe and the usual retention of normal morjihologic aspects of this gland make it unlikely that the observed deficiencies in gonadotrophic functions are ascribable to an inadequate vascular supply. It is becoming increasingly evident that the separate trophic functions of the anterior lobe can be selectively altered by appropriately placed lesions in the hypothalamus. The relationship of hypothalamic lesions to the thyrotrophic and adrenotrophic activities of the pituitary need not be considered here, because these seem to be independent mechanisms and exhibit no significant overlap with gonadal regulation. Evidence for the selective effect of localized hypothalamic lesions on pituitary trophic secretions has been presented by Bogdanove and Halmi (1953), Bogdanove, Spirtos and Halmi (1955), Ganong, Fredrickson and Hume (1955), and Bogdanove (1957). It
280
HYPOPHYSIS AND GONADOTROPHIC HORMONES
seems moreover, that such lesions may also selectively influence the individual gonadotrophins, and recently evidence has been provided that lesions in the ventral hypothalamus of sheep may abolish the behavioral manifestations of estrus without altering the ovarian cycle (Clegg, Santolucito, Smith and Ganong, 1958).
The study by Bogdanove and Halmi (1953) also confirms an observation noted by others (Desclin, 1942; May and Stutinsky, 1947; Stutinsky, Bonvallet and Dell, 1950) that lesions in the hypothalamus may lead to a considerable hypertrophy of the pars intermedia. Furthermore, Stutinsky, Bonvallet and Dell (1950) observed great enlargement of the sinusoids in the pars distalis following suitably placed hypothalamic lesions, and speculated that one means of hypothalamic mediation of hypophyseal function might be by way of vasomotor control of vessels in the pars distalis. As a modus operandi this has the drawbacks of nonspecificity and sluggishness. The view has not been given credence, but the observation merits study.
4. Transection of the Hypophyseal Stalk
An obvious experimental procedure in studying the extent of any control that the hypothalamus may impose on the anterior hypophysis is to interrupt all anatomic connections between them. This has been accomplished by either surgically transecting the stalk or occluding it with a silver clip. In either event the neural and vascular connections between the hypophysis and the brain are disrupted. Such procedures do, in fact, isolate the hypophysis from eveiy possible anatomical connection with the brain, save that of the systemic blood stream. Roundabout as the latter would be, even this avenue is available only in animals having a collateral blood supply to the anterior lobe. Present evidence indicates that this would include man and, with less certainty, monkey, dog, sheep, and rabbit. A consideration of primary importance in all surgical transections of the stalk concerns the demonstrated capacity of the disrupted vessels to regenerate and reestablish vascular connections. Harris (1936-1955) , Harris and .lacobsohn (1952) and Harris and Johnson (1950) have taken the in'ccaution
of placing a plate of impervious material between the severed ends of the stalk as a barrier to the regrowth of vessels. The severed neurons with nuclei in the hypothalamic ganglia undergo Wallerian degeneration.
Variations exist between species in the length of the pituitary stalk and in the anatomic relationship of the hypophysis to the base of the brain; consequently, it is often possible to divide the stalk at different levels, i.e., near the base of the brain (high) or near the pituitary (low). In those species, especially the rabbit, monkey, and man, which have a deep sella turcica and a relatively long infundibular stem, there is the likelihood that with high transection, portions of the portal circulation may remain intact. Man in particular is held to have a collateral blood supply to the pars distalis (Xuereb, Prichard and Daniel, 1954a, b) by way of the trabecular and the inferior hypophyseal arteries.
The level of stalk transection is also known to be important in terms of the diabetes insipidus which follows this operation. Severance in a plane near the hypophysis in rats (as many have already demonstrated) may result in only a moderate and transient polyuria, whereas after transection in a more proximal plane the polyuria is more often severe and permanent. A plausible explanation of these results is that after low transection, there occurs a compensatory development of neural lobe tissue at the proximal end of the severed stalk. Suprasellar reorganization of neurohypojihyseal tissue and restitution of neurohyiioi)hyscal functions have been described by Stutinsky (1951, 1953) and confirmed by Billenstien and Leveque (1955) and Benson and Cowie (1956) after hypophysectomy in the rat. (Jaupp and Spatz (1955) described comi)cnsatory development of a "suprasellar hyi:)ophysis" on the tuber cinereum following low transection of the stalk in rabbits. They found these growths to be composed of fibers rich in Gomori-positive granules. Some of the rabbits bearing such nodules came into late sexual maturity, which led the authors to surmise that these compensatory neural elements were providing gonadotrophic stimulation. Tlic nodules thev described are of
PHYSIOLOGY OF ANTERIOR HYPOPHYSIS
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great interest, but the imputation with respect to gonadotrophic functions is weak. No glandular parenchyma was found in the nodules. Moreover the rabbit's own pituitary, although isolated, remained in situ and, according to the authors, was undamaged in cytoarchitecture.
Stalk transection has led to extremely discordant findings with regard to the postoperative structure and function of the pars distalis, particularly in the earlier studies. In Dandy's (1940) often cited case of stalk transection in a young adult woman, the reproductive functions were not impaired and no symptoms of hypophyseal deficiency appeared, except a severe and permanent diabetes insipidus. Recently Russell (1956j and Eckles, Ehni and Kirschbaum (1958) observed varying but generally severe necrosis of the anterior lobe following stalk severance in man. The menstrual cycles ceased, and there was a notable regression of the gonads, adrenals, and thyroids; an increase in insulin sensitivity suggested a low output of somatotrophin as well. Quite unexpectedly either si)ontaneous lactation ensued or established milk flow persisted, suggesting output of LTH {vide infra). Russell and Eckles, and Ehni and Kirschbaum agreed that the extent of damage to the anterior lobe is associated with the level of stalk transection, this being most severe wdth low transection. Since in Dandy's patient the stalk was sectioned near the midpoint, it seems likely that the maintenance of the anterior pituitary functions he d£Si:iribes is explicable on the basis that a portion of the circulation to the pars distalis escaped damage. The fact that Russell's patients did not exhibit diabetes insipidus is in all probability attributable to extreme infarction of the anterior lobe and the poor state of the surviving fragments of this tissue.
Brooks (1938) found that stalk-transected rabbits, after a period of transitory gonadal regression, had normal appearing pituitaries and ovaries. These animals came into estrus and mated but failed to ovulate. He presumed that the operation had interfered with the LH-releasing mechanism. Others, too, in acute experiments have fully substantiated the fact that the postcoital ovulation in rabbits requires the presence
of an intact stalk (Westman and Jacobsohn, 1940; Brooks, Beadenkopf and Bojar, 1940). In stalk-transected rabbits which have been observed over protracted periods, extensive gonadal atrophy has usually supervened (Harris, 1937; Westman and Jacobsohn, 1940; Gaupp and Spatz, 1955). Nine of the 12 rabbits operated upon by Gaupp and Spatz showed extreme atrophy of the gonads and genitalia. The remaining 3 reached sexual maturity only after a delay of up to II/2 years.
Much of the pioneering work on stalk transection or occlusion in relation to adenohypophyseal function was done in dogs (Paulesco, 1908; Crowe, Gushing and Homans, 1910; Gushing and Goetsch, 1910) and monkeys (Karplus and Kriedl, 1910; ]\Iorawski, 1911). Gushing and his co-workers observed marked anterior lobe degeneration, and few of their dogs escaped "cachexia hypophysioprivia." In their words, "the resultant condition is almost the same as if this portion of the gland had actually been remo^'ed and then reimplanted like a graft, for the circulation is almost entirely cut off." These observations were thoroughly confirmed by Mahoney and Sheehan
( 1936) in 20 dogs with stalks occluded. Extensive infarction and central necrosis of the pars distalis with concomitant impairment of function was seen in all. Later workers, however, have observed very different sequelae: Keller and Hamilton
(1937) and Breckenridge and Keller (1948) found no deviations from the normal patterns of sexual functions in the majority of their stalk-sectioned dogs. The results in dogs must yet be regarded as inconclusive, inasmuch as regeneration of the portal vessels was not adecjuately controlled and the operation is a difficult one, which, at best, involves an indeterminate amount of trauma to the hypophyseal and median eminence areas.
Results have been more consistent in monkeys. Karplus and Kriedl (1910) and Morawski (1911) observed no untoward symptoms in stalk-transected monkeys and no obvious alteration in the histologic structure of the pars distalis; and Mahoney and Sheehan (1936) completely occluded the stalk in 20 monkeys with the same results, including no polyuria or polydipsia. At
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HYPOPHYSIS AND GONADOTROPHIC HORMONES
autopsy 3 days to 10 months later the hypophyses appeared normal and the vascularity of the anterior lobes was unimpaired, as shown by perfusion with carmine gelatin. These early findings have been substantiated by Harris and Johnson (1950), who made a suprasellar transection of the stalk in a large male monkey. This animal continued to ejaculate, and at the time of autopsy 89 days later the reproductive organs were normal in size and appearance. At autopsy the pars distalis appeared well vasculated and showed no abnormality in size or color. It was demonstrated that vascular continuity between the median eminence and the adenohypophysis had been re-established. From these particular observations one can deduce only that the severance of the neural connections to the hypothalamus are not important for the continuance of adenohypophyseal activity. Disturbance of posterior lobe functions was not observed ; this probably is accounted for by the fact that sufficient terminations of neurosecretory fibers exist proximal to the cut to sustain neurohypophyseal functions. As far as the guinea pig and rat are concerned, the widest possible divergence in results has been reported. In female stalksectioned guinea pigs Dempsey (1939) and Leininger and Ranson (1943) observed normal, lengthened, or absent estrous cycles. Tang and Patton (1951) severed the stalk in male guinea pigs and observed no gonadal atrophy. Their animals were maintained for 27 to 75 days, and although the portal vessels could have regenerated postoperatively, no verification of this was found on histologic examination of the region. Equally variable responses have been described in stalk-sectioned rats by Dempsey and Uotila (1940) and Dempsey and Searles (1943). They ascribed the abnormal cycles in such operated animals to operative trauma to the anterior pituitary. Using a parapharyngeal approach for sectioning the stalk in male and female rats, Barrnett and Greep (1951) and (Ireep and Barrnett (1951) found a very high incidence of severe gonadal atrophy along with generalized evidence of i)anhypopituitarism. These symptoms of anterior lobe dysfunction were consonant with the extensive infarction and necrosis of the pars distalis observed regularly following
the operation. They felt that the degree of gonadal dysfunction was relatable to the extent of damage to the blood supply of the pars distalis. The pituitaries showed no capacity for recovery, and there was no regeneration of the portal vessels as determined in specimens injected with India ink and studied in serial sections of the appropriate areas. Years before, Houssay and Giusti (1930) and Lascano-Gonzalez (1935) had reported on similar infarctions in toads following severance of the vessels to the pars distalis.
Much recent evidence has revealed that vascular infarction, necrosis, and dysfunction of the pars distalis are consequences of procedures which destroy the portal vessels. Daniel and Prichard (1956) studied the degree and localization of vascular lesions in the pars distalis of rats following the application of a fine cautery to all or a portion of the long hypophyseal portal vessels which pass down the ventral aspect of the stalk. Regions of the anterior lobe supplied by short portal vessels emanating from the lower portion of the neural stalk were undamaged. In an extension of their study of the vascular supply to the pituitary, Daniel and Prichard (1957a, b) observed in sheep that severing the stalk produced extensive necrosis of the anterior pituitary; the condition of the pituitary-dependent endocrine organs was not reported. The extent of damage to the pars distalis w^as remarkably similar to that seen in human cases of postpartum pituitary infarction.
Harris (1949) suggested that the discrepancies in findings following sectioning of the stalk might be reconciled by a study of the extent of regeneration of the portal vessels. Using a transtemporal approach, he sectioned the stalk in a series of rats, killed them at close intervals, and injected the vessels. He demonstrated that the portal vessels unquestionably regenerated in many cases" and that the regeneration was underway by 24 to 48 hours. In a follow-up study Harris (1950) inserted between the severed ends of the stalk an impervious l)late of varying composition. Results were judged by the effect on the estrous cycle. He concluded that the regeneration of portal vessels correlated with the ability of the pai's distalis to sustain cyclic gonadal func
PHYSIOLOGY OF ANTERIOR HYPOPHYSIS
283
tions. The fact that the pars distalis in rats receives its blood from the portal veins and that regeneration of these vessels means restoration of nutrient supply to this organ, would seem, on the face of it, to be a plausible explanation for resumption of secretory activity following stalk transection. However, there is strong evidence against this possibility. Grafts of the pituitary gland revascularized by vessels from other than the median eminence lose their ability to sustain ovarian functions other than progestational (Harris and Jacobsohn, 1952; Everett, 1954, 1956; NikitovitchWiner and Everett, 1957), and regain these functions when vascular continuity with the median eminence is restored (NikitovitchWiner and Everett, 1957, 1958a, b).
A series of recent reports has dealt with the capacity of stalk-sectioned ferrets to respond to the stimulus of added illumination. Thomson and Zuckerman (1953, 1954) and Zuckerman (1955» claimed that estrus supervened in 10 of 16 operated ferrets exposed to added illumination. In two of the animals which responded, careful study of serial sections of the operative site after India ink perfusion revealed no vascular connections between the pituitary and the median eminence. Thus, the essentiality of the portal vessels and whatever humors they might convey for the regulation of adenohypophyseal activities was called into question. Donovan and Harris (1956), on the contrary, found that stalk-transected ferrets in which regeneration of the portal vessels was-^recluded by a film barrier between the severed ends of the stalk did not exhibit light-induced estrus; 3 animals lacking the barrier responded with early estrus, but in each of them regeneration of a vascular link with median eminence was demonstrated. The question left unanswered by the latter experiments is whether the pituitaries in the animals which did not respond actually had the capacity to respond. Collateral evidence, such as reduced adrenal weight and decreased thyroid activity, suggests that the pituitaries may have been functionally incapacitated for reasons other than lack of a specific excitatory agent of hypothalamic origin. Campbell and Harris (1957) addressed themselves to this problem by studying the volume change of the rabbit pituitary after dividing the stalk. They found a reduction to 62-74 per cent of the normal volume for the whole gland, 68-83 per cent for the pars distalis, and 2627 per cent for the neural lobe. Because the extent of atrophy in operated animals was the same with or without plate insertion, they questioned the importance of ischemic damage. The rabbit, however, seems an unfortunate choice for study of this problem because Harris (1947) had already noted that the anterior lobe in this species has an arterial as well as a portal venous blood supply. Moreover, volume changes are not a crucial index of glandular competence. Breckenridge and Keller (1948) found no correlation between retention of sex functions and the size of the anterior lobe remnant in dogs which had been subjected to complete removal of the stalk and partial (graded) hypophysectomy. There was, however, a close correlation between maintenance of the genital structures and retention of normal cytoarchitecture of the remnant. The arrangement of nervous and vascular connections between the pituitary and the brain in birds is such that it is possible to section the infundibular stalk, leaving the portal vessels intact, or contrariwise, divide the vessels leaving the stalk undamaged, or by appropriate incision segregate the median eminence from the hypothalamus without disturbing either the portal vessels or the stalk. These experimental procedures have been carried out on the duck (Benoit and Assenmacher, 1953; Assenmacher and Benoit, 1953a, b). Stalk transection alone did not alter gonadal maturation in animals exposed to added illumination, but the response was completely blocked by sectioning either the median eminence or the anterior portal vessels ; in birds these vessels supply the cephalic lobe of the pars distalis. In spite of the impaired secretion of gonadotrophins, the functional capacity of the anterior lobe tissue was otherwise adequate as evidenced by the fact that the thyroids and adrenals were fully maintained. After severing both stalk and portal vessels in laying hens, Shirley and Nalbandov (1956a, b) observed gonadal atrophy, but no change in either the thyroids or adrenals. Benoit and his associates and Shirley and Nalbandov strongly favor the
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interpretation that hypothalamic agents are involved in the excitation of the hypophysis to secrete gonadotrophic hormones.
Mention has been made of the interesting preliminary observation by Eckles, Ehni and Kirschbaum (1958) of persistent lactation as a sequel to stalk transection in women with breast cancer. (Polyethylene platlets were inserted between the cut ends of the stalk.) In some women lactation was observed to continue for a year or longer. The menses ceased, hence it can be assumed that the secretion of FSH and LH was interrupted. Why then, was the secretion of LTH not also abated? It seems pertinent also to note here that galactorrhea has frequently been seen in female mammals and patients receiving tranquilizing drugs (Kehl, Audebert, Gage and Amarger, 1956; Polishuk and Kulcsar, 1956; Whitelaw, 1956; Meites, 1957; Sawyer, 1957). It would seem, in fact, that an inhibiting influence on LTH secretion had been removed by these procedures. These findings in man parallel the observations by Desclin (1950) and Everett (1954, 1956) that autografts of the pars distalis to the renal capsule secrete LTH selectively, and perhaps in increased quantities for several months (see also Nikitovitch-Winer and Everett, 1957, 1958a, b for further variants of this study of pituitary autografts). Perhaps the fact that pseudopregnancy often supervenes in rats under continued treatment with certain tranquilizing agents (Barraclough, 1957; Velardo, 1958; Barraclough and Sawyer, 1959j can be explained by an abnormal release of LTH from the pars distalis. Inherent in this evidence is the suggestion that the hypothalamus exercises either no influence or a "tonic" suppressive influence on the secretion of LTH by the pars distalis. Other recent evidence suggests, however, that the hypothalamus may also supply a stimulus for the release and possibly the j)roduction of prolactin (LTH). Oxytocin, a neurohypoi^hyseal hormone that is almost certainly elaborated in the hypothalamus (Scharrer and Scharrer, 1945, 1954a, b; Bargmann, 1949; Olivecrona, 1957), has been shown to lead to structural maintenance of the mammary gland (Benson and Folley, 1956, 1957). The resolution of such an interesting but bafflingly complex
hypothalamo-hypophyseal interrelationship must await much additional experimentation.
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