PROGRESS

IN ENDOCRINOLOGY

AND METABOLISM

Mechanism of Action of Luteinizing Hormone and Follicle-Stimulating Hormone on the Ovary In Vitro Cornelia P. Channing The mechanism of action of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) upon various cell types of the mammalian ovary is reviewed. Emphasis is placed upon in vitro studies using organ and cell culture as well as short-term incubations. FSH and LH actions upon the following ovarian functions are discussed: steroidogenesis and metabolism of the ovary as a whole and of the isolated follicle and its component cell types, the granulosa and thecal cells, as well as

and Alexander Tsafriri folliculogenesis and follicular growth, oocyte maturation, follicular rupture, and corpus luteum maintenance and steroidogenesis. The roles of gonadotropin receptors, AMP, prostaglandins, protein kinase, and protein synthesis in these LH and FSH actions are discussed. Intraovarian regulation of LH and FSH action is reviewed, including a discussion of the possible roles of follicular fluid inhibitors upon oocyte maturation and granulosa cell luteinization.

T

HE IMPORTANCE of pituitary luteinizing hormone (LH) and folliclestimulating hormone (FSH) in control of ovarian function is exemplified by the finding that if hypophysectomy is carried out prior to puberty the ovary fails to progress beyond the infantile state. Furthermore, if hypophysectomy is carried out during adult life the ovary regresses to its infantile state.’ LH and FSH, secreted prior to and during puberty and cyclically throughout adult reproductive life, are responsible for control of many ovarian functions, including: follicular growth past the antrum stage; maturation of the follicle, including estrogen secretion; final oocyte maturation; follicular rupture; and the formation, function, and maintenance of the corpus luteum. Since it appears that both gonadotropins may act upon different cell types of the ovary at different phases of the ovarian cycle, studies on the mechanism of action of LH and FSH upon the ovary should be carried out on isolated cell types and tissue compartments of the ovary. Eventually the actions should be put together as a functional unit within the context of the whole animal, taking into account how compartments within the ovary interact with one another. The purpose of this review is to discuss the mechanism of LH and FSH action in vitro upon ovarian function in general, as well as upon specific ovarian functions: (1) follicular maturation and steroidogenesis; (2) oocyte maturation; Abbreviations used in text: CAMP, cyclic adenosine 3’,5’-monophosphate; LH, luteinizing hormone; FSH, follicle-stimulating hormone; hCG, human chorionic gonadotropin; PGE, PGF, prostaglandins E and F, respectively; hMG. human menopausal gonadotropin: PMSG, pregnant mare serum gonadotropin: FFI, follicular fluid: BSA, bovine serum albumin: IBMX. isobutyl methyl xanthine. From the Department of Physiology, University of Maryland School of Medicine, Baltimore, Md. Receivedforpubiication June 2. 1976. Supported by USPHS Research Grants HD03315 and HD08834 from the National Institute of Child Health and Human Development, Grants M7452C and M7521 from the Population Council of New York, and grants from the World Health Organization and the Ford Foundation. Reprint requests should be addressed to Dr. Cornelia P. Channing, Department of Physiologv, University of Maryland School of Medicine, 660 West Redwood Street, Baltimore, Md. 21201. 0 1977 b.v Grune & Stratton, Inc. Metabolism, Vol. 26, No. 4 (April), 1977

413

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(3) follicular rupture; (4) granulosa-cell maturation and luteinimtion: and (5) maintenance of luteal function. An exhaustive review of the literature is not possible. The reader is referred to several excellent review articles and monographs that cover areas not detailed here.2 ” I. LH AND FSH ACTION

UPON

THE

INTACT

OVARY

Many studies on the mechanism of action of LH and FSH in vitro have been carried out employing whole ovaries. These will be reviewed separately, followed by a review of studies employing isolated ovarian cells and/or tissue compartments. Studies with intact or minced luteinized rat ovaries of the “Parlow type”‘* will be reviewed under the corpus luteum (section V below) since such ovaries are more than 95”,, corpus luteum. Some repetition of mechanistic steps is unavoidable. A generalized scheme of the mechanism of LH action (Fig. 1) upon the ovary starts out with binding to a specific LH receptor (gonadotropin binding to ovarian cells has recently been reviewed”). followed by a rapid stimulation of adenylate cyclase activity. (The role of AMP in gonadal function has recently been reviewed by Marsh.6) Stimulation of adenylate cyclase results in elevated levels of CAMP which then interact with the regulatory subunit of protein kinase, resulting in stimulation of enzymatic activity.5.6,20~23 In a series of subsequent reactions yet to be clearly defined, stimulation of steroidogenesis results. The nature of the steroid produced depends upon the cell type and species in question. In the case of corpus luteum, the principal steroid is usually progesterone, and in the case of thecal tissue. estrogen is the principal

1

I Fig. 1. by solid

Generalized arrows

(+)

and

mechanism inhibitory

of action actions

of LH on the ovary.

are indicated

by dotted

Stimulatory orrows

(-).

actions

are

indicated

LH AND

FSH ACTION

415

MECHANISM

steroid secreted. Ovaries of rats less than 5 days of age cannot bind lz51-human chorionic gonadotropin (hCG), but their ability to bind hCG increases as a function of age.24m26Accordingly, the ovary of the neonatal rat lacks the ability to respond to LH. However, prostaglandin E2 (PGE2) could increase CAMP formation in neonatal ovaries. By 7-12 days the ovaries acquire the ability to respond to LH with stimulation in CAMP production, along with the attainment of an ovarian protein kinase able to respond to CAMP.” A decrease in responsiveness of rabbit ovarian adenyl cyclase to LH was observed with advancing age.*’ A. Stimulation

of Steroidogenesis

The principal site of stimulatory action of LH in steroidogenesis is between cholesterol and pregnenolone,28,29 which is mediated by CAMP (see reviews by Marsh6q23). According to recent findings of Armstrong and his colleagues in the luteinized rat ovary, one site is a rapid stimulation of cholesterol esterase3’ and a second site is a stimulation of translocation of cholesterol from the cytoplasm into the mitochondrion3’ where the side chain is cleaved. There also appears to be a direct CAMP-mediated and protein-kinase-mediated stimulation of the side-chain cleavage enzyme itself involving a change in cytochrome P450 The rapid stimulation of cholesterol esterase by LH appears to enzyme. 23.32.33 be independent of the stimulation of transport of cholesterol into the mitochondrion. The net effect of both of these steps is to increase availability of cholesterol substrate to the mitochondrial side-chain cleavage enzyme. Both the action of LH and CAMP upon ovarian progesterone secretion are blocked by puromycin and cycloheximide,6s34m36 demonstrating an essential role of protein synthesis in CAMP and LH action in stimulation of progesterone biosynthesis. Protein synthesis is not required, however, for LH stimulation of CAMP production in granulosa cells or corpus luteum.7.37 Armstrong and his colleagues also demonstrated that LH stimulation of steroidogenesis in vitro is enhanced by a concurrent CAMP-mediated inhibition of cholesterol ester synthetase activity.38339 Exogenous progesterone and 20a-hydroxy-pregn-4-en3-one can also inhibit cholesterol ester synthetase. This would enhance the effectiveness of the LH by permitting more free cholesterol to be available for additional steroidogenesis. This inhibition of cholesterol ester synthetase brought about by LH was blocked by prior administration of cycloheximide in vivo, demonstrating a dependence upon protein synthesis.38 B. Stimulation

of Other Metabolic

Events

In prepubertal ovaries, LH administration in vivo and in vitro stimulates other metabolic events, namely, glucose uptake and lactic acid production8,40m43 Puromycin does not block the LH stimulation of glycolysis.40 The LH stimulatory action upon glycolysis can be mimicked by addition of CAMP or dibutryryl CAMP to the incubation medium. When both are at saturating doses the cyclic nucleotide and LH effects were not additive. Addition of theophylline, a phosphodiesterase inhibitor, enhances the ability of low doses of LH to stimulate lactic acid production.43 These findings support the concept that the LH stimu-

416

lation of glycolysis protein.

CHANNING

is mediated

by CAMP and does not require

AND TSAFRIRI

synthesis

of new

LH can also stimulate ornithine decarboxylase in prepubertal rat ovaries, of provided it is administered in vivo. 2o144This enzyme catalyzes the conversion ornithine to putrescine, which is used in the biosynthesis of polyamines. notably spermidine and spermine, which play a role in stabilization of nucleic acids and control of biosynthesis of nuclear proteins.45 LH stimulates the de novo synthesis of the enzyme by a mechanism which involves both RNA and protein synthesis. The LH stimulatory action is blocked by injection of puromycin, cycloheximide, or actinomycin D. Very young rats. before day 8, fAiled to respond to LH with an increase in ovarian decarboxylase activity. Thereafter, responsiveness to LH increased between days 9 and 20. More recent studies by Icekson et a1.46 demonstrated that the follicular tissue rather than the corpus luteum is the most responsive tissue to LH in terms of ornithine decarboxylase activity. In prepubertal rats Nilsson and Selstam47.48 were able to observe an LH and FSH stimulation of amino acid incorporation into ovarian protein and a stimulation of uptake of a nonmetabolizable amino acid, a-aminoisobutyrate. after in vivo administration of the hormones. This confirms and extends previous studies of Ahren et al.4y~50~5z and Noble and Kostyo.” Jarlstedt et al.j3 were also able to observe a stimulatory effect of LH and FSH upon uridine incorporation into RNA. In these studies they had to administer the LH and FSH in vivo in order to see an effect. Direct addition of gonadotropins in vitro were without effect. Dibutyryl CAMP and LH added in vitro also stimulated glycerol release from prepubertal ovaries,43 reflecting a net stimulation of lipolysis. The effects of LH were not as pronounced as were the effects of dibutyryl CAMP. Ovarian alkaline phosphatase can also be stimulated by LH and FSH addition in vitro.5J It appears that most of these diverse acute in vitro metabolic effects of LH on the ovary may be mediated by its stimulatory effect upon CAMP production since addition of dibutyryl CAMP in vitro can mimic many of these effects, including stimulation of glycolysis and alkaline phosphatase. and inhibition of ester synthetase.43,5”m57 The acute metabolic effects obtained by administration of LH in vivo which can not be replicated by administration of LH in vitro, such as stimulation of cholesterol esterase, cholesterol transport into the mitochondrion. ornithine decarboxylase. and amino acid incorporation into protein and uridine into RNA, could not be clearly associated with alterations in ovarian CAMP levels. Furthermore, addition of Dibutyryl CAMP in vitro was unable to stimulate these metabolic steps. This could mean that either these steps are not mediated by CAMP or they are mediated by CAMP and the in vitro systems employed are inadequate for observation of CAMP effects on these metabolic steps. Further research is needed to resolve this issue. A summary of these diverse metabolic effects of LH either apparently CAMPmediated, or not, is presented in Fig. I. C. Role of Prostaglandins No discussion of the mechanism without mention of prostaglandins. been reviewed.58m6’

of action of LH upon the ovary is complete Their actions on the ovaries have recently

LH AND FSH ACTION

MECHANISM

417

Kuehl et a1.62 proposed that prostaglandins of the E series are an essential intermediate in LH action upon the mouse ovary. They found that addition of a prostaglandin antagonist, 7-oxa- 13-prostynoic acid, can block LH action upon conversion of adenosine to CAMP. Other investigators were, however, unable to unequivocally demonstrate an essential role of prostaglandin E in LH action.20.37,63 Kolena and Channing,37 Marsh,63 and Lamprecht et al.*’ demonstrated that PGEz has a stimulatory effect beyond the maximal dose of LH in the stimulation of CAMP production in porcine granulosa cells, bovine corpus luteum, and prepubertal rat ovaries, respectively. Seven-oxa-13-prostynoic acid was unable to inhibit the LH stimulatory action upon CAMP production in porcine granulosa cells or in bovine corpus 1uteum.37*6’ Ovarian tissue can synthesize prostaglandins.“,67 LH can exert a stimulatory effect upon prostaglandin synthesis in rat ovarian homogenates,64 rat corpus luteum cultures,68 and rat and rabbit follicular tissue,6S*66,69,70 but not in granulosa cell cultures.66 In the case of rabbit follicular tissue both PGE and PGF synthesis are stimulated by LH; however, in the rat the rise of PGF is about 6-fold, whereas for PGE it is 30-fold.70 The physiologic relationship between prostaglandins of the E series and the mechanism of LH action therefore remains unclear. Prostaglandins of the E series can stimulate lactic acid production in the prepubertal rat ovary. ” Prostaglandin may be an essential intermediate in stimulatory action of LH in glycolysis since polyphoretin phosphate, an antagonist of prostaglandin action, can inhibit this LH stimulatory action.72 Whether or not the polyphoretin phosphate has other actions is not known. Perklev and Ahren7’,72 also found that high doses (100 pg/ml of 7-oxa-13prostynoic acid (an inhibitor of prostaglandin action) can inhibit prostaglandin stimulation of lactic acid production in prepubertal rat ovaries in vitro. At these high doses the prostynoic acid also inhibits protein synthesis in vitro. At lower doses the prostynoic acid cannot inhibit prostaglandin stimulation of lactic acid production and does not inhibit ovarian protein synthesis. These findings, as well as those of Channing 73 in monkey granulosa cell cultures, where addition of 50 pg/ml 7-oxa-13-prostynoic acid along with LH causes cellular necrosis, lead us to question the specificity of 7-oxa- 13-prostynoic acid as an inhibitor of prostaglandin action. D. Actions of FSH

The mechanism of action of FSH on the intact ovary in the rat has not been as extensively studied as the action of LH, perhaps because of the difficulty in obtaining a reproducible in vitro effect of FSH on ovarian tissue. An explanation for this may be that it has been difficult to obtain an FSH preparation convincingly devoid of LH activity and that FSH effects are restricted to a cell type which is usually present in small amounts in the ovary. Most of what is known about the mechanism of FSH action has been obtained with follicle and granulosa cell cultures and will be presented below in sections II and III. As in the case of LH, the first action of FSH involves binding to a membrane receptor which is principally located on the granulosa ce11.74,75FSH receptors in the granulosa cell are separate from the LH receptors (see section 1II.B below). The second action of FSH involves stimulation of adenyl cyclase, since addi-

418

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tion of FSH in vitro can stimulate adenyl cyclase76 as well as glucose uptake and lactic acid production in prepubertal rat ovaries.J”~77~7XPuromycin does not block the FSH effect upon glycolysis: however, puromycin does lower the basal rate of glucose uptake and lactate production, demonstrating that probably no new protein is needed for FSH stimulation of glycolysis. A similar situation is true for FSH stimulation of CAMP accumulation in the granulosa cell (see section III below). Saturating doses of LH and FSH are not additive in stimulation of adenyl cyclase’(’ in prepubertal ovaries and in porcine granulosa cells.” demonstrating that probably LH and FSH can act on the same adenyl cyclase but do so through separate receptors. Injections of FSH can lead to a marked stimulation of amino acid uptake by the prepubertal rat ovary.“7~J8.s2.56The stimulation of amino acid uptake measured in terms of uptake of a nonmetabolizable amino acid, cu-aminoisobutyric acid, occurrs in both the follicular and nonfollicular components of the ovary. Addition of FSH to the incubation medium in vitro did not, however, have any effects on amino acid uptake. The recent findings bl Moon et al.8” that FSH but not LH can stimulate aromatization of testosterone by ovarian explants from a hypophysectomized animal marks one of the first observations of an in vitro effect of FSH on estrogen synthesis. These investigators further demonstrated that the granulosa cell component of the ovary was most probably the target cell responding to the FSH.“‘,” In 1970 Channing observed a stimulatory effect of purified FSH upon progesterone secretion and morphological luteinization by monkey granulosa cell cultures (see section IllB below). E.

Recapitulation

In the prepubertal or follicular-phase ovary, LH binds to a membrane receptor demonstrable in thecal and granulosa cells as well as other cell types depending upon the species. The result is a stimulation of adenyl cyclase. The CAMP thus produced then interacts with CAMP-dependent protein kinase and activates it. In a series of reactions to be further clarified. the protein kinase activation leads to a stimulation of progesterone and estrogen synthesis. In addition, a number of other generalized metabolic steps are stimulated. including lactic acid production, glucose uptake, glucose oxidation. and RNA and protein synthesis. The LH stimulation of steroidogenesis involves a specitic stimulation of conversion of cholesterol to pregnenolone. The stimulation appears to involve three possible sites: namely. a stimulation of cholesterol esterase. a stimulation of cholesterol transport into the mitochondrion, and perhaps a direct stimulation of the side-chain cleavage enzyme itself. It appears that transport of cholesterol into the mitochondrion is the rate-limiting step in the enzyme reaction and a stimulation of this step and cholesterol esterase is sufficient to result in increased steroidogenesis. Other metabolic activities are stimulated by LH but may not involve CAMP as an essential intermediate. It is noteworthy that these other steps cannot be stimulated by LH added in vitro; the hormone must be injected in vivo in order for stimulation to occur. These steps include the stimulation of ornithine decarboxylase, amino acid transport, RNA synthesis, and cholesterol esterase.

LH AND

II. LH

FSH ACTION

AND

FSH

419

MECHANISM

CONTROL

OF

FOLLICULOGENESIS

AND

FOLLICULAR

GROWTH

This section will deal with the mechanism of action of LH and FSH upon folliculogenesis and follicular growth. In contrast to the next section, which deals with the acute metabolic effects of LH and FSH on the follicle, this section will deal with long-term actions of LH and FSH during the life of the follicle. In order to examine adequately the mechanism of action of LH and FSH on follicular growth it is necessary first to describe the normal events occurring during folliculogenesis and follicular growth, followed by an attempt to examine where LH and FSH act. A. Events During

Folliculogenesis

and Follicular

Growth

The formation of new germ cells by mitotic division of oogonia is completed in the majority of mammalian species examined either just before or shortly after birth. With cessation of mitotic activity, the oogonia enter the prophase of meiosis and thus become oocytes (see the review by Baker*-‘). Shortly after birth meiosis is arrested and the oocytes enter a “resting” dictyate stage. At the onset of the dictyate stage the oocyte is enclosed within a primordial follicle consisting of a single layer of cells. The origin of these enveloping cells is not clear. It has been suggested that they are derived from the surface epithelium of the genital ridge or the “germinal epithelium.“84J5 Witschix6 suggested an additional contribution of the subepithelial mesenchyme to follicular formation. Experimental and ultrastructural studies strengthened the possible contribution of the undifferentiated stroma. 85.117 More recent studies raised the possibility that cells of the rete ovarii are the source of granulosa cellsB8 and control follicle formation as well as the onset of meiosis. 89 Further development of the follicle involves the growth of the oocyte, an increase in the number of layers of granulosa cells, the appearance of a basement membrane surrounding them, and the differentiation of adjacent stromal cells into elongated thecal cells, forming a dense layer encircling the membrane. It was suggested that the proliferating granulosa cells produce inducing substances which stimulate the differentiation of the surrounding tissue into theta interna. During the second week of life in the mouse, concurrent with follicular growth, the thecal layer is formed. The vascular system differentiates, surrounds different follicles, and branches into the outer cortex. During weeks 3 and 4, follicle development progresses until there are small, medium, and large follicles present in the ovary, but the small follicles comprise about 90’1~ of the follicle population.9’ From infancy and throughout the reproductive period of the mature animal, small follicles are constantly growing and developing into more advanced structures. Peters and co-workers have conducted a detailed study of follicular development in the mouse ovary. Pedersen and Peters have proposed a classification of the components of the ovary.92 Pedersen93 investigated the growth of the granulosa cells by autoradiography after pulse labeling with 3H-thymidine. In the mature animal, the oocytes enclosed within small follicles are found to form a pool from which a number of follicles begin to grow during each day of the cycle. The time required for the development of a small follicle to an ovulatory follicle is about 19 days, i.e., four 5-day cycles. While the medium-sized follicles are continuously growing,

420

CHANNING

AND TSAFRIRI

the rate of their growth depends on the day of the cycle and varies from one type of follicle to another. The large follicles have the fastest growth rate, and this is not influenced by the stage of the cycle. The number of Graafian follicles is almost the same at all stages of the cycle, but their size changes: on day I only small Graafian follicles are seen, and these, apparently, degenerate during the first half of the cycle: on day 5 only the large Graafian follicles (type 7) are found. The follicles either ovulate the following night or degenerate. It seems that only the small Graahan follicles found on days 2 and 3 of the cycle will ovulate on the next estrus, while follicles which are either more advanced or retarded in their development at this time are doomed to atresia. Follicular development in the rat appears to closely resemble that in the mouse.y4.y5 More detailed accounts of follicular development and related subjects can be found in the proceedings of three recent symposia.9’.96,97 6. In Vitro

Effects

of Gonadotropins

on Follicular

Growth

I. Eflects on Fetal Ovaries Culture of isolated ovaries demonstrates that female germ cells can proceed through meiotic prophase in the absence of gonadotropic hormones in the culture medium.9X lo3 However, the possibility cannot be excluded that some factors (including gonadotropins) trigger the germ cell differentiation before the earliest stage at which the ovaries are explanted. Moreover. in mouse gonads cultured from the 14th or 16th days of gestation to a time equivalent to day 2 or 3 of life, the total number of oocytes is reduced.‘O? This may be due to lack of some tropic stimulus which is needed for oogonial mitoses and/or their progression to the leptotene stage of meiosis. This needed tropic stimulus may be acting directly on the oocytes or through its effect on differentiation of granulosa cells. Addition of gonadotropins to human fetal ovaries does not improve the histologic appearance of the germ cells or their progression through mitosis or meiotic prophase. Furthermore, the development of the ovarian stroma appears to be normal in the absence of tropic hormones.‘“’ 1. Efects

on Postnatal

Ovaries

In in vivo studies hypophysectomy or administration of antigonadotropin sera fail to completely arrest initial development of follicles: the ovaries of such animals still contain preantral follicles. 95 Therefore, the initiation of follicular growth up until the antral stage is commonly considered not to be dependent on gonadotropins. However. Eshkol et al. 1”4.‘05showed that follicular growth in I-15-day-old mice is abnormal in animals treated daily with antigonadotropin serum during the first 2 wk of life. In the antigonadotropin-treated mice the follicles have a poorly developed granulosa cell layer: the theta does not develop and the ovarian vascular system is retarded in its development. The normal growth of the oocytes. however, is not affected. When animals treated with the antigonadotropic preparation are injected simultaneously with FSH. the overall microscopic appearance of the ovary is similar to that of the control animals. Moreover, the total number of growing follicles is larger in the FSHtreated animals than in the untreated controls. However, the capillary system in

LH AND FSH ACTION

MECHANISM

421

the treated animals seems poorly developed in comparison to the untreated controls. When human menopausal gonadotropin (hMG; a mixture of LH and FSH activity) is administered simultaneously with the antigonadotropic serum, in addition to restoration of normal follicular organization and growth, the theta is well developed and the basement membrane is complete. Furthermore, hMG treatment also induces antrum development, which is not found in the in vitro untreated controls.‘@‘~‘05 In similar studies in the rat, administration of antisera to gonadotropins from day 5 to 15 of life failed to show any effect on early follicular growth.106J07 Studies on the effects of gonadotropins on postnatal ovaries in vitro have been reviewed by Fainstat. lo8 Addition of gonadotropins to the medium has been found to support the survival of explants of mouse and rat ovaries and to increase the number of developing follicles. In a more recent study lo9 mouse ovaries explanted on the second day of life and cultured for 12 days in a hormone-free medium were found to have few small granulosa cells surrounding the oocytes; the development of the stroma is inadequate and the theta does not develop. Addition of FSH (0.2 mg/ml, NIH-FSH-S-9) restores the granulosa layer but not the theta; however, an excessive degeneration of oocytes results. Addition of LH (0.2 mg/ml, NIH-LH-S-18) improves the overall histology of the ovary, leading to proliferation of granulosa and thecal cells, but to a lesser degree than in vivo. It was therefore suggested that normal follicular development in postnatal mice requires the presence of both FSH and LH.‘09 These in vitro studies support the in vivo findings of Eshkol et a1.‘04,‘05However, newborn rat ovaries in organ culture initiate folliculogenesis and secrete a small amount of estrogen in the absence of exogenous LH and FSH; furthermore, addition of exogenous LH and FSH does not alter estrogen secretion, demonstrating that early estogen secretion by neonatal rat ovary may not require gonadotropins.“O 3. Efects on Juvenile and Adult Ovaries During the juvenile period and before puberty is reached (days 15-20 and 35-40 in rodents), the serum gonadotropin levels are usually at low adult tonic levels and the ovaries are fully responsive to exogenous gonadotropins with follicular maturation and ovulation. In vitro studies on the effect of FSH and LH on follicular development in explants of 15-day-old mice were performed by Ryle and her collaborators. It seems that FSH stimulated follicle growth throughout the 6-day culture period, whereas, LH was effective only during the time interval between culture days 3 and 6. Using uptake of 3H-thymidine as a measure of hormone effects on follicular growth, it was found that FSH, but not LH, significantly enhances thymidine uptake during the first 2-4 days of the culture period.“2 However, 3 days of LH exposure resulted in enhanced 3H-thymidine incorporation during culture days 5 and 6. In contrast to FSH, which acts on small follicles, LH stimulates follicle growth only in larger follicles. ‘I2 The initial effect of LH following a 4-day culture seems to be an increase in the number of follicles with a well-developed theca.‘i3 The primary action of FSH on granulosa cells in the initial stages of follicular

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development has been confirmed by several investigators.i”?,‘“j.“?.“J In studies of gonadotropin binding by porcine granulosa cells, it has been shown that cells from small follicles bind more FSH than those from larger follicles.“’ On the other hand, the number of granuiosa cell LH-hCG receptors increases as the follicle matures.“5-“X LH hCG binding is stimulated by FSH in vitro in porcine granulosa cell cultures’20 and in vivo in the rat.“‘ Nevertheless. it has been shown that both FSH and LH are required to sustain normal follicular growth. ‘02.‘04~“2 A synergistic effect of FSH and LH was found on estrogen synthesis by explanted mouse ovaries.‘?’ This synergistic effect requires several days for its development and it has been demonstrated only during culture days 3 6. Only a few studies on follicular growth in ovarian explants from mature animals have been reported. Neal and Baker found that limited growth of follicles and oocyte maturation result from pregnant mares’ serum gonadotropin (PMSG: a hormone having FSH and LH activity) addition followed by hCG addition to explanted mouse ovarian fragments.‘“’ The same authors failed to induce follicular growth in explanted cortical fragments of human ovaries by addition of gonadotropins.“‘” C. Recapitulation The mechanism by which a primary follicle is recruited under the influence of gonadotropins from the nonproliferating pool and induced to grow still remains a mystery. There is no clear evidence for an obligatory role of gonadotropins on the fetal stages of oogenesis and normal ovarian stroma development. In vitro studies on explanted postnatal and juvenile ovaries seem to indicate a need for both FSH and LH even for the early stages of follicular development prior to antrum formation. Whether follicular growth at these immature stages represents the normal regulatory processes occurring during the estrous cycle is not clear. Nevertheless, studies on hypophysectomized animals demonstrate a need for gonadotropins for follicular growth beyond the antrum formation and for ovulation.‘“~‘z? III. LH AND FSH ACTIONS

ON METABOLISM

BY ISOLATED

A. Graafian

OVARIAN

AND STEROIDOGENESIS

CELL TYPES

Follicle

The basic steps in the mechanism of action of LH and FSH on isolated follicles are in general agreement with those employing intact prepubertal ovaries. Since the Graafian follicle is the principal ovarian source of androgen and estrogen, studies on the mechanism of action of LH and FSH in relation to estrogen biosynthesis will be examined. Since the follicle is composed of two steroidogenically active compartments, the granulosa and the thecal layer (Fig. 2), any metabolic activity (either steroidogenesis or general metabolism of the follicle) is a reflection of reactions taking place in both cell types. Since more is known about the influences of LH and FSH upon the granulosa ceils compared to the theta, the discussion of actions upon the follicle as a whole will be combined with what is known about actions upon isolated thecal tissue. A separate section (III-B below) will be devoted to granulosa cells. Any action of LH and FSH upon the Graafian follicle can be looked upon as

LH AND FSH ACTION

423

MECHANISM

OOCYTE

SMALL FOLLICLE

Fig. 2.

MEDIUM FOLLICLE

MATURATION

LARGE PREOVULATORY FOLLICLE

Diagramatic sketch of LH and FSH action during maturation of the Graafian follicle.

initiation of the luteinization process. Therefore, the process of luteinization will first be defined and the steps of action of LH and FSH upon the follicle outlined. I. Luteinization The luteinization process of the follicle can best be defined biochemically in terms of a concurrent increase in progesterone secretion and a decrease in estrogen secretion in the presence of the LH and FSH surge occurring immediately prior to ovulation. ‘o*36Depending on the species, some morphological changes start in granulosa cells, heralding the luteinization process within the follicle prior to ovulation. These changes include an increase in smooth endoplasmic reticulum, an increase in lipid droplets, and some mitochondrial changes which involve a change from lamelliform into villiform type of christae.‘23 After ovulation, morphological luteinization of the granulosa cell becomes fully expressed. Morphological luteinization involves an increase in cell size, cytoplasmic-nuclear ratio, accumulation of cytoplasmic eosinophilic granules, and lipid droplets. Thecal cell “luteinization” occurs earlier than granulosa cell luteinization in the life of a follicle. 2. LH and FSH Binding to Follicle The first step in the action of LH (or hCG) upon the follicle is binding to a thecal or/and granulosa cell receptor. I9 Isolated thecal tissue of mature porcine follicles can bind iodinated hCG more so than thecal tissue obtained from less mature smaller fo11icles.19 Rat thecal tissue can also bind LH or hCG.74J24.‘25 Granulosa cells can also bind iodinated hCG, with an increased number of binding sites per cell occurring as the follicle matures.‘9~1’7 Intact rabbit and rat

424

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AND TSAFRIRI

follicles can also bind iodinated hCG in vitro.‘26.“7 It is of interest that in the period after the LH surge when the follicle loses its responsiveness to exogenous LH or hCG there is no concomitant diminution in hCG binding.“’ This could indicate that the loss in follicular responsiveness to LH prior to ovulation is at a step after the LH-hCG receptor. FSH binding has not been demonstrated in thecal tissue.7J~“J~‘2X FSH does, however, bind to granulosa cells.“4~“’ It is difficult to explain the findings of Ahren et al.,4’who demonstrated that FSH but not LH could stimulate oxygen consumption by rat thecal tissue if there are no thecal receptors for FSH. It is possible that there are few FSH receptors in the thecal tissue which cannot be demonstrated by conventional autoradiographic techniques or, alternatively, it is possible that the thecal FSH receptors become saturated easily with endogenous hormone, making it difficult to demonstrate their existence employing exogenous labeled FSH. 3. Stimulation

of CA MP

Production

LH and FSH stimulate CAMP synthesis. steroidogenesis, and lactate production by isolated rat proestrous follicles’“~‘0~‘2y and by immature PMSGtreated rat follicles in vitro.jb.L7 Marsh et al.130 also observed an LH stimulation of isolated rabbit follicles. FSH of CAMP accumulation in steroidogenesis’j’ alone has an intrinsic ability to stimulate CAMP accumulation and steroidogenesis in the rat follicle since it is active even in the presence of a specific anti-8 ovine LH antiserum which neutralizes the LH contaminant of the FSH preparation.lO.36.56

4. Stimulation

of Steroidogenesis

Exposure of estrous-phase rabbit follicles to LH leads to the eventual luteinization of the follicles when they are transplanted into the kidney capsule.‘32 Since exposure of rat and rabbit follicles to dibutyryl CAMP has the same luteinizing action, it seems that CAMP mediates the action of LH on luteinization.‘33,‘34 Rat preovulatory follicles, isolated prior to the endogenous gonadoestradiol during 12-hr incubations.3h tropin surge, secrete predominantly Estradiol is the major radioactive product formed in vitro from W-acetate by of LH to rat proestrous follicles in human I33 and rabbit follicles.‘3’ Addition culture causes a striking increase in the rate of progesterone accumulation. This increase is accompanied during the first 4 hr of incubation by a smaller, though significant increase in the rate of accumulation of 17-hydroxyprogesterone, androstenedione, testosterone, and estradiol. 36,‘36Similar results are obtained for rabbit follicles.“’ “j 17/3_hydroxyandrogen secretion is stimulated at a low dose of LH. whereas estrogen and progestin are stimulated using higher levels of LH. Only a short exposure (1 set) of the follicles to LH (5 pg/ml) appears to be required for later stimulation of steroidogenesis. Compared to estrogen and progestin, l7hydroxyandrogens, including testosterone, are the most rapidly and dramatically stimulated. Addition of CAMP itself can also lead to a stimulation of rabbit an LH and follicular androgen secretion. 142Mills and Savard also observed FSH stimulation of follicular estrogen, androgen, and progesterone secretion

LH AND FSH ACTION

MECHANISM

425

as well as a stimulation in CAMP accumulation.‘3’.‘43 Puromycin and cycloheximide but not actinomycin D block the LH stimulatory effect on steroidogenesis in the isolated rabbit follicle, demonstrating that perhaps protein but not new mRNA synthesis is required for the LH stimulation of androgen synthesis.14’ Gorski and Padnos’44 also observed in preovulatory rabbit ovarian explants that a dose of actinomycin D which is capable of diminishing incorporation of 3H-cytidine into RNA to less than 10% of that of controls failed to inhibit LH stimulation of steroidogenesis, whereas cycloheximide and puromycin block the LH stimulation of steroidogenesis. On the other hand, LH-induced progesterone is synthesis by rat proestrous follicles35.36 and in bovine corpora lutea is blocked by protein-synthesis inhibitors as well as by actinomycin D. Actinomycin D abolishes effectively the stimulatory effect of LH on progesterone in rat follicles only if given within the first hour after exposure to LH. When the addition of actinomycin D is delayed until 2 hr, progesterone accumulation continues undisturbed for an additional 10 hr. Therefore, it was suggested that the actinomycin D-sensitive product (mRNA?) required for progesterone accumulation is synthesized in an adequate amount during the first 2 hr of LH action, and is stable for at least 10 hr.36 This difference in the sensitivity to actinomycin D of LH-induced steroidogenesis between mammalian species may reflect a difference in the control of the steroidogenie process. We have demonstrated that LH can stimulate progesterone secretion, and to a lesser degree estrogen secretion, by preovulatory porcine follicles as well as by 6-8 mm follicles obtained from PMSG-treated monkeys which were grown in rolling organ cultures for 48 hr (Channing and Tsafriri, unpublished observations). Moor and co-workers ‘45~‘47demonstrated a stimulatory effect of LH upon progesterone secretion by cultured sheep follicles. They also demonstrated that LH and CAMP added to the sheep follicle cultures lead to a decrease in estrogen secretion. ‘4X In cultured preovulatory sheep follicles 3flhydroxysteroid dehydrogenase appears first in the thecal layer and only later in the granulosa layer.‘46 Lindner et al.‘O observed that the addition of LH (as well as FSH plus anti-8 LH antiserum) to isolated preovulatory rat follicles exerts a small stimulatory effect upon estrogen secretion, followed by an inhibitory effect. A biochemical explanation of the inhibitory influence of LH on follicular estrogen secretion is not available. It is possible that follicular androgens and their metabolites may be aromatase inhibitors since LH stimulates follicular androgen secretion. Thompson et al. ‘49have demonstrated that reduced androgens can inhibit placental aromatase activity. An LH-induced decrease in follicular aromatase would explain the sharp drop in estrogen in the blood immediately upon initiation of the LH surge in the monkey150 and in other species. During late follicular growth LH and FSH act to stimulate hypertrophy of the theta prior to the granulosa.‘46~‘5’~‘52 As a result, estrogen is secreted by the follicle into the ovarian vein. ‘53.‘54In the preovulatory follicle the theta apparently can synthesize estrogen alone without granulosa cells, as demonstrated in vitro in the mare,‘55 sheep,ls2 human,‘56J56a and monkeyi56a and in vivo in the monkey’53,‘54 and mare. 157Observations by Hay and Moor’52 done in cultured Graafian sheep follicles support the concept that the theta is the principal

426

CHANNING

AND TSAFRIRI

source of estrogen. They found that early in culture at a time when the theta was rich and the granulosa cells lacked histochemically demonstable 3phydroxysteroid dehydrogenase the follicles secreted elevated amounts of estrogen. Other investigators, led by Falck, I58 think that the granulosa and theta interact together to synthesize estrogen. In the cow,IL9 the rabbit,‘38 and the hamster160 minces of theta and granulosa produce more estrogen mixed together compared to either cell type alone. Data from in vitro studies where the theta is minced up and mixed with granulosa should be interpreted with caution since broken cells may be present and enable unphysiologic mixing of enzymes, precursors, and products. This situation may not exist in vivo, where theta and granulosa are normally separated by a basement membrane. B. Granulosa

Cells

As a model cell for LH and FSH mechanism of action, the granulosa cell offers many advantages, a major one being that it is a homogenous cell type even at the ultrastructural level.“‘~‘6’~‘63 In addition, it changes its sensitivity to gonadotropins as a function of follicular maturation. The granulosa cells lie within the follicle suspended in an avascular compartment containing follicular fluid (Fig. 2). As the follicle grows and matures under the influence of FSH and LH (described above in section I), the granulosa cells divide and differentiate. Granulosa cells will1 eventually form the corpus luteum after ovulation. Granulosa cells from a variety of species will luteinize spontaneously in culture provided they are obtained from mature Graafian follicles. Since the early findings of Channing and Grieves in the mare,‘5L.‘64~‘66granulosa cell luteinization has been observed in cultures of granulosa cells of the pig,lb7 “O human 156,171 173 monkey,l61,174~177 rat,l?3.178 rabbit 162.163.179.180 mouse,181 and ~0w.l~~ ‘Hence the granulosa-cell culture system provides a good model for studying the mechanism of the luteinization process.‘83,‘84 Furthermore, granulosa cells obtained from less mature follicles can be made to luteinize in vitro by addition of appropriate gonadotropins or CAMP. The purpose of this section is to review the nature of these LH- and FSHinduced maturational changes in granulosa cells and the way in which they can result in eventual luteinization of the granulosa cells after ovulation. The expression of these maturational changes can be observed in short-term in vitro incubations and under longterm monolayer culture conditions (Fig. 3) in the presence and absence of LH, FSH, or a mixture of LH and FSH. 1. Properties

of Granulosa

Cells at Various Phases of Follicular

Maturation

a. Immature smallfollicles. Here the granulosa cells exist in a relatively undifferentiated state. Cultures of such cells obtained from the monkey.‘74,‘77 pig,‘68 mare,184~185and rabbit’79 are unresponsive to either LH or FSH added alone in terms of stimulation of progesterone secretion or morphological luteinization. They are also unable to respond to LH in terms of stimulation of CAMP production after a 30-min incubation period.‘83,‘86 The lack of responsiveness of these immature granulosa cells to LH can be explained by a lack of available LH receptors. Pig granulosa cells harvested from small follicles can bind only small quantities of ‘251-hCG or LH in vitro.“6.“7 This is also true

LH AND

Fig.

FSH

3.

sontation

cells

maturation.

cubations;

LH,

the presence LH

of

reproin porcine

during C,

in-

incubations

in

1 Pg/ml

were

30-min

ovine

Data

production

binding

follicu-

control

(NIH-oLH-S19).

CAMP

hCG

obtained

after

incubation. was

after

a

incubation

cell

cultures. are

after

using

a

in

4%hr

employed detail

and

Rigby.‘S

(Reproduced

permission

from

Tsafriri.“)

of

monolayer

Methods

described

Channinf

estimated

luteinization

estimoted

incubation

PRODUCTION(30YIHl

suspensions.

Morphological was

a

DF LH TO STIMULATE CAMP

ABILITY

Proges-

secretion 24-hr

for

and

terone

granulosa

427

MECHANISM

Diogromotic of changes

granulosa lar

ACTION

in

Ledwitz-

Channing

with

MORPHOLOG’CAL LUTEINIZAT’ON IN CULTURE‘4s MS)

NO

SMALL ‘I-ZMY)

NO

YES

MED’UM ‘3-SMLO

LARGE ‘6-12MM’

FOLLICLE SIZE

in the rat, since after injection of labeled LH or hCG or addition of the labeled hormone to ovarian slices in vitro granulosa cells in mature follicles but not small follicles bind the hormone.74,‘“9,‘90 In the case of FSH, in contrast to LH, it is a generally accepted concept that cells in smaller follicles are more responsive to FSH as compared to cells in Zeleznik et al.lY3 demonstrated in preliminary studies that larger follicles.“’ granulosa cells obtained from small porcine follicles can bind more FSH compared to cells obtained from more mature medium and large follicles. Changes in FSH responsiveness of porcine granulosa cells in terms of CAMP accumulation during follicular maturation have recently been examined by Lindsey and Channing. 19’ Ovine FSH (NIH-FSH-SlO) pretreated with antiserum to LH to inactivate contaminating LH was added to granulosa cells obtained from small (l-2-mm) medium (3-5-mm) and large (6-12-mm) porcine follicles. The cells were incubated for 30 min, followed by assay of cellular CAMP according to methods described previously.“’ Consistently, granulosa cells obtained from small follicles produced lO_20-fold more CAMP in response to l-100 pg/ml FSH compared to cells obtained from medium and large follicles.‘9’ The opposite was true for LH responsiveness: 19’cells harvested from large follicles responded to 1 pg/ml LH with a resultant increase in CAMP levels 5-20-fold greater than in the case of cells harvested from small follicles ‘83.19’ b. Medium-sized follicles. FSH as well as LH stimulates CAMP production by granulosa cells from medium-sized follicles of the pig.37.79 Granulosa cells obtained from medium-sized porcine’68 or monkey ‘76 follicles will not luteinize spontaneously in culture, but will do so if cultured with either LH or FSH or

428

CHANNING

AND

TSAFRIRI

dibutyryl cAMP’~‘.“” or PGE2.j8 LH can stimulate progesterone secretion after 6-- 12 hr by cells obtained from medium-sized porcine follicles if cells are grown in suspensions.‘” The LH stimulation of progesterone secretion is preceded by stimulation of CAMP production which lasts from 5 min to 223 hr.‘7.“y.‘X6 The increased responsiveness of these cells to LH compared to cells from small follicles can be accounted for by their attainment of an increased number ol LH_hCG binding sites_?b.IIb.I I8.188.IY3 IY7 N .th el er puromycin nor cycloheximide could inhibit LH stimulation of granulosa cell CAMP production.” During development of a medium follicle there is an increase in follicular fluid estrogen and in blood estrogen levels in the human and other species.“‘, 154~171~173,18?~198 Therefore, while they are in a “medium-sized follicle,” granulosa cells are exposed to an increased estrogen content plus LH, FSH. and other hormones.17? Estrogen levels of follicular fluid of medium-sized (3 5-mm) follicles from monkey”3~“J and human’7’~i7”~‘yX follicles may vary from 100 to 2000 ng/ml. Studies in the human have revealed that FSH is present in a greater number of small follicles (~8 mm) during or just after the peaks of FSH in peripheral plasma. During the middle follicular phase the concentration of both FSH and estradiol in the fluid of large follicles (>8 mm) is high. During the late follicular phase large follicles contain high amounts of progesterone in addition to estradiol, low levels of proactin. and concentrations of LH and FSH of about 30”, and 60”,,. respectively, of those found in plasma.‘7’~‘7’ This in vivo hormonal environment within the follicle is important to consider in an eventual discussion of what controls differentiation of the granulosa cell in vitro. McNatty and Sawers”’ went on to demonstrate that in the human only granulosa cells from follicles containing some FSH and high concentrations of estradiol undergo spontaneous mitosis in vitro. Mitoses can be stimulated in vitro by adding FSH and estradiol to the culture, providing the concentration of LH is low in the culture medium and in the follicle prior to cell harvest. Cells harvested from follicles containing LH. FSH, and high concentrations of estradiol secrete significantly more progesterone in culture than cells obtained from follicles which do not contain all three hormones. c. Largefollicles. By the time the follicle reaches the preovulatory phase and is a large follicle, the cell undergoes a 50-500-fold increase in the number of The increase in binding capacity appears LH-hCG binding sites. 7~~i’s-“7~‘87~‘93~‘96 to be due to an increase in the number of receptors per cell rather than due to a change in affinity for hCG or LH, since by Scatchard plot analysis the K, does not change as a function of follicular growth (Table I). as demonstrated by five groups of investigators (including C. Sakai and C.P. Channing, unpublished observations).‘x7,‘88,‘93 ly7 V,d Iues for the dissociation constants for hCG reported in Table 1 are given for cells obtained from large follicles. The K, in the case 01 the cells obtained from small and medium follicles does not differ significantly from the values shown for cells obtained from large follicles. Granulosa cells harvested at the time of ovulation show a decrease in their available LH-hCG receptors compared to cells harvested a few hours earlier,“~“’ possibly because the receptors become saturated with endogenous LH or the receptor becomes as suggested by Hunzicker-Dunn and Birnbaumer.“’ “inactivated,” Cells harvested from large follicles can also synthesize more CAMP and

LH AND FSH ACTION

MECHANISM

429

Table 1. Bindina Cdl Preparation

Affinities

Kn(M

of lodinated

hCG to Ovarian Cells

x lo-“)

Porcine

granuloso

8.3 f

Porcine

granuloso

2

Investigator

1.1

Ref.

Stouffer

et cd.

194

Zeleznik

et al.

193

Porcine

granuloso

1.3-1.8

Kammermon

Porcine

granuloso

3

Sakoi

and Ross

195

Porcine

granulosa

2.4

Lee

188

5.2

Lee and Ryan

226,227

and Channing

Unpublished observations

Luteinized ovarian Data data

buffered

membranes

of Kammerman

of Sakai

porcine

rat

and

gronuloso saline

cells from

containing

were

and Ross”’

Charming

obtained

(unpublished large

0.1%

(6-12.mm)

bovine

after

observations)

serum

o 5-hr were

incubation obtained

porcine

follicles.

albumin

according

The

of porcine after

cells

to methods

a 2-hr

were

granulosa incubation

incubated

detailed

in

elsewhere.

cells,

while

(37°C)

of

phosphate117,183

respond to exogenous LH with more CAMP production compared to cells obtained from smaller follicles’75~‘83~‘86~‘9’ F‘ ( lg. 3). This finding is also true in the presence of excess (3 mM) theophylline, an inhibitor of phosphodiesterase, demonstrating that changes in CAMP production in response to LH during follicular maturation are due to an increase of the ability of LH to stimulate production of CAMP rather than alterations in phophodiesterase activity. LH stimulation of granulosa cell adenylate cyclase is also elevated as the follicle matures.200 Most likely, the increased responsiveness to LH is due to the increase in LH-hCG receptors, although the possibility exists that adenylate cyclase activity may be regulated independent of the LH-hCG receptor. Recently, scanning electron micrographs of porcine granulosa cells at various stages of maturation within the follicle have given some insight into what may be going on in the granulosa cell membrane during follicular maturation.20” Cells present in large 6-12-mm preovulatory follicles contain a vast amount of microvilli, whereas cells present in l-2-mm small or 3%5-mm medium follicles contain few if any microvilli. Whether the increased LH-hCG binding sites in mature granulosa cells can be solely accounted for by the increased amount of surface area of the cell due to the appearance of the microvilli cannot be ascertained at the present time. Alternatively, the changes of surface of microvilli may reflect changes in the cell cycle and may not be involved in the maturation process per se. 2. Role ofcA MP in Luteinization Initiation as well as maintenapce of granulosa cells in the luteinized state requires CAMP as an essential intermediate. If granulosa cells harvested from preovulatory monkey follicles are incubated with 10 mm imidazole, a stimulator of phosphodiesterase, spontaneous morphological luteinization and progesterone secretion are inhibited.‘75 The stimulatory action of exogenous LH is also inhibited.“’ Addition of dibutyryl CAMP can stimulate morphological luteinization and progesterone secretion of granulosa cells from nonpreovulatory porcine,‘92 monkey,“’ and cowzo’ follicles. Addition of dibutyryl CAMP can stimulate further luteinization after an 8-day period in culture after

430

CHANNING

AND TSAFRIRI

luteinization has been lost.‘75 These findings have been extended by Gospodarowicz and Gospodarowiczzo2.203 m monolayer cultures of bovine corpus luteum cells. Futher support of a role of CAMP in formation of the corpus luteum is obtained in the finding that addition of theophylline, a phosphodiesterase inhibitor, enhances the ability of the monkey’74 and porcine”’ granulosa cells to secrete progesterone in the presence and absence of exogenous gonadotropins. 3. Follicular

Fluid Granulosa

Cell Luteinization

Inhibitor

As the follicle matures, some restraining controlling mechanism appears to act within the follicle to keep the granulosa cells from luteinizing until immediately prior to or after ovulation. A follicular fluid (FFI) “luteinization inhibitor” has been proposed to explain this situation. Ledwitz-Rigby and associates demonstrated that 20”,-50”” FFl obtained from small (1 -2-mm) porcine follicles, but not fluid obtained from large (6-12-mm) follicles or porcine serum, can inhibit granulosa cell luteinization in vitro, both morphologically and in terms of progesterone secretion.“9.204m206 The site of inhibitory action of the FFI appears to be on LH stimulation of CAMP production and not on binding of LH to preexisting LH-hCG receptors. 206The FFI from small procine follicles may lead to increased destruction of CAMP by a stimulation of phosphodiesterase since its inhibitory activity can be overcome by theophylline and it stimulates phosphodiesterase activity in porcine granulosa cells.206 Bernard has also demonstrated an inhibitory influence of bovine and porcine FFI upon rat granulosa cells in culture.207~208 However, Bernard did not examine the effects of FFl obtained from different sizes of follicles. In vivo studies by Dzuik ‘09 in the pig and by Hammond and Wodzicki”’ in the mare also support the existence of a FFl luteinization inhibitor. They slit the preovulatory follicle and removed the follicular fluid. In both instances the granulosa cells luteinize in the slit follicle before they do in sham-operated control follicles. 4. Role of Bound LH in Granulosa Cell Luteinization That it is the bound LH which is responsible for stimulating granulosa cell luteinization after ovulation or in culture can be ascertained by examination of the effects of removal of bound LH. We have demonstrated that granulosa cells incubated with 1 pg/ml ovine LH for 20 min preincubation followed by a I-hr incubation in the presence of a 1:50 dilution of anti-p ovine LH (kindly supplied by Dr. Koch of the Weizmann Institute) can inhibit the LH stimulation of CAMP accumulation.” If the cells are preincubated for 40 min with 1 pg/ml LH followed by a 60-90-min incubation with LH antiserum, the action of the previously bound hormone cannot be inhibited. This would indicate that bound LH can stimulate CAMP, but changes are induced between 20 and 40 min and the bound hormone (or whatever is available for removal by antiserum) is no longer required. This antiserum was shown by Koch et al.“’ to remove 70”,, of bound LH from rat ovarian slices. Recently we have demonstrated that this LH antiserum can remove 60:~~ of bound iodinated LH from porcine granulosa cells (Thanki and Channing, unpublished observations). Functional luteinization (spontaneous increase in progesterone secretion) of porcine granulosa cells ob-

U-l AND FSH ACTION

431

MECHANISM

tained from large preovulatory follicles is not inhibited during a 24-hr addition of LH antiserum” under conditions where the LH antiserum can remove 60% of bound LH and inhibit the action of bound LH in terms of stimulation of CAMP. This finding could not however, rule out a role of bound hormone in the induction of luteinization since the cells had previously been exposed to LH in vivo, probably for a time period of more than 20-40 min. A similar type of situation exists in hCG-induced ovulation in the mouse. If mice are given hCG and a period of 2 hr is allowed to elapse, followed by antiserum administration, the mice ovulate normally 12 hr later.2’2 Either the bound hormone is no longer needed to finish those processes once they are started, or it binds up irreversibly and induces irreversible changes which can proceed in the absence of further hormone. Prolonged maintenance of luteal function does, however, require bound hormone since injection of LH antiserum during the luteal phase of the cycle can decrease luteal progesterone in the sheep213 and rat214**15and can cause functional luteal regression in the mare.2’6 A role of bound LH in maintenance of granulosa cells in the luteinized state beyond l-2 days has been demonstrated in cultures of monkey granulosa cells obtained from large preovulatory follicles (obtained during the estrogen surge; Channing, unpublished observations). Such cells luteinize spontaneously initially in culture but lose part of their luteinized morphological traits and decrease in progesterone secretion rate after 6-8 days in culture. Addition of 1:50 and 1:5000 anti-human LH (obtained from the Hormone Distribution Office of the NIH) causes a diminution in progesterone secretion and a hastened loss in morphological signs of luteinization compared to control cultures. The antiserum does not cause irreversible damage to the cells. When the cells are washed to remove the antiserum after 4 days of exposure to it and a 4-day incubation in plain culture medium elapses, followed by subsequent addition of 100 ng hCG, the cells respond to the late addition of gonadotropin with a stimulation of morphological luteinization and progesterone secretion. This antiserum can also inhibit the stimulation of luteinization brought about by exogenous LH. 5. Granulosa Cell LH-hCG

Receptor Excess

In both the testis2” and adrenal 2’8 it has been shown that there is an excess of tropic hormone receptors beyond those required for maximal stimulation of testosterone secretion or corticosterone production. Whether this is true for the ovary has never been directly proven, since binding, CAMP production, and progesterone secretion have not been measured in the same type of granulosa cell suspension. The possibility of “receptor excess” has been alluded to indirectly based on studies using monkey and porcine granulosa cell cultures and suspensions. It was found that in porcine granulosa cell suspensions obtained from large follicles it takes 0.5 pg/ml of hCG to saturate the receptors,“’ whereas a dose of less than 0.01 pg/ml of hCG is required to maximally stimulate progesterone secretion in monkey granulosa cell cultures after 4-8 days in culture.‘74,‘75 In the present study, binding, CAMP accumulation, and progesterone secretion have all been examined in granulosa cell suspensions obtained from

CHANNING AND TSAFRIRI

432

medium-sized porcine follicles. Binding and CAMP production have been measured after a 30-min incubation period, whereas progesterone secretion has been measured after 24 hr, since a good response of progesterone secretion to LH takes at least 12 hr.3S.“9 In the granulosa cells of medium-sized follicles it is thought that the LH must induce a number of biochemical changes before an increase in progesterone secretion can be observed. Granulosa cell CAMP production was measured by a protein-binding assay as described previously.37,‘83 Progesterone was measured by radioimmunoassay2’9 using an antiserum against 6-OH progesterone linked to bovine serum albumin generously donated by Dr. Gordon Niswender of Colorado State University. The hCG binding was assessed using biologically active ‘251-labeled hCG as outlined previously.“7’96 In the cells shown in Fig. 4, hCG binding was not saturated until 500 and 1000 ng/ml hCG were added, whereas maximal stimulation of progesterone secretion occurred at about 5 ng/ml hCG, demonstrating that only about 5”,,- lo”,, of available receptors are required for the physiologic response. Maximal

GRANULOSA

20

IS-

c

IO,000

1

CELLS

FROM

PORCINE

s,ooo-

MEDldM

SIZED ,4C

FOLLICLES

-.

,rl, 0

‘.

‘.

PROGESTERONE

‘.

,’

‘=_._ /’ , c

/

I

8

I

n

,’

130

:’

Id

f

/

Fig. 4. Effect of various doses of hCG on lz51-hCG binding, CAMP levels, and progesterone secretion by a batch of porcine granulosa cells harvested from medium-sized (3-5-mm) follicles. Binding of “‘1-hCG and cellular CAMP levels were measured after 30 min, and progesterone secretion was measured after 24 hr of incubation. The granulosa cells were incubated with shaking in 5% CO1 in air at 37°C at a concentration of 1 x 10’ cells in 1.O ml Eagles medium containing 1% bovine serum albumin. (Methodologic details were previously reported.la3)

LH AND FSH ACTION

MECHANISM

433

CAMP production was brought about by only about 10 ng/ml hCG, about twice the dose required to maximally stimulate progesterone secretion. Therefore, there is only a modest excess of CAMP production beyond that required for the progesterone response. In the testis and adrenal there is a much larger excess of CAMP production beyond that required for the steroidogenic response. Perhaps in the ovary there is tighter coupling between CAMP and the response than in the testis or adrenal. It has been suggested by Robert Ryan (personal communication, 1976) that there may not be a “receptor excess.” The possibility exists that the lo/,-lo% of receptors required for the activation of the physiologic process are of very high affinity, and that these receptors are linked to adenylate cyclase, while the other 90%-997; have a lower affinity for hCG, about lo-” M, and are not linked to adenylate cyclase. It is possible that the l%-loo/, high-affinity receptors cannot be detected in the conventioanl Scatchard plot. The low/affinity receptors may be physiologically “inert” or serve as a storage depot for transfer of the hormone to the high-affinity receptors. If one compares the amount (picomoles) of hCG bound required to initiate the maximum basal response to the picomoles of CAMP produced and to the picomoles of progesterone produced, it is apparent that there is a significant amplification of the final response (progesterone secretion) by a factor of over 1300 (1365 and 1428 in two separate batches of granulosa cells). A quantitative look at such relationships between receptor binding and CAMP and progesterone production may be meaningful in situations such as follicular atresia, when one of these amplification sites may be deficient. Just how the parallel control of receptor formation, CAMP production, and progesterone secretion is brought about during follicular maturation is not known. 6. Gonadotropin Structural Requirements of the Granulosa Cell for LH-hCG Binding and Stimulation of Granulosa Cell Luteinization In binding studies employing porcine granulosa cells and “‘1-hCG and in biologic potency studies measuring progesterone secretion by monkey granulosa cell cultures, the ability of hormonal derivatives to inhibit hCG binding agreed with their ability to stimulate monkey granulosa cell luteinization as measured by stimulation of progesterone secretion over an g-day period (Table 2). The biologic response was measured over a longer time period (4-8 days) compared to binding, which was measured in 30 min. Nevertheless, there was agreement between binding and progesterone secretion. Sialic acid was not required for either binding or stimulation of progesterone secretion. Both binding and stimulation of progesterone secretion, however, required an intact hormone since subunits of hCG or LH could not inhibit binding of 12’I-hCG nor stimulate progesterone secretion. Bahl and associates 222-224have examined the sugar requirements of hCG for binding and biologic activity employing testis cells. Studies in the testis225 and in the luteinized rat ovary226,227 confirm the correspondence between binding and biologic activity of LH or hCG, asialo hCG, and their subunits in vitro. Studies of potencies of various derivatives of LH or hCG in vivo often do not yield agreement between binding and biologic activity due to the fact that asialo hCG and LH and hCG subunits may be degraded

434

CHANNING

Table

2.

Influence

Inhibit

“’

of Alterations

I-hCG

Binding

of the Ovine

and

the Ability

as Measured

LH and

hCG

to Bring

About

by Progestin

Molecule

to

Secretion Potency Compared lo Native hCG and LH in Stimulating Monkey Granulosa

Cells (%I 100

hCG

loo-150

(> 95%

the Ability

Luteinization

‘~5 I-hCG Binding Hormone

Asialo-hCG

TSAFRLRl

Ability to Inhibit by Porcine Gronuloro

Native

Upon

Granuloro

AND

Progertin

Cell

Secretion (%I

(n = 8)

100

(n=8)

(n = 6)

120

(n = 7)

desialyated)

a-hCG

subunit

.

meeting on the development the reproductiLe

197-l

sur le corps jaune,

uptake

pituitar!

crinol 52:5X5. lY71

of the corde I’hormone

V. Wagner

human

administratwn

hormone:

Nationale

de

Academic.

Morphol

in the

8: IX?. 1973

loque dc la Soci& et

and

12SI-labeled

Folia

Presl J. Po’rplsil J. I-igarova

Developmental

and maturit!

Gonadotroplns

biochemistry

C: Mode

(LH)

1972

of

gonadotropin.

1971

25. V:

ot

and follicle-stimulating

distribution

female. Copenhagen.

Nev. Yorh,

Biol Reprod

iuteinisante

S: Reproductive

(ed):

K: The

17. Hermier

Edin-

Excerpta

15. Moudgal

Ryle M: Gonado-

Development.

1970

H. Glasser

Amsterdam,

Gonadal

AC.

Ovarian

chorionic

sites in some organs

ing sites in the immature

trophins

human

gonadotropin

Grind!

Wiley

for

hormone

York,

CC.

V. Pospisil J, Wagner

J. tvldence

chorionic

1972

in

24. V.

gonadotropin BB.

in

13:30.

of

1976 I?.Sarena

AMP

Reprod

Res 30:79.

Sadler

in Fertility

Biol

female rat. EtTect of non-lahelled

maturation. in

role of cyclic

the immature

A: Regulation

IuteiniLatlon.

Segal S (eds): Advances

for

The

steroldogenesis.

human chorlonlc

ovulator!

og.

JM:

Marsh

01

1974 I I. Charming

Yorh.

1973, p I23

binding

division

Horm

Mechanisms.

vol 5. New

gonadotropic

differentiation

Pros

Control

Symposium.

1976

cultured

and

Recent

23.

gonadal

Barnea

of maturation

oocyte

cell.

S,

Academic,

AND TSAFRIRI

in

Winter

York,

A. Lieberman

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