Newborn pig ovarian tissue xenografted into Severe Combined Immunodeficient (SCID) mice acquires limited responsiveness to gonadotropins

In the pig ovary, the transition from primordial to primary and secondary ovarian follicles begins before birth, but antral follicles can be observed, for the first time, at ∼60-90 d of age. At approximately the same time, secondary follicles become responsive to gonadotropins, leading to the formation of antral follicles. Placing pieces of ovarian tissue under the kidney capsule of immunodeficient (SCID) mice allows the requirements for follicular recruitment and development to be studied. The objective of this study was to investigate if primordial follicles contained in ovarian fragments isolated from newborn piglets (36 ± 12 h old) and immediately transplanted under the kidney capsule of SCID mice, are able to become responsive to gonadotropins after 60 d (as in an unaltered animal). Ovarian fragments were transplanted under the kidney capsule of three groups of four female and four male SCID mice. The first group did not receive any hormonal treatment for 12 wk. The second group was treated from the 9th week with 1 IU of FSH/LH on alternating days for 3 wk, and the third group was treated with 5 IU Pregnant Mare Serum Ganadotropin (PMSG) 48 h before euthanasia. Primordial follicles contained in ovarian fragments isolated from newborn piglets developed only to the secondary stage. Therefore, development of gonadotropin responsiveness in ovarian fragments xenotransplanted in SCID mice was delayed compared to what occurs in the unaltered animal, and there was minimal response to exogenous gonadotropins.     

Oocyte bone morphogenetic protein 15, but not growth differentiation factor 9, is increased during gonadotropin-induced follicular development in the immature mouse and is associated with cumulus oophorus expansion

Bone morphogenetic protein (BMP) 15 and growth differentiation factor (GDF) 9 are oocyte-secreted growth factors that are critical local regulators of ovarian function and may be involved in preovulatory cumulus expansion. As cumulus expansion occurs in response to the ovulatory surge, the present study was designed: 1) to investigate whether GDF9 and BMP15 are regulated by gonadotropins in the mouse ovary; and 2) to visualize changes in both GDF9 and BMP15 immunostaining in response to gonadotropins. Immature 21-day-old mice were sequentially treated with recombinant human FSH (r-hFSH), 5 IU daily, at Days 21, 22, and 23 of life, then injected with 5 IU hCG at Day 24 of life. In response to r-hFSH, steady-state Bmp15 mRNA expression levels increased in both total ovaries and cumulus-oocyte complexes, whereas Gdf 9 mRNA levels did not. In addition, BMP15 protein levels increased in total ovaries. The GDF9 immunostaining was exclusively seen in growing oocytes in both control and gonadotropin-treated mice, whereas that of BMP15, which was also primarily seen in growing oocytes, exhibited important changes in response to gonadotropins. Strong BMP15 immunostaining was observed in the follicular fluid of atretic antral follicles after FSH treatment and in expanded, but not in compact, cumulus cells after hCG. The present results show for the first time that BMP15 levels increase during gonadotropin-induced follicular development, in parallel with oocyte maturation, and that this local factor is likely involved in cumulus expansion as previously suggested by studies in Bmp15-null mice.     

Growth differentiation factor 9 and bone morphogenetic protein 15 are essential for ovarian follicular development in sheep

The aim of this study was to test the hypothesis that both growth differential factor 9 (GDF9) and bone morphogenetic protein (BMP15; also known as GDF9B) are essential for normal ovarian follicular development in mammals with a low ovulation rate phenotype. Sheep (9-10 per group) were immunized with keyhole limpet hemocyanin (KLH; control), a GDF9-specific peptide conjugated to KLH (GDF9 peptide), a BMP15-specific peptide conjugated to KLH (BMP15 peptide), or the mature region of oBMP15 conjugated to KLH (oBMP15 mature protein) for a period of 7 mo and the effects of these treatments on various ovarian parameters such as ovarian follicular development, ovulation rate, and plasma progesterone concentrations evaluated. Also in the present study, we examined, by immunohistochemistry, the cellular localizations of GDF9 and BMP15 proteins in the ovaries of lambs. Both GDF9 and BMP15 proteins were localized specifically within ovarian follicles to the oocyte, thereby establishing for the sheep that the oocyte is the only intraovarian source of these growth factors. Immunization with either GDF9 peptide or BMP15 peptide caused anovulation in 7 of 10 and 9 of 10 ewes, respectively, when assessed at ovarian collection. Most ewes (7 of 10) immunized with oBMP15 mature protein had a least one observable estrus during the experimental period, and ovulation rate at this estrus was higher in these ewes compared with those immunized with KLH alone. In both the KLH-GDF9 peptide- and KLH-BMP15 peptide-treated ewes, histological examination of the ovaries at recovery (i.e., approximately 7 mo after the primary immunization) showed that most animals had few, if any, normal follicles beyond the primary (i.e., type 2) stage of development. In addition, abnormalities such as enlarged oocytes surrounded by a single layer of flattened and/or cuboidal granulosa cells or oocyte-free nodules of granulosa cells were often observed, especially in the anovulatory ewes. Passive immunization of ewes, each given 100 ml of a pool of plasma from the GDF9 peptide- or BMP15 peptide-immunized ewes at 4 days before induction of luteal regression also disrupted ovarian function. The ewes given the plasma against the GDF9 peptide formed 1-2 corpora lutea but 3 of 5 animals did not display normal luteal phase patterns of progesterone concentrations. The effect of plasma against the BMP15 peptide was more dramatic, with 4 of 5 animals failing to ovulate and 3 of 5 ewes lacking surface-visible antral follicles at laparoscopy. By contrast, administration of plasma against KLH did not affect ovulation rate or luteal function in any animal. In conclusion, these findings support the hypothesis that, in mammals with a low ovulation rate phenotype, both oocyte-derived GDF9 and BMP15 proteins are essential for normal follicular development, including both the early and later stages of growth.     

mRNA expression pattern of gonadotropin receptors in bovine follicular cysts

Follicular growth and steroidogenesis are dependent on gonadotropin binding to their receptors in granulosa and theca cells of ovarian follicles. The aim of the present study was to evaluate the expression patterns of follicle-stimulating hormone receptor (FSHR) and luteinizing hormone receptor (LHCGR) in ovarian follicular structures from cows with cystic ovarian disease (COD) as compared with those of regularly cycling cows. Relative real-time RT-PCR analysis showed that the expression of FSHR mRNA in granulosa cells was highest in small antral follicles, then decreased significantly as follicles increased in size, and was lowest in cysts. FSHR mRNA was not detected in the theca cells of any follicular category, including cysts. LHCGR mRNA expression in granulosa cells was significantly higher in large antral follicles than in cysts, and not detected in granulosa cells of small and medium antral follicles. In theca cells, the expression level of LHCGR mRNA in medium antral follicles was higher than in small and large antral follicles, whereas that in follicular cysts it was similar to those in small and medium antral follicles, but higher than that in large antral follicles. Our findings provide evidence that there is an altered gonadotropin receptor expression in bovine cystic follicles, and suggest that in conditions characterized by altered ovulation, such as COD, changes in the signaling system of gonadotropins may play a fundamental role in their pathogenesis.     

Ovarian tumors not induced by irradiation and gonadotropins in hypogonadal (hpg) mice

Hypogonadal (hpg/hpg) mice deficient in gonadotropin-releasing hormone were used to study gonadotropin involvement in ovarian tumorigenesis following gamma irradiation. In the first experiment, 30-day-old hpg/hpg and normal (+/-) littermate mice were irradiated. The same mice were killed 10-15 mo later, and autopsies were performed. Ovaries of irradiated hpg/hpg mice were devoid of oocytes, but retained follicular structures. Neither mesothelial adenomas nor granulosa cell tumors were observed. In contrast, all irradiated +/- mice formed mesothelial adenomas or granulosa cell tumors, or both. Therefore, oocyte death in the absence of gonadotropins did not initiate ovarian tumorigenesis. In the second experiment, irradiated and nonirradiated hpg/hpg and +/- mice were injected 3 times weekly for 180 days with either low or high doses of pregnant mare serum gonadotropin (PMSG) and human chorionic gonadotropin (hCG) in combination. Irradiation reduced ovarian mass and markedly reduced ovarian weight increase in response to exogenous gonadotropins. Follicular dissolution and stromal cell hypertrophy were observed in saline-treated and gonadotropin-treated +/- mice that had been irradiated, and in hpg/hpg mice given the high gonadotropin dose. Mesothelial adenoma formation was observed in 100% of saline-treated, 14% of low dose-treated, and 11% of high dose-treated +/- mice. No mesothelial adenomas developed in any hpg/hpg or nonirradiated +/- mice, despite gonadotropin-induced stromal luteinization. These results indicate that, in the absence of gonadotropins, irradiation leads only to the loss of oocytes. The presence of gonadotropins was necessary to promote follicular dissolution and stromal luteinization, but was insufficient to stimulate mesothelial adenoma formation.     

Development of antral follicles in cryopreserved cat ovarian tissue transplanted to immunodeficient mice

Ovarian cortex cryopreservation and xenotransplantation into immunodeficient mice represents a potential means for female germplasm conservation and an immediate model for investigation of folliculogenesis. The objectives of this study were to: (1) assess follicle survival after cryopreservation and transplantation of cat ovarian tissue into non-obese diabetic severely combined immunodeficient (NOD SCID) mice; and (2) evaluate the effects of gonadotropin treatments on follicular development in the transplanted tissue. Slices from the cat ovarian cortex were frozen and after thawing, transplanted under each kidney capsule of castrated male NOD SCID mice (eight xenografts in four mice). Sixty-two days after surgery, mice were randomly assigned (two per group) to gonadotropin-treated (eCG and hCG 88 h later) or control (saline-treated) groups. Twenty-four hours after the last injection, ovarian tissue was recovered and processed for histology. Fresh ovarian tissue from the same original source was similarly processed. Follicles were counted, measured, and classified as primordial, primary, secondary, or antral. Immunoreactive proliferating cell nuclear antigen (PCNA) stain was used to assess follicle viability. Microscopic examination revealed no evidence of necrosis or fibrosis. The grafts were well-vascularized, with follicles at all stages of development. Numbers of follicles in the transplanted tissue were markedly reduced compared to fresh tissue, with approximately 10% of follicles surviving freezing and transplantation procedures. Growing follicles positive for PCNA were found in all xenografts. Gonadotropin treatment did not alter the proportion of resting to growing follicles or mean follicle diameter by comparison with controls from untreated mice. By contrast, luteinization, but not ovulation, of antral follicles was observed only in grafts from treated mice. In summary, frozen-thawed cat ovarian cortex tissue not only survived xenotransplantation, it also contained follicles able to grow to antral stages. Exogenous gonadotropin treatment in this model resulted in luteinization of antral follicles but enhancement of follicular growth and ovulation did not occur.     

The neuroendocrine regulation of the human ovarian cycle.

The menstrual cycle is now thought to be mainly determined by the ovary itself, which sends various signals to the pituitary and the hypothalamus. The hypothalamus is an autonomous pacemaker, with a pulse frequency that is modulated by ovarian signals; in turn, it is indispensable to ovarian function. In women, the ovarian cycle produces a single mature oocyte each month from puberty to menopause. This follicle is rescued from atresia, the genetically controlled ovarian apoptosis (or "programmed cell death"), involving 99.9% of the follicles. Follicular growth and maturation are mostly independent of gonadotropins from the stage of primordial to antral follicles. A complete intraovarian paracrine system is implied in this gonadotropin-independent follicular growth and in the modulation of the action of gonadotropins in the ovary. Follicle-stimulating hormone (FSH) allows the rescue of a minority of follicles from atresia and is indispensable only for the final maturation of the preovulatory follicle during the follicular phase of the cycle. Luteinizing hormone (LH) is responsible for the final growth of the dominant follicle in the late follicular phase. the induction of ovulation during the LH peak, and the survival of the corpus luteum during the luteal phase. The cyclical variations of gonadotropins are under the control of ovarian steroids (estradiol and progesterone) and peptides (inhibins). The cycle length is determined by the duration of terminal follicular growth and by the fixed life span of the corpus luteum. The ovarian cycle can be monitored as well at the level of target tissues of steroids, such as the endometrium. In fact, the endometrial maturation is synchronized to follicular development, and this synchronization is indispensable for successful implantation of the embryo. The improving knowledge of follicular and endometrial physiology will allow the development of new treatments of infertility, the design of new contraceptive techniques, and a better tolerance of treatments using sex steroids.     

Gonadotropin uptake in genetic and irradiation models of ovarian tumorigenesis

Genetic and irradiation models of ovarian tumorigenesis were investigated for evidence that elevated gonadotropins have a role in tumorigenesis. Wx/Wv mice lack oocytes at birth, develop complex mesothelial adenomas by 6 mo, and additional ovarian tumor types later. Uptake of iodinated human chorionic gonadotropin (125I-hCG) was measured in mice aged 1 to 30 mo, and uptake iodinated human follicle-stimulating hormone (125I-hFSH) was measured in mice aged 1 to 12 mo. Gonadotropin uptake by Wx/Wv ovaries in vivo declined quickly and was undetectable by 6 mo. Irradiated ovaries rapidly lost oocytes and follicular structures, formed mesothelial adenomas by 5 mo, and later formed additional types of ovarian tumors. In the irradiation model, 125I-hCG uptake also declined quickly and was undetectable by 3 mo of age. Neither the surface nor the tubular epithelium of the mesothelial adenoma were consistently labeled by 125I-hCG in autoradiography studies with either model. Although these data do not exclude an acute role for gonadotropins in initiation of preneoplastic events, they do indicate that ovarian cells do not require chronic gonadotropin stimulation during subsequent tumorigenesis. These findings are discussed in relation to additional models of ovarian tumorigenesis.     

Intrabursal administration of the antiangiopoietin 1 antibody produces a delay in rat follicular development associated with an increase in ovarian apoptosis mediated by changes in the expression of BCL2 related genes

The angiopoietin (ANGPT) receptor (TEK) system plays a crucial role in blood vessel development and regression. To date, no reports have addressed the actions of the anti-ANGPT1 antibody on gonadotropin-stimulated follicular development and atresia in the ovary. Therefore, in this study we specifically investigated whether ANGPT1 plays a critical intraovarian survival role for gonadotropin-dependent folliculogenesis. In particular, we examined the effect of local administration of anti-ANGPT1 antibody on follicular development, apoptosis, and expression of BCL2 protein family members (BAX, BCL2, and BCL2L1), TNFRSF6, and FASLG in ovarian follicles from prepubertal eCG-treated rats. The inhibition of ANGPT1 caused an increase in the number of atretic follicles and a decrease in the number of both antral follicles (AFs) and preovulatory follicles in gonadotropin-treated rat ovaries. Taking into account that follicular atresia is mediated by apoptosis, we analyzed the effect of the antibody against ANGPT1 on programmed cell death. The inhibition of the action of ANGPT1 caused an increase both in the number of apoptotic granulosa cells in AFs and in the spontaneous DNA fragmentation of AFs cultured in serum-free medium. Besides, AFs obtained from rats treated with intraovarian antibodies against ANGPT1 showed both a decrease in BCL2 and an increase in BAX protein levels. Moreover, a reduction in the BCL2L1(L)/BCL2L1(S) ratio was observed in this group, with a reduction of BCL2L1(L) greater than that of BCL2L1(S), thus showing that the expression of these antiapoptotic proteins is lower in follicles from treated rats than in those from untreated ones. Our findings suggest that the inhibition of ANGPT1 activity causes an increase in the number of atretic follicles mediated by ovarian apoptosis through an imbalance in the ratio of antiapoptotic to proapoptotic proteins. This could take place through a paracrine effect on granulosa cells mediated by the TEK receptor in theca cells. Therefore, these data clearly indicate that ANGPT1 is necessary for follicular development induced by gonadotropins.     

The ovarian phenotype of the aromatase knockout (ArKO) mouse

Targeted disruption of exon 9 of the cyp19 gene gives rise to a non-functional aromatase enzyme incapable of converting androgens to oestrogens. The aromatase knockout (ArKO) mouse is, thus, characterised by a dysfunctional pituitary-gonadal axis, which manifests in non-detectable levels of oestrogen in serum. These mice also exhibit elevated levels of circulating gonadotrophins (luteinising hormone (LH) and follicle stimulating hormone (FSH)) and testosterone. The ArKO mouse is infertile due to folliculogenic disruption and a failure to ovulate. The age-dependent ovarian phenotype revealed a block in follicular development at the antral stage and a complete absence of corpora lutea. By 21–23 weeks of age haemorrhagic cystic follicles were present and by 1 year there were abnormal follicles, an absence of secondary and antral follicles and atretic primary follicles. Interstitial tissue remodelling was extensive and exemplified by an increase in collagen deposition and an influx of macrophages, coincident with the loss of follicles. In mice, maintained on a soy-free and, thus, phytoestrogen-free diet, the ovarian phenotype was accelerated and exacerbated. In conclusion, the ovarian phenotype of the ArKO mouse can be attributed to the altered hormonal environment brought about by the absence of aromatase and the failure of androgens to be converted to oestrogens in the presence of elevated gonadotropins.     

Modulation of ovarian ornithine decarboxylase activity during follicular differentiation/maturation

The role of gonadotropins and estrogen on the regulation of ovarian ornithine decarboxylase was studied during follicular differentiation/maturation. In intact immature rats follicular differentiation/maturation was initiated with sequential administration of estrogen and follicle stimulating hormone. Ornithine decarboxylase activity in response to human chorionic gonadotropin was markedly enhanced (2-fold) in rats with preovulatory antral follicles when compared to rats with non-ovulatory follicles. This increase could be attributed to the alteration in the turnover of the enzyme. Following follicle maturation the half life of the human chorionic gonadotropin stimulated ornithine decarboxylase was increased from 18 to 62 min. This increase in half life was associated with differentition of follicles. In the estrogen treated group (which does not induce follicular differentiation), the half life of the enzyme remained unaltered. The regulation of ornithine decarboxylase through the formation of protein inhibitor antizyme induced by diamino hexane, was unaltered during follicular differentiation.     

Knockout of luteinizing hormone receptor abolishes the effects of follicle-stimulating hormone on preovulatory maturation and ovulation of mouse graafian follicles

It is considered a dogma that a secretory peak of LH is indispensable as the trigger of ovulation. However, earlier studies on hypophysectomized rodents have shown that stimulation with recombinant FSH, devoid of any LH activity, is able to boost the final stages of follicular maturation and trigger ovulation. As the expression of ovarian LH receptors (LHRs) still persists after hypophysectomy, such studies cannot totally exclude the possibility that LHR activation is involved in the apparently pure FSH effects. To revisit this question, we analyzed in LHR knockout (LuRKO) mice the progression of folliculogenesis and induction of ovulation by human chorionic gonadotropin and human recombinant FSH treatments. The results provide clear evidence that follicular development and ovulation could not be induced by high doses of FSH in the absence of LHR expression. Ovarian histology and oocyte analyses indicated that follicular maturation did not advance in LuRKO mice beyond the antral follicle stage. Neither were ovulations detected in LuRKO ovaries after any of the gonadotropin treatments. The ovarian resistance to FSH treatment in the absence of LHR was confirmed by real-time RT-PCR and immunohistochemical analyses of a number of gonadotropin-dependent genes, which only responded to the treatments in wild-type control mice. Negative findings were not altered by estradiol priming preceding the gonadotropin stimulations. Hence, the present study shows that, in addition to ovulation, the expression of LHR is essential for follicular maturation in the progression from antral to preovulatory stage.     

The effects of immunizing sheep with different BMP15 or GDF9 peptide sequences on ovarian follicular activity and ovulation rate

The aims of these studies were to determine the abilities of antisera against different regions of ovine bone morphogenetic protein 15 (BMP15) and growth differentiation factor 9 (GDF9) to inhibit ovarian follicular activity, estrus (mating), and ovulation in sheep. The 9-15-mer peptides were conjugated to keyhole limpet hemocyanin (KLH) and used to generate antibodies against the flexible N-terminal regions of the mature protein as well as against regions in which dimerization of the protein or interaction with a type 1 BMP or a type 2 TGFB or BMP receptor was predicted to occur. Ewes (n = 10 per treatment group) were vaccinated with KLH or the KLH-BMP15 (n = 9 different peptides) or KLH-GDF9 (n = 10) peptides in Freund adjuvant at five consecutive monthly intervals. Overall, antisera generated against peptides that corresponded to amino acid residues 1-15 of the N-terminus of the BMP15 or GDF9 mature protein or GDF9 amino acid residues 21-34 were the most potent at inhibiting ovulation following primary and single booster vaccination. Several other BMP15 (8/9) or GDF9 (6/10) treatment groups, but not KLH alone, also produced significant reductions in the numbers of animals that ovulated, although 2, 3 or 4 booster vaccinations were required. Anovulation was commonly associated with the inhibition of normal ovarian follicular development and anestrus. The in vitro neutralization studies with IgG from the BMP15 or GDF9 immunized ewes showed that the mean inhibition of BMP15 plus GDF9 stimulation of (3)H-thymidine uptake by rat granulosa cells was approximately 70% for animals without corpora lutea (CL), whereas for animals with one to three CL or more than three CL, the inhibition was 24%-33% or 27%-42%, respectively. In summary, these data suggest that reagents that block the biological actions of BMP15 or GDF9 at their N-termini have potential as contraceptives or sterilizing agents.     

In vitro culture of secondary follicles and prematuration of cumulus–oocyte complexes from antral follicles increase the levels of maturation‐related transcripts in bovine oocytes

This study evaluates the levels of messenger RNA (mRNA) for eIF4E, PARN, H1FOO, cMOS, GDF9, and CCNB1 in oocytes from secondary and antral follicles at different stages of development. The effects of in vitro culture, in vitro prematuration, and in vitro maturation on the expression of these genes on oocytes were also analyzed. The results showed that mRNA levels for H1FOO, GDF9, and PARN were higher in oocytes from small, medium, and large antral follicles, respectively, than those seen in secondary follicles. Oocytes from small, medium, and large antral follicles had higher levels of CCNB1 than oocytes from secondary follicles. Oocytes from cultured secondary follicles had higher levels of GDF9, CMOS, PARN, eIF4E, CCNB1, and H1FOO than before culture. Prematured oocytes from small antral follicles had higher levels of mRNA for GDF9, PARN, and eIF4E than before culture. In addition, higher levels of cMOS and H1FOO were identified in prematured oocytes from medium antral follicles. In conclusion, follicular growth is associated with an increase in the expression of H1FOO, GDF9, CCNB1, and PARN. The culture of secondary follicles, prematuration, and maturation of oocytes from antral follicles increase the expression of eIF4E, PARN, H1FOO, cMOS, GDF9, and CCNB1.     

Interrelationship of growth differentiation factor 9 and inhibin in early folliculogenesis and ovarian tumorigenesis in mice

To investigate the interrelationship of inhibin alpha and growth differentiation factor 9 (GDF9) during early folliculogenesis, we generated mice lacking both inhibin alpha and GDF9. Our findings on these Inha Gdf9 double-mutant mice are as follows: 1). females develop ovarian tumors and a cachexia-like wasting syndrome, resembling mice lacking inhibin alpha alone. This indicates that the granulosa cells are competent to proliferate despite the lack of GDF9; 2). follicular development progresses to multiple-layer follicle stages before tumorigenesis. This demonstrates that the up-regulation of inhibin alpha in the Gdf9 knockout ovary directly prevents the proliferation of the granulosa cells at the primary follicle stage, an effect that is released in the absence of inhibin alpha; 3). a morphological theca forms around the preantral follicles with no detectable selective theca markers [i.e. 17alpha-hydroxylase (Cyp17), LH receptor (Lhr), and Kit]. These results indicate that the theca recruitment can occur independently of GDF9, but the differentiation of thecal cells is blocked; and 4). inhibin/activin subunits betaA, betaB, and Kit ligand (Kitl) mRNA are highly up-regulated, suggesting that the increased activins and KITL play functional roles in early folliculogenesis. Thus, GDF9 appears to function indirectly to regulate early granulosa cell proliferation and theca recruitment in vivo.     

Gonadotropin regulation of glutamate cysteine ligase catalytic and modifier subunit expression in rat ovary is subunit and follicle stage specific

We have observed that levels of the antioxidant glutathione (GSH) and protein levels of the catalytic and modifier subunits of the rate-limiting enzyme in GSH synthesis, GCLc and GCLm, increase in immature rat ovaries after treatment with gonadotropin. The goals of the present studies were to delineate the time course and intraovarian localization of changes in GSH and GCL after pregnant mare's serum gonadotropin (PMSG) and after an ovulatory gonadotropin stimulus. Twenty-four hours after PMSG, there was a shift from predominantly granulosa cell expression of gclm mRNA, and to a lesser extent gclc, to predominantly theca cell expression. GCLc immunostaining increased in granulosa and theca cells and in interstitial cells. Next, prepubertal female rats were primed with PMSG, followed 48 h later by 10 IU of hCG. GCLm protein and mRNA levels increased dramatically from 0 to 4 h after hCG and then declined rapidly. There was minimal change in GCLc. The increase in gclm mRNA expression was localized mainly to granulosa and theca cells of preovulatory follicles. To verify that GCL responds similarly to an endogenous preovulatory gonadotropin surge, we quantified ovarian GCL mRNA levels during the periovulatory period in adult rats. gclm mRNA levels increased after the gonadotropin surge on proestrus and then declined rapidly. Finally, we assessed the effects of gonadotropin on ovarian GCL enzymatic activity. GCL enzymatic activity increased significantly at 48 h after PMSG injection and did not increase further after hCG. These results demonstrate that gonadotropins regulate follicular GCL expression in a follicle stage-dependent manner and in a GCL subunit-dependent manner.