In vitro membrane desialylation in porcine granulosa cells unmasks functional receptors for follicle-stimulating hormone.

To evaluate the possible existence of masked gonadotropin-binding sites and the functional role of the binding sites in porcine granulosa cells (GCs), we characterized the effects of neuraminidase (NA) pretreatment on [125I]human LH and [125I]human FSH binding to porcine GCs and the functionality of the unmasked receptors exposed by NA pretreatment. NA pretreatment at 37 C for 30 min increased specific FSH binding (P less than 0.01) and had no effect on LH binding to the GCs harvested from follicles of different sizes. Analysis of equilibrium binding experiments revealed that NA increased the number of FSH-binding sites without altering the affinity. In addition, pretreatment with NA resulted in progressive loss of cell membrane-bound sialic acid (P less than 0.01). To evaluate whether the unmasked FSH receptors constitute functional gonadotropin-binding sites, we measured the amounts of cAMP and 17 beta-estradiol produced by cultured GCs pretreated with NA. cAMP formation by GCs pretreated with NA was significantly (P less than 0.02) greater than that by GCs pretreated with medium alone in the presence of ovine FSH. Moreover, 17 beta-estradiol formation significantly increased (P less than 0.05) in GCs pretreated with NA compared with that in GCs pretreated with medium alone. However, there was no difference between medium-treated and NA-treated GCs in cAMP formation stimulated by forskolin. We suggest that porcine GCs contain a population of masked FSH-binding sites exposed by in vitro pretreatment of NA, and the binding sites constitute functional receptors capable of increasing cAMP and 17 beta-estradiol formation with FSH stimulation. Moreover, a possible involvement of plasma membrane-associated sialic acid in the masking/unmasking mechanism of FSH-binding sites was suggested.

Possible involvement of protein kinase C in gonadotropin-induced ovulation in the rat ovary.

Recent reports indicate that protein kinase C may play an important role in the process of gonadotropin-induced ovulation in the ovary. In the present study, we examined the effect of the protein kinase C inhibitor, 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7), on LH-stimulated tissue type plasminogen activator (tPA) activity in cultured rat granulosa cells. Granulosa cells were obtained from PMSG-treated rats and cultured for 48 h in the presence or absence of H-7 (0.1-60 microM) with ovine LH (30 ng/ml), phorbol 12-myristate 13-acetate (10(-8) M), phorbol 12,13-dibutyrate (10(-8) M), or (Bu)2cAMP (5 mM). After culture, tPA activity in the conditioned medium was assayed by fibrin autography technique after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. H-7 (1.0-60 microM) inhibited LH-, phorbol 12-myristate 13-acetate-, or phorbol 12,13-dibutyrate-stimulated tPA activity dose dependently, and each ID50 was approximately 8 microM. However, H-7 did not inhibit (Bu)2cAMP-stimulated tPA activity. To investigate the effect of H-7 on the ovulatory process in vivo, PMSG-treated immature rats were injected with H-7 (10(-9)-10(-3) M) into the unilateral ovarian bursa just before human CG administration. After 24 h, the number of oocyte-cumulus complexes in the oviduct was counted. H-7 suppressed the number of oocytes released from treated ovaries dose-dependently. The light microscopical observation revealed that ovaries treated with H-7 contained a few corpora lutea and many large unruptured follicles. The results of the present study suggest that the suppressive effects of H-7 on human CG-induced ovulation might be partly due to the inhibition of tPA secretion by rat granulosa cells via protein kinase C inhibition.

Follicle-stimulating hormone induces functional receptors for basic fibroblast growth factor in rat granulosa cells.

In the present study we examined the existence of receptors for basic fibroblast growth factor (bFGF) and its regulation in rat granulosa cells. The binding of labeled bFGF to rat granulosa cells was dose-dependently displaced by unlabeled bFGF, but not other growth factors. FSH induced a dose-dependent increase in specific binding for bFGF to cultured rat granulosa cells. FSH treatment did not change the binding affinity (Kd, 2.8-3.0 x 10(-10) M) of the bFGF receptor, but increased the total number of bFGF-binding sites, whereas treatment with several steroid hormones had no effect on the specific binding of bFGF. Since cycloheximide, a protein synthesis inhibitor, inhibited the increase in bFGF binding induced by FSH, it is suggested that protein synthesis might be involved in the FSH stimulation of bFGF receptor induction. Furthermore, bFGF stimulated tissue plasminogen activator activity in a dose-dependent manner, and FSH-primed granulosa cells were more responsive to bFGF action, with a decrease in the ED50 from 5.0 to 1.5 ng/ml. The antibody against human bFGF neutralized the stimulatory effect of bFGF on tissue plasminogen activator secretion. The present study suggests that FSH induces functional receptors for bFGF in granulosa cells and that bFGF may play a role in the process of differentiation under the influence of FSH.

In vitro and in vivo bioactivity of recombinant human follicle-stimulating hormone and partially deglycosylated variants secreted by transfected eukaryotic cell lines.

Recent studies have shown that Chinese hamster ovary (CHO) cells transfected with the FSH subunit genes secrete bioactive FSH. Here, we determined the in vitro and in vivo bioactivity of recombinant FSH produced by CHO mutant cells deficient in the glycosylation enzyme N-acetylglucosamine transferase-I (NAGT-), resulting in glycoproteins with asparagine-linked (GlcNAc)2(Mannose)5 oligosaccharides, or mutant cells defective in sialic acid transport into the Golgi (ST-). In the latter, glycoproteins are secreted lacking terminal sialic acids. Determination of in vitro bioactivity, using the granulosa cell aromatase bioassay, indicated that both FSH variants are as active as FSH secreted by the wild type (WT) cells and purified pituitary FSH. Also, these normal and variant forms of FSH are equipotent in a radioligand receptor assay using rat testis membranes. However, the variant FSH molecules are more basic than the WT FSH as determined using a chromatofocusing column (pI: wild type 3.6-5.0, NAGT- greater than 7.0, ST- approximately 6.0 and greater than 7.0). Injection of immature estrogen-treated rats with WT FSH induced high aromatase activity in their granulosa cells whereas treatment with either one of the FSH variants was ineffective; the lack of in vivo activity of the FSH variants was correlated with rapid clearance of these molecules in serum. Thus, recombinant human FSH produced by cells deficient in NAGT-I or defective in sialic acid transport retains normal receptor binding and in vitro bioactivity, but exhibits minimal in vivo activity and a shortened half-life when compared to WT FSH, indicating the important role of terminal sugars for FSH action in vivo.

Basic fibroblast growth factor induction of granulosa cell tissue-type plasminogen activator expression and oocyte maturation: potential role as a paracrine ovarian hormone.

Gonadotropin-induced ovulation is associated with oocyte maturation and preovulatory increases of tissue plasminogen activator (tPA) expression. Basic fibroblast growth factor (bFGF), an angiogenic factor found in many organs including the ovary, modulates steroidogenesis in granulosa cells and increases PA activity in endothelial cells. Here studies were performed to examine the possible roles of bFGF as an intragonadal regulator of tPA expression and oocyte maturation. In cultured granulosa cells, bFGF caused a time-dependent (onset at 24 h) and dose-dependent (ED50 = 0.6 nM) increase (up to 5-fold) in tPA enzyme activity as measured by the fibrin overlay technique. Northern blot hybridization also revealed that treatment of cells with bFGF (2 nM) increased the level of the 22S tPA messenger RNA. Slot blot analysis indicated that the effects of bFGF were time dependent and dose dependent; tPA message levels increase before tPA activity levels. bFGF (0.6 nM) also significantly increased granulosa cell cAMP production in both the absence and presence of a phosphodiesterase inhibitor. In follicle-enclosed oocytes incubated for 24 h in media with or without increasing concentrations of LH or bFGF, germinal vesicle breakdown was observed in only 1.6% of controls, but 85% of LH (1 microgram/ml)-treated oocytes underwent maturation. Likewise, bFGF induced germinal vesicle breakdown (10-80%) over a dose range of 0.6 to 333 nM. In the same follicles, bFGF, like LH, also stimulated prostaglandin E production. These results, coupled with the identification of bFGF in growing follicles, suggest that bFGF acts as an intraovarian inducer of granulosa cell tPA gene expression and oocyte maturation.

Immunohistochemical localization of inhibin subunits in human corpora lutea during menstrual cycle and pregnancy.

The cellular localization of the alpha-, beta A-, and beta B-subunits of inhibin was studied in human corpora lutea (CL) during the menstrual cycle and pregnancy. Immunohistochemical techniques using antisera selective for each subunit were used to localize the polypeptide chains. Immunoreactive staining with antisera against the alpha-, beta A-, and beta B-subunits was mainly observed in granulosa luteal cells (luteal cells), whereas thecal luteal cells were faintly stained with antibodies selective for each inhibin subunit. The intensity of immunostaining with alpha-subunit antiserum in luteal cells increased from the early luteal phase to the midluteal phase and subsequently decreased toward the late luteal phase. Inhibin alpha-subunit in the retrogressive CL was not detected. Not only was the immunolocalization of both beta A- and beta B-subunits detected in the luteal cells of the CL of the cycle, but the relative intensity of immunostaining with beta-subunit antiserum changed in a pattern similar to that shown by the alpha-subunit. The luteal cells and thecal luteal cells in the CL of pregnancy exhibited positive staining with antisera directed against each inhibin subunit. The present findings provide further evidence that 1) immunoreactive inhibin subunits are present in luteal cells in the CL of the menstrual cycle; 2) the detectable level of each subunit changes in parallel with the function of the CL over the course of the menstrual cycle; and 3) the CL of pregnancy might be involved in serum inhibin elevation early in gestation.

Immunohistochemical localization of inhibin subunits in polycystic ovary.

The immunohistochemical localization of the alpha-, beta A-, and beta B-subunits of inhibin was examined in polycystic ovary (PCO). The granulosa cells in multicystic follicles in the PCO exhibited negative immunostaining for alpha-subunit and positive staining for beta A- and beta B-subunits. In contrast, the hyperplastic thecal cells exhibited distinct positive staining for alpha-, beta A-, and beta B-subunits. The luteinized stromal cells (stromal hyperthecosis) also exhibited positive staining for each inhibin subunit. The granulosa cells in the tertiary follicle exhibited negative staining for alpha-subunit and positive staining for beta A- and beta B-subunits. No staining for each inhibin subunit was observed in the thecal layer in the tertiary follicle. The present findings suggest that immunoreactive inhibin alpha-, beta A-, and beta B-subunits are present in hyperplastic thecal and stromal cells in PCO, and that granulosa cells in the follicular cyst of PCO might not produce inhibin, in contrast to the normal ovary.

Immunohistochemical localization of inhibin/activin subunits in human ovarian follicles during the menstrual cycle.

The immunohistochemical localization of the alpha-, beta A-, and beta B-subunits of inhibin was examined in human follicles during follicular growth. Immunoreactive staining with antisera against the alpha-, beta B-subunits was observed in follicular granulosa cells, whereas no staining for each inhibin subunit was observed in thecal or interstitial cells. In the preantral and small antral follicles, the granulosa cells exhibited positive immunoreactive staining with antisera against beta A- and beta B-subunits and negative immunostaining with antiserum against alpha-subunit. In medium-sized healthy antral follicles obtained during the midfollicular phase, positive immunostaining with antisera against alpha-, beta B-subunits was detected in the granulosa cells. In contrast, immunostaining for alpha-, beta A-, and beta B-subunits was not detected in the granulosa cells of similarly sized atretic follicles. The granulosa cells of preovulatory follicles revealed enhanced positive staining for the three inhibin subunits. The present findings suggest that immunoreactive inhibin subunits are present in the follicular granulosa cells during the menstrual cycle, and that the localization and intensity of immunostaining for each inhibin subunit might change during follicular development and maturation.

Expression of luteinizing hormone and chorionic gonadotropin receptor messenger ribonucleic acid in human corpora lutea during menstrual cycle and pregnancy.

In the present study, we examined the expression of LH and CG receptor messenger RNA (mRNA) in human corpora lutea (CL) during the menstrual cycle and pregnancy. Poly(A)-enriched RNA was extracted from CL and analyzed by Northern and slot blots, using a radiolabeled complementary RNA probe derived from the human LH receptor complementary DNA. Northern blot analysis indicated the presence of multiple LH receptor mRNA transcripts with molecular sizes of 8.0, 7.0 and 4.5 kilobases in human CL during the menstrual cycle. The predominant transcript was 4.5 kilobases in size. However, no hybridization signals were observed in nongonadal tissues (heart, liver, and kidney). Densitometric analyses revealed that the levels of LH receptor mRNA increased from early luteal phase to midluteal phase and subsequently decreased during late luteal phase. After the onset of menstruation, the LH receptor mRNA level was undetectable in the regressing CL. Moreover, radioligand receptor assay (RRA) showed a close parallelism between LH receptor mRNA levels and LH receptor content in CL throughout the menstrual cycle. LH receptor mRNA expression was also found in CL during early pregnancy. The level of LH receptor mRNA was relatively high in early pregnancy CL, whereas LH receptor content was low. Using in situ hybridization, LH receptor mRNAs were uniformly expressed in both large and small luteal cells during early and midluteal phase and early pregnancy, but not in regressing CL. In conclusion, these data demonstrate that the regulation of LH receptor content in human CL during luteal phase is associated with similar changes in the receptor message levels, suggesting the physiological roles for LH receptor mRNA during the menstrual cycle in the human. In addition, the expression of LH receptor mRNA was demonstrated in human CL during early pregnancy.

Apoptosis of human corpora lutea during cyclic luteal regression and early pregnancy.

To elucidate the role of apoptotic cell death in human corpus luteum (CL) regression, human CL during the menstrual cycle and early pregnancy were isolated and processed for biochemical (radio-labeling) analysis of DNA integrity. Total DNA extracted from human CL of the early luteal phase contained predominantly high mol wt DNA, whereas CL of the midluteal phase exhibited the appearance of DNA cleavage into low mol wt ladders characteristic of apoptosis. Although apoptotic DNA cleavage of human CL significantly increased from the midluteal phase to the late luteal phase (P < 0.05), CL of early pregnancy did not exhibit apoptotic DNA fragmentation by biochemical analysis. In situ analysis of DNA fragmentation revealed that both large and small luteal cells exhibited DNA cleavage in human CL of the midluteal and late luteal phases and in regressive CL. The present findings suggest that 1) human luteal regression may be mediated by apoptosis; and 2) CL of early pregnancy may be rescued from luteolysis through inhibiting the occurrence of apoptotic luteal cell death.

The vascular endothelial growth factor/fms-like tyrosine kinase system in human ovary during the menstrual cycle and early pregnancy.

In human ovaries, angiogenesis is known to be associated with the development of follicles and the formation of the corpus luteum (CL). A complex vascular network is formed within the thecal cell layer during follicular growth, and rapid neovascularization occurs toward the granulosa cell layer after ovulation. Vascular endothelial growth factor (VEGF) is a multifunctional cytokine, stimulating endothelial cell growth and enhancing microvascular permeability. A specific receptor for VEGF, fms-like tyrosine kinase (Flt-1), is expressed in vascular endothelial cells that mediates the action of VEGF. We examined the localization and expression of VEGF and Flt-1, using an immunohistochemical technique and RT-PCR analysis, in human follicles and corpora lutea during the normal menstrual cycle and early pregnancy. We measured concentrations of VEGF in extracts of human CL using an enzyme-linked immunosorbent assay during the luteal phase and early pregnancy. Immunostaining for VEGF was observed in granulosa cells from small antral follicles to preovulatory follicles. The staining was detected in thecal cells from medium-sized to preovulatory follicles. The intensity of the staining was gradually increased as a follicle grew. Flt-1 was localized in granulosa and thecal cells of preovulatory follicles as well as in endothelial cells. In the human CL, the intense staining for VEGF was observed in granulosa and thecal lutein cells, especially in the midluteal phase. The immunostaining for Flt-1 was faint in endothelial cells in the CL, whereas it was distinct in granulosa and thecal lutein cells. The concentrations of VEGF in lutein extracts were high in the early and midluteal phases and tended to decrease toward the late luteal phase. During early pregnancy, a measurable amount of VEGF was detected. RT-PCR analysis demonstrated that messenger ribonucleic acids encoding VEGF121, VEGF165, and Flt-1 were expressed in the CL. These results suggest that VEGF might have an autocrine role in the ovulatory process and luteal function as well as a paracrine role in angiogenesis.

Effects of oestrogen and prostaglandin F2 alpha on luteinizing hormone receptors in human corpora lutea.

The binding of 125I-labelled human LH (hLH) to the 2000 g subcellular fraction of human corpora lutea of the menstrual cycle was examined. Displacement studies demonstrated that 125I-labelled hLH was specifically bound in the 2000 g fraction of human luteal tissue. Specific binding of 125I-labelled hLH was demonstrated in all the corpora lutea examined except for two aged corpora lutea at an early proliferative phase of the cycle. The number of binding sites for hLH increased between the early to mid-luteal phase and decreased towards the late luteal phase. However, the apparent dissociation constant (Kd) in each corpus luteum did not vary throughout the menstrual cycle. In addition, the effects of treatment with diethylstilboestrol diphosphate (DES) and prostaglandin F2 alpha (PGF2 alpha) on the binding of 125I-labelled hLH to the 2000 g fraction of luteal tissue were investigated and the changes in hLH receptors were estimated by Scatchard analysis. The number of binding sites were 1.59 x 10(-14) mol/mg protein in control tissue, 0.86 x 10(-14) mol/mg protein in DES-treated luteal tissue and 2.95 x 10(-14) mol/mg protein in PGF2 alpha-treated luteal tissue. Thus, the binding sites for hLH decreased as a result of treatment with DES and increased by treatment with PGF2 alpha. In contrast, the apparent Kd in each luteal tissue revealed almost the same value (4.24 x 10(-10) mol/1) after treatment with DES or PGF2 alpha. The results of the present study suggest that oestrogen and prostaglandin might have an important role in modulating hLH receptor in human corpora lutea.

Cytokine modulation of inhibin secretion in cultured rat granulosa cells.

In the present study, we examined the effects of tumour necrosis factor alpha (TNF alpha) and interleukin-1 beta (IL-1 beta) on inhibin secretion by cultured rat granulosa cells using immunoblotting and two-site enzyme immunoassay for inhibin A (alpha-beta A dimer). FSH stimulated the secretion of the inhibin alpha-beta A dimer (32 kDa) by the cells in a dose-dependent manner. In addition to the predominant 32 kDa inhibin alpha-beta A dimer staining, staining of minor immunoreactive bands was also enhanced by FSH. TNF alpha alone did not have any effect on inhibin secretion. Immunoblot analyses using an antiserum against alpha-subunit and an antiserum against beta A-subunit revealed a dose-dependent inhibition by TNF alpha of FSH-stimulated secretion of inhibin by rat granulosa cells. Similarly, TNF alpha inhibited in a dose-dependent manner FSH-stimulated inhibin secretion when measured using a two-site enzyme immunoassay. IL-1 beta alone did not exert any effect on inhibin secretion but it inhibited FSH-stimulated inhibin release in a dose-dependent manner (using both immunoblotting and a two-site assay for inhibin A). The present observations suggest that TNF alpha and IL-1 beta inhibit gonadotrophin-stimulated inhibin production by cultured rat granulosa cells.

FSH and LH up-regulate epidermal growth factor receptors in rat granulosa cells.

Epidermal growth factor (EGF) modulates ovarian folliculogenesis and steroidogenesis and its binding sites have been demonstrated in the ovary. We investigated the localization of EGF-binding sites in the rat ovary, and the effects of FSH and LH on EGF binding to cultured granulosa cells. Autoradiographic localization of 125I-labelled mouse EGF-binding sites was demonstrated in the granulosa and luteal cells. Displacement study and Scatchard analysis showed that a single class of specific binding sites for 125I-labelled mouse EGF was present in the granulosa cells, obtained from the ovaries of immature rats treated with diethylstilbestrol. The number of binding sites and the apparent dissociation constant were 4336 binding sites/cell and 3.42 pmol/l respectively. The granulosa cells were cultured for 48 h at 37 degrees C in medium alone or with increasing amounts of ovine FSH (oFSH; 1-1000 micrograms/l). FSH treatment increased 125I-labelled mouse EGF binding to the granulosa cells in a dose-dependent manner. After culture with oFSH (100 micrograms/l) for 48 h, the cells were cultured in medium alone or with increasing amounts of ovine LH (oLH; 1-1000 micrograms/l) for an additional 48 h. LH treatment also increased 125I-labelled mouse EGF binding in a dose-dependent manner, compared with the control. However, neither FSH nor LH altered receptor-binding affinity. Furthermore, after culture with oFSH (FSH-primed) or oFSH followed by oLH (LH-primed), tissue plasminogen activator (tPA) activities in the conditioned media were examined by fibrin autography.(ABSTRACT TRUNCATED AT 250 WORDS)

The role of tyrosine kinase in gonadotropin-induced ovulation in the rat ovary.

OBJECTIVES: Our purpose was to elucidate the involvement of the tyrosine kinase pathway in gonadotropin-induced ovulation in the rat ovary. STUDY DESIGN: We investigated the effect of a tyrosine kinase inhibitor, tyrphostin, on the rat ovulatory process in vivo and in vitro. METHODS: In cultured rat granulosa cells, the effect of tyrphostin on LH-, dibutyryl cyclic AMP ((Bu)2cAMP)- or forskolin-stimulated tissue type plasminogen activator (tPA) activities was examined by using a fibrin autography technique. In an in vivo system, tyrphostin was injected into the bursal cavity of the ovary in pregnant mare serum gonadotropin-treated rats, just before human chorionic gonadotropin administration. After 24 h, the number of oocytes in the oviduct was counted and the tyrphostin-treated ovaries were examined histologically. RESULTS: Tyrphostin inhibited LH-stimulated tPA activity but did not affect (Bu)2cAMP- or forskolin-stimulated ones. In an in vivo study, tyrphostin suppressed oocyte release dose-dependently. Histological observations revealed that tyrphostin-treated ovaries contained many large unruptured follicles and a few corpora lutea. CONCLUSION: This study suggests that the suppressive effect of tyrphostin on ovulation may be partly due to tPA activity inhibition in the granulosa cells via the suppression of tyrosine kinase activity. Additionally, tyrosine kinase phosphorylation may be involved in gonadotropin-activated signaling systems in the rat ovulatory process.

[3H]naloxone binding sites in porcine ovarian follicles and corpora lutea during the ovarian cycle.

We demonstrated the presence of opioid receptors in the porcine ovary using [3H]naloxone. We also examined the change in the number of opioid receptors during follicular maturation. In addition, we found specific binding of [3H]naloxone in the porcine ovary using naloxone, beta-endorphin, methionine-enkephalin and dynorphin. The binding of [3H]naloxone to porcine granulosa cells and the 2000-g subcellular fraction of corpora lutea was examined to demonstrate the presence of specific [3H]naloxone binding in the porcine ovary. Binding of [3H]naloxone to porcine granulosa cells was displaced by cold naloxone and beta-endorphin but not by dynorphin and methionine-enkephalin. A similar phenomenon was also demonstrated in the 2000 g subcellular fraction of porcine corpora lutea. However, Scatchard analyses revealed a single class of high-affinity (Kd = 28.5 x 10(-9) mol/l) and low-capacity binding sites (Bmax = 30.5 fmol/5 x 10(6) cells) in porcine granulosa cells. Similar binding parameters were obtained in the 2000-g subcellular fraction of porcine luteal tissue (Kd = 28.3 x 10(-9) mol/l, Bmax = 59.3 nmol/kg protein). [3H]Naloxone binding sites in the porcine ovary showed binding characteristics similar to those of opioid receptors in other organs like brain, uterus and placenta. Furthermore, we demonstrated that the specific binding sites of [3H]naloxone in porcine granulosa cells decreased during follicular maturation. Opioid receptors have been detected in the uterus, placenta and Sertoli cell cultures in some species. However, there is no detailed study on opioid receptors in granulosa cells and luteal tissues in any species. Our data suggest a relationship between folliculogenesis and ovarian opioid peptides.(ABSTRACT TRUNCATED AT 250 WORDS)

Presence and localization of endothelin receptor in the rat ovary and its regulation by pituitary gonadotropins.

In the present study we examined the regulation of receptors for endothelin 1 (ET-1) in rat granulosa cells. We examined the localization and regulation of ET receptors in immature rat ovary and the effects of ET-1 on steroidogenesis in cultured rat granulosa cells. The ovaries used in autoradiography were derived from pregnant mare serum gonadotropin and human chorionic gonadotropin-treated immature rats. Granulosa cells were obtained from diethylstilbestrol-treated immature rats and incubated with 125I-ET-1. Granulosa cells were cultured with ET-1 in the presence or absence of ovine follicle-stimulating hormone. The concentrations of sex steroid hormones in conditioned media were measured by radioimmunoassay. The binding site for ET-1 was localized in the granulosa cells, but not in thecal and luteal cells. Follicle-stimulating hormone (FSH) induced a dose-dependent increase in specific binding for ET-1 to cultured rat granulosa cells. In contrast, luteinizing hormone (LH) induced a dose-dependent decrease in specific binding for ET-1 to cultured rat granulosa cells. Conversely, treatment with prolactin and several sex steroid hormones had no effects on the specific binding of ET-1. Treatment with ET-1 inhibited FSH-stimulated accumulation of progesterone and estradiol in cultured rat granulosa cells. The results indicate that both FSH and LH influence the expression of ET-1 receptor, and that ET-1 may play a regulatory role in the ontogeny of the granulosa cell.

Purification of high-molecular-weight follicle-stimulating hormone binding inhibitor in porcine follicular fluids.

We performed the purification of high-molecular-weight follicle-stimulating hormone binding inhibitor (FSHBI) from porcine follicular fluids. The FSHBI activities of high-molecular-weight fractions acquired by ultrafiltration of follicular fluids from small, medium and large follicles with Centriflo CF25 membrane cone were 277.2 +/- 24.6, 176.7 +/- 3.0 and 141.3 +/- 3.6 U, respectively. By affinity chromatography of CF25 retentate with a column of Blue Sepharose CL6B, 94.1 +/- 5.3% of FSHBI activity was recovered in the unretained fraction. The FSHBI in the unretained fraction was purified by anion-exchange chromatography with a column of Mono Q and gel filtration on Sephacryl S300HR. As a result of sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) of the final purified fraction, a single silver-stained band was observed at 310 kD under the non-reducing conditions. On the other hand, under the reducing conditions, SDS-PAGE revealed three bands at 178, 101 and 55 kD. A double reciprocal plot analysis of this substance showed competitive inhibition in FSH binding. The results of the present study suggest the existence of a 310 kD FSHBI composed of three subunits of 178, 101 and 55 kD in porcine follicular fluids.

Immunohistochemical localization of inhibin alpha- and beta A-subunits in the ovary of immature female rats.

Immunohistochemical localization of inhibin alpha- and beta A-subunits was examined in the ovaries of immature female rats. The granulosa cells in various sized ovarian follicles obtained from rats that were 10-24 days old exhibited positive staining for inhibin alpha- and beta A-subunits. The relative intensities of immunostaining for alpha- and beta A-subunits increased during follicular growth and maturation. Ova and internal thecal cells did not show any immunostaining for inhibin alpha- and/or beta A-subunits. These results suggest that granulosa cells of immature rat ovaries may produce inhibin from the 10th day after birth, and that an increase in the number of mature ovarian follicles results in an increase in inhibin production in the immature rat ovary during prepubertal development.

Immunohistochemical localization of inhibin-activin subunits in hydatidiform mole and invasive mole.

OBJECTIVE: To examine the cellular localization of each inhibin subunit in hydatidiform mole and invasive mole. METHODS: Tissues were collected and fixed in Bouin's solution and studied with the immunohistochemical technique avidin-biotin-peroxidase complex. RESULTS: In the villous trophoblasts of hydatidiform mole and invasive mole, distinct immunostaining for inhibin alpha-subunit was observed in proliferative syncytiotrophoblasts, but not in the cytotrophoblasts. Positive immunostaining for beta A- and beta B-subunits was observed in both syncytiotrophoblasts and cytotrophoblasts. In extravillous trophoblasts of the invasive mole, staining for beta A- and beta B-subunits was observed, whereas staining for alpha-subunit was not detected. CONCLUSION: Inhibin-activin subunits may be produced in the trophoblasts of hydatidiform mole and invasive mole, and inhibin and activin may play a role in trophoblastic proliferation and invasion.