Qualitative and quantitative changes in protein synthesis of bovine follicular cells during the preovulatory period.

To understand the mechanisms governing oocyte maturation better, the effects of the gonadotropin surge were studied on follicular cells of bovine preovulatory follicles. For this purpose, qualitative and quantitative changes in protein synthesis by both granulosa cells and cumulus cells were compared relative to the luteinizing hormone (LH) surge and the resumption of meiosis in the oocyte. Follicular cells were collected at different times before and up to 25 hr after the LH surge. For each individual preovulatory follicle, granulosa and cumulus cells were incubated separately for 3 hr with 3H-methionine or with 35S-methionine. Newly synthesized cytosolic proteins from granulosa and cumulus cells and proteins secreted into the medium were analyzed by polyacrylamide gel electrophoresis. The radioactivity was measured by liquid scintillation counting after slicing of the gels or revealed by fluorography. Three major peaks of the newly synthesized proteins, with molecular weights of 76, 56, and 30 kDa, were studied throughout the preovulatory period. After the LH surge, the overall level of protein synthesis increased in granulosa cells. In addition, the pattern of cytosolic proteins in granulosa cells changed, and, in particular, the relative synthesis of the 30 kDa peak decreased. These changes in cytosolic protein synthesis may be due to the action of LH since they could be reproduced in vitro in LH-stimulated granulosa cells. A predominant peak of 56 kDa was secreted by granulosa cells throughout the experimental period. No significant change was observed in proteins synthesized by cumulus cells under the same experimental conditions. The amounts of radioactivity incorporated into the three major proteins secreted by granulosa cells, however, were correlated significantly with the amounts of radioactivity incorporated by similar proteins synthesized by cumulus cells. These results indicate that cumulus cells respond differently from granulosa cells to the gonadotropin surge but not in an independent manner.     

Secretion of cumulus expansion-enabling factor (CEEF) in porcine follicles

The objective of this study was to find out whether porcine cumulus and mural granulosa cells can secrete cumulus expansion-enabling factor (CEEF). Culture drops of M-199 medium were conditioned with denuded porcine oocytes (1 oocyte/μl), cumulus cells from oocytectomized complexes (1 OOX/μl), pieces of mural granulosa isolated from preantral to preovulatory follicles (1000 cells/μl), or oviductal cells (1000 cells/μl) for 24 hr. The production of CEEF was assessed by the addition of mouse OOX and follicle-stimulating hormone (FSH) (1 μg/ml) to microdrops of the conditioned medium. After 16–18 hr, expansion of the mouse OOX was scored on a scale of 0 to 4 by morphologic criteria. Mouse OOX did not expand in nonconditioned FSH-supplemented medium. Immature porcine oocytes produced +3 to +4 expansion of the mouse OOX. Granulosa cells isolated from preantral and early antral follicles and cumulus cells isolated from all stages of follicle development constitutively secreted CEEF under in vitro conditions. Mural granulosa cells of small, medium, and preovulatory (PMSG) follicles also secreted CEEF in vitro; however, FSH or leutenizing hormone (LH) stimulation was essential for this secretion. Hormonally induced secretion of CEEF was accompanied by expansion of the mural granulosa itself. Granulosa cells isolated from follicles of gilts 20 hr after PMSG and human chorionic gonadotropin (hCG) administration did not produce CEEF and did not expand in response to FSH and LH in vitro. CEEF activity also was found in the follicular fluid of small antral follicles, was reduced in medium follicles, and was not detectable in PMSG-stimulated follicles. However, CEEF activity was reestablished in the follicular fluid of preovulatory follicles by hCG injection, conceivably due to increased production of CEEF by cumulus cells. We conclude that (1) porcine cumulus and mural granulosa cells are capable of CEEF production in vitro and (2) autocrine secretion of CEEF by cumulus cells is involved in regulation of porcine cumulus expansion both in vitro and in vivo. Mol. Reprod. Dev. 49:141–149, 1998. © 1998 Wiley-Liss, Inc.     

Control of in vitro maturation of bovine cumulus-oocyte complex by preovulatory granulosa cells

The present study was designed to investigate (1) the influence of the secretions of follicular cells on the in vitro maturation of bovine cumulus-oocyte complexes (COCs) and (2) the origin of the factors controlling the metabolic function of cumulus cells during the preovulatory period. Preovulatory granulosa cells were collected from synchronized heifers either before or 7–9 hr after the luteinizing hormone (LH) surge, and their secretions were recovered after a 3 hr incubation. Follicular fluids (FFs) originating from the same follicles and sera from the same animals were also collected. The effects of FFs, sera, and secretions of granulosa cells on COC metabolism were compared during 24 hr of culture. FF stimulated cumulus expansion, progesterone secretion, and overall protein synthesis by COCs but decreased the amount of a major protein of 28 kDa. The time at which FF was collected influenced both cumulus expansion and protein synthesis by COCs. The effects of FF on COC metabolism were detected at the lowest protein concentration studied (0.073 mg/ml) and could be mimicked with serum, but only at a protein concentration 100-fold higher. The inhibitory effect of FF and serum on the amount of the 28 kDa protein was reproduced with the secretions of granulosa cells, acting at protein concentrations five- and 500-fold lower, respectively. However, the secretions of granulosa cells enhanced slightly cumulus expansion and had no effect on progesterone secretion and overall protein synthesis by COCs. These results suggest that COC metabolism is influenced both by endocrine and by local factors secreted by granulosa cells in response to gonadotropins. The paracrine control of COC metabolism by preovulatory granulosa cells could be exerted not only via intercellular contacts but also via substances secreted in FF. © 1993 Wiley-Liss, Inc.     

Knockout of pentraxin 3, a downstream target of growth differentiation factor-9, causes female subfertility

The ovulatory process is tightly regulated by endocrine as well as paracrine factors. In the periovulatory period, extensive remodeling of the follicle wall occurs to allow the extrusion of the oocyte and accompanying cumulus granulosa cells. Growth differentiation factor-9 (GDF-9) and bone morphogenetic protein-15 (BMP-15) are secreted members of the TGFbeta superfamily that are expressed beginning in the oocyte of small primary follicles and through ovulation. Besides its critical role as a growth and differentiation factor during early folliculogenesis, GDF-9 also acts as a paracrine factor to regulate several key events in preovulatory follicles. By analyzing GDF-9-regulated expression profiles using gene chip technology, we identified TNF-induced protein 6 (Tnfip6) and pentraxin 3 (Ptx3 or PTX3) as novel factors induced by GDF-9 in granulosa cells of preovulatory follicles. Whereas Tnfip6 is induced in all granulosa cells by the LH surge, Ptx3 expression in the ovary is specifically observed after the LH surge in the cumulus granulosa cells adjacent to the oocyte. PTX3 is a member of the pentraxin family of secreted proteins, induced in several tissues by inflammatory signals. To define PTX3 function during ovulation, we generated knockout mice lacking the Ptx3 gene. Homozygous null (Ptx3(-/-)) mice develop normally and do not show any gross abnormalities. Whereas Ptx3(-/-) males are fertile, Ptx3(-/-) females are subfertile due to defects in the integrity of the cumulus cell-oocyte complex that are reminiscent of Bmp15(-/-)Gdf9(+/-) double mutant and BMP type IB receptor mutant mice. These studies demonstrate that PTX3 plays important roles in cumulus cell-oocyte interaction in the periovulatory period as a downstream protein in the GDF-9 signal transduction cascade.     

Distribution of ganglioside GM3 in the rat ovary after gonadotropin stimulation.

Gangliosides are ubiquitous membrane components in mammalian cells and are suggested to play important roles in various cell functions, such as cell-cell recognition, differentiation and transmembrane signalling. Ovaries have been shown to contain GM3 as a major ganglioside. To study GM3 distribution during gonadotropin stimulation in the hypophysectomized rat ovary, ovarian sections and cultured granulosa cells were stained with specific monoclonal antibody against GM3. Interstitial cells of follicles of immature hypophysectomized rat ovary expressed ganglioside GM3. Theca cells of early antral follicles but not primary follicles expressed GM3. No granulosa cells of these follicles expressed GM3. When a surge dose of FSH/LH was injected, Graafian follicles were formed and GM3 expression was detected in granulosa cells of these follicles. After ovulation, cumulus cells kept expressing GM3 in the ampulla region of ovulated oviduct. The follicles did not show GM3 expression in their granulosa cells after an ovulatory dose of FSH/LH. At 48 h after in vitro culture with FSH/LH of granulosa cells from preantral follicles, GM3 was expressed to a detectable extent on the outer part of the granulosa layer. Finally, at 72 h after culture, all granulosa cells became positive to anti-GM3 antibody. These data suggest that the expression of ganglioside GM3 in the hypophysectomized rat ovary is spatiotemporally regulated by FSH/LH during follicular development and ovulation.     

The effect of stage of estrous cycle and follicular maturation on ovarian inhibin production in sheep

Twenty-four Scottish Blackface ewes (mean weight 50.0 +/- 0.1 kg with ovulation rate 1.3 +/- 0.1) were randomly divided into 4 groups of 6 animals. Under general anesthesia, following the collection of a timed sample of ovarian venous blood, the ovaries of these animals were collected either on Day 10 of the luteal phase or 12, 24, and 48 h after a luteolytic dose of a prostaglandin (PG) F2 alpha analogue (cloprostenol 100 micrograms i.m.) administered on Day 10. All follicles greater than 3 mm were dissected from the ovaries and incubated in Medium 199 (M199) at 37 degrees C for 2 h, following which the granulosa cells were harvested and incubated in triplicate for 24 h in M199 with or without ovine FSH or ovine LH. Plasma and culture media samples were assayed for inhibin, estradiol (E2), androstenedione (A4), and testosterone (T) by specific RIA. After correcting for hematocrit, ovarian secretion rates were calculated from the product of the plasma concentration and flow rate. The rate of ovarian inhibin secretion during the luteal phase was similar from ovaries categorized on the basis of presence of luteal tissue (1.0 +/- 0.3 and 0.9 +/- 0.5 ng/min for CL present and absent, respectively), confirming that the ovine CL does not secrete appreciable amounts of inhibin. Inhibin secretion was higher (p less than 0.05) at 12 h after PG-induced luteolysis but not at 24 or 48 h compared to values for luteal phase control ewes. Although ovaries containing large estrogenic follicles (greater than or equal to 4 mm in diameter and classified as estrogenic from in vitro criteria) secreted the most inhibin (55%; p less than 0.05), both ovaries containing large nonestrogenic follicles (33%) and small (11%; less than 4 mm in diameter) follicles secreted appreciable amounts of inhibin. This contrasted strongly with E2 where greater than 80% of the steroid was secreted by large estrogenic follicles. The rate of ovarian inhibin secretion was positively correlated (p less than 0.05) with the rate of E2, A4, and T secretion. Overall, there was no significant effect of stage of cycle on follicular inhibin content after 2 h incubation in vitro, release of inhibin by follicles incubated in vitro, or synthesis of inhibin by granulosa cells cultured in vitro. FSH and LH had no effect on the production of either inhibin or estradiol by cultured granulosa cells. Follicular diameter was positively correlated (p less than 0.001) with follicular inhibin and steroid release. Follicular inhibin content after 2 h incubation in vitro was more highly correlated with inhibin release by incubated follicles (r = 0.7; p less than 0.001) than with inhibin synthesis by granulosa cells in vitro (0.4; p less than 0.01).(ABSTRACT TRUNCATED AT 400 WORDS)     

Role of the epidermal growth factor network in ovarian follicles

The LH surge causes major remodeling of the ovarian follicle in preparation for the ovulatory process. These changes include reprogramming of granulosa cells to differentiate into luteal cells, changes in cumulus cell secretory properties, and oocyte maturation. This review summarizes published data in support of the concept that LH stimulation of ovarian follicles involves activation of a local epidermal growth factor (EGF) network. A model describing this property of LH signaling and its branching to other signaling modules is discussed. According to this model, LH activation of mural granulosa cells stimulates cAMP signaling, which, in turn, induces the expression of the EGF-like growth factors epiregulin, amphiregulin, and betacellulin. These growth factors function by activating EGF receptors in either an autocrine/juxtacrine fashion within the mural layer, or they diffuse to act on cumulus cells. Activation of EGF receptor signaling in cumulus cells, together with cAMP priming, triggers oocyte nuclear maturation and acquisition of developmental competence as well as cumulus expansion. This model has important implications for ovarian physiology and for the development of new strategies for the pharmacological control of ovulation and for gamete maturation in vitro.     

Follicle and oocyte morphology in ewes after treatment with insulin in the late follicular phase

Stress reduces fertility in ruminants. Various experimental models, such as insulin-induced hypoglycaemia, have been used to investigate the mechanisms involved, and have revealed abnormal LH profiles (both pulse and surge secretion). This disruption affects follicular function and it is proposed there may be negative consequences on subsequent oocyte morphology. Insulin (5 iu/kg), administered to ewes in the late follicular phase, induced hypoglycemia for 10 h, decreased estradiol concentrations for 8-12 h and delayed the LH surge by 15 h. Although the diameters of dominant follicles just before ovulation were not affected, granulosa cells had fewer pycnotic nuclei, less apoptosis and increased proliferation 16-17 h after the LH surge. Nevertheless, we did not observe gross ultra-structural differences in nuclear, cytoplasmic or cumulus maturity between oocytes from insulin-treated and control animals. This suggests that reduced LH pulsatility and a delay in the LH surge may only produce very subtle changes in gross oocyte morphology, imperceptible by electron microscopy.     

Meiotic state of bovine oocytes is regulated by interactions between cAMP,cumulus, and granulosa

Bovine oocytes are arrested at the prophase of first meiotic cell cycle. Meiosis resumes in oocytes of pre-ovulatory follicles upon LH surge. However, oocytes from secondary follicles spontaneously resume meiosis in the absence of hormones if removed from the follicle and cultured in vitro. The nature of meiotic arrestor in bovine follicles is poorly understood. In this study we investigated the role of cell-cell interactions between granulosa and cumulus cells and the oocyte in mediating maintenance of meiotic arrest by cAMP. We sorted oocytes as granulosa-cumulus oocyte complexes (GCOC) if surrounded with cumulus cells attached to a large granulosa investment or cumulus oocytes complexes (COC) if surrounded with cumulus cells only and investigated the role cAMP in maintenance of meiotic arrest in these oocytes under various conditions. In hormone- and serum-free medium both GCOC and COC enclosed oocytes resumed meiosis. When [cAMP](i) was elevated with addition of invasive adenylate cyclase (iAC) GCOC enclosed oocytes were maintained in the prophase with intact germinal vesicle (GV) while COC enclosed oocytes underwent GV breakdown (GVBD). iAC elevated [cAMP](i) in both types of oocytes to the same level. If oocytes were liberated from the cumulus and granulosa cells, they re-initiated meiosis in serum and hormone free medium, but remained in the GV stage if iAC was added to the medium. Untreated GCOC and COC enclosed oocytes extruded first polar body at the same frequency in hormone-supplemented media. GCOC and COC enclosed oocytes but not denuded oocytes (DO) cultured without somatic cells acquired developmental competence if cultured in hormone-containing medium. It is concluded that maintenance of meiotic arrest is regulated by the interplay of [cAMP](i), and cumulus and granulosa cells.     

Preovulatory biosynthesis and granulosa cell secretion of immunoreactive oxytocin by goat ovaries

Oestrous cycles of goats were synchronized hormonally. Immunoreactive oxytocin was undetectable (less than 0.1 ng/mg protein) in media from granulosa cells isolated before the LH surge for small (1-2 mm), medium (3-5 mm) and large (greater than 5 mm diameter) follicles when cultured for 24 h without or with added hormones. Granulosa cells from large and medium, but not small, follicles isolated 6-12 h after spontaneous preovulatory LH surges secreted high concentrations of oxytocin (4-12 ng/mg protein). Addition of PGE-2 (1 microgram/ml) caused a further significant (P less than 0.05) increase in oxytocin secretion by cultured granulosa cells, whereas PGF-2 alpha, FSH and LH were ineffective when added to culture media. Ovarian venous blood and granulosa cells were collected at 0, 6, 12 or 18 h after GnRH injection in hormonally synchronized goats. Peripheral serum LH values were increased significantly in all but 2 of 22 goats within 2 h of GnRH injection. At the earliest sampling time after GnRH (6 h), ovarian venous levels of oxytocin were increased significantly from basal levels of 0.4 pg/ml to 2.4 pg/ml. Oxytocin concentrations in follicular fluid increased from a basal value of 67 pg/ml to 155 pg/ml by 6 h and to 372 pg/ml by 18 h after GnRH injection. Oxytocin secretion by cultured granulosa cells was not increased significantly by 6 h (0.1 ng/mg protein) but rose to 1.4 and 3.5 ng/mg protein at 12 and 18 h, respectively. Approximately parallel increases occurred in progesterone in ovarian venous blood and granulosa cell culture media over the same time period. (ABSTRACT TRUNCATED AT 250 WORDS)     

Developmental and hormonal regulation of cumulus expansion and secretion of cumulus expansion-enabling factor (CEEF) in goat follicles

The objective of this article was to study the developmental and hormonal regulation of cumulus expansion and secretion of cumulus expansion-enabling factor (CEEF) in goat follicles. M-199 medium was conditioned for 24 hr with cumulus-denuded oocytes (DOs), oocytectomized complexes (OOXs), or mural granulosa cells (MGCs) from goat follicles of different sizes. Mouse OOXs and eCG were added to culture drops of the conditioned medium and cumulus expansion was scored at 18 hr of culture to assess CEEF production. While mouse OOXs did not expand, goat OOXs underwent full cumulus expansion when cultured in nonconditioned eCG-supplemented M-199 medium. When cultured in nonconditioned medium containing 10% follicular fluid (FF) from goat medium (2-4 mm) and small (0.8-1.5 mm) follicles, 71-83% mouse OOXs expanded; but expansion rates decreased (P < 0.05) at either lower or higher FF concentrations. FF from large (5-6 mm) follicles did not support mouse OOX expansion at any concentrations. While medium conditioned with DOs from follicles of all the three sizes supported expansion of 80-90% mouse OOXs, medium conditioned with mature DOs had no effect. While cumulus cells from follicles of all the three sizes secreted CEEF in the absence of gonadotropins, MGCs from large follicles became gonadotropin dependent for CEEF production. Both FSH and LH stimulated CEEF production by large follicle MGCs, but FSH had a shorter half-life than LH to expand mouse OOXs. Few meiosis-incompetent goat oocytes from small follicles underwent cumulus expansion when cultured in medium conditioned with goat DOs or cocultured with goat COCs from medium follicles. It is concluded that (1) goat cumulus expansion is independent of the oocyte; (2) the limited CEEF activity in FF from large follicles was due mainly to the inability of MGCs in these follicles to secret the factor in absence or short supply of gonadotropins; (3) the cumulus expansion inability of the meiosis incompetent goat oocytes was due to the inability of their cumulus cells to respond to rather than to produce CEEF.     

Secretion of 17 alpha-hydroxyprogesterone, androstenedione, and estrogens by porcine granulosa and theca interna cells in culture

The steroid secreting activities of dispersed granulosa and theca interna cells from preovulatory follicles of prepubertal gilts 72 h after pregnant mare's serum gonadotropin treatment (750 IU) were compared. The cells were cultured for 24 h with or without steroid substrate (10(-8) to 10(-5) M progesterone, 17 alpha-hydroxyprogesterone, or androstenedione), FSH (100 ng/mL), LH (100 ng/mL), and cyanoketone (0.25 microM, an inhibitor of 3 beta-hydroxysteroid dehydrogenase). Granulosa cells cultured alone secreted mainly progesterone. Theca interna cells secreted mainly 17 alpha-hydroxyprogesterone and androstenedione, with secretion being markedly enhanced by LH. In the presence of cyanoketone, which inhibited endogenous progesterone production, theca interna but not granulosa cells were able to convert exogenous progesterone to 17 alpha-hydroxyprogesterone and androstenedione, and exogenous 17 alpha-hydroxyprogesterone to androstenedione and estradiol-17 beta in high yield. The secretion of the latter steroids from exogenous substrates was unaffected by LH. Theca interna cells secreted more estradiol-17 beta than did granulosa cells in the absence of aromatizable substrate, but estradiol-17 beta secretion by the latter was markedly increased after the addition of androstenedione. These apparent differences in steroid secreting activity between the cell types suggest that the enzymes responsible for conversion of C21 to C19 steroids, i.e., 17 alpha-hydroxylase and C17,20-lyase, reside principally in the theca interna cells. However, aromatase activity appears to be much higher in granulosa cells.     

Synaptosomal-associated protein 25 gene expression is hormonally regulated during ovulation and is involved in cytokine/chemokine exocytosis from granulosa cells

During ovulation, granulosa cells and cumulus cells synthesize and secrete a wide variety of factors including members of the IL cytokine family via the process of exocytosis. Exocytosis is controlled by the soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor complex consisting of proteins residing in the vesicle membrane and the plasma membrane. One of the soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor proteins, synaptosomal-associated protein (SNAP)25, is expressed abundantly in neuronal cells and is also induced transiently in the rat ovary in response to LH. Therefore, we sought to determine the molecular mechanisms controlling ovarian expression of the Snap25 gene, and the role of SNAP25 in exocytosis of secreted factors, such as ILs from cumulus cells and granulosa cells. In preovulatory follicles of equine (e) chorionic gonadotropin (CG)-primed mice, expression of Snap25 mRNA was negligible but was induced markedly 8 h after human (h) CG stimulation. In Pgr null mice Snap25 mRNA and protein levels were significantly lower at 8 h after hCG compared with wild-type mice. To analyze the molecular mechanisms by which progesterone receptor regulates this gene, a 1517-bp murine Snap25 promoter-luciferase reporter construct was generated and transfected into granulosa cell cultures. Three specificity protein (SP)-1/SP-3 sites, but not consensus activator protein 1 or cAMP response element sites, were essential for basal and forskolin/phorbol 12-myristate 13-acetate-induced promoter activity in granulosa cells. The induction was significantly suppressed by PGR antagonist, RU486. Treatment of cumulus oocyte complexes or granulosa cells with FSH/amphiregulin, LH, or forskolin/phorbol 12-myristate 13-acetate-induced elevated expression of Snap25 mRNA and increased the secretion of eight cytokine and chemokine factors. Transfection of granulosa cells with Snap25 small interfering RNA significantly reduced the levels of both SNAP25 protein and the secretion of cytokines. From these results, we conclude that progesterone-progesterone receptor-mediated SNAP25 expression in cumulus oocyte complexes and granulosa cells regulates cytokine and chemokine secretion via an exocytosis system.     

Bovine theca and granulosa cells interact to promote androgen production

Pieces of theca interna or follicle wall (theca interna + attached granulosa cells), obtained from bovine preovulatory follicles prior to the surge of luteinizing hormone (LH) and cultured for 3 days, secreted androstenedione. Luteinizing hormone, but not follicle-stimulating hormone (FSH), increased production of androstenedione 3 to 4-fold. In both the presence and absence of LH, follicle wall preparations secreted about 4-fold more androstenedione than did equivalent amounts of theca interna tissue. Isolated granulosa cells produced only negligible quantities of androstenedione, which suggests that they may contribute to the greater production of androstenedione by follicle wall by supplying progestin precursor to the theca cells. The addition of pregnenolone or progesterone to isolated theca interna increased the secretion of androstenedione, but pregnenolone was by far the more effective precursor. This suggested that the delta 5 (delta 5) pathway is the preferred pathway for androstenedione synthesis by bovine theca cells and that granulosa cells might supply progestin precursor in the form of pregnenolone. Follicle wall and granulosa cell cultures secreted 2 and 7 times more pregnenolone, respectively, than did theca cultures. Luteinizing hormone, but not FSH, increased production of pregnenolone by the follicle wall, whereas the gonadotropins had no effect on secretion by either granulosa or theca cells. Since exogenous testosterone enhanced the production of pregnenolone by granulosa cells, thecal androgen (which is stimulated by LH) may increase the ability of granulosa cells to make pregnenolone and explain the stimulatory effect of LH on pregnenolone secretion by follicle wall.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mouse oocytes promote proliferation of granulosa cells from preantral and antral follicles in vitro.

Evidence is now emerging that the oocyte plays a role in the development and function of granulosa cells. This study focuses on the role of the oocyte in the proliferation of (1) undifferentiated granulosa cells from preantral follicles and (2) more differentiated mural granulosa cells and cumulus granulosa cells from antral follicles. Preantral follicles were isolated from 12-day-old mice, and mural granulosa cells and oocyte-cumulus complexes were obtained from gonadotropin-primed 22-day-old mice. Cell proliferation was quantified by autoradiographic determination of the 3H-thymidine labeling index. To determine the role of the oocyte in granulosa cell proliferation, oocyte-cumulus cell complexes and preantral follicles were oocytectomized (OOX), oocytectomy being a microsurgical procedure that removes the oocyte while retaining the three-dimensional structure of the complex or follicle. Mural granulosa cells as well as intact and OOX complexes and follicles were cultured with or without FSH in unconditioned medium or oocyte-conditioned medium (1 oocyte/microliter of medium). Preantral follicles were cultured for 4 days, after which 3H-thymidine was added to each group for a further 24 h. Mural granulosa cells were cultured as monolayers for an equilibration period of 24 h and then treated for a 48-h period, with 3H-thymidine added for the last 24 h. Oocyte-cumulus cell complexes were incubated for 4 h and then 3H-thymidine was added to each group for an additional 3-h period. FSH and/or oocyte-conditioned medium caused an increase in the labeling index of mural granulosa cells in monolayer culture; however, no differences were found among treatment groups.(ABSTRACT TRUNCATED AT 250 WORDS)     

Synthesis and secretion of sulphated glycoproteins by rabbit oviduct explants in vitro

The ability of rabbit oviduct explants to incorporate radiolabelled precursors into specific secretory products was investigated. Ampullary and isthmic oviduct segments were cultured in the presence of [3H]glucosamine or [35S]sodium sulphate. Medium samples were analysed for the presence of secreted, labelled macromolecules. Explants incorporated the [3H]glucosamine and secreted labelled glycoproteins in vitro. SDS gel electrophoresis and subsequent fluorographic analysis of culture medium demonstrated a differential secretion of glycoproteins between the ampulla and the isthmus. Although ampullary tissue secreted a greater amount of labelled glycoproteins during the sampling period, the major secretory constituent of Mr approximately 66,000 was common to both oviduct segments. Tissue incubated with [35S]sodium sulphate also secreted a labelled glycoprotein or subunit of Mr approximately 66,000. The results indicate that rabbit oviduct explants are capable of synthesis and secretion of specific sulphated glycoproteins in vitro and that there is a difference in the type and amount of secretion produced between the two oviduct segments.     

Changes in expression of inhibin subunits in the cyclic golden hamster (Mesocricetus auratus) and the regulation of inhibin alpha subunit production by luteinizing hormone

In the present study, changes in localization of each inhibin subunit in the ovary were investigated during the estrous cycle of the golden hamster. The effect of LH surge on changes in localization in inhibin alpha subunit in the ovary was also investigated. Inhibin alpha subunit was localized in granulosa cells of various stages of follicles throughout the estrous cycle. Inhibin alpha subunit was also present in numerous interstitial cells on days 1 and 2 (day 1 = day of ovulation), but the number of positive interstitial cells was fewer on days 3 and almost disappeared on day 4 of the estrous cycle. Newly formed luteal cells were also positive for inhibin alpha subunit on days 1 and 2. On the other hand, positive reactions for inhibin beta A and beta B subunits were only present in the granulosa cells of healthy antral follicles. However, a positive reaction for inhibin beta B subunit in peripheral mural granulosa cells disappeared on days 3 and 4 of the estrous cycle. Treatment with LHRH-AS at 1100 h on day 4 completely blocked the luteinizing hormone (LH) surge and ovulation, although relatively high concentrations of plasma follicle-stimulating hormone (FSH) were maintained throughout the experiment. There were few positive reactions for inhibin alpha subunit in theca and interstitial cells 24 hr after LHRH-AS injection. The effect of LHRH-AS treatment was blocked by a single injection of 10 IU human chorionic gonadotropin. These results suggest that the major source of dimeric inhibin in the cyclic hamster was granulosa cells of healthy antral follicles. Different distribution pattern of inhibin beta A from beta B subunits in large antral follicles on days 3 and 4 of the estrous cycle suggests different secretion patterns of inhibin A from B on these days. Furthermore, the LH surge may be an important factor to induce production of inhibin alpha subunit in interstitial cells of the cyclic hamster.