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E. V. YOUNGLAI

Different cell types of the ovarian follicle have been used to study steroid biosynthetic pathways. These investigations involved the use of tissue culture (Channing & Grieves, 1969) and incubations in vitro with specific buffers (Ryan & Short, 1965; Ryan, Petro & Kaiser, 1968). The activity of these cells in biological fluids has not been extensively studied. The aim of the present investigation was, therefore, to compare plasma and follicular fluid as incubating media for the biochemical study of the 3β-hydroxysteroid dehydrogenase and aromatizing activities of granulosa cells from equine ovarian follicles obtained at oestrus.

Radioactive steroids of high specific activity were purchased from the Radiochemical Centre, Amersham, Bucks., and were checked for purity before use. Reproductive tracts were obtained at slaughter from four mares judged to be in oestrus by gross examination of the uterus, cervix and corpus luteum using the criteria outlined by Channing (1969). At the

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A. C. Hynes, M. T. Kane and J. M. Sreenan

Granulosa cell-inhibitory factor (GCIF), a low molecular weight factor from bovine follicular fluid, inhibits the proliferation of bovine granulosa cells in vitro and the growth of large follicles in rats in vivo. In this study the effects of (1) immunization of rats against GCIF on follicular growth and (2) immunization of sheep against GCIF on ovulation rate were studied. The ability of antiserum from sheep immunized against GCIF to reduce the inhibitory effect of GCIF on bovine granulosa cell proliferation in culture was also examined. Immunization of rats against GCIF increased the number of large follicles (P < 0.001) but decreased the number of small follicles (P < 0.05) per ovary. Ovarian mass (P < 0.05) and uterine wet (P < 0.05) and dry (P < 0.01) masses were increased in immunized rats. Immunization of sheep against GCIF, followed by boosting over two breeding seasons, increased ovulation rate (P < 0.01). Addition of antiserum from sheep immunized against GCIF reduced or abolished the inhibitory effect of GCIF on granulosa cell proliferation (P < 0.01). These data provide further evidence that GCIF has an important role in controlling follicle growth and ovulation in vivo.

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R. S. Carson, D. M. Robertson and J. K. Findlay

Summary. The ability of antral follicular fluid obtained from sheep follicles to inhibit 3T3 fibroblasts maintained for 48 h in concentrations of fetal calf serum optimal for cell growth was examined. Addition of pooled follicular fluid to cultures resulted in a dose-dependent and reversible inhibition of [3H]thymidine incorporation. Serum from ovariectomized ewes, fetal calf serum, bovine inhibin, oestradiol-17β, testosterone, cortisol or progesterone were without effect over a range of doses. Treatment of pooled follicular fluid with charcoal–dextran did not reduce inhibitory activity which was only partly removed by heating at 85°C. Fluid obtained from large follicles (>5 mm) was more potent as an inhibitor than was fluid obtained from smaller (<5 mm) follicles. Gel chromatography of pooled fluid resolved two peaks of inhibitory activity associated with material of M r ∼ 180 000 and <10 000 respectively. No inhibitory activity was evident in fractions of serum from ovariectomized ewes chromatographed in an identical manner. These results indicate that ovine follicular fluid contains two components able to inhibit reversibly mitosis of 3T3 fibroblasts in vitro.

Keywords: fibroblast mitosis in vitro; inhibition; ovarian follicular fluid

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G Maillet, A Benhaim, H Mittre and C Feral

Follicular atresia is characterized by a rapid loss of granulosa cells and, to a lesser extent, theca cells, via apoptosis. The aim of this study was to investigate the possible involvement of theca cell secretions in the regulation of apoptosis of rabbit granulosa cells. The annexin-V binding method based on externalization of phosphatidylserine to the outer layer of plasma membrane during apoptosis was used to detect apoptotic granulosa cells in flow cytometry. Regulation of apoptosis of granulosa cells was studied in three different culture systems: (i) isolated cultured granulosa cells, (ii) granulosa cells obtained from cultured preovulatory follicles and (iii) granulosa cells co-cultured with theca cells. The results of this study indicate that: (i) the rate of apoptosis of granulosa cells was significantly reduced when granulosa cells were co-cultured with theca cells or obtained from cultured preovulatory follicles in comparison with isolated cultured granulosa cells; (ii) FSH exerts its anti-apoptotic effect only on granulosa cells issued from cultured preovulatory follicles; (iii) ovarian steroids do not affect the percentage of isolated apoptotic granulosa cells; and (iv) the occurrence of an apoptotic process in rabbit theca cells could be upregulated in vitro by hCG and an analogue of the gonadotrophin second messenger cAMP. The results of this study indicate that in rabbits (i) steroids were ineffective in vitro in protecting isolated granulosa cells against apoptosis in comparison with observations in vivo in rats, and (ii) the presence of theca cells was efficient to reduce granulosa cell apoptosis but not sufficient to allow the anti-apoptotic effect of gonadotrophins observed in cultured follicles.

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F. P. Daen, E. Sato, K. Naito and Y. Toyoda

The aim of this study was to analyse the substance(s) present in porcine follicular fluid that promotes cumulus expansion and male pronucleus formation in pig oocytes matured and fertilized in vitro. The follicular fluid was separated into four fractions by ultracentrifugation at 220 000 g at 10°C for 48 h. Oocyte–cumulus-cell complexes obtained from prepubertal gilts were cultured in vitro for 48 h in each of the fractions after reconstitution with a modified Krebs–Ringer bicarbonate solution. Each fraction containing oocyte–cumulus complexes was then fertilized in vitro with frozen–thawed and preincubated epididymal boar spermatozoa. After 24 h of culture, oocytes that matured in fraction I showed a marked expansion of the surrounding cumulus, as did those matured in pig follicular fluid. However, complexes cultured in the remaining fractions exhibited very little or no expansion. No difference was observed in the degree of expansion caused by follicular fluid collected from small, medium or large follicles. Further analysis of fraction I by HPLC gave several subfractions, one of which (22) induced a significantly greater expansion of the cumulus than did others (P < 0.01). Fraction 1 also induced higher rates of male pronuclei formation (62%) than did fractions 2, 3 and 4 (22%, 35% and 30%, respectively). The rate of male pronuclei formation in pig follicular fluid from large follicles was significantly lower (33%) than in fluid from small or medium follicles (89% and 78%, respectively). Two subfractions (22 and 23) derived from HPLC of fraction 1 produced the highest rate of male pronuclei formation compared with the other subfractions (which showed very minimal rates of male pronuclei formation). The active factor(s) responsible for cumulus expansion was heat stable at 100°C for 15 min, resistant to freezing and thawing, and did not lose its activity completely when subjected to proteinase K digestion. The molecular mass of the active factor(s) was < 6.5 kDa, as measured by HPLC.

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E. K. Asem and R. P. Novero

Experiments were conducted in vitro to examine the effect of epidermal growth factor (EGF) and transforming growth factor α (TGF-α) on the production of fibronectin (deposited, secreted into medium and cell-associated) by hen granulosa cells isolated from the largest (F1; about 35 mm in diameter, mature) and third largest (F3; 15–20 mm in diameter) preovulatory follicles, as well as from a pool of immature small yellow follicles (6–8 mm in diameter). The cells were incubated in culture wells coated with type IV collagen or in wells without collagen coating, and the amounts of fibronectin produced were measured using a specific ELISA. The total amount of fibronectin produced by unstimulated cells was greatest in wells containing F1 cells and increased with time. The amount of fibronectin deposited by unstimulated cells was greatest in wells containing F1 cells and was much higher in collagen-coated wells than in uncoated wells. Both EGF and TGF-α increased the quantity of fibronectin deposited by granulosa cells in collagen-coated and uncoated wells. Fibronectin secreted into the medium by unstimulated cells also increased with the stage of follicular maturation and was enhanced by EGF and TGF-α. The quantity of cell-associated fibronectin in granulosa cells in collagen-coated and uncoated wells was also increased by these growth factors. Type IV collagen did not have any appreciable effect on the amount of fibronectin present in the incubation medium or on cell-associated fibronectin. Because of its marked effect on deposited fibronectin, there were greater total quantities of fibronectin in culture wells coated with type IV collagen. These results demonstrate that EGF and TGF-α stimulate the production and deposition of fibronectin by chicken granulosa cells. The results also suggest that in combination with collagen IV, these growth factors can regulate the formation of the extracellular matrix (for example, basal lamina) of the hen ovarian follicle.

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B. D. BAVISTER, R. G. EDWARDS and P. C. STEPTOE

Positive identification of the sperm midpiece and tail in the vitellus establishes beyond reasonable doubt that pronucleate eggs are undergoing fertilization. Previously Edwards, Bavister & Steptoe (1969) tentatively identified sperm midpieces in pronucleate human eggs fertilized in vitro. Unequivocal evidence of midpieces and tails in eggs undergoing fertilization is now presented. Oocytes recovered from two ovaries excised 3 hr previously were cultured in a mixture containing three parts follicular fluid : one part of Bavister's medium (Bavister, 1969). Four samples of follicular fluid were used, three being straw-coloured and a fourth, which was slightly pink, probably came from an atretic follicle. After 36 to 37 hr in culture, the eggs were washed through two or three changes of Bavister's medium, the dilution of the follicular fluid being approximately 1:100. Ejaculated spermatozoa were washed twice in this medium and then added to some of the eggs at a concentration of
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C Robert, D Gagne, JG Lussier, D Bousquet, FL Barnes and MA Sirard

As the expression of the LH receptor (LH-R) in granulosa cells is thought to be associated with later stages of folliculogenesis, this study was undertaken to evaluate the presence of LH-R mRNA as a suitable marker for developmental competence of oocytes. Granulosa cells and cumulus-oocyte complexes (COCs) were recovered from cows that had received ovarian stimulation. The COCs were subjected to embryo production procedures in vitro to assess the embryonic potential of the oocyte, and the corresponding granulosa cells were used to evaluate the presence of LH-R mRNA by RT-PCR. The presence of LH-R transcripts in granulosa cells is not a key characteristic of a follicle bearing a competent oocyte, although a higher proportion of oocytes reach the blastocyst stage when LH-R mRNA is detected in the granulosa cells. Different LH-R isoforms were cloned and sequence discrepancies among six of the isoforms enabled the design of specific oligonucleotides to study the presence of the isoforms in different follicular cells. All LH-R transcripts studied and the 80 kDa protein product corresponding to the full length receptor were found in granulosa cells of small (< 4 mm) and large (> 5 mm) follicles. When the granulosa cells were cultured, the transcripts were downregulated by the culture conditions; downregulation was more acute in granulosa cells from small follicles. The addition of LH to the culture media enhanced LH-R mRNA downregulation. The presence of several LH-R transcript isoforms was tissue specific and in the theca cells LH-R mRNA was restricted mainly to cells from larger follicles. This finding indicates that the expression and the splicing of LH-R mRNA are regulated in a cell-specific and follicular size-specific manner.

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MA Driancourt, K Reynaud, R Cortvrindt and J Smitz

Evidence from mouse mutants indicates that the Kit gene encoding KIT, a receptor present on the oocyte and theca cells, and the Mgf gene encoding KIT LIGAND, the ligand of KIT, are important regulators of oogenesis and folliculogenesis. Recently, in vitro cultures of fetal gonads, of follicles and of oocytes have identified specific targets for the KIT-KIT LIGAND interaction. In fetal gonads, an anti-apoptotic effect of KIT-KIT LIGAND interactions on primordial germ cells, oogonia and oocytes has been demonstrated. In postnatal ovaries, the initiation of follicular growth from the primordial pool and progression beyond the primary follicle stage appear to involve KIT-KIT LIGAND interactions. During early folliculogenesis, KIT together with KIT LIGAND controls oocyte growth and theca cell differentiation, and protects preantral follicles from apoptosis. Formation of an antral cavity requires a functional KIT-KIT LIGAND system. In large antral follicles, the KIT-KIT LIGAND interaction modulates the ability of the oocyte to undergo cytoplasmic maturation and helps to maximize thecal androgen output. Hence, many steps of oogenesis and folliculogenesis appear to be, at least in part, controlled by paracrine interactions between these two proteins.

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Y. Cognie, F. Benoit, N. Poulin, H. Khatir and M. A. Driancourt

Booroola ewes have a major gene that affects ovulation rate. Gene expression has consequences on ovarian somatic cells but it is unknown whether it also affects germ cells in the adult ovary. Hence, the present study examined (1) whether oocyte growth was similar in Fec B Fec B and Fec + Fec + oocytes during preantral and antral follicular growth, (2) whether the patterns of proteins neosynthesized by oocytes of these two genotypes were identical, (3) whether the ability of the oocytes to resume meiosis was unaffected by genotype and (4) whether, after IVF, oocytes from both genotypes could develop to the blastocyst stage at similar rates. Histological examination of the respective sizes of the oocyte and of the follicle demonstrated that oocytes were larger in Fec B Fec B versus Fec + Fec + preantral follicles. Resolution of the proteins neosynthesized by Fec B Fec B and Fec + Fec + oocytes by one-dimensional PAGE and image analysis demonstrated that quantitative (but not qualitative) differences could be observed between genotypes for bands at 74, 59, 35 and 25 kDa. In addition, a genotype by oocyte size interaction was detected for two additional bands at 45 and 43 kDa. After 24 h of culture in vitro in TCM-199 plus 100 ng ml−1 FSH plus 10% sheep follicular fluid, oocytes from Fec B Fec + follicles gained the ability to resume meiosis at a smaller size and a higher proportion of them reached metaphase II irrespective of the size class studied compared with Fec + Fec + follicles. In addition, the developmental rate of eggs after IVF was also affected by follicle size and genotype, since Fec B Fec + oocytes originating from 1.0–3.5 mm follicles had a greater ability (P < 0.05) to develop to the blastocyst stage than Fec + Fec + oocytes. It is concluded that the Fec B gene, in addition to its effects on granulosa cell maturation, also affects oocyte development and function. Whether these alterations are related requires further investigation.