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M Endo, R Kawahara-Miki, F Cao, K Kimura, T Kuwayama, Y Monji and H Iwata

Antrum formation and estradiol (E2) secretion are specific features of oocyte and granulosa cell complexes (OGCs). This study investigates the effect of E2 on the in vitro development of bovine OGCs derived from early antral follicles as well as on the expression of genes in granulosa cells (GCs). The supplementation of culture medium with either E2 or androstenedione (A4) improved the in vitro development of OGCs and the nuclear maturation of enclosed oocytes. When OGCs were cultured in medium containing A4, developmentally competent OGCs secreted more E2 than OGCs that were not competent. In addition, fulvestrant inhibited the effect of both E2 and A4 on OGCs development. Comprehensive gene expression analysis using next-generation sequence technology was conducted for the following three types of GCs: i) GCs of OGCs cultured for 4 days with E2 (1 μg/ml; E2(+)), ii) GCs of OGCs cultured for 4 days without E2 (E2(−)) or iii) OGCs that formed clear antrum after 8 days of in vitro culture in medium containing E2 (1 μg/ml; AF group). GCs of the E2(+) group had a similar gene expression profile to the profile reported previously for the in vivo development of large follicles. This genetic profile included factors implicated in the up-regulation of E2 biosynthesis and down-regulation of cytoskeleton and extracellular matrices. In addition, a novel gene expression profile was found in the AF group. In conclusion, E2 impacts the gene expression profile of GCs to support the in vitro development of OGCs.

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Sergio Romero and Johan Smitz

Epiregulin mediates LH ovulatory effects in vitro. This study evaluated the use of epiregulin as an alternative to hCG/epidermal growth factor (EGF) stimulus upon cultured ovarian follicles in contrast to isolated cumulus–oocyte complexes (COCs). Pre-antral mouse ovarian follicles were cultured for 12 days and final maturation was induced by administration of 0.65 nM EGF or 100 nM epiregulin without or with 1.2 IU/ml hCG. Results showed that both EGF or epiregulin as sole stimulators are poor inducers of mucification/expansion of cumulus cells and oocyte meiotic reinitiation in follicle-enclosed COCs (25±17 and 22±16% GVBD respectively; versus 97±4 and 90±15% GVBD by control hCG/EGF and hCG/epiregulin respectively; mean±s.d). Furthermore, EGF or epiregulin did not induce follicle luteinisation: progesterone production was marginally increased and oestradiol was incompletely shut down. Supposing that the sub-normal progesterone secretion was a potential cause for incomplete meiosis in this model, effectiveness of progesterone supplementation and addition of a progesterone receptor inhibitor (RU486) were evaluated on meiotic resumption. Progesterone was not found to be a major regulator of meiosis in this mouse model. Epiregulin induced meiosis more effectively in COCs isolated from cultured preovulatory follicles in a secondary culture well. In conclusion, epiregulin has similar effects as EGF upon fully grown follicles. Used as a sole stimulator of periovulatory events in intact cultured follicles, both are poor inducers of follicle luteinisation and oocyte maturation. By contrast, epiregulin is as efficient as hCG/EGF, when used as meiotic stimulator for COCs isolated from the follicular environment (mural granulosa and theca cells; and conditioned medium).

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JL Tremoleda, T Tharasanit, HT Van Tol, TA Stout, B Colenbrander and MM Bevers

It has been suggested that preculturing immature oocytes in a manner that maintains them in meiotic arrest may improve cytoplasmic maturation and, thereby, the eventual developmental competence of oocytes matured in vitro. This study examined the ability of follicular cells to maintain meiotic arrest in equine oocytes. Cumulus-oocyte complexes (COCs) recovered from dead mares were cultured for 38 h in M199 either attached to, or together with, different follicle wall components, as follows: (1) attached to the follicle wall, (2) cocultured with separated follicle wall, (3) attached to membrana granulosa (COCG), (4) COCGs cocultured with sheets of theca cells, (5) COCGs cultured in theca-cell conditioned medium, and (6) control COCs without any follicle wall components. When oocytes were cultured attached to their follicle wall, 79% remained in the GV stage throughout the 38 h incubation. However, when oocytes were cocultured with separate pieces of follicle wall, meiosis resumed and a similar proportion of oocytes progressed to metaphase II (79%) as under control conditions (84%). Only 16% of oocytes cultured while still attached to the membrana granulosa (COCGs) maintained the GV stage, whereas when COCGs were cocultured with theca cells or in theca-cell conditioned medium, significantly more oocytes remained in the GV stage (64 and 52%, respectively), indicating that theca cells secrete a meiosis-inhibiting factor. The effect of FSH on the meiosis-inhibiting activity of follicular cells was investigated by culturing COCs attached to the follicle wall and COCGs in the presence or absence of theca cells in medium containing FSH. Addition of 0.05 iu recombinant human FSH ml(-1) to the culture medium did not affect nuclear maturation and failed to overcome the suppressive effect exerted by the follicle wall or by theca cells, despite the fact that mRNA for the FSH receptor was found using RT-PCR in both cumulus and granulosa cells. These results demonstrate that the maintenance of meiotic arrest in equine oocytes during culture can be promoted by theca cells, which appear to act via a secreted inhibitory factor that cannot be suppressed or counteracted by FSH.

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A L Johnson, Christine Ratajczak, Morgan J Haugen, Han-Ken Liu and Dori C Woods

Tumor necrosis factor-related apoptosis inducing ligand (TRAIL) represents one of several cytokine members of the tumor necrosis factor superfamily reported to initiate apoptosis in a wide range of transformed, but not most normal, cell types. The present studies were conducted to evaluate the potential for TRAIL to promote apoptotic cell death in differentiated granulosa cells collected from hen preovulatory follicles. While mRNA encoding critical components (including TRAIL) required for a functional extrinsic cell death pathway are expressed in granulosa cells, TRAIL treatment by itself fails to induce either caspase-3 activity or a decrease in cell viability. On the other hand, preculture of cells with the conventional chemotherapeutic, cisplatin, or the 20S proteosome inhibitor, Z-LLF-CHO, sensitizes granulosa cells to TRAIL as evidenced by enhanced caspase-3 activity after 4 h of culture and loss of cell viability after 24 h when compared with either cisplatin or Z-LLF-CHO treatment alone. Moreover, the sensitizing effect of Z-LLF-CHO on TRAIL-induced loss of cell viability is prevented by the selective caspase-8 inhibitor, Z-IETD-FMK. Interestingly, TRAIL mRNA expression is elevated both in prehierarchal follicles undergoing spontaneous atresia and in prehierarchal follicles induced to undergo atresia for 6 h in vitro. In summary, the data demonstrate the presence of a functional TRAIL signaling pathway in hen granulosa cells, and are consistent with the possibility that TRAIL signaling may directly or indirectly participate in the process of follicle atresia in vivo.

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Large intact follicles and granulosa cells were obtained from sheep ovaries at various stages of the oestrous cycle and were cultured in vitro for 7 days. The daily output of steroid hormones into the culture medium was determined using a procedure capable of measuring a wide range of steroids including immunoreactive oestrogen, testosterone, androstenedione, progesterone, 20α-hydroxy-pregn-4-en-3-one, 17α-hydroxyprogesterone, 17α,20α-dihydroxy-pregn-4-en-3-one, pregnenolone, 17α-hydroxypregnenolone and certain hydrogenated metabolites.

Intact follicles explanted from sheep on Day 4 of the cycle produced oestrogen (40 ng/mg follicular tissue per 24 hr) during the first 3 days in culture followed by large amounts of testosterone (100 ng/mg per 24 hr) and finally 17α-hydroxylated progestin. Follicles explanted at midcycle produced constant amounts of oestrogen (30 ng/mg per 24 hr) throughout the culture period; no other steroids were produced in significant quantities. The highest levels of oestrogen (75 ng/mg per 24 hr) were produced by follicles which had been explanted from sheep on Day 14 of the cycle. Oestrogen production declined rapidly in follicles explanted just before oestrus (late Day 15) and was replaced by the production of testosterone and 17α-hydroxylated progestin. The production of oestrogen and testosterone was very low in follicles explanted at oestrus; these follicles produced a transient peak of 17α-hydroxypregnenolone (30 ng/mg per 24 hr) followed by large amounts of progesterone (300 ng/mg per 24 hr) and 20α-hydroxy-pregn-4-en-3-one (250 ng/mg per 24 hr).

Monolayer cultures of granulosa cells produced only progesterone, 20α-hydroxy-pregn-4-en-3-one and pregnenolone, thus implicating the cells of the theca interna as the principal source of oestrogen, androgen and 17α-hydroxylated progestin.

Our findings indicate that the biosynthesis of oestrogen in the cells of the theca interna involve a sequence of steps including pregnenolone → 17α-hydroxypregnenolone→17α-hydroxyprogesterone→ testosterone or androstenedione→ oestrogen. During the transformation of follicles from oestrogen to progesterone secretors, steroid synthetic capacity is transferred from the theca interna to the membrana granulosa. The accumulation first of testosterone and then of 17α-hydroxypregnenolone suggests that the aromatase and then the desmolase systems limit steroid production in the theca interna during the period of transformation.

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K.M. Henderson and P. Franchimont

Summary. No detectable amounts of inhibin were produced by cultured ovarian stroma or luteal tissue. Follicular tissue produced inhibin in vitro and removal of the granulosa cells from the follicle wall caused inhibin production to fall by 80%. Granulosa cells alone had the greatest ability of any ovarian cell type to produce inhibin in vitro, and are probably the major site of follicular inhibin production.

Cyproterone acetate at concentrations of 35 and 350 μM inhibited basal and testosterone (3·5 μM)-stimulated inhibin production by cultured intact follicle wall and granulosa cells. In addition, each concentration of cyproterone acetate inhibited progesterone but not oestradiol-17β production by the follicle wall and granulosa cell cultures. The synthetic, non-aromatizable androgens, methylestrenolone and mesterolone, at concentrations of 5 and 25 μM, mimicked the effect of testosterone and stimulated granulosa cell inhibin production, methylestrenolone being the more potent. These findings provide further evidence that androgens regulate follicular inhibin and progesterone production and that these may be receptor-mediated processes, and suggest that inhibin production may be a general property of androgenic compounds.

Preliminary examination of the physicochemical characteristics of inhibin indicated that the inhibin activity of bovine granulosa cell culture medium was (a) retained by an Amicon XM100A filter with a nominal molecular weight cut-off point of 100 000; and (b) destroyed by heating to 80°C for 30 min.

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Eric E Nilsson, Jacob Stanfield and Michael K Skinner

Follicle assembly is the process by which groups or ‘nests’ of oocytes break down to form primordial follicles. The size of the primordial follicle pool is the major determinant of the reproductive lifespan of a female. Previously, progesterone (P4) has been shown to inhibit follicle assembly, while tumor necrosis factor-α (TNFα) has been shown to promote the apoptosis that is necessary for follicle assembly. The present study examines how TNFα and progesterone interact to regulate primordial follicle assembly. Ovaries were collected from newborn rats and placed in organ culture to examine the actions of P4 and TNFα. P4 was found to decrease primordial follicle assembly and increase the percentage of unassembled oocytes both in vitro and in vivo. TNFα treatment did not change the proportion of assembled follicles in cultured ovaries, but blocked the ability of P4 to inhibit follicle assembly. Microarray analysis of the ovarian transcriptome revealed that progesterone treatment of the ovaries altered the expression of 513 genes with 132 only expressed after P4 treatment and 16 only expressed in control ovaries. The majority of genes were up-regulated greater than twofold over control, with a small subset of 16 genes down-regulated. Categories of genes affected by P4 are described including a group of extracellular signaling factors. The progesterone receptors expressed at the time of follicle assembly included the surface membrane progesterone receptors PGRMC1, PGRMC2, and RDA288. The nuclear genomic P4 receptor was not expressed at appreciable levels. Progesterone increased the expression of several genes (TANK, NFκB, Bcl2l1, and Bcl2l2) involved in a signaling pathway that promotes cell survival and inhibits apoptosis. Observations indicate that P4 acts through the surface membrane progesterone receptors to regulate primordial follicle assembly, and that TNFα can override the inhibitory actions of P4 on follicle assembly. A major mechanism involved in the actions of P4 is an increase in cell survival genes and inhibition of the apoptosis pathway. Observations provide insight into the hormonal regulation of primordial follicle assembly and lead to novel approaches to potentially manipulate follicle assembly and reproductive capacity.

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YH Choi, CC Love, DD Varner, JA Thompson and K Hinrichs

Two different culture media (TCM-199 and follicular fluid), two activation treatments (10 and 50 micromol calcium ionophore l(-1)) and three culture periods with cycloheximide were evaluated to find effective culture conditions for activation of cumulus-free equine oocytes. Oocytes were collected by scraping the follicle walls of ovaries obtained from an abattoir. Oocytes with expanded cumuli were matured at 38.2 degrees C in a humidified atmosphere of 5% CO(2) in air, in either TCM-199 with 10% fetal bovine serum (FBS) and 5 microU FSH ml(-1), or in 100% follicular fluid derived from a preovulatory follicle 24 h after injection of hCG. After 40--42 h of in vitro maturation, oocytes were denuded by gentle pipetting in TCM-199 plus 10% FBS with hyaluronidase. Oocytes with intact cytoplasmic membranes (n = 398; 94% presumed metaphase II) were treated in protein-free PBS with 10 or 50 micromol calcium ionophore l(-1) for 5 min. After washing, the oocytes were cultured in TCM-199 containing 10% FBS and 10 microg cycloheximide ml(-1) for 6 h, in cycloheximide for 6 h and then in cycloheximide-free medium for 18 h, or in cycloheximide for 24 h. The oocytes were fixed and evaluated by fluorescence microscopy. Oocytes with pronucleus I--II (dense to decondensing chromatin), pronucleus III--IV (decondensed chromatin) or progressing towards the first cleavage division were considered activated. The activation rate for oocytes matured in TCM-199 was significantly (P < 0.05) higher than for oocytes matured in follicular fluid (49% (99/204) versus 35% (60/171), respectively; P < 0.05). Culture with cycloheximide for 24 h resulted in a significantly higher rate of activation (67%, 74/111) than did the 6 h (33%, 44/136) or 6 h plus 18 h (32%, 41/128) treatments. The highest rate of activation (82%) was observed in oocytes matured in TCM-199, treated with 50 micromol calcium ionophore l(-1) and cultured with cycloheximide for 24 h.

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Isolated Graafian follicles cultured intact were used as a test-system for the meiosis-inducing action of hormonal preparations and for analysing the mediation of this hormone effect.

When enlarged follicles were explanted from rats on the day of pro-oestrus before 14.00 hours, i.e. before the preovulatory lh-surge, the oocytes remained in the dictyate state of meiosis throughout an 18-hr culture period. Completion of the first meiotic division could be induced by addition of lh or prostaglandin E2 to the culture medium, or by microinjection of dibutyryl cyclic AMP into the antrum of the cultured follicle. Addition of hcg or fsh to the culture medium was also effective, though possibly due to lh-like activity present in these preparations. Prostaglandin F, at 2·8 × 10−5 m, was only partly effective; prolactin, progesterone, 20α-dihydroprogesterone, oestradiol-17β, linolenic acid and adenosine-5′-monophosphate were completely ineffective. The maturation-inducing action of lh was not blocked by cyanoketone, an inhibitor of steroid synthesis.

Addition of lh to the culture medium stimulated the formation of cyclic AMP by the isolated follicles. Exogenous cyclic AMP enhanced protein kinase activity in the supernatant fraction of follicular homogenates.

It is proposed that the action of lh on oocyte maturation involves the mediation of the adenyl cyclase/cyclic AMP system and possibly of the prostaglandins. An action of steroids could not thus far be implicated. The experimental model described permits study of the mechanism of the meiosis-inducing action of lh under controlled conditions in vitro.

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J. Prépin and N. Hida

Summary. Fetal ovaries of 14·5-day-old rats were cultured for periods of up to 19 days in control medium or in medium conditioned by the preliminary culture of testes from fetal or young rats. In all ovaries, after 12 days of culture in either medium, epithelial cords were noted having an aspect identical to that of seminiferous cords present in fetal testes explanted at 14·5 days and also cultured for 12 days, i.e. the epithelial cords appeared in ovaries when there was no 'male' or testicular influence.

The appearance of histological preparations suggested that the disappearance of the germ cells might bring about a reorganization of the follicular cells in epithelial cords during the differentiation period of the first follicles. With ovaries cultured in conditioned medium, degeneration of the germ cells was more marked, follicles were rare and intra-ovarian cords were greater in number than in ovaries cultured in control medium. The ovaries thus transformed produced the anti-Miillerian hormone (AMH) although they lacked the "germinostatic activity" normally developed by testes of fetal or young rats. This germinostatic activity prevents the multiplication of oogonia when the testes and ovaries are co-cultured in vitro. The transformed ovaries therefore do not have all the functional capacities of fetal testes.

Keywords: ovarian cords; anti-Müllerian hormone; oocyte depletion; germinostatic activity; male influence; rat