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Introduction Ovarian antral follicles require gonadotropin stimulation for development and ovulation whereas preantral follicles seem capable of development in the absence of follicle-stimulating hormone (FSH) and luteinizing hormone (LH
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ABSTRACT
In-vivo changes in steroidogenesis in preantral hamster follicles following exposure to the LH surge could be mimicked by stimulation with exogenous LH in vitro. Luteinizing hormone given only during the first hour (media were changed every hour) of a 6-h incubation promptly increased the concentration of androstenedione and adenosine 3′:5′-monophosphate (cAMP) in the media and this was followed by a gradual decline to <20% of the peak value; progesterone in media was not detectable with a single LH stimulation. However, LH given every hour increased progesterone and cAMP concentrations in the media throughout the period of incubation, but the transient increase and subsequent decline in androstenedione was still observed. The decline in androstenedione release by preantral follicles was apparently due to a lack of steroid precursor and not to either inhibition of hydroxyl-lyase or lack of LH or cAMP stimulation. Exogenous dibutyryl cAMP (dbcAMP) and 8-Br-cAMP mimicked the effects of LH on the pattern of follicular androstenedione release into the media; however, dbcAMP and 8-Br-cAMP did not increase concentrations of progesterone in vitro. In preantral follicles, LH stimulated cAMP release into the media and apparently inhibited phosphodiesterase activity, since methyl isobutylxanthine (MIX) did not potentiate the effect of LH on cAMP. Follicle-stimulating hormone also increased androstenedione and cAMP in the media of the preantral follicles in a manner similar to that of LH, except that between 4 and 6 h of incubation the release of androstenedione and cAMP was less than that produced by stimulation with LH. Interestingly, FSH and MIX stimulated androstenedione and cAMP release by preantral follicles in a manner similar to that induced by LH alone.
These results indicate that LH stimulation of preantral follicles in vitro induces an androstenedione– progesterone shift which is mediated by cAMP. The decline in androstenedione release by the preantral follicle in vitro appears to be due to a lack of appropriate steroid precursors and not to an inhibitory action of LH on androgen synthesis.
J. Endocr. (1987) 114, 55–63
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ABSTRACT
The present study describes the acute changes in steroids and human chorionic gonadotrophin (hCG) and FSH binding of preantral follicles induced by the gonadotrophin surges on the day of pro-oestrus. Preantral follicles were isolated by microdissection before (09.00–10.00 h), during (15.00–16.00 h) and after (21.00–22.00 h) the LH surge on pro-oestrus. Follicles at each time-period were pooled and steroid concentrations and gonadotrophin receptors determined. Before the LH surge, concentrations of progesterone and androstenedione were 40·8 ± 6·1 (s.e.m.) and 10·7 ± 3·9 fmol/follicle respectively. At the peak of the LH surge, progesterone and androstenedione concentrations in preantral follicles increased to 848 ± 186 and 129 ± 33 fmol/follicle respectively. Immediately after the LH surge, progesterone increased to 1238 ±97 fmol/follicle whereas androstenedione declined to 13·3 ±2·1 fmol/follicle. Oestradiol was less than 6 fmol/follicle throughout these periods. Binding of hCG and FSH to preantral follicles increased after the surge (hCG, 56± 2·6 c.p.m./follicle; FSH, 29·8 ± c.p.m./follicle) when compared with values obtained before the surge (hCG, 15·8± 4·0 c.p.m./follicle; FSH, 14·1 ± 1·9 c.p.m./follicle). Also, hCG binding increased significantly (P<0·05) from 09.00 to 21.00 h (56 ±2·6 c.p.m./follicle).
In order to ascertain which follicular compartments were affected by the LH and FSH surges on pro-oestrus, granulosa cells and thecae from preantral follicles were isolated and steroid concentrations and LH and FSH binding measured. Both thecal and granulosal concentrations of androstenedione were significantly (P<0·01) higher at the peak of the LH surge (15.00 h) than at 09.00 h (before the surge) and 21.00 h (after the surge). The granulosal concentration of progesterone was lowest at 09.00 h, highest at 15.00 h and remained increased at 21.00 h. Thecal progesterone concentrations did not increase significantly until 21.00 h at pro-oestrus. Binding of hCG to theca increased significantly (P<0·01) at 15.00 and 21.00 h when compared with values at 09.00 h. Binding of FSH to theca was not detected at 09.00 h and was low at 15.00 and 21.00 h. Binding of hCG and FSH to granulosa cells did not change significantly throughout this period on pro-oestrus.
These results indicate that the gonadotrophin surges on pro-oestrus induce significant stepwise increases in thecal LH binding in preantral follicles and that these coincide with parallel increases in follicular progesterone. Thus the gonadotrophin surges on pro-oestrus may be a signal regulating the onset of steroidogenesis and growth of preantral follicles.
J. Endocr. (1987) 114, 49–54
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ABSTRACT
The neonatal rat ovary is completely devoid of antral follicles until the twelfth day of age. During this period the ovary becomes steroidogenically active and responsive to gonadotrophins. The aim of this study was to correlate the onset of ovarian androgen and oestrogen production in vitro with the first appearance of distinct granulosa and theca cells. Although ovarian aromatase activity increased significantly on day 7 of age, ovarian oestrogen production was limited by low progesterone and testosterone production until day 12 of age. Increased aromatase activity on day 7 and androgen production on day 12 were coincident with the first appearance of granulosa and theca cells respectively. These functional and morphological changes were not associated with significant alterations in ovarian weight or concentrations of LH or FSH in serum.
J. Endocr. (1986) 110, 87–92
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Institute, Cary, NC, USA). Results Immunohistochemistry revealed the presence of FGF17 predominantly in oocytes and granulosa cells of preantral and antral follicles ( Fig. 1 ). Staining was predominant in the nucleus of oocytes in preantral follicles, and
Department of Mathematics, Imperial College London, London SW7 2AZ, UK
Laboratory of Neuroendocrinology, Department of Neurobiology, The Babraham Institute, Babraham, Cambridge CB2 4AT, UK
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Department of Mathematics, Imperial College London, London SW7 2AZ, UK
Laboratory of Neuroendocrinology, Department of Neurobiology, The Babraham Institute, Babraham, Cambridge CB2 4AT, UK
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Department of Mathematics, Imperial College London, London SW7 2AZ, UK
Laboratory of Neuroendocrinology, Department of Neurobiology, The Babraham Institute, Babraham, Cambridge CB2 4AT, UK
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Department of Mathematics, Imperial College London, London SW7 2AZ, UK
Laboratory of Neuroendocrinology, Department of Neurobiology, The Babraham Institute, Babraham, Cambridge CB2 4AT, UK
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Department of Mathematics, Imperial College London, London SW7 2AZ, UK
Laboratory of Neuroendocrinology, Department of Neurobiology, The Babraham Institute, Babraham, Cambridge CB2 4AT, UK
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Department of Mathematics, Imperial College London, London SW7 2AZ, UK
Laboratory of Neuroendocrinology, Department of Neurobiology, The Babraham Institute, Babraham, Cambridge CB2 4AT, UK
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Department of Mathematics, Imperial College London, London SW7 2AZ, UK
Laboratory of Neuroendocrinology, Department of Neurobiology, The Babraham Institute, Babraham, Cambridge CB2 4AT, UK
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Department of Mathematics, Imperial College London, London SW7 2AZ, UK
Laboratory of Neuroendocrinology, Department of Neurobiology, The Babraham Institute, Babraham, Cambridge CB2 4AT, UK
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reflects the arrest of follicle development in the late antral stages but recent work from our group ( Webber et al. 2003 ), and that of Erickson and colleagues ( Maciel et al. 2004 ), has highlighted a disorder of early, preantral follicle development
Human and Animal Physiology Group, Department of Animal Sciences, Wageningen University, Haarweg 10, 6709 PJ Wageningen, The Netherlands
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Human and Animal Physiology Group, Department of Animal Sciences, Wageningen University, Haarweg 10, 6709 PJ Wageningen, The Netherlands
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Human and Animal Physiology Group, Department of Animal Sciences, Wageningen University, Haarweg 10, 6709 PJ Wageningen, The Netherlands
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Human and Animal Physiology Group, Department of Animal Sciences, Wageningen University, Haarweg 10, 6709 PJ Wageningen, The Netherlands
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Human and Animal Physiology Group, Department of Animal Sciences, Wageningen University, Haarweg 10, 6709 PJ Wageningen, The Netherlands
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(++). Preantral follicles Healthy granulosa
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Abstract
Gonadotrophin-releasing hormone (GnRH) and its agonists are implicated in the local control of rat ovarian function. We have evaluated the effects of long-term administration of different doses of GnRH agonist (GnRH-Ag) in vivo (a) on reproductive cyclicity and follicular development, (b) on peripheral gonadotrophin and steroid concentrations and (c) on in vitro cAMP and progesterone production by the follicles in response to stimulatory doses of FSH or LH (1 μg/ml). GnRH-Ag (0·2, 1 or 5 μg/day) administration for 28 days had a profound impact on the oestrous cycle of rats as revealed by vaginal cytology. GnRH-Ag treatment caused a decrease in ovarian and uterine weights, which correlated very well with the decrease in the number of follicles present in the ovary. GnRH-Ag (5 μg/day) reduced the number of early preantral follicles and there was complete disappearance of early as well as late antral follicles. However, a dose of 1 μg GnRH-Ag/day was effective in the complete demise of only late antral follicles with a significant attenuation in the number of early antral follicles. There was an enhancement in serum LH concentrations in response to the highest dose of GnRH-Ag administration with serum FSH concentrations declining in rats treated with the two higher doses. However, serum prolactin concentrations were attenuated only in rats treated with the highest dose of GnRH-Ag. GnRH-Ag treatment decreased serum progesterone and oestradiol concentrations. Preantral follicles obtained from the rats treated with 0·2 or 1 μg GnRH-Ag/day resulted in an attenuated response to LH-or FSH-stimulated progesterone production, whereas antral follicles showed an exaggerated response to the stimulatory doses of FSH in vitro. Antral follicles obtained from the rats treated with 0·2 μg/day showed a robust decrease in cAMP accumulation in response to LH with a slight decrease only with FSH. In contrast, preantral follicles obtained from GnRH-Ag-treated rats did not show any significant attenuation in cAMP production. These data suggest that GnRH-Ag exerts a direct inhibitory effect on follicular development and steroidogenesis and as a result it interferes with the normal oestrous cyclicity in the rat.
Journal of Endocrinology (1995) 146, 349–357
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The contribution of specific follicle populations to dimeric inhibin production and inhibin subunit mRNA expression by the rat ovary has been investigated in two model systems, granulosa cells isolated from 25-day-old diethylstilboestrol (DES)-treated rats and post-natal rat ovaries, dispersed in culture or whole ovaries, using specific two-site immunoassays and 'real time' PCR. Media from FSH-stimulated granulosa cell cultures fractionated by gel filtration and RP-high performance liquid chromatography revealed two predominant peaks of alpha subunit activity which were attributed to alpha subunit and 31 k dimeric inhibin-A. The corresponding inhibin-B levels were low. FSH stimulation did not alter the ratio of inhibin-A:alpha subunit produced by granulosa cells. All three inhibin subunit mRNAs were expressed by granulosa cells, with eight-fold more alpha subunit mRNA relative to either of the beta subunits. Administration of DES to immature rats prior to the isolation of granulosa cells from the ovary led to beta(A) and beta(B) mRNA expression being down-regulated in the absence of any significant change in alpha subunit expression by the granulosa cells. Inhibin-A, -B and -alpha subunit were produced by basal and stimulated cultures of ovarian cells prepared from 4-, 8- and 12-day-old rats, indicating that primary, preantral and antral follicles contribute to total inhibin production. Consistent with these results, follicles within these ovaries expressed all three inhibin subunit mRNAs, with maximal expression observed in the ovaries of 8-day-old rats. The appearance of antral follicles in the ovary at day 12 led to a decline in the mRNA levels of each of the subunits but was most evident for the beta subunits. There was a profound influence of secondary preantral follicles on dimeric inhibin-A production, with FSH stimulation increasing inhibin-A relative to alpha subunit levels in cultures of ovarian cells prepared from 8-day-old rats. Thus, preantral follicles exposed to FSH contribute significantly to beta(A) subunit production by the ovary. In contrast, primary and preantral follicles did not produce inhibin-B in response to FSH stimulation. Transforming growth factor-beta (TGF-beta) enhanced, in a time-dependent manner, the production of the inhibin forms by ovarian cells in culture, although inhibin-B production was not responsive until day 8. The simultaneous treatment of ovarian cell cultures with FSH and TGF-beta elicited the greatest increases in production of all the inhibin forms. In summary, ovaries of 4-, 8- and 12-day-old rats expressed inhibin subunit mRNAs and produced dimeric inhibin-A and -B and free alpha subunit. Preantral follicles (day-8 ovarian cell cultures) were particularly sensitive to stimulation by FSH and TGF-beta and had a substantial capacity for inhibin production. The production of oestrogen by follicles may be instrumental in regulating inhibin production given that beta subunit mRNA expression was down-regulated by DES. The mechanisms by which inhibin-A and inhibin-B are individually regulated are likely to be similar during the post-natal period, when folliculogenesis is being established, and diverge thereafter, when inhibin-A becomes the predominant form in the fully differentiated ovary.
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IGFs regulate gonadotrophin-stimulated proliferation and differentiation of granulosa and theca cells in vitro. However, the detailed pattern of mRNA expression of IGFs in bovine follicles remains controversial. The objectives of this study were therefore to describe the temporal and spatial pattern of expression of mRNA encoding IGF-I, IGF-II and the type 1 IGF receptor in bovine follicles in vivo. The expression of mRNA encoding IGF-II was detected in theca tissue from around the time of antrum formation up to and during the development of dominance. No IGF-II mRNA expression was detected in granulosa cells. In the majority of follicles we were unable to detect mRNA encoding IGF-I in either granulosa or theca tissue from follicles at any stage of development. Occasionally low amounts of mRNA encoding IGF-I were detected in the theca externa and connective tissue surrounding some follicles. Type 1 IGF receptor mRNA was detected in both granulosa and theca cells of preantral and antral follicles. Expression was greater in granulosa tissue compared with theca tissue. We also measured IGF-I and -II mRNA in total RNA isolated from cultured granulosa and theca cells using reverse transcriptase PCR. In contrast to the in vivo results, IGF-II mRNA was detected in both granulosa and theca tissue. IGF-I mRNA was detected in theca tissue and in very low amounts in granulosa cells. Using a specific IGF-I RIA we were unable to detect IGF-I immunoreactivity in granulosa conditioned cell culture media. Using immunohistochemistry we detected IGF-I immunoreactivity in some blood vessels within the ovarian stroma. We conclude from these results that IGF-II is the principal intrafollicular IGF ligand regulating the growth of bovine antral follicles. In preantral follicles the expression of mRNA encoding type 1 IGF receptor but absence of endogenous IGF-I or -II mRNA expression, highlights a probable endocrine mechanism for the IGF regulation of preantral follicle growth.
