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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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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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final magnification of X400 for primordial and primary follicles, and X100 for preantral, antral, and Graafian follicles and corpus luteum. Photographs of ovarian follicles were analyzed using Image Pro Plus for Windows (version 1.3.2; Media Cybernetics
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.e.m. , (C) Mann–Whitney test and (D) Welch's t- test, * P < 0.05. LIF decreased the initial recruitment and the growth preantral follicles in rat To determine whether or not LIF participates in the the initial recruitment of follicular
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Binding sites for IGF-I, insulin, and GH were localized by in situ binding of (125)I-labelled hormones to the different compartments of the sow ovary. Binding sites for IGF-I were detected in oocytes, granulosa and thecal cells of healthy and atretic follicles as well as in the antrum and the stroma. Competition of (125)I-labelled IGF-I with IGF-I, insulin and an analogue of IGF-I (Long R(3)IGF-I), which allowed discrimination between binding to binding proteins from binding to type-I receptors, suggested that type-I receptors were present in granulosa cells of healthy follicles, whilst binding in other compartments was mainly due to binding proteins. Binding of insulin was revealed in oocytes, granulosa and theca interna cells of healthy preantral and antral follicles, and, to a lesser extent, in theca externa and stromal cells, and was still observed in granulosa cells of atretic follicles. Labelling with (125)I-labelled bovine GH was demonstrated in oocytes, granulosa cells, theca interna cells, and, although less intense, in theca externa and stromal cells. It disappeared in granulosa cells during atresia. Binding sites for GH were detected at all follicular stages, from preantral to preovulatory stages, but the intensity of labelling in granulosa cells was more intense in preantral than in large follicles. These data support the participation of insulin, GH and IGF-I in oocyte maturation, follicular growth and stromal cell function in swine.
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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.
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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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SUMMARY
Follicular development and serum gonadotrophin levels were studied in female rats after neonatal androgen administration.
After injection of 1250 μg testosterone propionate (TP) on day 5 after birth the composition of the follicular population was altered: at nearly all ages the number of pre-antral follicles (follicular volume 2–20 × 105 μm3) was lower than in oil-treated rats, in some cases the number of small antral follicles (21–249 × 105 μm3) was also lower. Furthermore levels of serum follicle-stimulating hormone and luteinizing hormone were decreased from day 7 to day 20 suggesting that the high gonadotrophin levels before day 20 are of importance for normal follicular development.
In contrast, final follicular maturation in TP-treated rats was enhanced; at day 35 more large antral follicles (follicular volume ≥ 500 × 105μm3) were present in TP-treated rats than in oil-treated rats. The presence of more large antral follicles was accompanied by higher plasma oestradiol concentrations, higher uterine weights and advanced vaginal opening.
These results demonstrate an inhibition of normal follicular growth and an acceleration of ovarian maturation after neonatal androgen administration.
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To investigate the influence of ovariectomy on the physiological process of neovascularization of ovarian follicles, we harvested follicles with a theca interna at 48 h after pregnant mare's serum gonadotropin (PMSG) treatment of Syrian golden hamsters, for subsequent transplantation onto striated muscle tissue of chronically implanted skinfold chambers of non-ovariectomized PMSG-synchronized hamsters and bilaterally ovariectomized hamsters. Non-ovariectomized non-PMSG-treated animals served as controls. During a 2 week period after transplantation, neovascularization of these freely transplanted grafts was quantified in vivo by assessment of the newly developed microvascular follicular network, its microvessel density, the diameter of microvessels and their volumetric blood flow using fluorescence microscopic techniques. At day 3 after transplantation, capillary sprouts could be observed in all groups studied, finally developing a complete glomerulum-like microvascular network within 5-10 days. In ovariectomized animals, however, vascularization of follicular tissue was found to be accelerated and enhanced when compared with the follicles in both groups of non-ovariectomized animals. This was associated with significantly higher capillary blood perfusion, which may in part represent the graft's adaptive response to pro-angiogenic stimuli due to elevated gonadotropin levels, but might in particular be mediated by gonadotropin-induced release of vasoactive substances. Interestingly, small preantral follicles lacking a theca interna failed to vascularize in either of the groups. In conclusion, the study demonstrates that ovariectomy improves neovascularization and microcirculation of freely transplanted ovarian follicles.
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Abstract
Inhibin is a putative gonadal paracrine factor produced by FSH-stimulated granulosa cells. To assess the paracrine function of inhibin further, preantral follicles (approx. 240 μm) with attached thecal cells were isolated mechanically from immature rat ovaries and cultured individually in vitro for 5 days in medium containing homologous serum and FSH. After 5 days, follicles had grown to preovulatory size (approx. 470 μm) with a commensurate increase in oestradiol secretion but not progesterone. Immunoneutralization of endogenous inhibin resulted in a significant decrease in oestradiol secretion and an increase in progesterone accumulation. When antiserum-treated follicles were supplemented with exogenous inhibin, oestradiol secretion was restored and progesterone accumulation was reduced. Aromatase substrate (androstenedione) levels were too low to measure, regardless of antiserum treatment. However, follicles treated with inhibin antiserum in the presence of exogenous androstenedione also exhibited oestradiol levels similar to untreated controls while progesterone accumulation remained elevated. We interpret these data as evidence that inhibin secreted by FSH-stimulated granulosa cells exerts a physiologically significant paracrine function in the follicle wall. Based on previous observations that inhibin stimulates androgen synthesis by isolated thecal/interstitial cells, it is proposed that granulosa-derived inhibin promotes thecal androgen synthesis and hence oestrogen synthesis during preovulatory follicle development. The antiserum-induced increase in progesterone accumulation is most probably explained by reduced metabolism of C21 steroid substrate to androgen in thecal/interstitial cells deprived of inhibin. It is concluded that inhibin is a physiological modulator of follicular steroidogenesis which exerts its effect at the level of thecal cell androgen synthesis.
Journal of Endocrinology (1994) 140, 437–443
