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PubMed1995

[Human folliculogenesis and local regulation].

It is well known that gonadotropin controls a major part of follicular development. However, the mechanism of local regulation under the control of gonadotropins in still unclear. In this study, we focused on the local regulation of steroidogenesis, growth factors and cell proliferation to evaluate the human follicular development. To assess steroidogenesis, it is important to detect the expression of steroidogenic enzymes in the granulosa and theca cells during folliculogenesis. We initially tried to find out the transcription factor Ad4BP that binds the Ad4 site and regulates the function of steroidogenic enzyme. By immunohistochemistry, the expression of Ad4BP was confirmed sporaf1p4lly in preantral granulosa cells. In the antral follicles, the expression of Ad4BP was observed both in the granulosa and theca cell. According steroidogenic enzyme, we evaluated temporal and spatial localization of cholesterol side chain cleavage (scc), 3 beta hydroxysteroid dehydrogenase (3 beta HSD), 17 alpha hydroxylase (17 alpha) and aromatase, and steroid receptors. In briefly, the localizations of scc, 3 beta HSD and 17 alpha were observed in preantral follicles and the mRNA expressions of these enzymes were confirmed in the theca cell by in situ hybridization method. Expression of aromatase was generally observed in only one follicle (antral or mature follicle) per case in mid proliferative to premenstrual phase. The localization of androgen and estrogen receptor was observed in the antral follicle granulosa cells, and estrogen receptor was detected only in aromatase positive follicles. These results suggested that Ad4BP initially controls the function of steroidogenic enzymes and steroidogenic enzymes gradually express from primary follicles to mature follicles. At antral follicle stage, steroid metabolism completes to produce testosterone. When aromatase and estrogen receptor express in antral follicle, this antral follicle develops as the dominant follicle and produces estradiol to promote follicle maturation. We therefore speculate that the expression of aromatase and estrogen receptor have an important role for the selection of dominant follicle in human. According growth factors for follicular development, it has been demonstrated to be important in the biological activity in the ovary. In this study, we examined the localization of EGF, TGF alpha and their receptor (EGFR). The localization of EGF was not confirmed both mRNA and protein level through follicular development. On the other hand, the localization and expression of TGF alpha was confirmed in theca cells and EGFR in granulosa cells at antral stage.(ABSTRACT TRUNCATED AT 400 WORDS)

3-Hydroxysteroid Dehydrogenases↗

PubMed1993

Insulin-like growth factor-I stimulates the expression of 3 beta-hydroxysteroid dehydrogenase messenger ribonucleic acid in ovarian theca-interstitial cells.

Evidence accumulating in the literature supports the hypothesis that insulin-like growth factor-I (IGF-I) may play a role in stimulating differentiation of the ovarian theca-interstitial cells (TIC) during early follicular development. IGF-I has been shown to synergistically enhance the stimulation of androgen biosynthesis and to increase LH binding in TIC. The purpose of the present studies was to examine the role of IGF-I in TIC differentiation by determining the effects of IGF-I on 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) mRNA expression in TIC stimulated to differentiate in vitro. TIC were isolated from the ovaries of hypophysectomized immature rats by Percoll gradient centrifugation and cultured in the presence and absence of LH and IGF-I for up to 6 days. At various times cytoplasmic RNA was extracted from the TIC, and 3 beta-HSD mRNA was measured by specific assay using reverse transcription followed by the polymerase chain reaction. Amplification of 3 beta-HSD mRNA using primers designed to distinguish between the type I and type II 3 beta-HSD gene products revealed that the TIC expressed primarily the type I gene. Increasing concentrations of LH (0-1 microgram/ml) stimulated a dose-related increase in 3 beta-HSD mRNA that was approximately 3-fold at 100 ng/ml of LH. Addition of IGF-I (30 ng/ml) increased 3 beta-HSD mRNA approximately 2-fold over TIC treated with LH alone.(ABSTRACT TRUNCATED AT 250 WORDS)

3-Hydroxysteroid Dehydrogenases↗

PubMed1997

Expression of vascular endothelial growth factor (VEGF) during folliculogenesis and corpus luteum formation in the human ovary.

Vascular endothelial growth factor (VEGF) has been suggested to be involved in angiogenesis and microvascular hyperpermeability. We examined immunohistochemically the expression of VEGF in the granulosa and theca cells, along with that of proliferating cell nuclear antigen (PCNA), in the vascular endothelium during the course of follicular development and corpora lutea formation in human ovaries. The immunolocalization of VEGF in these cells was compared with that of another putative angiogenic factor, basic fibroblast growth factor (bFGF). The granulosa cells in the primordial and primary follicles were VEGF negative, but at the preantral stage, the granulosa cells showed weakly positive immunostaining for VEGF. However, the VEGF immunostaining in the granulosa cells was weak throughout the folliculogenesis. In contrast, the theca interna cells of developing follicles showed strong staining for VEGF, which was well correlated with the PCNA positivity in the vascular endothelial cells in the thecal layer. In the atretic follicles, the granulosa and theca cells were VEGF negative. In the corpora lutea, VEGF was strongly expressed in both granulosa and theca lutein cells in the early luteal phase when the PCNA positivity in the endothelium increased, but the VEGF staining in these cells became weak in the mid- and late luteal phases. Accordingly, the PCNA positivity in the vascular endothelium was well correlated with the expression of VEGF in the theca cells during follicular development and atresia, and that in the granulosa and theca lutein cells in corpora lutea formation and regression. In addition, the immunolocalization of VEGF was different from that of bFGF.

Biopsy↗

PubMed2002

Estrogen receptor alpha and beta expression in theca and granulosa cells from women with polycystic ovary syndrome.

A defining characteristic of dominant follicles is high estradiol concentrations. Abnormal expression of estrogen receptors (ERs) could contribute to poor follicular development and ovulatory failure in polycystic ovary syndrome (PCOS). The aim of this study was to determine whether there are differences in ERalpha and ERbeta expression in granulosa cells (GC) and theca cells (TC) from women with PCOS, compared with regularly cycling women. GC and TC were obtained by microdissection from 12 polycystic and 23 normal ovaries. ERalpha and ERbeta mRNA and protein expression were measured by semiquantitative RT-PCR and Western blot, respectively. In control ovaries, both GC and TC ERalpha mRNAs were higher in small antral (SA) than in dominant follicles. ERalpha mRNA was similar in PCOS and size-matched control follicles. In control follicles, ERalpha protein concentrations were higher in GC than in TC. In GC, the ERalpha concentrations were comparable among SA, dominant, and PCOS follicles. In TC, ERalpha concentrations were lower in dominant follicles but were markedly increased in PCOS. In control ovaries, GC and TC expression of ERbeta mRNA was higher in SA, compared with dominant follicles. In PCOS, ERbeta mRNA was intermediate between SA and dominant follicles in both GC and TC. In GC, the ERbeta protein concentrations followed the same pattern as mRNA expression; but in TC ERbeta, protein in PCOS was equivalent to that in dominant follicles. The results of this study demonstrate that there are significant alterations in the expression of ERalpha and ERbeta in PCOS that may be related to abnormal follicular development.

Adult↗

PubMed1986

Ovarian steroidogenesis during follicular maturation in the domestic fowl (Gallus domesticus).

The steroidogenic potential of various physiological compartments within the ovary of the hen were examined using in vitro systems. Three-hour incubations of individual whole small follicles (less than 1 mm-1 cm) or 100,000 collagenase-dispersed theca cells of the five largest ovarian follicles (F1-F5) were conducted in 1 ml of Medium 199 at 37 degrees C in the presence and absence of luteinizing hormone (LH) (0.39, 0.78, 1.56, 3.13 and 6.25 ng), progesterone (5 ng), and dehydroepiandrosterone (DHEA, 5 ng). Steroid output was measured by radioimmunoassay of incubation media. Progesterone was not produced by small follicles although they are a major source of DHEA and estradiol and a significant source of androstenedione. Output of DHEA, androstenedione and estradiol was highly stimulated by LH. The substrate for androstenedione and estradiol in small follicles is probably DHEA. Output of DHEA and androstenedione in theca cells of F2-F5 was stimulated by LH in a dose-related manner. A dose-response relationship between estradiol output and the concentration of LH in media was not apparent in theca cells from F2-F5. Steroidogenesis in theca tissue of large follicles occurs predominantly via the delta 4 pathway. The ability of these theca cells to metabolize progesterone to androstenedione is lost between 36 and 12 h before ovulation. Their ability to metabolize DHEA to androstenedione is still present 12 h before ovulation. Aromatase activity is significantly reduced between 36 and 12 h before ovulation. These data indicate that both large and small follicles can be stimulated by LH. The small follicles are the major source of estrogen. As the large yolky follicles mature, steroidogenesis shifts from the delta 5 to the delta 4 pathway. By 12 h before ovulation, the F1 follicle has lost the ability to convert progesterone to androstenedione. The inability of the largest ovarian follicle to convert progesterone to androstenedione contributes at least in part to the preovulatory increase in the plasma concentration of progesterone that generates the preovulatory LH surge by positive feedback.

Androstenedione↗

PubMed1979

Cyclic changes in the ovary of the rainbow trout, Salmo gairdneri, with special reference to sites of steroidogenesis.

In the maturation cycle of ovaries of cultured rainbow trout, three periods can be distinguished: (1) a period of ovulation and previtellogenesis (January-May), (2) a period of exogenous vitellogenesis (May-November/December), and (3) a period of maturation of oocytes (November/December-January). Enzyme cytochemical and electron microscopical data indicate that stroma cells (i.e., interstitial cells and special theca cells) and granulosa cells represent sources of steroids. Steroidogenesis in stroma cells is found throughout the annual cycle, reaching a peak activity in January and February. Weak steroidogenic activity is observed in the granulosa cells of exogenous vitellogenic follicles and young postovulatory follicles. Possible functions of steroids secreted by stroma cells and granulosa cells are discussed.

3-Hydroxysteroid Dehydrogenases↗

PubMed2005

Gonadotropin regulation of glutamate cysteine ligase catalytic and modifier subunit expression in rat ovary is subunit and follicle stage specific.

We have observed that levels of the antioxidant glutathione (GSH) and protein levels of the catalytic and modifier subunits of the rate-limiting enzyme in GSH synthesis, GCLc and GCLm, increase in immature rat ovaries after treatment with gonadotropin. The goals of the present studies were to delineate the time course and intraovarian localization of changes in GSH and GCL after pregnant mare's serum gonadotropin (PMSG) and after an ovulatory gonadotropin stimulus. Twenty-four hours after PMSG, there was a shift from predominantly granulosa cell expression of gclm mRNA, and to a lesser extent gclc, to predominantly theca cell expression. GCLc immunostaining increased in granulosa and theca cells and in interstitial cells. Next, prepubertal female rats were primed with PMSG, followed 48 h later by 10 IU of hCG. GCLm protein and mRNA levels increased dramatically from 0 to 4 h after hCG and then declined rapidly. There was minimal change in GCLc. The increase in gclm mRNA expression was localized mainly to granulosa and theca cells of preovulatory follicles. To verify that GCL responds similarly to an endogenous preovulatory gonadotropin surge, we quantified ovarian GCL mRNA levels during the periovulatory period in adult rats. gclm mRNA levels increased after the gonadotropin surge on proestrus and then declined rapidly. Finally, we assessed the effects of gonadotropin on ovarian GCL enzymatic activity. GCL enzymatic activity increased significantly at 48 h after PMSG injection and did not increase further after hCG. These results demonstrate that gonadotropins regulate follicular GCL expression in a follicle stage-dependent manner and in a GCL subunit-dependent manner.

Animals↗

PubMed2005

Aromatic hydrocarbon receptor (AhR) in the porcine theca and granulosa cells: effect of TCDD, PCB 126 and PCB 153 on the expression of AhR.

OBJECTIVE: In our previous papers, we demonstrated that TCDD and PCBs (both coplanar PCB 126 and non-coplanar PCB 153) caused the decreased estradiol secretion in cultured pig ovarian follicles. However, the mechanism of action is not clearly understood. Moreover, to our knowledge, the expression of AhR in pig follicular cells was not described yet. METHODS: In order to elucidate if TCDD, PCB 126, and PCB 153 can disrupt ovarian steroidogenesis via AhR-dependent mechanism, granulosa and theca cells were isolated from pig ovarian small follicles and subsequently treated with 32 pg/ml of TCDD, 100 ng/ml of PCB 153, 100 pg/ml of PCB 126 in an in vitro culture. After 3-hour exposure to the chemicals, cells were prepared for immunohistochemical analysis or collected for immunobloting, and the effects of TCDD and PCBs on the levels of AhR protein were studied. RESULTS: Under basal conditions, AhR staining showed a diffuse cytoplasmic pattern of distribution as well in granulosa and theca cells. However, immunocytochemical labeling of AhR in theca cells was less evident and only few cells displayed cytoplasmic staining. Immunoblotts prepared from granulosa and theca cell lysates detected a strong band of 120 kDa indicating AhR protein expression. The level of AhR expression detected by Western blot analysis was higher in granulosa cells than in theca cells. Intensive cytoplasmic and nuclear labeling corresponding to AhR protein in TCDD and PCB 126 treated granulosa cells was observed. In PCB 153 treated cells, the level of AhR staining was comparable with control. Western blot analysis showed a strong band in TCDD-treated grnulosa cells and only slightly differences compared with control in those exposed to PCB 126. The effect of both PCBs on immunocytochemical AhR staining in theca cells was less evident. Immunoblott analysis did not reveal any substantial differences between the control and TCDD- and PCBs-treated theca cells. CONCLUSIONS: This study has shown different AhR expression in porcine theca and granulosa cells, in respect of both its intracellular localization and cellular distribution. Both, dioxin and dioxin-like PCB activated AhRs in granulosa but not in theca cells. Differences in AhR-responsiveness of granulosa and theca cells to TCDD and dioxin-like PCB 126 are probably connected with the differences of these cells in estradiol secretion and different proliferative potential.

Animals↗

PubMed1990

Insulin-like growth factor-I selectively stimulates cholesterol side-chain cleavage expression in ovarian theca-interstitial cells.

Evidence accumulating in the literature supports the concept that insulin-like growth factor I (IGF-I) may be an important local regulator of ovarian function. Recent studies have demonstrated that IGF-I synergistically augments LH stimulation of theca-interstitial cell (TIC) androgen biosynthesis. The purpose of the present studies was to begin to elucidate the molecular mechanisms of the interaction between IGF-I and LH. TIC were purified from ovaries of hypophysectomized immature rats by Percoll gradient centrifugation. When isolated TIC (5 x 10(6) viable cells per dish) were cultured (4 days) in serum-free medium, low amounts (less than 10 ng/ml) of androsterone were produced. Basal androsterone production was not changed by incubation with IGF-I (30 ng/ml). Treatment with LH (50 ng/ml) caused an 85-fold stimulation of androsterone synthesis that was further increased 2.1-fold by concomitant treatment with IGF-I. Immunoblot analysis demonstrated that untreated TIC contained low levels of 17 alpha-hydroxylase/C17-20 lyase enzyme (P450(17 alpha] that were unchanged by incubation with IGF-I alone. LH treatment increased P450(17 alpha) content 5.5-fold and coincubation with LH plus IGF-I increased P450(17 alpha) content 16-fold above control levels. Cholesterol side chain cleavage enzyme (P450scc) was readily detected in immunoblots from untreated TIC. P450scc content was increased 2.6-fold by LH treatment and 4.2-fold by LH plus IGF-I. Interestingly, IGF-I alone induced a 2-fold increase in P450scc. To determine if the increases in P450scc content were associated with increased enzyme activity, progesterone production was measured.(ABSTRACT TRUNCATED AT 250 WORDS)

Androgens↗

PubMed2003

Stromal cells of the human postmenopausal ovary display a distinctive biochemical and molecular phenotype.

The stroma of the human postmenopausal ovary is postulated to produce androgens, but evidence for and against this idea exits in the literature. The purpose of this study was to determine whether key steroidogenic enzymes involved in androgen synthesis are expressed in the postmenopausal ovarian stroma. Stromal cells were isolated from postmenopausal ovaries and expression for genes involved in steroidogenesis [steroidogenic acute regulatory protein (StAR), P450scc, 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) P450c17, and P450c27] as well as for several growth factor binding proteins [gremlin, IGF binding protein-4, follistatin, and secreted frizzled-related protein (sFRP)-1 and -4], were compared with cultured human theca cells and dermal fibroblasts. Production of steroids (pregnenolone, progesterone, and hydroxysterol metabolites) and the metabolism of [3H] pregnenolone by ovarian stromal cells were also assessed. Isolated ovarian stromal cells from different subjects had a uniform morphology within and across cultures. Quantitative real time RT-PCR revealed that StAR, P450scc, and 3 beta-HSD transcripts were, respectively 30, 25, and 45 times more abundant in theca cells than in stromal cells. Mean levels of P450scc and 3 beta-HSD transcripts in stromal cells were similar to those found in dermal fibroblasts, whereas StAR transcripts in stromal cells were 285-fold more abundant than in fibroblasts. There was no significant expression of P450c17 in ovarian stromal cells or fibroblasts ( approximately 2000-fold less than in theca cells). Western analysis demonstrated the presence of the 30-kDa StAR mature protein in the cultured stromal cells, whereas P450c17 protein was not detectable. Ovarian stromal cells did not metabolize [3H] pregnenolone in the presence or absence of 8-Br-cAMP. Furthermore, pregnenolone and progesterone secretion by stromal cells was also undetectable, even in the presence of 22-hydroxycholesterol. P450c27 protein was detected in ovarian stromal cells and its metabolic products (i.e. 27-hydroxycholesterol and cholestenoic acid) were found in the culture media, reflecting functional cholesterol 27-hydroxylase activity. Follistatin, gremlin, IGF binding protein-4, and sFRP-1 and -4 transcripts were detected in the stromal cells in relative amounts significantly higher than theca cells, but not significantly different from fibroblasts, except for sFRP-1, which was significantly higher in stromal cells. Our observations demonstrate that stromal cells of the postmenopausal ovary have a signature biochemical and molecular phenotype that can be distinguished from fibroblasts. These cells do not appear to have significant steroidogenic potential in vitro, but they do metabolize cholesterol into hydroxysterols. We conclude that the predominant stromal cells of the postmenopausal ovary are not a significant site of androgen biosynthesis.

Carrier Proteins↗

PubMed1998

Recombinant luteinizing hormone as a survival and differentiation factor increases oocyte maturation in recombinant follicle stimulating hormone-supplemented mouse preantral follicle culture.

The effects of recombinant luteinizing hormone (rLH) and recombinant follicle stimulating hormone (rFSH) both separately and together were analysed on the in-vitro maturation of a well-defined class of mouse early preantral follicles. Metaphase-II (MII) oocytes were only obtained when rLH or rFSH or a combination of both hormones was added to a standard rich culture medium at constant doses throughout the culture period. Without gonadotrophins only 8% of the follicles reached day 12 of culture. Follicle survival was optimal (between 90 and 100%) when rFSH was used; significantly fewer follicles were kept intact when rLH was added as the only supplement (54%; P < 0.001). Theca cells played a fundamental role in follicle survival when rLH was the only supplement. Presence of rLH significantly influenced the formation of antral-like cavities: on day 12, 59% of follicles cultured with rLH + rFSH had a large cavity, as compared with only 21% in cultures with rFSH only (P < 0.0001). The rHCG stimulated germinal vesicle breakdown rate from the different gonadotrophin-supplemented cultures on day 12 was not significantly different (FSH: 89%; LH: 70%; FSH/LH: 83%). However, addition of rLH to rFSH did significantly improve (P < 0.05) the completion of the first meiotic division up to the MII stage (FSH: 46%; LH: 39%; FSH/LH: 76%). Interfollicle differences in steroid production were very large and depended on the presence of theca cells and on the kind of gonadotrophin supplemented. Without theca cells, oestrogen concentrations in rFSH-supplemented cultures were 100 times lower than their theca-containing counterparts during the entire culture period. From culture day 9 onwards most conditioned media had increased basal progesterone concentrations. This study points up the in-vitro role of LH on the differentiation of follicle-like structures and the oocyte's meiotic maturation and suggests an important role for theca cells in providing modulators of in-vitro gonadotrophin action.

Animals↗

PubMed1993

Differentiation of ovarian theca-interstitial cells in vitro: regulation of 17 alpha-hydroxylase messenger ribonucleic acid expression by luteinizing hormone and insulin-like growth factor-I.

Insulin-like growth factor-I (IGF-I) has been shown to synergistically augment LH-stimulated androgen biosynthesis in ovarian theca-interstitial cells (TIC). Additional evidence suggests that IGF-I may play a role in stimulating TIC differentiation during early follicle development. The purpose of the present studies was to examine the role of IGF-I in TIC differentiation by determining the effects of IGF-I on P450(17 alpha) mRNA expression in TIC differentiating in vitro. TIC isolated from the ovaries of hypophysectomized immature rats by Percoll gradient centrifugation were cultured with and without LH and IGF-I for up to 6 days. At various times, cytoplasmic RNA was extracted from the TIC, and P450(17 alpha) mRNA was measured by a specific assay, using reverse transcription and the polymerase chain reaction. LH stimulated a dose-related increase in P450(17 alpha) mRNA, with an ED50 comparable to that for androsterone biosynthesis (33.0 +/- 3.8 ng/ml), but significantly less than the ED50 for cAMP accumulation (385 +/- 0.1 ng/ml). IGF-I alone did not stimulate P450(17 alpha) mRNA, but in the presence of LH (100 ng/ml) stimulated a dose-related (ED50, 4.1 +/- 1.6 ng/ml) increase (3-fold) in P450(17 alpha) mRNA. IGF-I did not alter the ED50 for LH stimulation of P450(17 alpha) mRNA (36.85 +/- 1.1 ng/ml). Detailed time-course studies revealed that IGF-I did not alter the 20-h lag phase before LH caused an increase in TIC androsterone biosynthesis; however, IGF-I stimulated a more rapid increase in androsterone than LH alone. In the presence of LH alone, P450(17 alpha) mRNA levels remained low for approximately 18 h, then increased rapidly to maximum levels by 30 h where they were maintained through 48 h. Concomitant treatment with LH plus IGF-I did not alter the 18-h lag phase, but P450(17 alpha) mRNA levels increased to approximately 2-fold higher levels than with LH alone. The results of our studies demonstrate that IGF-I increases the expression of P450(17 alpha) mRNA in TIC and support the hypothesis that IGF-I may play a role in stimulating thecal differentiation in developing follicles.

Actins↗

PubMed2006

Expression of gap junctional connexins 26, 32, and 43 mRNA in ovarian preovulatory follicles and corpora lutea in sheep.

The objective of the current study was to evaluate the expression of connexins (Cx)26, Cx32, and Cx43 mRNA in granulosa and theca cells during the peri-ovulatory period (experiment 1) and in the corpus luteum (CL) during the estrous cycle (experiment 2) and during prostaglandin F2alpha (PGF)-induced luteal regression (experiment 3) in FSH-treated ewes. In experiment 1, Cx26, Cx32, and Cx43 mRNA was expressed in granulosa and theca cells, and expression of Cx32 and Cx43 mRNA, but not Cx26, was greater (p<0.001) in granulosa than in theca cells throughout the peri-ovulatory period. Expression of Cx43 mRNA in granulosa and theca cells decreased (p<0.01) 24 h after hCG treatment. In experiment 2, expression of Cx26 mRNA in the CL tended to be greater (p<0.06) on day 10 than on days 5 or 15, but expression of Cx43 mRNA was greater (p<0.01) on day 5 than on days 10 and 15 of the estrous cycle. In experiment 3, expression of Cx26, but not Cx32 or Cx43 mRNA decreased (p<0.001) during PGF-induced luteal regression. In all 3 experiments, expression of Cx32 mRNA was much less than Cx26 and Cx43 mRNA. Moreover, Cx32 mRNA expression was unchanged during the peri-ovulatory period or during several stages of luteal development and PGF-induced regression of the CL. Thus, we have shown that the mRNA expression pattern of Cx26 and Cx43 changes during peri-ovulatory period and during several stages of the luteal development. This suggests that Cx26 and Cx43 play a role in ovarian tissue remodeling during the critical time around ovulation and throughout luteal tissue growth, differentiation, and regression in sheep.

Animals↗

PubMed2006

Follicle-stimulating hormone (FSH) stimulates the expression of Pin1, a peptidyl-prolyl isomerase, in the bovine granulosa cells.

A peptidyl-prolyl isomerase, Pin 1, has been shown to play a role in the regulation of cell cycle progression, both in vitro and in vivo. However, the involvement of Pin 1 during follicular development is not well understood. The aim of this study was first to investigate the expression of Pin 1 mRNA in the granulosa and theca cells of the follicle at different developmental stages of follicles in the bovine ovary, and second, to examine the effects of follicle-stimulating hormone (FSH) and estradiol (E2) on the expression of Pin 1 in the cultured bovine granulosa cells. Follicles were classified into four groups based on the diameter (dominant follicles >8.5mm in diameter, subordinate follicles <8.5mm in diameter) and the relative levels of E2 and progesterone (P4) (E2:P4>1, estrogen active; E2:P4<1, estrogen inactive): i.e. preovulatory dominant follicles (POFs); E2 active dominant follicles (EADs); E2 inactive dominant follicles (EIDs); small follicles (SFs). The expression of the Pin 1 gene was significantly increased in the granulosa cells of EADs as compared with those of other follicles, whereas its expression in theca cells did not differ among follicles at different developmental stages. The concentration of 5 ng/ml FSH alone and the combination of 1 ng/ml E2 and 5 ng/ml FSH stimulated the expression of the Pin 1 gene in bovine granulosa cells. Our data provide the first evidence that Pin 1 expression in the granulosa cells but not the theca cells changes during follicular development, and that FSH stimulate the expression of the Pin 1 gene. These results suggest that Pin 1 regulates the timing of cell proliferation and may act as an intracellular signal responder in the granulosa cells during bovine follicle development.

Animals↗

PubMed1999

The modulation of gonadotrophic hormone action on the ovary by paracrine and autocrine factors.

It has been hypothesized that the physiological basis of follicle selection is the differential expression of factors, which modulate the action of gonadotrophins on follicular cells, at key points during the process of follicle development. The aim of this research was to test this hypothesis by identifying factors that can enhance or attenuate the action of the gonadotrophins in stimulating follicle development using both in vivo and in vitro models. Experiments in vivo utilized sheep with an ovarian autotransplant to allow intra-arterial infusion of putative local factors and exposure of the ovary to high local concentrations. Experiments in vitro utilized physiological serum-free cell culture systems for both granulosa and theca cells that allow gonadotrophin-induced differentiation in vitro. The putative local factors tested included insulin-like growth factor-I (IGF-I LR3 analogue), transforming growth factor alpha (TGF alpha) or epidermal growth factor (EGF) and inhibin A. IGF-I stimulated both cellular proliferation and hormone production by both granulosa and theca cells in vitro and similarly stimulated ovarian follicle development and ovarian androgen and oestradiol secretion in vivo. Both TGF alpha and EGF stimulated granulosa and thecal cell proliferation in vitro in a dose-responsive manner and concomitantly inhibited hormone production, whereas intra-arterial infusion of TGF alpha in vivo resulted in induction of atresia in large antral follicles and an acute fall in ovarian hormone secretion. Inhibin A in vitro augmented gonadotrophin stimulated androgen and oestradiol production by thecal and granulosa cells, respectively, but had no effect on cell number. Paradoxically, intra-arterial infusion of inhibin A resulted in an acute depression in ovarian steroid secretion. This depression, however, was also associated with an acute depression in circulating FSH concentrations. In conclusion, these data provide strong support for the hypothesis that factors can modulate the action of gonadotrophins on follicular cells to augment (IGF-I, inhibin A) or inhibit (TGF alpha/EGF) granulosa and thecal cell differentiation. The challenge for the future in this area of research is to understand how these factors interact to enable one follicle to be selected from an ovulatory cohort.

Animals↗

PubMedDate not supplied

Effect of PCB 126 and PCB 153 on incidence of apoptosis in cultured theca and granulosa cells collected from small, medium and large preovulatory follicles.

The aim of the presented study was to evaluate the effects of PCB 126 and PCB 153 on granulosa and theca cell apoptosis. Granulosa and theca cells were collected from small, medium, and large preovulatory porcine follicles and cultured as monolayers. Cells were initially cultured for 24 h to allow attachment to the plates. Media were changed and 100 pg/ml PCB 126 or 100 ng/ml PCB 153 were added. After 48 h, granulosa and theca cells were fixed for assessment of the number of apoptotic cells utilizing a Hoechst staining technique or frozen for measurement of caspase-3 activity. Media were collected for testosterone concentration analysis from theca cell cultures or estradiol from granulosa cell cultures. Neither PCB 153 nor PCB 126 had an effect on testosterone secretion by theca cells collected from small and medium size follicles, while both PCBs decreased testosterone secretion by large follicles. The decrease in testosterone secretion by large follicles under the influence of both PCBs was paralleled by a suppression of caspase-3 activity and a decreased incidence of apoptotic bodies. Neither of the PCBs had an effect on estradiol secretion by granulosa cells collected from small and medium size follicles, while both PCBs increased estradiol in granulosa cells collected from large follicles. PCB-associated increased estradiol secretion by granulosa cells collected from large follicles was accompanied by suppression of caspase-3 activity and a decreased incidence of apoptotic bodies. In conclusion, we have presented evidence that in preovulatory follicles PCBs inhibit both theca and granulosa cells apoptosis. Therefore, an exposure to PCBs may cause alterations in the pattern of terminal differentiation of follicles and attenuate spontaneous elimination of atretic follicles.

Animals↗

PubMed2004

Gonadotropins enhance caspase-3 and -7 activity and apoptosis in the theca-interstitial cells of rat preovulatory follicles in culture.

Apoptosis causes the elimination of ovarian germ cells and the atretic degeneration of ovarian follicles. Here we have used cultured rat preovulatory follicles to examine the regulation of effector caspase-3 and -7 in follicles undergoing apoptosis in the presence or absence of gonadotropins or IGF-I. Culturing follicles in the presence or absence of serum resulted in the induction of apoptosis of granulosa cells (GC), which was accompanied by effector caspase activation. Surprisingly, the addition of the survival factors LH or FSH, but not IGF-I, further increased caspase-3 and -7 activity. Immunohistochemistry studies of the LH- and FSH-treated follicles indicated that cleaved caspase-3 was predominantly localized to the peripheral theca-interstitial cells (TIC). Western blot analysis and caspase-3 and -7 activity assays of the separated follicular compartments confirmed that both LH and FSH treatments significantly enhance caspase-3 and -7 activity in TIC. The elevation in caspase-3 and -7 activity in TIC was accompanied by an increase in apoptosis. Interestingly, LH and FSH also induced an increase in caspase-3 and -7 activity in GC; however, this increase was accompanied by a decrease in apoptosis. Finally, we demonstrate that in freshly isolated preovulatory follicles and in antral follicles in intact ovaries, the expression level of procaspase-3 is significantly higher in TIC than in GC. Thus, LH and FSH have a dual effect on the cultured rat preovulatory follicle: an antiapoptotic effect on GC and a proapoptotic effect on TIC.

Animals↗

PubMed2004

Identification of genes involved in apoptosis and dominant follicle development during follicular waves in cattle.

We hypothesize that granulosa and theca cells from growing dominant follicles, with relatively high intrafollicular concentrations of estradiol, have a greater expression of genes involved in inhibiting apoptosis pathways and lower expression of genes involved in apoptosis pathways than growing subordinate follicles with lower estradiol concentrations. Using the well-characterized bovine dominant follicle model, we collected granulosa and theca cells from individual dominant and the largest subordinate follicle 3 days after initiation of a follicular wave in four animals. Based on ultrasound analysis, both follicle types were in the growth phase at the time of ovariectomy. However, dominant follicles were larger (9.8 +/- 1.0 versus 7.6 +/- 0.6 mm in diameter, P < 0.05) and had greater intrafollicular concentrations of estradiol (132.2 +/-3 8.5 versus 24.1 +/- 12.1 ng/ml, P < 0.05), compared with the largest subordinate follicles. We used bovine cDNA microarrays, which contained a total of 1400 genes, including a subset of 53 genes known to be involved in apoptosis pathways, to determine which apoptosis and marker genes from each of the four dominant versus subordinate follicles were potentially differentially expressed. Using a low stringency-screening criterion, 22 genes were identified. Quantitative real-time polymerase chain reaction confirmed that 16 of these genes were differentially expressed. Our novel results demonstrate that the high intrafollicular concentrations of estradiol in growing dominant follicles were positively associated with enhanced expression of mRNAs in granulosa cells for aromatase, LH receptor, estradiol receptor beta, DICE-1, and MCL-1, compared with granulosa cells from subordinate follicles (all survival-associated genes). In contrast, the relatively low intrafollicular concentrations of estradiol in growing subordinate follicles were positively associated with enhanced expression of mRNAs in granulosa cells for beta glycan, cyclo-oxygenase-1, tumor necrosis factor alpha, caspase-activated DNase, and DRAK-2, and in theca cells for beta glycan, caspase 13, P58(IPK), Apaf-1, BTG-3, and TS-BCLL, compared with granulosa or theca cells from dominant follicles (genes that are all associated with cell death and/or apoptosis). We suggest that that these genes may be candidate estradiol target genes and that they may be early markers for the final stages of follicle differentiation or initiation of apoptosis and thus selection of dominant follicles during follicular waves.

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