The late induction of prostaglandin G/H synthase-2 in equine preovulatory follicles supports its role as a determinant of the ovulatory process

PGs are important mediators of the ovulatory process and prostaglandin G/H synthase-2 (PGHS-2) is a key rate-limiting enzyme in the PG biosynthetic pathway. To determine whether PGHS-2 is regulated in equine follicles before ovulation and, if so, to characterize its time course of induction, preovulatory follicles were isolated during estrus, 0, 12, 24, 30, 33, 36, and 39 h after an ovulatory dose of hCG (n = 5 follicles/time point). Cellular extracts were obtained from preparations of follicle wall (theca interna with attached granulosa cells), isolated granulosa cells, and theca interna and were analyzed by Western blot using specific anti-PGHS antibodies. Immunohistochemistry was used to characterize the in situ localization of PGHS-2 protein in preovulatory follicles, and follicular fluid concentrations of PGE2 and PGF were determined. The results showed the induction of PGHS-2, but not PGHS-1, in equine follicles before ovulation. The PGHS-2 protein (72,000 mol wt) was undetectable 0, 12, and 24 h post-hCG, first became apparent at 30 h, and reached maximal levels 39 h after hCG treatment. The induction of follicular PGHS-2 was localized exclusively in granulosa cells, and a pronounced staining was observed in the perinuclear region. Follicular fluid concentrations of PGE2 and PGF were low and not different between 0-33 h, but levels were increased at 36 and 39 h post-hCG (P < 0.01). Thus, the time course of PGHS-2 induction in equine follicles (30 h post-hCG) is clearly distinct from those previously observed in rat (4 h post-hCG) and bovine (18 h post-hCG) preovulatory follicles. Interestingly, in all three species, the interval from PGHS-2 induction to follicular rupture is highly conserved (approximately 10 h). Therefore, the progressively delayed expression of PGHS-2 in species with longer ovulatory processes supports its role as a molecular determinant of the species-specific length of the ovulatory process.     

Levels of messenger ribonucleic acid for cholesterol side-chain cleavage cytochrome P-450 and 3 beta-hydroxysteroid dehydrogenase in bovine preovulatory follicles decrease after the luteinizing hormone surge

During the follicular/luteal phase shift in steroidogenesis, follicular steroid production changes from predominantly estradiol and androgen secretion before the LH surge to decreased androgen and estrogen and increased progesterone after the LH surge. Our objective was to determine whether changes in progesterone production by the preovulatory follicle are effected via changes in mRNA levels for the steroidogenic enzymes cholesterol side-chain cleavage cytochrome P450 (P450scc) and 3 beta-hydroxysteroid dehydrogenase/delta 5-delta 4-isomerase (3 beta HSD). Bovine preovulatory follicles were obtained in the early follicular phase (n = 9 follicles), the midfollicular phase (n = 4), or the late follicular phase (after the LH surge, but before ovulation; n = 5). Total RNA extracted from granulosa cells and theca interna at the time of cell isolation or after 24 or 72 h of culture in control or LH-containing medium was subjected to Northern analysis, and autoradiographs were scanned densitometrically. P450scc mRNA levels in granulosa cells were high in the early follicular phase and decreased by 96% after the LH surge (P < 0.05). 3 beta HSD mRNA levels in granulosa cells were 4.2-fold higher in early vs. late follicular phase (P < 0.01). In theca interna, 3 beta HSD mRNA levels were 3.6- and 2.6-fold higher in the early vs. the mid- and late follicular phase (P < 0.05), but levels of P450scc mRNA did not differ significantly with stage of follicular development. After granulosa cells had been cultured for 24 h in control or LH-containing medium, P450scc and 3 beta HSD mRNA had declined dramatically compared to mRNA levels at the time of cell isolation during the early follicular phase (P < 0.01). However, after 72 h in control or LH-containing medium, an increase in P450scc and 3 beta HSD mRNA was observed relative to levels at 24 h (P < 0.01). After 72 h of culture, the signal for P450scc and 3 beta HSD mRNA in granulosa cells exposed to LH was higher than the signal detected in cultures without LH (P < 0.01). Similar changes in message for P450scc were observed in cultured thecal cells. Thus, the previously observed increases in production of progesterone by bovine theca interna and granulosa cells obtained after vs. before the LH surge cannot be explained by an increase in message for P450scc and 3 beta HSD.(ABSTRACT TRUNCATED AT 400 WORDS)     

Expression of messenger ribonucleic acid (mRNA) encoding 3beta-hydroxysteroid dehydrogenase delta4,delta5 isomerase (3beta-HSD) during recruitment and selection of bovine ovarian follicles: identification of dominant follicles by expression of 3beta-HSD mRNA within the granulosa cell layer

The objective of the present study was to examine changes in expression of mRNA encoding 3beta-hydroxysteroid dehydrogenase delta4,delta5 isomerase (3beta-HSD) during recruitment and selection of bovine ovarian follicles. Dairy heifers (4-5/time period) were ovariectomized at 12, 24, 36, 48, 60, 72, 84, or 96 h after initiation of the first follicular wave (Time 0) following estrus. Expression of 3beta-HSD mRNA was localized by in situ hybridization and quantified by image analysis. Expression of 3beta-HSD mRNA was first detected in theca interna cells of preantral follicles with a well-developed theca layer and in granulosa cells of follicles > or = 8 mm in diameter. Regardless of stage of follicular wave, expression of 3beta-HSD mRNA in granulosa cells of follicles > or = 8 mm was correlated with follicular size (r = 0.665; p < 0.01). The 36-h time period appeared to be a transition period for selection since dominant follicles were detected by size and expression of 3beta-HSD mRNA in some cows but not in others. By 48 h after wave initiation, dominant follicles could be identified by both size and expression of 3beta-HSD mRNA. Expression of mRNA for 3beta-HSD in theca cells was higher (p < 0.05) at 24 h than at 12 h and remained elevated thereafter through 96 h. In contrast to theca cells, expression of mRNA for 3beta-HSD was undetectable within granulosa cells at 12 and 24 h. At 36 h, 3beta-HSD mRNA was expressed in granulosa cells of healthy follicles > or = 8 mm, and expression was higher (p < 0.05) at 48 h compared with 36 h. Expression of 3beta-HSD mRNA levels increased further in granulosa cells (p < 0.05) at 84 and 96 h compared to 48 h. Upon detection of mRNA for 3beta-HSD in granulosa cells, high levels of expression were always found in one (dominant) follicle/cow with the exception of two cows at 36 and 84 h that expressed 3beta-HSD mRNA in two large healthy follicles. Expression of 3beta-HSD mRNA was also detectable in granulosa cells of a few large atretic follicles in which remnant granulosa cells appeared to be luteinized. Healthy follicles expressed higher (p < 0.05) levels of 3beta-HSD mRNA in both theca and granulosa cells than did atretic follicles. Expression of 3beta-HSD mRNA in theca cells was higher (p < 0.01) in dominant follicles than in other subordinate healthy follicles. These results indicate that only selected dominant follicles express 3beta-HSD mRNA within granulosa cells, and expression increased in both thecal and granulosa cells during the follicular wave. Therefore, expression of 3beta-HSD mRNA within granulosa cells may be associated with the mechanism of selection of the dominant follicle during a follicular wave and may be required for maximum steroid production during follicular dominance.     

Follicle size-dependent induction of prostaglandin G/H synthase-2 during superovulation in cattle

Under physiological conditions, prostaglandin G/H synthase-2 (PGHS-2) is induced in bovine preovulatory follicles by the endogenous surge of gonadotropins. To characterize the pattern of follicular PGHS-2 expression during superovulation in cattle, heifers were treated with exogenous FSH and ovulation was induced with hCG. Animals were ovariectomized 0, 18, and 24 h post-hCG, and extracts of follicles > or = 6 mm were analyzed by Western blotting. Follicular fluid concentrations of prostaglandin (PG) E2, PGF2alpha, progesterone, and estradiol-17beta were determined by RIAs, and the morphology of the cumulus oocyte complex was examined. Results showed that PGHS-2 protein was absent in all follicles isolated at 0 h post-hCG (n = 119) and in small follicles (6 to < 8 mm) isolated between 0 and 24 h post-hCG (n = 27 follicles). In contrast, 12.3% of medium (8 to < 10 mm) and 43.7% of large (> or = 10 mm) follicles were PGHS-2-positive at 18 h post-hCG, and these percentages rose at 24 h to 45.9% and 91.0% in medium and large follicles, respectively (p < 0.05). Follicular fluid concentrations of PGE2 and PGF2alpha were low in follicles isolated at 0 h and increased only in PGHS-2-positive follicles isolated 24 h post-hCG (p < 0.05). Concentrations of progesterone and estradiol-17beta at 0 h were 28.2 +/- 5.8 and 291.8 +/- 13.0 ng/ml, respectively, and a shift from estradiol-17beta to progesterone dominance (luteinization) occurred at 24 h post-hCG only in PGHS-2-positive follicles. Also, expansion of the cumulus oocyte complex was detected at 24 h post-hCG only in PGHS-2-positive follicles. Lack of PGHS-2 induction in follicles of ovulatory size (> 8 mm) was associated with an apparent failure to respond to hCG (absence of luteinization and cumulus expansion). Collectively, these results demonstrate the presence of a time- and follicle size-dependent induction of PGHS-2 in bovine follicles during superovulatory treatment and suggest that PGHS-2 expression can be used as a marker for follicular commitment to ovulation during ovarian hyperstimulation protocols.     

Cortical spreading depression protects against subsequent focal cerebral ischemia in rats

Specific, high-affinity angiotensin II (A II) receptors were observed on granulosa and thecal cells of preovulatory ovarian follicles from immature PMSG-treated rabbits. Scatchard analysis of 125I-[Sar1,Ile8]A II binding to freshly prepared cells was indicative of only one class of binding sites. Kd values were 0.26 +/- 0.11 nM and 0.18 +/- 0.02 nM, densities of A II receptors were 0.06 +/- 0.02 fmol/10(5) cells and 0.08 +/- 0.01 fmol/10(5) cells for granulosa and thecal cells, respectively. When cells were incubated for 48 h with hCG, Kd values were of the same order of magnitude, but the amount of A II receptors was increased 2-fold in granulosa and 4-fold in theca. Using subtype specific ligands (Losartan for AT1 and PD 123319 for AT2) in competitive binding experiments, A II receptors were found to be of the AT1 type on both granulosa and thecal cells freshly prepared or incubated 48 h in vitro. These results establishing the existence of high affinity AT1 receptors on the two cell types of the rabbit preovulatory follicles contrast with previous observations showing the presence of AT2 receptors on granulosa or theca from several species.     

Differential changes in inhibin A, activin A, and total alpha-subunit levels in granulosa and thecal layers of developing preovulatory follicles in the chicken

Accumulating evidence implicates inhibins and activins as endocrine and local regulators of follicular development in mammals, and it was recently confirmed that inhibin/activin alpha and betaA genes are also expressed in the avian ovary. To investigate the potential involvement of these proteins in the chicken ovary, thecal and granulosa layers of the four largest follicles (F1-F4) and the most recent postovulatory follicle were collected from hens (10/group) killed 4, 12, and 20 h before the expected time of F1 ovulation. Inhibin A and activin A concentrations of tissue extracts (expressed per mg DNA) were measured using validated two-site enzyme-linked immunosorbent assays; total immunoreactive inhibin alpha-subunit (ir-alpha) was also measured by heterologous RIA (Monash assay). Inhibin A and ir-alpha were largely confined to the granulosa layer, whereas activin A was much more abundant in the thecal layer. Granulosa inhibin A contents were similar in F4 and F3, but increased approximately 40-fold from F3-F1 (P < 0.0001). As such, the F1 granulosa layer was by far the richest source of inhibin A in the chicken ovary, but contained very little activin A. Total ir-alpha in granulosa was much more abundant than inhibin A and increased only 3-fold from F4-F1 (P < 0.001). Activin A in both granulosa and theca showed little variation between F1 and F4 follicles (by ANOVA, P > 0.05). The inhibin A content of F1 granulosa was maximal 12 h before ovulation and had fallen approximately 6-fold (P < 0.0001) within 8 h, suggesting an inhibitory effect of the preovulatory LH surge on the F1 capacity to synthesize inhibin A. Inhibin A, activin A, and ir-alpha were all less in the postovulatory follicle compared with F1 before ovulation (P < 0.0001). In conclusion, application of the present two-site enzyme-linked immunosorbent assays to the chicken ovary revealed 1) divergent tissue distribution of inhibin A and activin A within preovulatory follicles, and 2) differential regulation of granulosa cell production of inhibin A and activin A dimers during preovulatory follicular development. These findings of dynamic changes in inhibin A, activin A, and total ir-alpha support the hypothesis that these proteins subserve regulatory roles during preovulatory follicular development in the hen.     

Follicle-stimulating hormone and luteinizing hormone/chorionic gonadotropin stimulation of vascular endothelial growth factor production by macaque granulosa cells from pre- and periovulatory follicles

Granulosa cells in the ovulatory follicle express messenger ribonucleic acid encoding vascular endothelial growth factor (VEGF), an agent that may mediate the neovascularization of the developing corpus luteum, but it is not known whether luteinizing granulosa cells synthesize and secrete VEGF during the periovulatory interval. Studies were designed to evaluate the effects of an in vivo gonadotropin surge on VEGF production by macaque granulosa cells (study 1) and to test the hypothesis that gonadotropins act directly on granulosa cells to regulate VEGF production (study 2). Monkeys received a regimen of exogenous gonadotropins to promote the development of multiple preovulatory follicles. Nonluteinized granulosa cells (i.e. preovulatory; NLGC) and luteinized granulosa cells (i.e. periovulatory; LGC) were aspirated from follicles before and 27 h after an ovulatory gonadotropin bolus, respectively. Cells were either incubated for 24 h in medium with or without 100 ng/mL hCG (study 1) or cultured for 6 days in medium with or without 100 ng/mL hCG or 0.1, 1, 10, and 100 ng/mL of recombinant human LH (r-hLH) or r-hFSH (study 2). Culture medium was assayed for VEGF and progesterone. In study 1, LGC produced 8-fold greater levels of VEGF than NLGC (899 +/- 471 vs. 111 +/- 26 pg/mL, mean +/- SEM; P < 0.05). In vitro treatment with hCG increased (P < 0.05) VEGF production by NLGC to levels that were not different from the LGC incubated under control conditions. In vivo bolus doses of r-hCG (100 and 1000 IU) and r-hFSH (2500 IU) were equally effective in elevating granulosa cell VEGF production. In study 2, in vitro treatment with r-hFSH, r-hLH, and hCG markedly increased (P < 0.05) VEGF and progesterone production by the NLGC in a dose- and time-dependent manner. By comparison, the three gonadotropins (100 ng/mL dose) only modestly increased VEGF and progesterone production by LGC. These experiments demonstrate a novel role for the midcycle surge of gonadotropin (LH/CG or FSH) in primates to promote VEGF production by granulosa cells in the periovulatory follicle. Further, the data demonstrate that FSH-like as well as LH-like gonadotropins directly stimulate VEGF synthesis by granulosa cells.     

Insulin-like growth factor binding protein -2 and -4 messenger ribonucleic acid expression in bovine ovarian follicles: effect of gonadotropins and developmental status

This work is concerned with the role of insulin-like growth factor binding protein (IGFBP)-2 and -4 in the regulation of IGF bioactivity in bovine follicles during the development of dominance. We measured the expression of IGFBP-2 and -4 messenger RNA (mRNA) in small (1-4 mm) gonadotropin-sensitive follicles and medium (4-8 mm) and large (>8 mm) gonadotropin-dependent follicles using in situ hybridization. In healthy nonatretic bovine follicles, IGFBP-2 and -4 mRNA expression was confined to granulosa and theca tissue, respectively. Moreover, during the development of follicular atresia, there were distinct changes in the temporal and spatial expression of these genes. IGFBP-2 immunoactivity was localized in granulosa tissue and the basement membrane of healthy preantral follicles, whereas IGFBP-4 immunoactivity was localized in both theca and granulosa tissue. Of particular interest was the lack of IGFBP-2 mRNA expression in large (>8 mm) gonadotropin-dependent follicles, an observation that was confirmed by the lack of immunoreactive IGFBP-2 in these follicles. The regulation of IGFBP-2 and -4 mRNA expression in granulosa and theca cells was analyzed using a serum-free cell culture system. FSH inhibited the expression of IGFBP-2 mRNA in granulosa cells, whereas LH stimulated IGFBP-4 mRNA expression in theca cells. Our results provide evidence for the existence of different roles for IGFBP-2 and -4 in the developing follicle.     

The effect of LH, FSH and pregnant mares' serum gonadotrophin on ornithine decarboxylase activity in thecal and granulosa tissue during follicular growth and atresia in laying hens (Gallus domesticus)

The effect of ovine LH, porcine FSH and pregnant mares' serum gonadotrophin (PMSG) on the activity of ornithine decarboxylase activity in theca and granulosa tissue during folliculogenesis in laying hens is described. The changes in the activity of ornithine decarboxylase induced by hormonal challenge was used to measure the sensitivity of the tissue to the hormone. Thecal tissue from small (< 6 mm) follicles showed a large increase in the activity of ornithine decarboxylase 3 h after treatment with LH, FSH and PMSG, in vivo, whereas ornithine decarboxylase activity in thecal tissue from large (> 8 mm) preovulatory follicles and atretic follicles did not respond to any of the hormonal treatments. Ornithine decarboxylase activity in granulosa tissue from the largest preovulatory follicle increased significantly 3 h after treatment with LH and PMSG in vivo; no effect was observed with FSH. Granulosa tissue from the third largest and fifth largest preovulatory follicles were refractory to the hormonal treatments. Basal activity of ornithine decarboxylase in granulosa tissue from preovulatory follicles increased as the follicles approached ovulation, whereas the activity in thecal tissue from the same follicles decreased. The difference in sensitivity of thecal tissue from small and large preovulatory follicles towards gonadotrophin treatment in vivo is correlated with the difference in the observed rate of atresia occurring within the two groups of follicles. Atresia is the common fate for small follicles, whereas it is a rare event for large preovulatory follicles under normal physiological conditions.(ABSTRACT TRUNCATED AT 250 WORDS)     

Steroidogenic factor-1 expression is transiently repressed and c-myc expression and deoxyribonucleic acid synthesis are induced in rat granulosa cells during the periovulatory period

The expression of steroidogenic factor 1 (SF-1), cytochrome P450 aromatase (P450arom), and cytochrome P450 cholesterol side-chain cleavage (P450scc) was examined during the periovulatory period. Immature rats were injected with eCG to induce development of ovarian follicles to the preovulatory stage. At 48 h after the eCG injection, the LH surge was simulated by an injection of an ovulatory dose of hCG, and RNA was isolated at 0, 2, 4, 6, 8, and 24 h after hCG injection. The mRNA levels for SF-1, P450arom, and P450scc were relatively high in total ovarian RNA samples from eCG-treated rats. Levels of SF-1 and P450arom mRNA decreased within 2 h after injection of hCG. The SF-1 mRNA levels gradually increased from 4 to 24 h. Aromatase levels remained undetectable until 24 h after hCG. P450scc mRNA levels increased throughout the 24-h period after hCG. Levels of SF-1 and P450arom, but not P450scc, mRNA were also reduced in RNA samples from isolated granulosa cells at 4 h after hCG treatment relative to those from eCG-treated rats. In situ hybridization analysis also revealed that hCG uniformly suppressed SF-1 mRNA levels an all granulosa cells compared to those of eCG-treated controls. The relationship of SF-1 expression to immediate/early gene expression and cell cycle traverse was also examined. C-myc mRNA levels were induced by up to 10-fold at 4 h after hCG injection. Similarly, DNA synthesis, as measured by the percentage of granulosa cells that incorporated 5'-bromodeoxyuridine, was increased from 16 +/- 4% in eCG-treated rats to 61 +/- 7% at 4 h after hCG treatment (p < 0.05). This study provides the novel finding that SF-1 expression is transiently repressed to very low levels in response to the LH surge. Further, these studies suggest that granulosa cells traverse the cell cycle before becoming terminally differentiated luteal cells.     

Immunocytochemical comparison of cultured normal epithelial prostatic cells with prostatic tissue sections

The cytologic localization and cellular levels of myc oncoprotein in the human ovary during follicular growth, regression and atresia were examined by the avidin/biotin immunoperoxidase method with a specific antibody to myc oncoprotein. In primordial follicles, only the oocyte showed intense immunostaining for myc protein, whereas the granulosa cells were negative for the staining. In preantral follicles, both the oocyte and granulosa cells were moderately immunostained for myc protein. In antral and preovulatory follicles, there was no appreciable staining for myc protein in the granulosa or theca cells, while myc protein staining in the oocyte persisted with less intensity. It is of interest that myc protein expression in granulosa cells was apparent only during the preantral follicle stage. Corpora lutea during the early and mid luteal phase were negative for myc protein staining, whereas in regressing corpora lutea during the late luteal phase, peripheral theca lutein cells adjacent to the central core of scar tissue were immunostained for myc protein. Corpora albicans showed no staining for myc protein. In atretic follicles, granulosa cells and theca interna cells demonstrated positive staining for myc protein. Ovarian stromal cells were negative for the immunostaining throughout the menstrual cycle. This demonstrates that myc protein is expressed in a stage-limited manner in the human ovary during follicular growth and regression. The abundant expression of myc protein in the oocyte at the primordial and preantral follicle stages and in the granulosa cells at the preantral follicle stage suggests a role for myc expression in the initial growth of the oocyte as well as in the autonomous growth of granulosa cells during the preantral stage seemingly independent of gonadotropic stimulation. Furthermore, notable expression of myc protein in the granulosa cells and theca interna cells of atretic follicles and in the peripheral theca lutein cells of regressing corpora lutea implies the possible participation of myc expression in remodelling the ovarian local tissue following atresia and luteolysis in the human ovary.     

Expression of inhibin alpha and inhibin/activin beta A subunits in the granulosa layer of the large preovulatory follicles of the hen

The expression of the mRNA for the inhibin/activin subunits (alpha and beta A) in the granulosa layer of the five largest preovulatory follicles of the hen was investigated. Total RNA from the granulosa layer of the F5 (the fifth largest) to F1 (the largest) follicles was extracted and analyzed by Northern blot analysis using homologous chicken inhibin alpha and beta A subunit cDNA probes. RNA loading was quantified by a cDNA probe of bovine 18S rRNA. Results showed that for the chicken inhibin alpha subunit mRNA signals (n = 3), the mean relative intensity for the F1, F2, F3, and F4 follicles was 0.50 +/- 0.10 ( +/- SEM,), 0.52 +/- 0.08, 0.59 +/- 0.06, and 0.81 +/- 0.04, respectively, compared to a mean relative intensity of 1.00 (p < 0.05) for the F5 follicle. For the beta A subunit mRNA signals (n = 3), the mean relative intensity for the F5, F4, F3, and F2 follicles was 0.25 +/- 0.06, 0.28 +/- 0.15, 0.40 +/- 0.17, and 0.48 +/- 0.10 (p < 0.05) for the F1 follicle. The inhibin alpha subunit was also estimated to be more abundantly expressed among follicles in the granulosa layer than was the beta A subunit. Our data indicate that the expression of inhibin alpha and beta A subunits is differentially regulated in the hen granulosa layer during follicular development. Expression of the alpha subunit is reduced with follicular development whereas inhibin beta A subunit expression is dramatically enhanced. In addition, the granulosa layer of the large preovulatory follicles may produce more inhibin alpha subunit than beta A subunit, and the F1 follicle may be the primary source of the beta A subunit for dimeric inhibin and/or activin in the hen.     

The antiovulatory effect of the antiprogestin onapristone could be related to down-regulation of intraovarian progesterone (receptors)

The present study was undertaken to investigate intraovarian mechanism(s) for the antiovulatory effect of Onapristone (ON), because antiprogestins possessing the same antiprogestational activity and inhibiting the preovulatory LH surge to the same extent differ in their antiovulatory potency. Ovulation was induced by treating immature female rats with pregnant mare serum gonadotropin (PMSG) for folliculogenesis and hCG for the induction of ovulation. The animals were treated twice with ON (200 mg/kg 42 h and 48 h after PMSG) and killed at different times. The ovulation rate was assessed by counting the number of ova in the fallopian tubes and uteri. Blood and ovaries were collected for radioimmunoassay (RIA) of steroid hormones and histological analysis for 3beta-hydroxysteroid dehydrogenase (3beta-HSDH), 17beta-hydroxysteroid dehydrogenase (17beta-HSDH), progesterone (PR), estrogen (ER) and androgen (AR) receptors. Treatment with ON totally blocked ovulation and the progesterone (P4) surge was significantly diminished in comparison to the control (6-8 h post-hCG), whereas androgen levels remained unaffected. The decreased P4 concentrations correlated well with a reduced staining intensity of 3beta-HSDH in granulosa cells of tertiary follicles. Moreover, we observed a down-regulation of PR in granulosa cells of tertiary follicles. Additionally, in secondary and tertiary follicles the expression of AR between 0 and 6 h after hCG was reduced. These results suggest that the antiovulatory effect of the antiprogestin ON is related to down-regulation of intraovarian progesterone, caused by attenuated 3beta-HSDH activity and PR expression. One can thus assume that intraovarian P4 is an important factor for the induction of ovulation. An effect of ON on the staining intensity of 17beta-HSDH in theca and granulosa cells could not be observed at any time. In conclusion, the inhibition of ovulation induced by the antiprogestin, ON, could be related to decreased intraovarian progesterone production through reduced 3beta-HSDH activity and the down-regulation of PR.     

Changes in extracellular matrix components and steroidogenic enzymes during growth and atresia of antral ovarian follicles in the sheep

To investigate the involvement of extracellular matrix (ECM) in folliculogenesis in the sheep, parallel changes in ECM components and key steroidogenic enzymes were studied by quantitative immunohistochemistry and immunoblotting during follicular growth and atresia. Growth of ovarian follicles from 1 to 5 mm in diameter was characterized by a progressive increase in P450 cholesterol sidechain cleavage levels in both thecal (p < 0.001) and granulosa cells (p < 0.001), an increase in P450 aromatase levels in granulosa cells of follicles larger than 3.5 mm (p < 0.001), and an increase in levels of P450 17 alpha-hydroxylase C17,20 lyase (P450(17 alpha)) in the theca interna. In addition, during follicular growth, a change in localization of cells expressing P450(17 alpha) within the theca interna was observed, positive cells being sparse within the theca interna of small follicles and specifically located close to the basal laminae in large follicles. In parallel, follicular growth was associated with an increase in levels of type I collagen in granulosa cell layers (p < 0.01) and an increase in levels of fibronectin (p < 0.05), particularly the specific ED-A alternatively spliced variant of fibronectin, in the theca externa. Follicular atresia was characterized by a loss of P450 aromatase in granulosa cells (p < 0.001) and a decrease in levels of P450(17 alpha) in the theca interna (p < 0.05). Simultaneously, levels of fibronectin (p < 0.05), particularly the ED-A variant of fibronectin, decreased in the theca externa of atretic follicles. Within the wall of granulosa cells, levels of fibronectin (p < 0.05), laminin, type IV collagen, and heparan sulfate proteoglycans strongly increased during follicular atresia. Overall, these results show that follicular growth and atresia were associated with distinct changes in levels of ECM components, suggesting that ECM components may play a role in the regulation of proliferation, differentiation, and apoptosis of follicular cells.     

In situ hybridization of high density lipoprotein (scavenger, type 1) receptor messenger ribonucleic acid (mRNA) during folliculogenesis and luteinization: evidence for mRNA expression and induction by human chorionic gonadotropin specifically in cell types that use cholesterol for steroidogenesis

The present studies were undertaken to examine the expression of the high density lipoprotein (HDL) receptor, SR-B1 messenger RNA (mRNA) in ovarian cell types during folliculogenesis and luteinization using in situ hybridization histochemistry and to examine its hormonal regulation using Northern blots. For the in situ study for HDL receptor mRNA localization, 21-day-old rats were treated with 50 IU PMSG, and ovaries were collected 0, 24, and 56 h postinjection. At 56 h, animals were treated with a single dose of hCG, and ovaries were subsequently collected at 6-, 12-, 24-, and 72-h and 5-day intervals. In addition, on day 4 of pseudopregnancy, a second dose of 50 IU hCG or saline was administered, and ovaries were collected at 12, 24, and 48 h to determine the induction of the expression of HDL receptor mRNA. The results of in situ hybridization histochemistry showed that in the immature ovary, HDL receptor mRNA is associated with theca interna and interstitial cells (stroma). The mRNA expression in these cell types increased with PMSG treatment, but no signal was detected in the granulosa cells. Northern blot analysis also showed a marked increase in mRNA content in thecal and interstitial cells during follicular development. During luteinization, the intensity of the signal began to appear in the luteinized granulosa cells. With the completion of luteinization, the signal in the corpus luteum tissue became more intense. Further treatment with hCG increased the HDL receptor mRNA content compared with that in the saline-treated control. These results demonstrate that the cholesterol-using cell types of the ovary, namely the interstitial cells, thecal cells, and fully luteinized granulosa cells are endowed with the HDL receptor mRNA, which provides credence to the functional significance of the role of HDL receptor SR-B1 in cholesterol transport and ovarian steroidogenesis.