Local regulation of primate granulosa cell aromatase activity

Granulosa cells produce inhibin and activin, proteins implicated in the local regulation of preovulatory follicular development. To assess interactions among FSH, LH, inhibin and activin on primate granulosa cell aromatase activity, we studied primary granulosa cell cultures from the ovaries of the common marmoset (Callithrix jacchus), a monkey with an ovarian cycle similar in length to the human cycle. The distinctive action of activin was augmentation of gonadotropin-responsive aromatase activity throughout antral follicular development. FSH-stimulated aromatase activity in granulosa cells from immature follicles was augmented many fold by picomolar amounts of activin. In cell cultures from preovulatory follicles, the presence of activin stimulated basal aromatase activity in the absence of gonadotropin, as well as augmenting the action of LH. Thus, locally produced activin has the potential to modulate aromatase activity in developing ovarian follicles. By contrast, inhibin or inhibin -subunit purified from bovine follicular fluid had minimal effects on aromatase activity. The only significant effect was slight suppression of FSH-inducible aromatase activity in granulosa cells from immature follicles at an inhibin concentration of 100 ng/ml. The finding that inhibin has a negligible effect on aromatase activity in granulosa cells from mature follicles suggests that it is unlikely to exert a physiologically significant influence on aromatase activity in vivo. However, evidence from other studies suggests that inhibin might affect aromatization indirectly through acting locally to modulate thecal androgen (aromatase substrate) production. Therefore, both inhibin and activin have the potential to contribute at different levels to paracrine and autocrine regulation of follicular oestrogen synthesis.     

The effect of stage of estrous cycle and follicular maturation on ovarian inhibin production in sheep

Twenty-four Scottish Blackface ewes (mean weight 50.0 +/- 0.1 kg with ovulation rate 1.3 +/- 0.1) were randomly divided into 4 groups of 6 animals. Under general anesthesia, following the collection of a timed sample of ovarian venous blood, the ovaries of these animals were collected either on Day 10 of the luteal phase or 12, 24, and 48 h after a luteolytic dose of a prostaglandin (PG) F2 alpha analogue (cloprostenol 100 micrograms i.m.) administered on Day 10. All follicles greater than 3 mm were dissected from the ovaries and incubated in Medium 199 (M199) at 37 degrees C for 2 h, following which the granulosa cells were harvested and incubated in triplicate for 24 h in M199 with or without ovine FSH or ovine LH. Plasma and culture media samples were assayed for inhibin, estradiol (E2), androstenedione (A4), and testosterone (T) by specific RIA. After correcting for hematocrit, ovarian secretion rates were calculated from the product of the plasma concentration and flow rate. The rate of ovarian inhibin secretion during the luteal phase was similar from ovaries categorized on the basis of presence of luteal tissue (1.0 +/- 0.3 and 0.9 +/- 0.5 ng/min for CL present and absent, respectively), confirming that the ovine CL does not secrete appreciable amounts of inhibin. Inhibin secretion was higher (p less than 0.05) at 12 h after PG-induced luteolysis but not at 24 or 48 h compared to values for luteal phase control ewes. Although ovaries containing large estrogenic follicles (greater than or equal to 4 mm in diameter and classified as estrogenic from in vitro criteria) secreted the most inhibin (55%; p less than 0.05), both ovaries containing large nonestrogenic follicles (33%) and small (11%; less than 4 mm in diameter) follicles secreted appreciable amounts of inhibin. This contrasted strongly with E2 where greater than 80% of the steroid was secreted by large estrogenic follicles. The rate of ovarian inhibin secretion was positively correlated (p less than 0.05) with the rate of E2, A4, and T secretion. Overall, there was no significant effect of stage of cycle on follicular inhibin content after 2 h incubation in vitro, release of inhibin by follicles incubated in vitro, or synthesis of inhibin by granulosa cells cultured in vitro. FSH and LH had no effect on the production of either inhibin or estradiol by cultured granulosa cells. Follicular diameter was positively correlated (p less than 0.001) with follicular inhibin and steroid release. Follicular inhibin content after 2 h incubation in vitro was more highly correlated with inhibin release by incubated follicles (r = 0.7; p less than 0.001) than with inhibin synthesis by granulosa cells in vitro (0.4; p less than 0.01).(ABSTRACT TRUNCATED AT 400 WORDS)     

Identification of calcitonin expression in the chicken ovary: influence of follicular maturation and ovarian steroids

Calcitonin (CALCA), a hormone primarily known for its role in calcium homeostasis, has recently been linked to reproduction, specifically as a marker for embryo implantation in the uterus. Although CALCA expression has been documented in several tissues, there has been no report of production of CALCA in the ovary of any vertebrate species. We hypothesized that the Calca gene is expressed in the chicken ovary, and its expression will be altered by follicular maturation or gonadal steroid administration. Using RT-PCR, we detected Calca mRNA and the calcitonin receptor (Calcr) mRNA in the granulosa and theca layers of preovulatory and prehierarchial follicles. Both CALCA and Calca mRNA were localized in granulosa and thecal cells by confocal microscopy. Using quantitative PCR analysis, F1 follicle granulosa layer was found to contain significantly greater Calca mRNA and Calcr mRNA levels compared with those of any other preovulatory or prehierarchial follicle. The granulosa layer contained relatively greater Calca and Calcr mRNA levels compared with the thecal layer in both prehierarchial and preovulatory follicles. Progesterone (P(4)) treatment of sexually immature chickens resulted in a significantly greater abundance of ovarian Calca mRNA, whereas estradiol (E(2)) or P(4) + E(2) treatment significantly reduced ovarian Calca mRNA quantity. Treatment of prehierarchial follicular granulosa cells in vitro with CALCA significantly decreased FSH-stimulated cellular viability. Collectively, our results indicate that follicular maturation and gonadal steroids influence Calca and Calcr gene expression in the chicken ovary. We conclude that ovarian CALCA is possibly involved in regulating follicular maturation in the chicken ovary.     

Activin A and gonadotropin regulation of follicle-stimulating hormone and luteinizing hormone receptor messenger RNA in avian granulosa cells.

Activin A regulation of the expression of mRNA for the LH receptor, FSH receptor, and the inhibin alpha subunit as well as the effect of activin A on the secretion of progesterone were investigated in chicken granulosa cell cultures. Granulosa layers were isolated from the F(1) and F(3) + F(4) follicles from five hens, pooled according to size, dispersed, and cultured for 48 h. In experiment 1 (n = 3 replications), granulosa cells were cultured with or without highly purified ovine (o) FSH at 50 ng/ml and in the presence of 0, 10, or 50 ng/ml of recombinant chicken activin A. Experiment 2 (n = 4 replications) followed the same protocol as experiment 1, except that oFSH was replaced with oLH. Results from these experiments showed that addition of activin A to the granulosa cell cultures had no effect on the expression of mRNA for the inhibin alpha subunit or the FSH receptor, but it did affect the expression of mRNA for the LH receptor. Treatment of F(3) + F(4) granulosa cells with LH stimulated the expression of mRNA for the LH receptor; however, when LH was combined with either dose of activin A, this induction was prevented. The highest dose of activin A with or without LH resulted in decreased expression of the LH receptor compared to the untreated controls in the F(3) + F(4) cell cultures. Progesterone secretion by the granulosa cells from both follicle sizes was not altered by activin A. In experiment 3 (n = 3 replications), the effect of activin A on the growth of granulosa cells was examined with the following treatments: 0, 10, or 50 ng/ml of activin A; 50 ng/ml of either oLH or oFSH; and oLH or oFSH combined with 10 ng/ml of activin A. The highest dose of activin reduced the rate of granulosa cell proliferation in both follicle types. Growth of F(1) and F(3) + F(4) granulosa cells was stimulated by the addition of either gonadotropin, and the presence of 10 ng/ml of activin A with either gonadotropin did not alter this proliferation, except for the LH-treated F(3) + F(4) granulosa cells, in which the increase in proliferation was prevented. The results suggest that activin A could act as a local factor that regulates follicular maturation by preventing excessive or untimely LH receptor expression.     

Messenger RNA and protein expression analysis of betaglycan in the pituitary and ovary of the domestic hen

Betaglycan was originally characterized as the type III receptor for TGFbeta, yet recent research has indicated that betaglycan can serve as an accessory receptor for inhibin. To understand better the action of inhibin in avian follicular development, we have investigated the expression of betaglycan in the pituitary gland and ovary of the hen. In experiments 1 and 2, betaglycan mRNA was detected at 6 kilobases (kb) by Northern blot analysis (n = 5) in chicken pituitary, granulosa, and theca layers and whole ovary. Expression of betaglycan was greatest in the pituitary gland in experiment 1 and greater in the granulosa layer of small yellow follicles (SYF) compared with the granulosa layer of larger follicles. In experiment 2, betaglycan mRNA was more abundantly expressed in the theca layer compared with the granulosa layer for all follicle sizes, although there was no significant difference in betaglycan expression in the theca layer among follicle sizes. In experiment 3, immunohistochemical analysis revealed betaglycan protein in the anterior pituitary as well as in the ovary (n = 4) and SYF (n = 4). Colocalization studies revealed a high abundance of cells within the anterior pituitary expressing both betaglycan and FSH (n = 4). Betaglycan protein was found in the granulosa layer; however, markedly enhanced staining was observed in the theca layer of ovarian follicles. Our results provide evidence for expression of betaglycan mRNA and protein colocalization with FSH in the anterior pituitary, consistent with known inhibin effects. Ovarian localization of betaglycan, particularly in the theca layer, suggests a paracrine role for inhibin in the hen.     

Changes in plasma inhibin A levels during sexual maturation in the female chicken and the effects of active immunization against inhibin alpha-subunit on reproductive hormone profiles and ovarian function

Inhibins and activins are firmly implicated in the control of pituitary FSH secretion and ovarian follicular development in mammals. As in mammals, inhibin A and activin A are expressed in the preovulatory follicles of birds, and a defined ovulation cycle for inhibin A has recently been demonstrated in the laying hen. To investigate further the role of inhibin-related proteins in developing pullets, circulating concentrations of inhibin A, inhibin B, total immunoreactive inhibin alpha-subunit (ir-alpha), activin A, LH, FSH, and progesterone were measured from the juvenile state through to sexual maturity in 22 birds. In the 11 birds assigned to control groups, plasma inhibin A levels were low from 7 to 13 wk of age rising about threefold to a peak at Week 19 after which levels fell slightly to a plateau level characteristic of adult hens. Plasma inhibin A levels were negatively correlated with FSH (r = -0. 33; P: < 0.001) and positively correlated with progesterone (r = 0. 67; P: < 0.001) and ir-alpha (r = 0.53; P: < 0.001). Plasma ir-alpha levels were much higher than inhibin A levels although the relative differences varied with age. Plasma levels of inhibin B and activin A were below assay detection limits at all times. The remaining group of 11 birds was actively immunized (IMM) against a synthetic chicken inhibin alpha-subunit peptide (amino acids 1-26). The IMM generated circulating antibodies that bound native bovine inhibin A but altered neither plasma FSH nor progesterone levels relative to control birds at any stage of development nor the timing of first oviposition in week 19. Apart from a transient decline 1 wk after primary IMM, plasma LH concentrations did not differ from controls. Comparison of the numbers and size-class distribution of ovarian follicles at 29 wk showed an approximate twofold increase in the number of 8- to 9.9-mm-diameter follicles (control; 1.82 +/- 0.44 vs. IMM; 3.91 +/- 0.89; P: < 0.05), a size class that corresponds to follicles that have just joined the preovulatory hierarchy. The numbers of growing follicles in other size-classes and the sizes of hierarchical F(1)-F(7) follicles were not altered by IMM. However, the number of postovulatory follicles increased (control 3.73 +/- 0. 20 vs. IMM 5.55 +/- 0.28; P: < 0.01), and significantly more (P: < 0. 02) immunized hens laid two eggs within a 24-h period on at least one occasion (control 1 of 11 vs. IMM 9 of 11). The IMM increased (P: < 0.05) activin A content of F(1) and F(2) theca layers and decreased (P: < 0.05) activin A content in F(3) and F(4) granulosa layers, raising the possibility of a local intraovarian role of activin in mediating the response to IMM. These findings support a role for inhibin A in regulating the entry of follicles into the preovulatory hierarchy in the chicken, although further studies are required to establish the mechanism by which inhibin IMM increases the rate of follicle selection and ovulation without raising plasma FSH.     

Consequences of the presence of the Booroola F gene on the intraovarian insulin-like growth factor system and terminal follicular maturation in Mérinos d'Arles ewes

In sheep, the presence of the Booroola F gene has several important consequences for ovarian function. This study investigated the consequences of the presence of the F gene for the insulin-like growth factor (IGF) system in the ewe ovary. Studies were undertaken in ovaries from F+ and ++ Mérinos d'Arles ewes to determine 1) the levels of type I IGF receptors and IGF binding proteins (IGFBPs) in follicular cells by quantitative autoradiography of [(125)]-IGF-I binding sites on ovarian sections; 2) the pattern of intrafollicular IGFBPs, by Western-ligand blotting on follicular fluids; and 3) the effects of IGF-I and FSH on proliferation and differentiation of granulosa cells in vitro, assessed by progesterone secretion and cytochrome P450 side-chain cleavage (P450(scc)) expression. The amounts of type I IGF receptors were similar in F+ and ++ follicular cells; however, at the same follicular size, F+ healthy follicles contained lower concentrations of IGFBPs smaller than 40 kDa (particularly IGFBP-2) than ++ healthy follicles. In vitro, in basal conditions as well as in IGF-I- or FSH-stimulated conditions (or both), granulosa cells from F+ follicles had a lower proliferative activity, secreted higher amounts of progesterone, and expressed higher levels of P450(scc) than granulosa cells from ++ follicles of the same size. When F+ and ++ preovulatory follicles were compared at the end of the follicular phase, IGFBPs <40 kDa concentrations were slightly higher, and responsiveness of granulosa cells to FSH in vitro was lower in F+ than in ++ follicles, suggesting that terminal maturation of F+ follicles, although precocious, was less complete than it was in ++ follicles. The early decrease in intrafollicular IGFBPs <40 kDa concentrations observed in F+ antral follicles, which likely leads to an early increase in IGF bioavailability, may at least partly account for the increased ovulation rate that characterizes F-carrier ewes.     

Rapid changes in the expression of inhibin alpha-, beta A-, and beta B-subunits in ovarian cell types during the rat estrous cycle

Distributions of inhibin alpha-, beta A-, and beta B-subunits in different ovarian compartments were studied in cycling female rats by in situ hybridization with complementary RNA probes and using immunohistochemical localization with antibodies selective for each inhibin subunit. Consistent with earlier studies showing inhibin production by granulosa cells of maturing follicles, we also detected mRNAs for inhibin alpha-, beta A-, and beta B-subunits in granulosa cells of these follicles. However, based on immunohistochemistry and in situ hybridization, we found that inhibin alpha- is not only expressed in granulosa cells of mature follicles but in follicles at all stages of maturation, including primary to tertiary follicles. A number of primordial follicles also contained alpha mRNA and immunodetectable alpha-subunit. Interestingly, theca interna and interstitial gland cells contained inhibin alpha mRNA and alpha-subunit. Low levels of inhibin alpha immunoreactivity as well as specific hybridization to the complementary inhibin alpha mRNA probe were observed in newly formed luteal tissue. beta-Subunits, on the other hand, were detected exclusively in granulosa cells of healthy tertiary follicles. The changes in expression of inhibin alpha-, beta A-, and beta B-subunits were more pronounced during the follicular phase of the cycle: inhibin alpha reached its highest level in granulosa cells, theca interna, and interstitial gland cells a few hours after the LH/FSH surge, while at the same time the beta-subunits decreased dramatically in granulosa cells of mature follicles. Immediately before ovulation (estrus 0200 h), the alpha-subunit sharply declined in preovulatory follicles and was present mainly in granulosa cells from nonovulatory follicles at various stages of maturation. At that time, the beta A- and beta B-subunits could not be detected in preovulatory follicles but were localized mainly in small tertiary follicles (less than 300 microns). Unlike for the alpha- and beta B-subunits, beta A mRNA and immunoreactivity was present in large tertiary follicles (approximately 600 microns) immediately before ovulation. The present findings support the hypothesis that a decrease in inhibin production could be responsible for the secondary FSH surge observed early on estrus. This could be initiated by a change in the ratios of activin-inhibin production by decreasing first, the levels of beta-subunits, second, the levels of alpha-subunit, and third, by a resurgence of activin A produced mainly by granulosa cells from large tertiary follicles.(ABSTRACT TRUNCATED AT 400 WORDS)     

Decreased oocyte DAZL expression in mice results in increased litter size by modulating follicle-stimulating hormone-induced follicular growth

While the germ cell-specific RNA binding protein, DAZL, is essential for oocytes to survive meiotic arrest, DAZL heterozygous (het) mice have an increased ovulation rate that is associated with elevated inhibin B and decreased plasma follicle-stimulating hormone (FSH). The relationship between decreased oocyte DAZL expression and enhanced follicular development in het mice was investigated using in vitro follicle cultures and in vivo modulation of endogenous FSH, by treating mice with inhibin and exogenous FSH. In vitro, follicles from het mice are more sensitive to FSH than those of wild-type (wt) mice and can grow in FSH concentrations that are deleterious to wild-type follicles. In vivo, despite no differences between genotypes in follicle population profiles, analysis of granulosa cell areas in antral follicles identified a significantly greater number of antral follicles with increased granulosa cell area in het ovaries. Modulation of FSH in vivo, using decreasing doses of FSH or ovine follicular fluid as a source of inhibin, confirmed the increased responsiveness of het antral follicles to FSH. Significantly more follicles expressing aromatase protein confirmed the earlier maturation of granulosa cells in het mice. In conclusion, it is suggested that DAZL expression represses specific unknown genes that regulate the response of granulosa cells to FSH. If this repression is reduced, as in DAZL het mice, then follicles can grow to the late follicular stage despite declining levels of circulating FSH, thus leading to more follicles ovulating and increased litter size.     

Inhibin production by bovine ovarian tissues in vitro and its regulation by androgens

No detectable amounts of inhibin were produced by cultured ovarian stroma or luteal tissue. Follicular tissue produced inhibin in vitro and removal of the granulosa cells from the follicle wall caused inhibin production to fall by 80%. Granulosa cells alone had the greatest ability of any ovarian cell type to produce inhibin in vitro, and are probably the major site of follicular inhibin production. Cyproterone acetate at concentrations of 35 and 350 microM inhibited basal and testosterone (3.5 microM)-stimulated inhibin production by cultured intact follicle wall and granulosa cells. In addition, each concentration of cyproterone acetate inhibited progesterone but not oestradiol-17 beta production by the follicle wall and granulosa cell cultures. The synthetic, non-aromatizable androgens, methylestrenolone and mesterolone, at concentrations of 5 and 25 microM, mimicked the effect of testosterone and stimulated granulosa cell inhibin production, methylestrenolone being the more potent. These findings provide further evidence that androgens regulate follicular inhibin and progesterone production and that these may be receptor-mediated processes, and suggest that inhibin production may be a general property of androgenic compounds. Preliminary examination of the physicochemical characteristics of inhibin indicated that the inhibin activity of bovine granulosa cell culture medium was (a) retained by an Amicon XM100A filter with a nominal molecular weight cut-off point of 100 000; and (b) destroyed by heating to 80 degrees C for 30 min.     

Follicle-stimulating hormone regulation of inhibin alpha- and beta(B)-subunit and follistatin messenger ribonucleic acid in cultured avian granulosa cells

FSH regulation of inhibin alpha-, beta(B)-subunit and follistatin mRNA was investigated in cultured chicken granulosa cells, which were isolated and pooled according to size from the F(4) + F(5) follicles, small yellow follicles (SYF), and large white follicles (LWF). In experiment 1 (four replicate experiments), granulosa cells were cultured, and the effect of FSH (50 ng/ml) on the growth of cells from the different follicles was examined at 24 and 48 h of culture. Cell viability was >95% for all of the granulosa cell cultures at 24 and 48 h. At 24 h, the number of granulosa cells in both the FSH-treated and the untreated cultures for all follicle types was numerically greater than the number of cells originally plated. At 48 h, FSH-treated cultures for all follicle types had twice (P: < 0. 05) the number of cells as the untreated cultures. In experiment 2 (three replicate experiments), FSH increased expression of the mRNA for inhibin alpha-subunit in LWF granulosa cells at 4 and 24 h to detectable levels and increased inhibin alpha-subunit protein accumulation to detectable levels by 24 h in granulosa cells from the LWF. FSH also increased (P: < 0.05) mRNA levels for the inhibin alpha-subunit at 4 and 24 h in SYF granulosa cells and at 24 h in F(4) + F(5) granulosa cells. The effects of FSH on follistatin and ss(B)-subunit were variable with respect to follicle development and culture duration. These results suggest that FSH plays an important role in stimulating the production of mRNA and protein for the inhibin alpha-subunit in small prehierarchical follicles.     

Characterization of integrin expression in the mouse ovary

Integrin alpha:beta heterodimers mediate cell contacts to the extracellular matrix and initiate intracellular signaling cascades in response to a variety of factors. Integrins interact with many determinants of cellular phenotypes and play roles in controlling the development, structural integrity, and function of every type of tissue. Despite their importance, little is known about the regulation of integrin subunits in the mammalian ovary and how they function in folliculogenesis. To determine their relevance to ovarian physiology, we have studied the expression of integrin subunit mRNAs by Northern blot analysis and in situ hybridization in ovaries of wild-type, growth differentiation factor 9 (Gdf 9) knockout, FSHbeta (Fshb) knockout, and inhibin alpha (Inha) knockout mice. Integrin alpha6 mRNA is expressed in oocytes and granulosa cells of single-layer follicles and in oocytes and theca cells of multilayer follicles. Integrin alpha6 is highly expressed in Gdf 9 knockout ovaries, which are enriched in oocytes and primary (single layer) follicles because of a block at this stage of follicular development. Integrin alpha(v) mRNA is most highly expressed in the granulosa cells of multilayer growing follicles, and therefore only low levels of expression are detectable in the Gdf 9 knockout ovaries. Integrin beta1 mRNA exhibits a broad expression pattern in ovaries, including oocytes, granulosa cells, theca cells, and corpora lutea. Integrin beta3 mRNA is expressed in theca and interstitial cells and is upregulated in corpora lutea. It is nearly undetectable in ovaries of Fshb knockout mice, which develop preantral follicles but have no luteal cells. Integrin beta5 mRNA is predominantly expressed in granulosa cells of multilayer follicles. It is expressed at high levels in the Fshb knockout mice and in a compartmentalized manner in the granulosa cell/Sertoli cell tumors that develop in the Inha knockout mice. Specific integrins are associated with ovarian cellular phenotypes in mice, which raises intriguing possibilities as to integrin functions in oocyte competence, follicular development, luteinization, and granulosa cell proliferation.     

Structural and hormonal changes during follicular maturation in the ovary of the domestic goose

Follicle maturation in the ovary of sexually mature domestic geese in the spring reproductive cycle was investigated by histological methods and steroid-RIA. The single-layer granulosa of primary follicles temporarily transformed in the growing white follicles into several layers or a simple membrana granulosa with nuclei at several different levels in the cell. In the yolky follicles the granulosa represents a cuboidal epithelium (F4-F3) and subsequently a high cylindrical epithelium (F1). The originally connective tissue-like cells of the theca interna show a glandular proliferation in the largest white (F7) and the small yolky follicles (F6-F5). Glandular cell nests in the theca externa are typical in the generation of small white follicles and are absent in the wall of yolky follicles. Progesterone-content in the follicular wall (granulosa + theca) is the highest in the F1-F2 and F6-F5 types and is low in small white follicles (F8, F9 and F10). E2 concentration shows only slight variations between F1-F10. TEST content shows a slight increase between F1 and F3 and is high in medium-sized white follicles (F8-F9). The results suggest that in addition to the granulosa, the theca interna is also capable of an intensive progesterone synthesis.     

中国荷斯坦奶牛卵巢卵泡细胞的免疫组织化学研究

目的和方法应用七种特异性抗体(sGCα、sGCβ、nNOS、FOXO1、PDE4、PKB/Akt和Inhibinα)对中国荷斯坦奶牛卵巢卵泡细胞进行免疫组织化学定位。结果表明PKB主要存在于原始卵泡、腔前卵泡和有腔卵泡颗粒细胞,黄体细胞中没有检测到;FoxO1主要位于原始卵泡、腔前卵泡和有腔卵泡颗粒细胞;PDE4仅位于部分有腔卵泡的颗粒细胞;抑制素α、nNOS、sGCα和β存在于各级卵泡的颗粒细胞层,其中sGCα和β主要存在于原始卵泡和腔前卵泡的颗粒细胞。结论这七种蛋白的阶段和细胞特异性表达表明它们与中国荷斯坦卵巢卵泡的发育、闭锁和黄体化过程有着密切的关系。     

Localization of androgen receptor and cytochrome P450 aromatase in the follicle and corpus luteum of the porcine ovary

The following study was undertaken to localize androgen receptors (AR) and aromatase cytochrome P450 (P450arom) in porcine ovarian tissue because ovarian androgens may act locally to modulate follicular and luteal function in various species. Androgen receptor was detected immunohistochemically in granulosa and theca cells of preantral as well as in growing antral follicles. The most intensive staining was observed in the antral granulosa layer. Luteinizing granulosa cells of preovulatory follicles, and luteal cells from the early and midluteal phases stained weakly for the androgen receptor. Fully regressed corpora lutea in the early follicular phase of the next cycle did not stain for androgen receptor. In contrast, granulosa cells were very weakly stained for aromatase in early stages of follicular development. The P450arom was maximally expressed with the same intensity in mural and antral layers in large ovulatory follicles. Corpora lutea from the early luteal phase showed positive staining, whereas those from midluteal phase did not stain for aromatase, some cells of regressed corpora lutea unexpectedly exhibited aromatase staining.     

Changes in the secretion of inhibin and steroid hormones during induced follicular atresia after hypophysectomy in the rat

Sequential changes in the function of antral follicles during the period of follicular atresia were investigated after hypophysectomy (Hypox) at 1100 hr on proestrus. Within 6 hours after Hypox, concentrations of progesterone (P), testosterone (T) and estradiol-17 beta (E) decreased abruptly in ovarian venous plasma (OVP) and follicles showed a reduced ability to ovulate. Six hours after Hypox, ovulation was still induced by human chorionic gonadotropin (hCG) in all animals but with significantly fewer number of oocytes compared to the group given hCG at 1100 hr on the day of proestrus. Nine hours after Hypox, several granulosa cells of all large follicles (greater than 400 microns in diameter) exhibited morphological signs of atresia. Twelve hours after Hypox, all large and medium sized (200-400 microns in diameter) follicles showed advanced stages of atresia and almost all follicles failed to ovulate in response to hCG. Inhibin activity in OVP declined more slowly compared to the profiles of steroid hormones and 53% of the initial inhibin activity was still maintained at 18 hours after the operation. Inhibin activity further decreased to 7% of the initial level at 24 hours and was undetectable by 48 hours after Hypox. These results suggest that fully developed Graafian follicles gradually lose their ability to secrete inhibin in contrast to the rapid decrease in secretion of steroid hormone during the process of atresia.     

Expression of retinol-binding protein and cellular retinol-binding protein in the bovine ovary

Retinol (vitamin A) is essential for reproduction, and retinoids have been suggested to play a role in ovarian steroidogenesis, oocyte maturation, and early embryonic development. Retinol is transported systemically and intercellularly by retinol-binding protein (RBP). Within the cell, cellular retinol-binding protein (CRBP) functions in retinol accumulation and metabolism. Since the actions of retinoids are mediated, in part, by retinoid-binding proteins, the objective of this study was to investigate cell-specific expression of RBP and CRBP in the bovine ovary. Immunocytochemical analysis (ICC) localized RBP to the thecal and granulosa cell layers of antral and preantral follicles with the most intense staining in the cells of large, healthy follicles. The tunica adventitia of arterial blood vessels also exhibited RBP staining. Immunostaining of CRBP was most intense in the granulosa cells of preantral follicles and present, but diminished, in thecal and granulosa cells of antral follicles. Within the corpus luteum, both proteins were observed in large luteal cells, but only RBP was observed in small luteal cells. Northern blot analysis demonstrated that thecal and granulosa cells from antral follicles and luteal tissue expressed RBP and CRBP mRNA. Synthesis and secretion of RBP by thecal cells, granulosa cells, and luteal cells were demonstrated by immune-complex precipitation of radiolabeled RBP from the medium of cultured cells or explants, followed by SDS-PAGE and fluorography. Follicular fluid was collected from small (<5 mm) and large (8-14 mm) follicles, pooled according to follicular size, and analyzed for retinol, RBP, estradiol-17beta, and progesterone. Concentrations of retinol, RBP, and estradiol were greater in the fluid of large follicles. Results demonstrate retinoid-binding protein expression by bovine ovaries and provide physical evidence that supports the concept that retinoids play a role in ovarian function.     

Prostaglandin levels in peripheral and follicular plasma, the isolated theca and granulosa layers of pre- and postovulatory follicles, and the myometrium and mucosa of the shell gland (uterus) during a midsequence-oviposition of the hen (Gallus domesticus)

Prostaglandins (PG) F and E were measured by radioimmunoassay in peripheral, uterine and follicular plasma and in the theca and granulosa layers of the five largest preovulatory and the three largest postovulatory follicles, and in the myometrium and mucosa. Plasma and tissues were collected 16, 12, 8 and 4 h before and immediately after a midsequence oviposition that was accompanied by the next ovulation. PGF concentrations in the peripheral and uterine plasma increased at oviposition with a concomitant, 16-fold increase in plasma PGF concentrations of the largest preovulatory (F1) follicle. There was a gradual increase in PGF concentrations in the theca layers during follicular maturation, with the large increases occurring 12 h before oviposition in most follicles. The highest and the second highest concentrations were observed at oviposition in the F1 and the largest postovulatory (R1) follicles. In contrast, there were no specific changes in PGF concentrations in the granulosa layers of the follicles in relation to oviposition or follicular maturation. PGE concentrations in the theca layers of the F2 and F1 follicles were greater than in other follicles, while concentrations in the granulosa layer of all the follicles remained low. PGF concentrations in the myometrium and mucosa increased 8 h before oviposition but abruptly decreased at oviposition. These results suggest that the primary source of the increase in plasma PGF at oviposition are the theca layers of the F1 and R1 follicles and that PGs may be involved in uterine contractions for oviposition and in the ovulation process.