Gonadotropin regulation of RIP140 messenger ribonucleic acid expression in the rat ovary

Female mice null for receptor-interacting protein 140 (RIP140) are infertile because of the failure of follicle rupture. The present study examined gonadotropin regulation of RIP140 expression in immature rat ovary. Treatment with PMSG increased ovarian RIP140 mRNA and protein levels. In contrast, hCG treatment rapidly inhibited RIP140 mRNA and protein levels within 1-3 h. RIP140 mRNA was detected in theca cells of growing follicles in untreated ovary and in granulosa cells in PMSG-treated ovary. Interestingly, hCG treatment reduced RIP140 mRNA levels in granulosa cells of preovulatory follicles, but not of growing follicles. Neither treatment of immature rats with diethylstilbestrol in vivo nor of immature granulosa cells with FSH in vitro affected RIP140 mRNA levels. Treatment of immature granulosa cells with 17beta-estradiol in vitro, however, stimulated RIP140 mRNA levels. In cultured preovulatory granulosa cells, RIP140 mRNA levels were stimulated at 1 h and then declined to below control levels by 3 h after LH treatment. Treatment with MDL-12,330A, an inhibitor of adenylate cyclase, or chelerythrine chloride, an inhibitor of protein kinase C (PKC), inhibited LH-stimulated RIP140 gene expression. Furthermore, forskolin or TPA treatment for 1 h mimicked the stimulatory action of LH, indicating the involvement of both adenylate cyclase and PKC pathways. These results demonstrate the stimulation by PMSG and inhibition by hCG of RIP140 expression in granulosa cells of preovulatory follicles in the rat ovary.     

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.     

Localization of preovulatory expression of plasminogen activator inhibitor type-1 and tissue inhibitor of metalloproteinase type-1 mRNAs in the rat ovary.

Proteinases and their inhibitors control follicular connective tissue remodeling associated with follicular rupture. We examined the regulation and cellular localization of plasminogen activator inhibitor type-1 (PAI-1) and tissue inhibitor of metalloproteinase type-1 (TIMP-1) mRNAs by in situ hybridization. [35S]UTP-labeled RNA probes were hybridized to ovarian sections of eCG-primed immature rats treated with hCG. Before hCG stimulation of ovulation, very low expression of PAI-1 mRNA was observed in theca cells. After hCG administration, expression of PAI-1 mRNA was increased in theca cells of most antral follicles, whereas expression in granulosa cells was limited to preovulatory follicles and only to areas where the basal membrane was dissociated. Before hCG treatment, low expression of TIMP-1 mRNA was observed in theca cells, but not in granulosa cells. After hCG treatment, TIMP-1 mRNA was greatly stimulated in theca cells irrespective of follicle size, while the expression in granulosa cells was limited to large antral follicles. The present study demonstrates cell-specific expression of PAI-1 and TIMP-1 mRNAs in the LH/hCG-stimulated ovary, thus confirming the localized control of preovulatory proteolysis by coexpression of both enzymes and their respective inhibitors.     

Localization of relaxin in the pig follicle during preovulatory development

The avidin-biotin immunoperoxidase method and antisera to purified porcine relaxin were used to localize relaxin in sections of follicles from pregnant mare's serum gonadotropin (PMSG)/human chorionic gonadotropin (hCG)-primed pigs during preovulatory development. Prepubertal pigs were treated i.m. with PMSG (750 IU) and 72 h later with hCG (500 IU) to induce follicular development and ovulation. Follicles were collected from untreated gilts or from gilts 24, 48, 60, 72, 84, 96, or 108 h after PMSG treatment. Light immunostaining in the theca interna was observed early in follicular development, at 48 and 60 h post-PMSG. At 72 h post-PMSG, relaxin immunostaining in the theca interna of the preovulatory follicle was more intense. After hCG treatment, the intense thecal immunostaining persisted and was apparent 84 and 96 h after PMSG. At about 6 h prior to expected ovulation (108 h post-PMSG), there was thinning of the follicle wall and a reduction in relaxin immunostaining in the theca interna. Immunoactive relaxin was not detected in follicles from untreated gilts, follicles 24 h post-PMSG, small healthy or atretic follicles, or in granulosa cells, theca externa or ovarian stroma, at any of the time points studied. These studies support the hypothesis that the theca interna is the primary source of follicular relaxin and provide further evidence for a paracrine role for relaxin in the ovulatory process.     

Plasminogen activator activity in cumulus-oocyte complexes of gonadotropin-treated rats during the periovulatory period

Two types of plasminogen activator (tissue-type, tPA; urokinase-type, uPA) have been demonstrated in ovarian granulosa cells, but only tPA activity was found in denuded oocytes. Immature rats were treated subcutaneously with 20 IU pregnant mare's serum gonadotropin (PMSG) to stimulate follicle maturation, followed 2 days later by an injection of 10 IU human chorionic gonadotropin (hCG) to induce ovulation. Cellular plasminogen activator activities were determined by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis followed by a fibrin-overlay technique. Cumulus-oocyte complexes from rats before and after PMSG treatment contained low amounts of tPA, but not uPA, activity. After hCG treatment, tPA activity showed a time-dependent increase, reaching a maximum at 24 h after injection. At 12 and 24 h after hCG treatment, uPA activity was also detected. The appearance of high molecular weight lysis zones further suggested the formation of plasminogen activator-inhibitor complexes. Morphological analysis indicated that the increases in oocyte tPA activity were correlated with the extent of cumulus cell expansion and dispersion. In denuded oocytes, tPA activity also progressively increased during the periovulatory period to a maximum at 24 h after hCG treatment. In contrast, neither uPA activity nor activator-inhibitor complex was detected. Secretion of the proteases was measured in the conditioned media of cumulus-oocyte complexes cultured for 24 h in vitro. Substantial increases in tPA release were found in complexes obtained at 8 and 12 h after hCG injection, with lower secretion from complexes obtained at 24 h after hCG treatment.(ABSTRACT TRUNCATED AT 250 WORDS)     

Types I and IV collagenolytic and plasminogen activator activities in preovulatory ovarian follicles

During ovulation, enzymatic degradation of the extracellular matrix occurs within and around the graafian follicles. In this study, the activities of several different proteolytic enzymes were measured in the culture media of follicles taken from pregnant mare serum gonadotropin (PMSG)-primed immature rats. At 52 h after PMSG, the follicles were cultured for 2 to 15 h in media with or without human chorionic gonadotropin (hCG). Type I collagenase activity in hCG-stimulated follicles gradually increased within 6 h to 3.3-fold above that of the controls. Relatively pure populations of granulosa cells produced type I collagenase to a similar extent. Likewise, type IV collagenase increased 3.8-fold by 6 h after exposure of the follicles to hCG. In contrast, plasminogen activator activity increased by 3.9-fold at 2 h after hCG, but was negligible at 4, 6, and 15 h after incubation. These results suggest that plasminogen activator may activate both type I and type IV collagenase in hCG-stimulated ovulatory follicles.     

Gonadotropin surge-induced up-regulation of the plasminogen activators (tissue plasminogen activator and urokinase plasminogen activator) and the urokinase plasminogen activator receptor within bovine periovulatory follicular and luteal tissue

This study examined the effect of the preovulatory gonadotropin surge on the temporal and spatial regulation of tissue plasminogen activator (tPA), urokinase plasminogen activator (uPA), and uPA receptor (uPAR) mRNA expression and tPA, uPA, and plasmin activity in bovine preovulatory follicles and new corpora lutea collected at approximately 0, 6, 12, 18, 24, and 48 h after a GnRH-induced gonadotropin surge. Messenger RNAs for tPA, uPA, and uPAR were increased in a temporally specific fashion within 24 h of the gonadotropin surge. Localization of tPA mRNA was primarily to the granulosal layer, whereas both uPA and uPAR mRNAs were detected in both the granulosal and thecal layers and adjacent ovarian stroma. Activity for tPA was increased in follicular fluid and the preovulatory follicle apex and base within 12 h after the gonadotropin surge. The increase in tPA activity in the follicle base was transient, whereas the increased activity in the apex was maintained through the 24 h time point. Activity for uPA increased in the follicle apex and base within 12 h of the gonadotropin surge and remained elevated. Plasmin activity in follicular fluid also increased within 12 h after the preovulatory gonadotropin surge and was greatest at 24 h. Our results indicate that mRNA expression and enzyme activity for both tPA and uPA are increased in a temporally and spatially specific manner in bovine preovulatory follicles after exposure to a gonadotropin surge. Increased plasminogen activator and plasmin activity may be a contributing factor in the mechanisms of follicular rupture in cattle.     

消炎痛对大鼠成熟滤泡破裂的影响——形态学观察

本工作的目的是,观察前列腺素合成的抑制物——消炎痛对大鼠排卵的抑制作用,并从形态学的角度对已排卵的以及排卵受阻的滤泡壁成份进行比较,藉此,探讨卵巢自身的前列腺素在滤泡破裂机制中的作用。25—27天龄Wistar大鼠用PMSG与HCG诱导成熟,其中部分动物作为对照,排卵率为100%;另一部分动物皮下注射消炎痛3.0mg/只,94%的动物被抑制排卵。处于动情前期的自然成熟鼠,注射消炎痛5mg/只或7.5 mg/只,排卵抑制率达100%,而对照鼠均排卵。从诱导成熟及自然成熟的大鼠卵巢组织切片所见,对照鼠已排卵滤泡的裂口处的白膜与膜层的结缔组织纤维断裂;被消炎痛抑制排卵的动物,卵母细胞因不能突破膜层或白膜而受阻。于电镜下见到,对照动物滤泡裂口附近泡壁的白膜细胞及外膜细胞出现退化、解体;与此同时,存在于这两种组织巾的胶元蛋白纤维也呈溶解现象。实验组排卵被阻的滤泡,白膜下与外膜细胞中间仍存在大量胶元蛋白纤维,因而使白膜与外膜层成为阻挡卵母细胞排出的屏障。另外,内膜与颗粒细胞的高尔基体发达,粗糙的内质网趋向光滑,说明这两类细胞分泌功能旺盛。关于前列腺素对胶元蛋白纤维降解的可能作用以及动物经消炎痛处理后仍产生类固醇激素的可能性均进行了讨论。     

Ultrastructural alterations associated with the initiation of follicular atresia

Summary To identify and describe ovarian follicles committed to undergo follicular degeneration (atresia), immature rats were primed with pregnant mare serum gonadotropin (PMSG). After PMSG treatment, preovulatory follicles develop but subsequently degenerate. Prior to the appearance of pyknotic nuclei (Stage I of atresia), degenerative changes were observed in focal areas of the granulosa cell layer. These changes include blebbing of the cytoplasm and alterations in the shape of the granulosa cells. The appearance of these degenerative changes coincides with a decrease in ovarian concentrations of estradiol and testosterone. Since estrogens and androgens maintain the follicle, the decline in estradiol and testosterone could be responsible for the further degenerative alterations that lead to complete deterioration of the preovulatory follicle. In Stage I atretic follicles, lysosome-derived autophagic vacuoles develop and macrophages invade both the thecal and granulosa cell layers. The combined actions of the autophagic vacuoles and macrophages could destroy both the granulosa-cell and thecal layers and thereby transform the preovulatory follicle into an ovarian cyst.     

Effect of indomethacin on ovarian follicle rupture--a morphological observation

The purpose of this investigation was to study the role played by indomethacin in blocking ovulation. Immature Wistar rats induced to maturation by PMSG and HCG and normal mature rats were used. Changes in follicle wall of preovulatory follicles occurred after indomethacin treatment were studied both by light and electron microscopy, and were compared with those in controls. 94% of PMSG and HCG stimulated rats, then followed indomethacin injection (3 mg/rat), were inhibited to ovulate; while rats only given hormonal stimulation ovulated in 100%. Adult females in proestrus were treated with indomethacin in doses either of 5 mg or 7.5 mg, none of them ovulated. Whereas, ova were found in the ampullae of normal controls. Ovarian histological examinations of indomethacin treated rats showed that ovum frequently went through the stratum granulosa, however, the theca or the albuginea failed to rupture. The electron microscopy examinations showed that a large amount of collagen fibers scattered under the albuginea layer and interwove with cells of albuginea and theca externa. These two layers, due to containing abundant collagen fibers, thus became barriers for an ovum escaping from a follicle. Follicle walls near the gap of ovulated follicles in controls only had a small quantity of collagen fibers which were more or less with obscure appearance. Cytolysis in albuginea and theca externa layers was also noted. Theca interna cells and granulosa cells, with well developed Golgi bodies and more smooth endoplasmic reticulum in experimental rats revealed that these two tissue components still had a normal endocrine function in spite of receiving indomethacin treatment. The possible effects of prostaglandins on degradation of collagen fibers and contraction of preovulatory follicles were also discussed.     

Secretion of cumulus expansion-enabling factor (CEEF) in porcine follicles

The objective of this study was to find out whether porcine cumulus and mural granulosa cells can secrete cumulus expansion-enabling factor (CEEF). Culture drops of M-199 medium were conditioned with denuded porcine oocytes (1 oocyte/μl), cumulus cells from oocytectomized complexes (1 OOX/μl), pieces of mural granulosa isolated from preantral to preovulatory follicles (1000 cells/μl), or oviductal cells (1000 cells/μl) for 24 hr. The production of CEEF was assessed by the addition of mouse OOX and follicle-stimulating hormone (FSH) (1 μg/ml) to microdrops of the conditioned medium. After 16–18 hr, expansion of the mouse OOX was scored on a scale of 0 to 4 by morphologic criteria. Mouse OOX did not expand in nonconditioned FSH-supplemented medium. Immature porcine oocytes produced +3 to +4 expansion of the mouse OOX. Granulosa cells isolated from preantral and early antral follicles and cumulus cells isolated from all stages of follicle development constitutively secreted CEEF under in vitro conditions. Mural granulosa cells of small, medium, and preovulatory (PMSG) follicles also secreted CEEF in vitro; however, FSH or leutenizing hormone (LH) stimulation was essential for this secretion. Hormonally induced secretion of CEEF was accompanied by expansion of the mural granulosa itself. Granulosa cells isolated from follicles of gilts 20 hr after PMSG and human chorionic gonadotropin (hCG) administration did not produce CEEF and did not expand in response to FSH and LH in vitro. CEEF activity also was found in the follicular fluid of small antral follicles, was reduced in medium follicles, and was not detectable in PMSG-stimulated follicles. However, CEEF activity was reestablished in the follicular fluid of preovulatory follicles by hCG injection, conceivably due to increased production of CEEF by cumulus cells. We conclude that (1) porcine cumulus and mural granulosa cells are capable of CEEF production in vitro and (2) autocrine secretion of CEEF by cumulus cells is involved in regulation of porcine cumulus expansion both in vitro and in vivo. Mol. Reprod. Dev. 49:141–149, 1998. © 1998 Wiley-Liss, Inc.     

Production of prostaglandins by porcine preovulatory follicular tissues and their roles in intrafollicular function

Prostaglandin production in vitro by theca and granulosa cells isolated from prepubertal pig ovaries was quantified in order to investigate the role of prostaglandins in intrafollicular function. Prepubertal gilts were slaughtered without treatment (O h, control) or treated with 1000 IU pregnant mare's serum gonadotropin (PMSG) and slaughtered at 36 or 72 h, or at 75 h following treatment with 500 IU of hCG at 72 h. Theca and granulosa cells were isolated from preovulatory follicles and cultured for 24 h alone or with follicle-stimulating hormone (FSH) or luteinizing hormone (LH). In vitro accumulation of 6-keto-prostaglandin F1 alpha (6-keto-PGF1 alpha), prostaglandin E2 (PGE2) and prostaglandin F2 alpha (PGF2 alpha) was measured by radioimmunoassay. On a per follicle basis theca produced more of each prostaglandin (approx. 10-fold) than granulosa at each stage of follicular development; production by each tissue type increased with development of the follicle, responding to administration of gonadotropin (PMSG) in vivo. Neither tissue type was generally responsive to further gonadotropin stimulation in vitro. However, production of PGE2 by granulosa cells was increased by addition of gonadotropin, particularly LH, in vitro, with the greatest response observed in tissue obtained at 36 and 72 h after PMSG. There were no functional correlates between prostaglandin production and steroidogenesis by either tissue type and we conclude that prostaglandins do not have an obligatory role in follicular steroidogenesis. However, these data provide additional circumstantial evidence for a role of PGE2 in granulosa cell luteinization, and possibly in ovulation. The data also indicate that prostaglandins derived from thecal tissue in relatively large quantities may play an important role in ovulation.     

Expression of basigin, an inducer of matrix metalloproteinases, in the rat ovary

The extensive tissue remodeling that occurs during follicular development, ovulatory rupture, and the formation and regression of the corpus luteum (CL) requires local degradation of the extracellular environment by matrix metalloproteinases (MMPs). This report characterizes the expression pattern of basigin (Bsg), a putative regulator of MMP induction, in the rat ovary. An induced superovulation model (eCG/hCG) was used in immature rats to evaluate Bsg expression profiles in ovaries collected during the follicular phase, the preovulatory period, and the luteal lifespan. Levels of Bsg mRNA were unchanged through follicular growth (0-48 h post-eCG) and increased during postovulatory luteinization (24 and 48 h post-hCG; P < 0.01). Bsg expression persisted into pseudopregnancy (4-8 days post-hCG) and after functional luteal regression (12 days post-hCG). The profile of Bsg expression during regression of the CL was examined using a model of induced luteolysis. Both functional and structural regression was associated with a decline in Bsg expression levels. Bsg mRNA and protein localized to the theca of preovulatory follicles (12 h post-hCG) and formative and functional CL (24 h-8 days post-hCG). Bsg expression profiles in the induced ovulation and CL regression models were similar to observations made in naturally cycling mature rats. In the cycling ovary, Bsg signaling localized to newly forming CL, the theca of preovulatory follicles, and appeared to be lower in CL from previous estrous cycles. A putative regulatory mechanism of Bsg expression was identified using an in vitro model; treatment of cultured granulosa cells with hCG significantly augmented Bsg mRNA expression levels. The processes of ovulation and luteogenesis may be facilitated by Bsg expression and its induction or regulation of the MMPs.     

Immunocytochemical localization of aromatase in immature rat ovaries treated with PMSG and hCG, and in pregnant rat ovaries

Immunocytochemical localization of aromatase cytochrome P-450 was examined in immature rat ovaries treated with pregnant mare serum gonadotropin (PMSG) and human chorionic gonadotropin (hCG), and in pregnant rat ovaries. It is well known that PMSG and hCG treatments induce ovulation about 12 h after hCG injection. At 24 h after hCG injection, many antral follicles were recognized in immature rat ovaries and only the granulosa cells in the antral follicles were stained weakly with the anti-aromatase antibody. At 0 to 9 h after hCG injection, in addition to the antral follicles, some large Graafian follicles could be observed in the rat ovaries, and the granulosa cells of these follicles were positively stained for aromatase. Each follicle was surrounded by the basal lamina which shows lineally distinct positive reaction against anti-laminin antibody. At 12 h after hCG injection, some large Graafian follicles without oocyte were weakly positive to the anti-aromatase antisera, and the outline of their basal lamina stained with anti-laminin antibody became irregular in shape and fragmentous. At 15 to 18 h after hCG injection, the luteinized cysts could be seen, and the granulosa-lutein cells of these cysts were almost negative for aromatase. Fragmentous reaction to the anti-laminin antibody was observed around the luteinized cysts. In the ovaries of day 4 in pregnancy, only the granulosa cells of the large antral follicles were weakly stained, but corpora lutea negatively reacted to the anti-aromatase antibody. At 7 to 19 days in gestation, both the granulosa cells of antral follicles and pregnant luteal cells were positively stained against aromatase antisera.(ABSTRACT TRUNCATED AT 250 WORDS)     

Induction of maturation in follicle-enclosed oocytes: the response to gonadotropins at different stages of follicular development

Antral follicles, isolated from either nontreated or pregnant mare's serum gonadotropin (PMSG)-primed 27-day-old rats, were incubated in the absence or the presence of either luteinizing hormone (LH), follicle-stimulating hormone (FSH), or forskolin. The effect of these agents on oocyte maturation and cyclic adenosine 3',5'-monophosphate (cAMP) accumulation was studied and compared. Both gonadotropins, LH and FSH, as well as forskolin, effectively induced maturation of oocytes enclosed by large antral follicles isolated from PMSG-primed rats. On the other hand, we found that maturation of oocytes enclosed by small antral follicles, isolated from nonprimed and PMSG-primed rats, could be induced by either FSH or forskolin but not by LH. cAMP determinations revealed that, in spite of the inability of LH to induce oocyte maturation, elevated concentrations of the nucleotide were detectable in small antral follicles exposed to this gonadotropin. Since granulosa cells isolated from the large but not the small antral follicles were stimulated by LH to generate cAMP, the elevation of cAMP concentrations in the small antral follicle apparently represented the response of the theca cells to this gonadotropin. Since it is the ability of the granulosa cells to interact with the hormone that determines whether or not oocyte maturation will occur, we suggest that the granulosa, but not the theca cells, mediate LH action to induce oocyte maturation.     

Immunocytochemical Localization of Aromatase in The Ovary of Superovulated Cattle,Pigs and Sheep

The localization of aromatase, an enzyme converting androgens to estrogen, in the ovaries of superovulated cattle, pigs and sheep was studied immunocytochemically in the preovulatory and postovulatory period using anti-human placental aromatase cytochrome P-450 antiserum. Immunostaining for aromatase was detected in the granulosa cells of preovulatory follicles of all species studied. Theca interna cells were stained in preovulatory follicles in the pig but not in cattle and sheep. Interstitial gland cells, cumulus cells and oocytes were unstained in all species. In cattle and pig the corpora lutea were unstained whereas they displayed staining in the sheep. Preantral and small antral follicles were unstained during both the preovulatory and postovulatory period in all species. It is concluded that granulosa cells of preovulatory follicles are the main residence for aromatase activity in superovulated cattle, pig and sheep, whereas the activity of theca interna and corpora lutea is species specific.     

Immunocytochemical localization of aromatase in immature rat ovaries treated with PMSG and hCG,and in pregnant rat ovaries

Summary Immunocytochemical localization of aromatase cytochrome P-450 was examined in immature rat ovaries treated with pregnant mare serum gonadotropin (PMSG) and human chorionic gonadotropin (hCG), and in pregnant rat ovaries. It is well known that PMSG and hCG treatments induce ovulation about 12 h after hCG injection.At 24 h after hCG injection, many antral follicles were recognized in immature rat ovaries and only the granulosa cells in the antral follicles were stained weakly with the anti-aromatase antibody. At 0 to 9 h after hCG injection, in addition to the antral follicles, some large Graafian follicles could be observed in the rat ovaries, and the granulosa cells of these follicles were positively stained for aromatase. Each follicle was surrounded by the basal lamina which shows lineally distinct positive reaction against anti-laminin antibody. At 12 h after hCG injection, some large Graafian follicles without oocyte were weakly positive to the anti-aromatase antisera, and the outline of their basal lamina stained with anti-laminin antibody became irregular in shape and fragmentous. At 15 to 18 h after hCG injection, the luteinized cysts could be seen, and the granulosa-lutein cells of these cysts were almost negative for aromatase. Fragmentous reaction to the anti-laminin antibody was observed around the luteinized cysts.In the ovaries of day 4 in pregnancy, only the granulosa cells of the large antral follicles were weakly stained, but corpora lutea negatively reacted to the anti-aromatase antibody. At 7 to 19 days in gestation, both the granulosa cells of antral follicles and pregnant luteal cells were positively stained against aromatase antisera. The luteal cells were increased in size during pregnancy. And weakly positive reaction was detected on day 7 of pregnancy, then the immunoreaction became stronger in the corpora lutea on day 15 and 19 of pregnancy.The localization of aromatase was immunocytochemically examined in immature rat ovaries treated with PMSG and hCG injection, and the reaction of the granulosa cells of the antral follicles against anti-aromatase antibody became strongly positive about 12 h before ovulation and the became very weak suddenly after ovulation. In rat-ovaries, the pregnant corpora lutea was positively stained for aromatase after day 7 of pregnancy.This study was supported by Grants from the Ministry of Education, Science and Culture, Japan, and from USPHS Research Grants HD04945, USA     

Expression of link protein during mouse follicular development.

To gain insight into the role of link protein in ovarian follicle development, we used immunohistochemistry to determine the patterns of link protein expression in mouse ovary in response to gonadotropin stimulation. Polyclonal antibodies were raised against link protein purified from bovine cartilage. Stimulation of immature mice with gonadotropins increased link protein expression in the granulosa layer of large preovulatory follicles. The number and intensity of immunostained cells increased over 2 hr after hCG injection. Cumulus cells stained link protein mainly in the extracellular matrix, whereas mural granulosa cells showed marked deposits of link protein in the cytoplasm. Link protein expression persisted in luteinized granulosa cells after ovulation and in corpora lutea. Link protein staining was also present in the theca cells and oocytes, which was a consistent finding regardless of gonadotropin treatment. The staining intensity was negated by treatment with hyaluronidase, suggesting that the link protein is bound to hyaluronic acid. On Western blotting, a reacting protein species of about 42 kD was seen in the gonadotropin-treated ovarian extract. The precise cellular distribution of link protein in mouse ovary was determined for the first time by an immunohistochemical method in this study. (J Histochem Cytochem 47:1433-1442, 1999)     

Androgen synthesis during follicular development: evidence that rat granulosa cell 17-ketosteroid reductase is independent of hormonal regulation

Although androgens have been implicated in follicular atresia, ovarian follicular androgen synthesis is required for preovulatory follicular growth. To localize the site(s) of androgen biosynthesis and to obtain a better understanding of the regulation of the androgenic pathway(s) in rat ovarian follicles we examined the relative abilities of developing follicles to accumulate specific androgens [testosterone (T) and dihydrotestosterone (DHT)] using both radioimmunoassay (RIA) and 3H-substrate metabolism techniques. Small antral and preovulatory follicles were obtained from control or human chorionic gonadotropin (hCG)-primed immature rats, respectively (Richards and Bogovich, 1982). Small antral follicles, theca and granulosa cells produced little immunoassayable androgen (T + DHT) when incubated with or without 8-bromo-cAMP. In contrast, preovulatory follicles and theca produced more androgen than small antral tissues and in a manner acutely stimulable by cAMP. Granulosa cells produced little androgen under these conditions. Inclusion of [3H] androstenedione in the incubates yielded increased accumulation of [3H] T and [3H] DHT for all small antral and preovulatory tissues. Indeed, granulosa cells from both small antral and preovulatory follicles possessed a remarkable ability to accumulate [3H] T. This ability was not altered by hypophysectomy or subsequent treatment with estradiol and/or follicle-stimulating hormone (FSH). These results suggest that 17-ketosteroid reductase may be a constitutive enzyme in granulosa cells.