Differential effects of amount of feeding on cell proliferation and progesterone production in response to gonadotrophins and insulin-like growth factor I by ovarian granulosa cells of broiler breeder chickens selected for fatness or leanness.

Strain differences in reproductive performance were demonstrated between broiler breeder female chickens selected for growth (GL line) or for food conversion efficiency (FC line) and the improvement in reproductive performance due to feed restriction also differed significantly. Feed allowance effects on the maturation of ovarian follicles, the incidence of atresia and egg production differed between the two lines exposed to similar feeding protocols. Feed restriction reduced body weights significantly and to a similar extent in both GL and FC lines. The number of normal and atretic yellow follicles was significantly higher under ad libitum feeding and in GL line than it was in the FC line. In both lines, feed restriction decreased multiple ovulation and increased egg production. In culture, granulosa cells from the three largest follicles (F1, F2 and F3) increased progesterone production in response to LH, FSH and insulin-like growth factor I but responses were different between the GL and FC lines fed either ad libitum or restricted diets. Granulosa cells from the two or three largest follicles in GL and FC (ad libitum) lines produced similar amounts of progesterone in response to LH, FSH and insulin-like growth factor I whereas, in restricted birds, the progesterone production was of the rank order F1 > F2 > F3 in both lines. The responsiveness of the GL line fed ad libitum was higher for LH than for either FSH or insulin-like growth factor I but in the GL line fed a restricted diet, it was high for all the hormones. In the FC line, responses to LH, FSH or insulin-like growth factor I were high in ad libitum-fed birds, but low in birds fed a restricted diet for all hormones. Insulin-like growth factor I combined with LH or FSH significantly increased the progesterone production of granulosa cells from birds fed restricted diets of both lines and this effect increased with increasing follicular size. There was a lack of interaction between insulin-like growth factor I and LH or FSH in the regulation of progesterone production by birds of both lines fed ad libitum. Insulin-like growth factor alone or in combination with LH or FSH increased granulosa cell proliferation in birds fed ad libitum more than it did in birds fed restricted diets. The greater proliferation rate of granulosa cells of chickens fed ad libitum, in response to insulin-like growth factor I alone or in combination with gonadotrophins, leading to the simultaneous differentiation of two or three large follicles with high progesterone production in response to LH or insulin-like growth factor I, accelerates the rate of maturation of follicles. This may also be the major cause of erratic and multiple ovulations in broiler breeder female chickens fed ad libitum. In conclusion, insulin-like growth factor I, alone or in combination with LH or FSH, is an important component in the control mechanisms for follicular development in broiler breeder hens. It is this component that is targeted by feed allowance and inadvertently altered by selection for growth.     

Evidence for gonadotropin-releasing hormone receptor mRNA expression by estrogen in rat granulosa cells

The hormonal regulation of ovarian gonadotropin-releasing hormone (GnRH) receptor mRNA expression has been examined by in situ hybridization in hypophysectomized immature rats. In hypophysectomized rats, GnRH receptor mRNA expression is localized in the interstitial cells. After diethylstilbestrol treatment, most follicles grow to form early antral follicles and express GnRH receptor mRNA in the peripheral part of the granulosa layer, indicating that the expression in the growing follicles is estrogen-dependent. Only weak or no expression of the receptor mRNA is detectable in the atretic follicles of hypophysectomized rats, whereas very strong expression has been observed in the granulosa cells of atretic follicles of intact immature rats. Administration of testosterone or a GnRH agonist, both of which are atretic agents for ovarian follicles, to hypophysectomized rats markedly increases the apoptotic cell death of the granulosa cells but fails to induce GnRH receptor mRNA expression. The co-administration of these agents with diethylstilbestrol causes the granulosa cells of atretic follicles to express the receptor mRNA very strongly, suggesting that this mRNA expression in the atretic follicles is also estrogen-dependent. On the other hand, expression of the receptor mRNA in the ovarian interstitial cells is not affected by hypophysectomy or hormone treatments. All of these results clearly indicate that estrogen is essential for the expression of ovarian GnRH receptor mRNA in the granulosa cells of atretic follicles and growing follicles, whereas the expression in the interstitial cells is estrogen-independent.     

Regulation of matrix metalloproteinase-19 messenger RNA expression in the rat ovary

Matrix metalloproteinases (MMPs) are instrumental in the constant tissue remodeling in the ovary. An induction of MMP-19 mRNA in periovulatory follicles has been reported in mouse ovaries. However, little is known about MMP-19 expression during the follicular and luteal periods or about the ovarian regulation of MMP-19 mRNA expression. We examined the expression pattern of MMP-19 mRNA during various reproductive phases and the periovulatory regulation of MMP-19 mRNA in the rat ovary. In gonadotropin-primed, immature rat ovaries, levels of MMP-19 mRNA transiently increased during both follicular growth and ovulation. The MMP-19 mRNA was localized to the theca-interstitial layer of growing follicles and to the granulosa and theca-interstitial layers of periovulatory follicles. A similar expression pattern of MMP-19 mRNA in periovulatory follicles was observed in ovaries from naturally cycling adult rats. Accumulation of MMP-19 mRNA was detected in regressing corpus luteum. The regulation of MMP-19 mRNA expression during the periovulatory period was investigated via in vivo studies and through in vitro culture studies on follicular cells. The hCG-induction of MMP-19 mRNA was mimicked by treating granulosa cells, but not theca-interstitial cells, from preovulatory follicles with LH or activators of the protein kinase (PK) A or PKC pathways. Cycloheximide blocked the LH- or forskolin-induced MMP-19 mRNA expression, demonstrating the requirement for new protein synthesis. In contrast, blocking activation of the progesterone receptor or prostaglandin synthesis had no effect on the increase in MMP-19 mRNA expression. In conclusion, the induction of MMP-19 mRNA suggests an important role of this proteinase during follicular growth, ovulation, and luteal regression.     

mRNA expression pattern of gonadotropin receptors in bovine follicular cysts

Follicular growth and steroidogenesis are dependent on gonadotropin binding to their receptors in granulosa and theca cells of ovarian follicles. The aim of the present study was to evaluate the expression patterns of follicle-stimulating hormone receptor (FSHR) and luteinizing hormone receptor (LHCGR) in ovarian follicular structures from cows with cystic ovarian disease (COD) as compared with those of regularly cycling cows. Relative real-time RT-PCR analysis showed that the expression of FSHR mRNA in granulosa cells was highest in small antral follicles, then decreased significantly as follicles increased in size, and was lowest in cysts. FSHR mRNA was not detected in the theca cells of any follicular category, including cysts. LHCGR mRNA expression in granulosa cells was significantly higher in large antral follicles than in cysts, and not detected in granulosa cells of small and medium antral follicles. In theca cells, the expression level of LHCGR mRNA in medium antral follicles was higher than in small and large antral follicles, whereas that in follicular cysts it was similar to those in small and medium antral follicles, but higher than that in large antral follicles. Our findings provide evidence that there is an altered gonadotropin receptor expression in bovine cystic follicles, and suggest that in conditions characterized by altered ovulation, such as COD, changes in the signaling system of gonadotropins may play a fundamental role in their pathogenesis.     

IGF-Ⅰ及其受体、IGF结合蛋白-2和LH受体mRNA在卵泡中的表达

利用原位杂交和原位DNA－3’末端标记的方法研究了胰岛素样生长因子河（IG－I）、IGF－I受体、IGF结合蛋白－2、和促性腺激素受体的信使核糖核酸（mRNA）在不同生长与闭锁阶段的大鼠卵巢卵泡中表达的变化。结果表明：IGF－I主要在正常生长的初级卵泡、窦前卵泡和小窦状卵泡中表达。在各生长与成熟阶段的卵泡中都检测到IGF－I受体mRNA,闭锁卵泡的IGF－I受体表达降低。窦前与窦状的生长和闭锁卵泡均表达IGFBP－2。促卵泡激素（FSH）受体在窦前和小窦状卵泡的表达水平比其在大卵泡中的高。窦前与小窦状卵泡仅在膜细胞中表达黄体生成素（LH）受体mRNA,大卵泡的膜细胞与颗粒细胞均表达LH受体,在闭锁卵泡中仅在膜细胞中观察到LH受体的信号。综上结果,提示IGF－I,IGF－I受体和FSH受体在窦前和小窦状卵泡中的协同表达对卵泡的早期发育有重要作用。LH受体mRNA特异地在大卵泡的颗粒细胞中表达可能与优势卵泡选择相关。     

Ovarian follicle vascularization in fasted pig

The authors have investigated in the different classes of ovarian follicles the vascular area, the blood vessel distribution, the vascular endothelial growth factor (VEGF) mRNA expression and the VEGF secretion during equine chorionic gonadotropin (eCG) induced follicle growth in prepubertal gilts fed ad libitum or fasted. Immunohistochemistry staining of Von Willebrand factor showed that fasting caused a dramatic increase in the vascular area of medium-large tertiary follicles. The increase involved the two concentric vessel networks and the area between them that, becoming crossed by several anastomosis, modified the whole vessel architecture. Both in situ hybridization and in vitro culture experiments demonstrate that granulosa cells from medium-large follicles are engaged in a copious VEGF production upon eCG stimulation both in gilts fed ad libitum or fasted. More surprisingly, the production of VEGF becomes diffuse amongst theca cells of fasted animals thus recruiting a compartment that in condition of normal feeding regimen appears nearly quiescent. In conclusion, the data presented describe a local angiogenic process that develops in the follicle wall of growing antral follicle in case of acute severe food restriction. The mechanism, essentially confined to follicles that potentially approach ovulation, appears to assume the meaning of a local compensatory mechanism that may help maintaining adequate nutrient delivery to follicles that undergo ovulation.     

Differential effect of long-term food restriction on fatty acid synthase and leptin gene expression in rat white adipose tissue.

Long-term food restriction (85%, 70% and 50% of ad libitum energy intake for one month) induced a substantial fall in serum leptin concentration and leptin mRNA levels in epididymal white adipose tissue in rats. Surprisingly, this suppression was not reversed by refeeding ad libitum for 48 h. The reduction in serum leptin concentration and leptin mRNA level did not strictly correlate with reduction in fat or body mass. Unlike serum leptin concentration and epididymal adipose tissue leptin mRNA levels, fatty acid synthase activity, fatty acid synthase protein abundance and fatty acid synthase mRNA levels increased significantly in white adipose tissue after refeeding rats subjected to food restriction. The increase in serum insulin concentration was observed in all groups on different degrees of food restriction and refed ad libitum for 48 h compared to controls. A decrease in serum insulin concentration was found in the rats not refed before sacrifice. Long-term food restriction did not significantly affect serum glucose concentrations in either refed or non-refed rats. The data reported in this paper indicate that there is no rapid rebound in serum leptin concentration or leptin gene expression in contrast to the increase in serum insulin concentration and fatty acid gene expression in white adipose tissue of rats refed ad libitum after one month's food restriction.     

Thrombin generation and presence of thrombin receptor in ovarian follicles

Prothrombin, once converted to its enzymatically active form (i.e., thrombin), induces a broad spectrum of cellular responses in both vascular and avascular tissues. Bovine ovarian granulosa cells isolated from healthy follicles of various sizes contain both prothrombin mRNA and immunologically reactive prothrombin that appears to be identical to prothrombin in follicular fluid and plasma. When tissue factor, the primary physiological activator of thrombin generation in plasma, is used to initiate thrombin formation, the profile of prothrombin-to-thrombin conversion is similar in follicular fluid and plasma. The conclusion that biologically functional prothrombin is synthesized by granulosa cells is further supported by evidence that mRNA for gamma-glutamyl carboxylase, an enzyme essential for the vitamin K-dependent posttranslational modification of prothrombin, is expressed in granulosa cells in a manner similar to prothrombin mRNA. Thrombin's biological effects are mediated through selective proteolytic cleavage and activation of specific receptors. Bovine granulosa cells possess thrombin receptor (PAR-1) mRNA, and as seen with prothrombin mRNA and gamma-glutamyl carboxylase mRNA, cells isolated from small follicles possess more PAR-1 mRNA than cells from large follicles. Thrombin receptor expression by cells in close proximity to an active thrombin-generating system suggests that these factors may be important mediators of cellular function in the ovarian follicle.     

Estrogen receptor protein and mRNA expression in the ovary of sheep

Using immunohistochemistry and in situ hybridization, we attempted to identify the estrogen receptor (ER) protein and messenger RNA (mRNA) in sheep ovaries during the follicular phase of the estrous cycle. Monoclonal anti-ER antibodies H222 and 1D5 were used for localizing estrogen receptor on ovarian cryo-sections. Labeling for ER was found over the nuclei of surface epithelium, interstitial tissue, and granulosa cells of small as well as large ovarian follicles. In the preantral and small antral follicles, intense nuclear ER labeling was observed in mural granulosa cells and particularly in cumulus/granulosa cells surrounding the oocyte. In the large healthy looking follicles, greater diversity in labeling for ER was observed, which is characterized by mixed populations of granulosa cells expressing positive and more or less negative nuclear labeling. Such a pattern of labeling was particularly evident in follicles showing the signs of atresia. Generally, more intense nuclear staining was localized in granulosa cells proximal to basal membrane. In situ hybridization studies revealed the presence of ER mRNA in ovarian tissue. Autoradiographic visualization localized ER mRNA expression over the granulosa cells of healthy follicles of all sizes. Level of hybridization signal was comparable in mural and cumulus granulosa cells. In atretic follicles, the level of hybridization signal in granulosa cells was comparable to that of healthy follicles. A relatively weaker level of labeling was observed in granulosa cells dispersed in follicular antrum in follicles with advanced atretic lesions. Theca cells expressed a lower level of labeling than granulosa cells. Specificity of labeling for both ER protein and mRNA in ovary was proved by parallel probing the ovine uterus. Ovine ER recognition by both H222 and 1D5 antibodies was also proved by immunoblotting. These studies demonstrate the presence of the estrogen receptor and its messenger RNA in the sheep ovary and suggest an autocrine/paracrine role of estradiol and its receptor in the regulation of ovarian follicle development in sheep. Mol. Reprod. Dev. 48:53–62, 1997. © 1997 Wiley-Liss, Inc.     

Effect of Monochromatic Light on Expression of Estrogen Receptor (ER) and Progesterone Receptor (PR) in Ovarian Follicles of Chicken

Artificial illumination is widely used in modern poultry houses and different wavelengths of light affect poultry production and behaviour. In this study, we measure mRNA and protein abundance of estrogen receptors (ERs) and progesterone receptors (PRs) in order to investigate the effect of monochromatic light on egg production traits and gonadal hormone function in chicken ovarian follicles. Five hundred and fifty-two 19-wk-old laying hens were exposed to three monochromatic lights: red (RL; 660 nm), green (GL; 560 nm), blue (BL; 480 nm) and control cool white (400–760 nm) light with an LED (light-emitting diode). There were 4 identical light-controlled rooms (n = 138) each containing 3 replicate pens (46 birds per pen). Water was supplied ad libitum and daily rations were determined according to the nutrient suggestions for poultry. Results showed that under BL conditions there was an increase in the total number of eggs at 300 days of age and egg-laying rate during the peak laying period. The BL and GL extended the duration of the peak laying period. Plasma melatonin was lowest in birds reared under BL. Plasma estradiol was elevated in the GL-exposed laying hens, and GL and BL increased progesterone at 28 wk of age. In the granulosa layers of the fifth largest preovulatory follicle (F5), the third largest preovulatory follicle (F3) and the largest preovulatory follicle (F1), ERα mRNA was increased by BL and GL. Treatment with BL increased ERβ mRNA in granulosa layers of F5, F3 and F1, while GL increased ERβ mRNA in F5 and F3. There was a corresponding increase in abundance of the proteins in the granulosa layers of F5, with an increase in PR-B, generated via an alternative splice site, relative to PR-A. Treatment with BL also increased expression of PR mRNA in all of the granulosa layers of follicles, while treatment with GL increased expression of PR mRNA in granulosa layers of SYF(small yellow follicle), F5 and F1. These results indicate that blue and green monochromatic lights promote egg production traits via stimulating gonadal hormone secretion and up-regulating expression of ERs and PRs. Changes in PR-B protein suggest that this form of the progesterone receptor is predominant for progesterone action in the granulosa layers of preovulatory follicles in chickens during light stimulation.     

Localization of an activin/activin receptor system in the porcine ovary.

The aim of this study was to locate a possible activin/activin receptor system within porcine ovaries containing functional corpora lutea. In situ hybridization was used to assess the gene expression of beta(A)- and beta(B)-activin subunits, and immunohistochemical studies were done to detect activin-A protein and activin receptor type II. mRNA expression of the beta(A)- and beta(B)-activin subunits was found in the granulosa from the unilaminar follicle stage onward, in the developing thecal layer of multilaminar and small antral follicles, in the theca interna of mid-sized antral follicles, in corpora lutea, and in the ovarian surface epithelium. Immunoreactive activin A protein could be detected at the same ovarian sites, but in thecal tissue of small antral follicles only. This protein was also demonstrated at the peripheral zone of oocytes from multilaminar and antral follicles. A positive immunoreaction for activin receptor was found in granulosa cells from multilaminar and older follicles and in oocytes from the earliest stages of follicular development onward. In late multilaminar follicles and in antral follicles, the oolemma was stained. Except for small antral follicles, a positive activin receptor immunoreaction was absent in the follicular theca. Activin receptor immunoreaction was furthermore present in corpora lutea and in the ovarian surface epithelium. It is concluded that, within porcine ovaries containing functional corpora lutea, an activin/activin receptor system is present in all intact follicles, the corpora lutea and the surface epithelium. Within follicles, granulosa and theca cells are the main sites of activin synthesis, while oocytes and granulosa cells are the main activin binding sites.     

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.     

Insulin-like growth factor binding protein 4 expression parallels luteinizing hormone receptor expression and follicular luteinization in the primate ovary

It has been suggested that locally produced insulin-like growth factor binding protein 4 (IGFBP4) inhibits ovarian follicular growth and ovulation by interfering with IGF action. According to this hypothesis, IGFBP4-expressing follicles should demonstrate atresia, whereas healthy dominant follicles should be devoid of IGFBP4. Alternatively, according to this view, there could be constitutive expression of the inhibitory IGFBP4 but selective expression of an IGFBP4 protease in dominant follicles, allowing the follicle to mature and ovulate because of degradation of the binding protein. To examine these views concerning the role of IGFBP4 in primate follicular selection, we analyzed cellular patterns of IGFs 1 and 2, IGFBP4, and the IGFBP4 protease (pregnancy-associated plasma protein A [PAPP-A]) mRNA expression in ovaries from late follicular phase rhesus monkeys using in situ hybridization. The IGF1 mRNA was not detected, but the IGF2 mRNA was abundant in theca interna and externa of all antral follicles and was present in the granulosa of large preovulatory and ovulatory follicles. The IGFBP4 mRNA was selectively expressed by LH receptor (LHR) mRNA-positive theca interna cells of healthy antral follicles (defined by aromatase and gonadotropin receptor expression) and by LHR-expressing granulosa cells found only in large preovulatory and ovulatory follicles (defined by size and aromatase expression). The PAPP-A mRNA was abundant in granulosa cells of most follicles without obvious relation to IGFBP4 expression. Ovarian IGFBP4 mRNA levels were markedly increased after treatment with the LH analog, hCG, whereas IGF2 and PAPP-A mRNAs were not significantly altered. In summary, IGFBP4 expression appears to be associated with follicular selection, not with atresia, in the monkey ovary. The IGFBP4 is consistently expressed in healthy theca interna and in luteinized granulosa cells, likely under LH regulation. The IGFBP4 protease, PAPP-A, is widely expressed without apparent selectivity for IGFBP4-expressing follicles or for dominant follicles. These observations suggest that IGFBP4 or an IGFBP4 proteolytic product may be involved with LH-induced steroidogenesis and/or luteinization rather than with inhibition of follicular growth.     

Expression of mRNA of lipoprotein receptor related protein 8, low density lipoprotein receptor, and very low density lipoprotein receptor in bovine ovarian cells during follicular development and corpus luteum formation and regression

Lipoproteins in the plasma are the major source of cholesterol obtained by the ovarian theca and granulosa cells for steroidogenesis. In this study, we have identified mRNA expression in bovine theca and granulosa cells of two lipoprotein receptors, low density lipoprotein receptor (LDLr) and very low density lipoprotein receptor (VLDLr) in granulosa cells from small antral follicles through preovulatory follicles and in theca cells from large and medium sized antral follicles. In the corpus luteum (CL) both these receptors were found in the developing and differentiating stages whereas only mRNA for VLDLr was detected in the regression stage. This study also described for the first time, the presence of lipoprotein receptor related protein (LRP8) in granulosa cells from small antral follicles through preovulatory follicles and in theca cells from large and medium sized antral follicles. This may indicate a role of LRP8 in cholesterol delivery to steriodogenic cells. LRP8 was not detected in any of the CL stages. The roles of the LDLr superfamily in lipid transport to ovarian cells and its participation in follicular and CL development and regression is discussed.