Comparison of effects of leptin and ghrelin on porcine ovarian granulosa cells

The aim of our studies was to compare the roles of leptin and ghrelin in the direct control of proliferation, apoptosis, and secretory activity by porcine ovarian cells. In our in vitro experiments, we analyzed the effects of leptin and ghrelin treatments (at 0, 1, 10, or 100 ng/mL medium) on the accumulation of proliferation-related peptides (PCNA, cyclin B1, MAP kinase [MAPK]) and apoptosis-associated peptides (Bax, caspase 3, p53), and on progesterone secretion by cultured porcine granulosa cells, using immunocytochemistry, SDS PAGE-Western immunoblotting, and radioimmunoassay (RIA). Leptin stimulated proliferation (PCNA, cyclin B1, MAPK), apoptosis (Bax, p53), and progesterone secretion. Ghrelin promoted proliferation (PCNA, cyclin B1, MAPK) and progesterone secretion but suppressed apoptosis (Bax, caspase 3, p53). These observations suggest that both leptin and ghrelin directly control proliferation, apoptosis, and secretory activity by porcine ovarian cells. At the level of the ovary, in contrast to the hypothalamo-hypophysial system, leptin and ghrelin may have similar action in promoting granulosa cell proliferation and progesterone secretion, but they may be antagonistic to one another (leptin, stimulator; ghrelin, inhibitor) in controlling apoptosis.     

Effects of ghrelin and its analogues on chicken ovarian granulosa cells

The aim of these in vitro experiments was (1) to examine the effects of ghrelin on the basic functions of ovarian cells (proliferation, apoptosis, secretory activity); (2) to determine the possible involvement of the GHS-R1a receptor and PKA- and MAPK-dependent post-receptor intracellular signalling cascades; (3) to identify the active part of the 28-amino acid molecule responsible for the effects of ghrelin on ovarian cells. We compared the effect of full-length ghrelin 1-28, a synthetic activator of GHS-R1a, GHRP6, and ghrelin molecular fragments 1-18 and 1-5 on cultured chicken ovarian cells. Indices of cell apoptosis (expression of the apoptotic peptide bax and the anti-apoptotic peptide bcl-2), proliferation (expression of proliferation-associated peptide PCNA), and expression of protein kinases (PKA and MAPK) within ovarian granulosa cells were analysed by immunocytochemistry. The secretion of progesterone (P(4)), testosterone (T), estradiol (E(2)) and arginine-vasotocin (AVT) by isolated ovarian follicular fragments was evaluated by RIA/EIA. It was observed that accumulation of bax was increased by ghrelin 1-28, GHRP6 and ghrelin 1-18, but not by ghrelin 1-5. Expression of bcl-2 was suppressed by addition of ghrelin 1-28, GHRP6 and ghrelin 1-5, but promoted by ghrelin 1-18. The occurrence of PCNA was reduced by ghrelin 1-28, GHRP6, ghrelin 1-18 and ghrelin 1-5. An increase in the expression of MAPK/ERK1, 2 was observed after addition of ghrelin 1-28, GHRP6 and ghrelin 1-18, but not ghrelin 1-5. The accumulation of PKA decreased after treatment with ghrelin 1-28 and increased after treatment with GHRP6 and ghrelin 1-18 but not ghrelin 1-5. Secretion of P(4) by ovarian follicular fragments was decreased after addition of ghrelin 1-28 or ghrelin 1-5 but stimulated by GHRP6 and ghrelin 1-18. Testosterone secretion was inhibited by ghrelins 1-28 and 1-18, but not by GHRP6 or ghrelin 1-5. Estradiol secretion was reduced after treatment with ghrelin 1-28 but stimulated by ghrelins 1-18 and 1-5; GHRP6 had no effect. AVT secretion was stimulated by ghrelin 1-28, GHRP6 and ghrelin 1-18, but inhibited by ghrelin 1-5. The comparison of the effects of the four ghrelin analogues on nine parameters of ovarian cells suggest (1) a direct effect of ghrelin on basic ovarian functions-apoptosis, proliferation, steroid and peptide hormone secretion; (2) that the majority of these effects can be mediated through GHS-R1a receptors; (3) an effect of ghrelin on MAPK- and PKA-dependent intracellular mechanisms, which can potentially mediate the action of ghrelin at the post-receptor level; (4) that ghrelin residues 5-18 may be responsible for the major effects of ghrelin on the avian ovary.     

The isoflavone daidzein directly affects porcine ovarian cell functions and modifies the effect of follicle‐stimulating hormone

The key biological active molecule of soya is the isoflavone daidzein, which possesses phytoestrogenic activity. The direct effect of soya and daidzein on ovarian cell functions is not known. This study examined the effect of daidzein on basic porcine ovarian granulosa cell functions and the response to follicle‐stimulating hormone (FSH). We studied the effects of daidzein (0, 1, 10 and 100 μm ), FSH (0, 0.01, 0.1, 1 IU/ml) and combinations of FSH (0, 0.01, 0.1, 1 IU/ml) + daidzein (50 μm ) on proliferation, apoptosis and hormone release from cultured porcine ovarian granulosa cells and ovarian follicles. The expression of a proliferation‐related peptide (PCNA) and an apoptosis‐related peptide (Bax) was analysed using immunocytochemistry. The release of progesterone (P4) and testosterone (T) was detected using EIA. Leptin output was analysed using RIA. Daidzein administration increased granulosa cell proliferation, apoptosis and T and leptin release but inhibited P4 output. Daidzein also increased T release and decreased P4 release from cultured ovarian follicles. Follicle‐stimulating hormone stimulated granulosa cell proliferation, apoptosis and P4, T and leptin release. The addition of daidzein promoted FSH‐stimulated apoptosis (but not proliferation) but suppressed FSH‐stimulated P4, T and leptin release. Our observations of FSH action confirm previous data on the stimulatory effect of FSH on ovarian cell proliferation, apoptosis and steroidogenesis and demonstrate for the first time the involvement of FSH in the upregulation of ovarian leptin release. Our observations of daidzein effects demonstrated for the first time that this soya isoflavone affected basic ovarian cell functions (proliferation, apoptosis and hormones release) and modified the effects of FSH. Daidzein promoted FSH action on ovarian cell proliferation and apoptosis and suppressed, and even inverted, FSH action on hormone release. The direct action of daidzein on basic ovarian cell functions and the ability of these cells to respond to FSH indicate the potential influence of soya‐containing diets on female reproductive processes via direct action on the ovary.     

Plant polyphenols can directly affect ovarian cell functions and modify toluene effects

The influence of toluene alone and in combination with plant polyphenols apigenin, daidzein or rutin on viability, proliferation (proliferating cell nuclear antigen accumulation), apoptosis (Bax accumulation) and release of progesterone (P), testosterone (T) and estradiol (E) in cultured porcine ovarian granulosa cells was evaluated. Toluene reduced ovarian cell viability, proliferation and E release; it promoted P release, demonstrating no effect on apoptosis or T output. Apigenin alone failed to affect cell viability, proliferation, apoptosis and P and T release, but stimulated E release, promoting the inhibitory action of toluene on proliferation, preventing and even reversing the stimulatory effect of toluene on apoptosis and P. Daidzein alone reduced cell viability and promoted T release, preventing and reversing the stimulatory effect of toluene on cell proliferation. Rutin administration reduced cell viability and E output, promoting the inhibitory action of toluene on cell viability and stimulatory effect on P release, and preventing the inhibitory action of toluene on E release. Toluene reduced apigenin- and rutin-induced E release, promoting action of daidzein on cell viability. These observations suggest the action of toluene and plant polyphenols on ovarian cell functions and the functional interrelationships between these molecules in the ovary.     

Relationship between apoptosis and proliferation in granulosa and theca cells of cystic follicles in sows

To investigate the causes of the occurrence and persistence of porcine cystic follicles, we evaluated the apoptosis and proliferation of follicular cells in these cysts. Apoptotic frequencies were examined by TUNEL assay and the expression of apoptosis regulators (XIAP, bax, bc1-2 and caspase-3) by immunohistochemistry, Western blotting and real-time quantitative PCR; cell proliferation activity was evaluated by PCNA immunohistochemistry and proliferation of in vitro cultured granulosa and theca cells. The low apoptotic frequency and weak proliferative activity were found in cystic follicles. Low frequency of apoptosis might be associated with decreased amounts of apoptotic-related factors (bax and caspase-3) and increased amounts of anti-apoptotic factors (XIAP and bcl-2) in cystic follicles. Significantly lower proliferation activity was detected in granulosa and theca cells from cystic follicles, and lesser PCNA-positive cells were found in cystic follicles. Our results indicate that the programmed cell death and cell proliferation system were altered in cystic follicles. The disorder between apoptosis and proliferation was responsible for maintaining a static condition without degeneration, which leads to the long-term persistence of follicles. These findings provide important novel insights into the pathogenesis of follicular cysts in sows.     

Bisphenol A attenuates thyroxine‐induced apoptosis in ovarian granulosa cells of pigs

Bisphenol A (BPA) is a chemical of high production volume that is used widely in many industries and is known as a xenooestrogen and anti‐thyroid hormone endocrine disrupter. There is little information regarding the effects of BPA in the presence of thyroid hormone on porcine granulosa cell development. Thus, the primary granulosa cells were treated with thyroxine (T4, 10 nM), BPA (10 µM) or T4 plus BPA; we subsequently evaluated the effects of T4 or BPA on 17β‐estradiol synthesis, cellular proliferation and apoptosis. Our data showed that BPA significantly increased the accumulation of 17β‐estradiol and promoted granulosa cell proliferation, whereas T4 significantly decreased 17β‐estradiol and had no effect on cellular proliferation. In addition, it was noteworthy that T4 treatment induced apoptosis in porcine granulosa cells and BPA co‐incubation attenuated T4‐induced apoptosis as shown from flow cytometric assay analysis. We hypothesized that BPA attenuates T4‐induced apoptosis by regulating 17β‐estradiol accumulation and oestrogen receptor‐mediated signalling pathways. In conclusion, our results demonstrated that T4 affected 17β‐estradiol accumulation and induced cellular apoptosis, but did not affect granulosa cell proliferation. Exposure to BPA increased 17β‐estradiol accumulation, promoted granulosa cell proliferation and attenuated T4‐induced apoptosis in porcine granulosa cells in vitro.     

miR-495-3p对山羊卵巢颗粒细胞功能的影响

旨在探究miR-495-3p对山羊卵巢颗粒细胞功能的影响及作用机制.本研究选取健康的3～4月龄大足黑山羊母羊,收集卵巢颗粒细胞,利用miR-495-3p模拟物(mimics)和抑制物(inhibitor)构建过表达和抑制模型,通过流式细胞术检测细胞凋亡和周期,ELISA分析颗粒细胞的雌二醇(E2)和孕酮(P4)分泌,采...     

Consumption of bee pollen affects rat ovarian functions

The aim of this study was to examine possible effects of bee pollen added to the feed mixture (FM) on rat ovarian functions (secretion activity and apoptosis). We evaluated the bee pollen effect on the release of insulin‐like growth factor I (IGF‐I) and steroid hormones (progesterone and estradiol), as well as on the expression of markers of apoptosis (Bcl‐2, Bax and caspase‐3) in rat ovarian fragments. Female rats (n = 15) were fed during 90 days by FM without or with rape seed bee pollen in dose either 3 kg/1000 kg FM or 5 kg/1000 kg FM. Fragments of ovaries isolated from rats of each group (totally 72 pieces) were incubated for 24 h. Hormonal secretion into the culture medium was detected by RIA. The markers of apoptosis were evaluated by Western blotting. It was observed that IGF‐I release by rat ovarian fragments was significantly (p < 0.05) decreased; on the other hand, progesterone and estradiol secretion was increased after bee pollen treatment at dose 5 kg/1000 kg FM but not at 3 kg/1000 FM. Accumulation of Bcl‐2 was increased by bee pollen added at 3 kg/1000 kg FM, but not at higher dose. Accumulation of Bax was increased in ovaries of rats fed by bee pollen at doses either 3 or 5 kg/1000 kg FM, whilst accumulation of caspase‐3 increased after feeding with bee pollen at dose 5 kg/1000 kg FM, but not at 3 kg/1000 kg FM. Our results contribute to new insights regarding the effect of bee pollen on both secretion activity (release of growth factor IGF‐I and steroid hormones progesterone and estradiol) and apoptosis (anti‐ and pro‐apoptotic markers Bcl‐2, Bax and caspase‐3). Bee pollen is shown to be a potent regulator of rat ovarian functions.     

The Influence of Ovarian Stromal/Theca Cells During In Vitro Culture on Steroidogenesis,Proliferation and Apoptosis of Granulosa Cells Derived from the Goat Ovary

Early follicular development is closely related to oocyte‐granulosa cells‐ovarian stromal cells/theca cells. The aim of the present study was to investigate the effects of ovarian cortical, medullary stromal and theca cells on oestradiol and progesterone biosynthesis, proliferation and apoptosis of goat ovary granulosa cells in vitro. Using Transwell coculture system, we evaluated steroidogenesis, cell proliferation and apoptosis, and some molecular expressions regarding steroidogenic enzyme, luteinizing hormone receptor and apoptosis‐related genes in granulosa cells. The results indicated that ovarian stromal/theca cells were able to stimulate oestradiol and progesterone production, promote cell proliferation and inhibit apoptosis of granulosa cells. Among all the three kinds of cells, theca cells affected strongly on granulosa cell function, and ovarian medullary stromal cells had the weakest effect on granulosa cells. These findings would provide an important knowledge of cell interaction among follicular cells during follicular development.     

Of humans and canines: Immunohistochemical analysis of PCNA, Bcl-2, p53, cytokeratin and ER in mammary tumours

Mammary tumours are the most common neoplasms in humans and canines. Human and canine mammary tumours share several important epidemiological, clinicopathological and biochemical features. Development of mammary tumours involves accumulation of mutant cells caused by excessive proliferation and insufficient apoptosis or dysregulation of cellular differentiation. The present study was therefore designed to investigate the expression of proliferation, differentiation, and apoptosis associated proteins together with expression of estrogen receptors (ER) in both human and canine mammary tumours. Thirty breast cancer patients categorized as pre- and postmenopausal, and 30 mammary gland tumours obtained from bitches were included in this study. The expression of proliferating cell nuclear antigen (PCNA), Bcl-2, p53, cytokeratin and ER in tumour tissues and adjacent tissues were investigated using immunohistochemical staining. While the expression of PCNA, Bcl-2, p53 and ER was significantly increased, expression of cytokeratin was significantly lower in both human as well as canine mammary tumours compared to corresponding adjacent tissues. The magnitude of the changes was however more pronounced in premenopausal patients compared to postmenopausal patients. The changes in proliferation, apoptosis and differentiation associated proteins in human and canine mammary tumours validate use of the canine model to understand the molecular mechanisms of mammary carcinogenesis.     

Canine mammary carcinomas: influence of histological grade,vascular invasion,proliferation, microvessel density and VEGFR2 expression on lymph node status and survival time

Spontaneous invasive non‐inflammatory canine mammary carcinomas (CMC) and their regional lymph nodes (LN) were analysed (n = 136). Histological grade (HG) and vascular invasion (VI) in the tumours and lymph node status were recorded. Proliferation index (PI), microvessel density (MVD) and vascular endothelial growth factor receptor 2 (VEGFR2) expression were estimated using anti‐proliferating cell nuclear antigen (PCNA), anti‐von Willebrand factor and anti‐Flk‐1, respectively. Eighteen months follow‐up was performed (34 bitches). Tumours of different grades showed differences regarding PI, Flk‐1/integrated optical density (Flk‐1/IOD) and MVD. Every feature showed significant association with LN status through bivariate analyses. From multivariate analyses, VI and Flk‐1/IOD were selected to predict LN status. Data revealed that the probability of a CMC‐bearing bitch to remain alive at 1, 4, 5 and 14–18 months was 0.91, 0.87, 0.81 and 0.77, respectively. Besides LN status, VI was the only feature positively correlated with survival time, although a trend to shorter survival of animal patients bearing high expressing VEGFR2 CMC was noted.     

The effects of baicalein on canine osteosarcoma cell proliferation and death

Flavonoids are a group of modified triphenolic compounds from plants with medicinal properties. Baicalein, a specific flavone primarily isolated from plant roots (Scutellaria baicalensis), is commonly used in Eastern medicine for its anti‐inflammatory and antineoplastic properties. Previous research shows greater efficacy for baicalein than most flavonoids; however, there has been little work examining their effects on sarcoma cells, let alone canine cells. Three canine osteosarcoma cell lines (HMPOS, D17 and OS 2.4) were treated with baicalein to examine cell viability, cell cycle kinetics, anchorage‐independent growth and apoptosis. Results showed that osteosarcoma cells were sensitive to baicalein at concentrations from approximately 1 to 25 μM. Modest cell cycle changes were observed in one cell line. Baicalein was effective in inducing apoptosis and did not prevent doxorubicin cell proliferation inhibition in all the cell lines. The mechanism for induction of apoptosis has not been fully elucidated; however, changes in mitochondrial permeability supersede the apoptotic response.     

氯丙嗪对大鼠离体培养卵巢颗粒细胞凋亡的影响

本试验旨在通过氯丙嗪对颗粒细胞凋亡影响的研究,探讨氯丙嗪对雌性大鼠性腺毒性的作用机制。对未成熟的Wistar大鼠卵巢颗粒细胞进行原代培养,用不同浓度的氯丙嗪(0、0.1、1、10 μmol/L)染毒细胞,细胞培养24 h。染毒结束后采用MTT法检测细胞相对活力,荧光染料Hoechest 33258检测颗粒细胞的凋亡变化,RT-PCR检测凋亡调控基因Bax、Bcl-2和P53 mRNA的表达。在本试验所设置的剂量范围内,与对照组比较,氯丙嗪能极显著促进颗粒细胞凋亡(P＜0.01),呈浓度依赖关系;RT-PCR检测则显示氯丙嗪能引起Bcl-2、Bax、P53 mRNA表达水平和Bax mRNA/Bcl-2 mRNA值升高,除低剂量组的Bax mRNA表达水平和Bax mRNA/Bcl-2 mRNA值无明显改变外,其余各组与对照组相比均差异极显著(P＜0.01)。氯丙嗪可显著抑制大鼠卵巢颗粒细胞活力,诱导颗粒细胞凋亡。     

GDF9 affects the development and tight junction functions of immature bovine Sertoli cells

Transforming growth factor‐β (TGFβ) superfamily are critical regulators of germ cell development that act as extracellular ligands of the signal transduction pathways regulating proliferation, apoptosis and other aspects of cell behaviour. As a member of the TGF‐β superfamily, growth differentiation factor 9 (GDF9) plays a critical role in ovarian follicular development and the ovulation rate in females; however, its role in the testis has not been well elucidated. The aim of this study was to investigate the expression of GDF9 and its receptor genes BMPRII and ALK5 in prepuberal bovine Sertoli cells (SCs). In addition, we assessed the effects of GDF9 on immature SCs apoptosis, the cell cycle and tight junction functions. We found that GDF9 and its receptor genes BMPRII and ALK5 were expressed in immature SCs. Exogenous GDF9 significantly promoted SCs proliferation and inhibited the apoptosis of SCs by significantly upregulating Cyclin E (cell cycle) and bcl‐2 (anti‐apoptosis) mRNA expression and downregulating caspase‐3 (pro‐apoptosis) mRNA expression. Meanwhile, exogenous GDF9 significantly decreased the value of transepithelial electrical resistance by significantly downregulating claudin‐11 mRNA expression and influencing the distribution of occludin. In conclusion, this study reveals that GDF9 is a key regulator of bovine SCs through the modulation of the cell cycle, apoptosis and tight junction functions.     

MiR‐222 inhibits apoptosis in porcine follicular granulosa cells by targeting the THBS1 gene

Apoptosis of granulosa cells affects follicular atresia and reproduction and is regulated by miRNAs and the expression of certain genes. For the present study, we investigated the regulatory relationship between microRNA‐222 (miR‐222) and THBS1 in porcine follicular granulosa cells (pGCs) and its effects on apoptosis to provide empirical data for developing methods to improve pig fecundity. Results revealed that miR‐222 promotes the proliferation of pGCs. MiRNA mimics and luciferase reporter assays revealed that miR‐222 functions as an anti‐apoptotic factor in pGCs. MiR‐222 mimics in pGCs result in the upregulation of the anti‐apoptotic BCL‐2 gene, down‐regulation of the proapoptotic caspase‐3 gene, and inhibition of apoptosis. MiR‐222 inhibitors reduced BCL‐2 and had no significant effect on caspase‐3. MiR‐222 mimics promoted estrogen levels. Inhibition of THBS1 inhibited pGC apoptosis. Transfection of THBS1‐siRNA reduced the proapoptotic BAX gene. MiR‐222 can directly target the 3′‐untranslated region of the THBS1 gene. MiR‐222 mimics suppressed THBS1 mRNA and proteins, but these were upregulated by the miR‐222 inhibitor. Transfection of THBS1‐siRNA resulted in the inhibition of the miR‐222 inhibitor, which suggests that miR‐222 inhibits pGC apoptosis by targeting THBS1. These findings suggest that miR‐222 and THBS1 play important roles in follicular atresia, ovarian development, and female reproduction.     

猫脂肪间充质干细胞及其条件培养基对庆大霉素致损猫肾细胞增殖和凋亡的影响

试验旨在探究猫脂肪间充质干细胞（adipose mesenchymal stem cells,AD-MSCs）及其条件培养基（adipose mesenchymal stem cells conditioned medium,AD-MSCs-CM）对庆大霉素致损的猫肾细胞（crandell rees feline kidney,CRFK）增殖和凋亡的影响及机制。试验分为空白组、庆大霉素（gentamicin,GM）组、AD-MSCs组和AD-MSCs-CM组;用Ⅰ型胶原酶消化法分离猫AD-MSCs,并用流式细胞术鉴定分离的细胞;以0、2、4、8 mmol/L GM完全培养基处理CRFK,并于12、24和48 h用CCK-8法检测细胞活性,筛选最适造模浓度;致损的CRFK分别与猫AD-MSCs及AD-MSCs-CM共培养,于24、48和72 h后用CCK-8法检测细胞增殖活性,24 h后通过Annexin V流式细胞术检测细胞凋亡率,最后通过实时荧光定量PCR检测细胞周期蛋白D1（cyclin d1,CCND1）、细胞增殖核抗原（proliferative nuclear antigen,PCNA）、Bax和Bcl-2基因的表达水平。结果显示,分离培养的猫AD-MSCs在体外培养条件下呈典型的梭型,高表达MSCs表面标记物CD44、CD90和CD105,不表达白细胞表面标记物CD45;AD-MSCs和AD-MSCs-CM均能促进GM致损CRFK的增殖并抑制其凋亡,其中AD-MSCs和AD-MSCs-CM可通过上调增殖相关基因CCND1、PCNA和抗凋亡基因Bcl-2的表达并降低促凋亡基因Bax的表达而发挥促增殖、抑凋亡作用。试验成功分离得到猫AD-MSCs,并证实AD-MSCs及其条件培养基在体外能促进GM致损CRFK的增殖和迁移,为猫AD-MSCs治疗猫急性肾损伤提供依据。     

Antitumour effects of Liporaxel (oral paclitaxel) for canine melanoma in a mouse xenograft model

Paclitaxel, a member of the taxane family, exhibits antitumour effects by targeting the microtubules in cancer cells. Recently, oral paclitaxel has been developed to overcome the side effects of intravenous paclitaxel administration in human patients. The objective of this study was to investigate the antitumour effects of oral paclitaxel in vitro and in vivo. Three weeks after inoculation, oral paclitaxel (25 and 50 mg/kg) or saline was administered every week for three consecutive weeks. To explore the underlying mechanism, tumour angiogenesis was examined by immunohistochemistry with an anti‐CD31 antibody. Tumour cell apoptosis was detected by Terminal deoxynucleotidyl transferase dUTP Nick‐End Labeling assay, and cell cycle arrest was confirmed by western blot analysis. Oral paclitaxel treatment of canine melanoma cells exerted mediated antiproliferative effects and mediated cell cycle arrest in vitro. In animal experiments, after oral paclitaxel administration, the average tumour size decreased to approximately 30% of that in the control. Histologically, oral paclitaxel showed anti‐angiogenic effects and induced the apoptosis in tumour tissues. Oral paclitaxel also downregulated the intratumoural expression of cyclin D1 and inhibited cell proliferation. The study findings support potential application of oral paclitaxel as a novel chemotherapeutic strategy to treat canine melanoma. This is the first study to investigate the potential of oral paclitaxel as a therapeutic drug against canine tumours.     

Role of ghrelin in regulating rabbit ovarian function and the response to LH and IGF-I

The aim of these in vivo and in vitro studies was to examine the role of ghrelin in the control of plasma hormone concentrations, the proliferation, apoptosis and secretory activity of ovarian granulosa cells and the response of these cells to hormonal treatments. Female rabbits were injected with ghrelin (10 μg/animal/day for one week before ovulation induced by 25 IU PMSG and 0.25 IU LHRH). On the day of ovulation, blood samples were collected and analyzed for concentrations of progesterone (P₄), testosterone (T), estradiol (E₂), estrone-sulphate (ES), insulin-like growth factor I (IGF-I) and leptin (L) by RIA. Some control and ghrelin-treated animals were killed in the periovulatory period, their ovaries were weighed and granulosa cells were isolated and cultured for 2 d. Cell proliferation (expression of PCNA) and apoptosis (expression of TdT) were evaluated by immunocytochemistry and TUNEL respectively. Secretion of P₄, T, E₂, IGF-I, and prostaglandin F (PGF) by granulosa cells cultured with and without LH or IGF-I (1, 10 or 100 ng/ml medium) was assessed by RIA. The remaining control and treated animals were kept until parturition, while the number, viability and body weight of pups were recorded.