Changes in the expression of toll-like receptor mRNAs during follicular growth and in response to lipopolysaccharide in the ovarian follicles of laying hens

The aim of this study was to determine the changes in the mRNA expression of Toll-like receptors (TLRs) in hen ovarian follicles during follicular growth and in response to lipopolysaccharide (LPS). White follicles and the fifth largest to largest follicles (WF and F(5)-F(1), respectively) were collected from laying hens. To examine the effects of LPS, the laying hens were treated intravenously with LPS (1 mg/kg BW) 0, 3, 6, 12 and 24 h before examination. Expressions of TLRs and IL-1beta in the theca and granulosa layers were examined by semi-quantitative RT-PCR. Immunocytochemistry was performed to identify immunoreactive TLR-4. The theca layer expressed TLR-2, TLR-4, TLR-5 and TLR-7, whereas the granulosa layer expressed only TLR-4 and TLR-5. The expression of TLR-4 and TLR-5 in the theca layer increased significantly during follicular growth. In the granulosa layer, the expression of TLR-5 increased, but that of TLR-4 was unchanged. Expression of TLR-4 increased significantly during the period of 6 to 12 h of LPS treatment in the theca layer and during the period of 12 to 24 h in the granulosa layer of F(3). Immunoreaction products for TLR-4 were observed in theca interna and granulosa layers of WF and F(5)-F(1), with the greater amount observed in the theca interna. LPS treatment significantly increased expression of IL-1beta in the theca layer after 3 h and in the granulosa layer during the period of 12 to 24 h. These results suggest that TLRs are expressed in ovarian follicles and that TLR-4 and TLR-5 expression increase with the growth of follicles. Enhanced expression of TLR-4 and IL-1beta by LPS in the theca and granulosa layers suggests possible roles of TLR in recognition of microorganisms.     

大豆黄酮对伊莎鸡卵泡发育及其P450arom基因表达的影响

实验探讨了大豆黄酮(DAI)对伊莎鸡卵泡发育及其芳香化酶(P450arom)mRNA表达的影响。实验选取16只产蛋后期伊莎鸡,等分为对照组和DAI处理组。对照组饲喂基础日粮,实验组在基础日粮中添加10 mg/kgDAI。实验持续7周后,分离排卵前卵泡(F1、F2、F3……)的颗粒层及小黄卵泡和大白卵泡,通过RT-PCR法检测P450arom mRNA表达的相对丰度。结果表明:DAI明显提高了伊莎鸡小黄卵泡和大白卵泡的数量,P450arommRNA在伊莎鸡不同发育阶段卵泡中的表达存在差异,部分卵泡P450arom mRNA表达的相对丰度显著增加。因此,在产蛋后期伊莎鸡基础日粮中添加DAI可增加不同发育阶段卵泡的数目,上调部分卵泡中与发育相关的基因表达以促进卵泡发育。     

In vitro rapid clearance of infectious bursal disease virus in peripheral blood mononuclear cells of chicken lines divergent for antibody response might be related to the enhanced expression of proinflammatory cytokines

Infectious bursal disease (IBD) is an acute and highly contagious viral disease of young chickens caused by infectious bursal disease virus (IBDV). An effective way to control IBDV would be to breed chickens with a reduced susceptibility to IBDV infection. In the present work, we used chickens selected for high and low specific responses to sheep red blood cells (SRBC) (H and L, respectively) to assess the susceptibility of differential immune competent animals to IBDV infection. The peripheral blood mononuclear cells (PBMCs) of high SRBC line (HL) and low SRBC line (LL) were infected with IBDV and viral RNA loads were determined at different time post-IBDV infection. Chicken orthologues of the T helper 1 (Th1) cytokines, interferon-γ (IFN-γ) and interleukin-2 (IL-2); a Th2 cytokine, IL-10; a pro inflammatory cytokine, IL-6; the CCL chemokines, chCCLi2, chCCLi4 and chCCLi7; colony stimulating factor, GM-CSF; and a anti-inflammatory cytokine, transforming growth factor β-2 (TGFβ-2) were quantified. The expression of chCCLi2, chCCLi4 and chCCLi7 was significantly higher in L line as compared to H line. However, in H line the viral RNA loads were significantly lower than in L line. Therefore, the upregulated chemokines might be associated with the susceptibility to IBDV. The expression of IFN-γ, IL-2 and IL-6 was significantly higher in H line as compared to L line. We assume that the higher proinflammatory cytokines expression in H line might be related to the rapid clearance of virus from PBMCs. Significantly higher levels of IL-10 and TGFβ-2 mRNAs in L line might be related to the pathogenesis of IBDV. In conclusion, selection for antibody responses appears to influence the expression profiles of chemokines and cytokines against IBDV. Further, the selection for high SRBC response might improve the immuno-competence of chickens against IBDV.     

Expression of aquaporin 4 in the chicken ovary in relation to follicle development

In the mammalian ovary, aquaporins (AQPs) are thought to be involved in the regulation of fluid transport within the follicular wall and antrum formation. Data concerning the AQPs in the avian ovary is very limited. Therefore, the present study was designed to examine whether the AQP4 is present in the chicken ovary, and if so, what is its distribution in the ovarian compartment of the laying hen. Localization of AQP4 in the ovarian follicles at different stage of development was also investigated. After decapitation of hens the stroma with primordial follicles and white (1–4 mm), yellowish (4–8 mm), small yellow and the three largest yellow pre‐ovulatory follicles F3‐F1 (F3 < F2 < F1; 20–36 mm) were isolated from the ovary. The granulosa and theca layers were separated from the pre‐ovulatory follicles. The AQP4 mRNA and protein were detected in all examined ovarian compartments by the real‐time PCR and Western blot analyses, respectively. The relative expression of AQP4 was depended on follicular size and the layer of follicular wall. It was the lowest in the granulosa layer of pre‐ovulatory follicles and the highest in the ovarian stroma as well as white and yellowish follicles. Along with approaching of the largest follicle to ovulation the gradual decrease in AQP4 protein level in the granulosa layer was observed. Immunoreactivity for AQP4 was present in the granulosa and theca cells (theca interna ≥ theca externa > granulosa). The obtained results suggest that AQP4 may take part in the regulation of water transport required for follicle development in the chicken ovary.     

Expression of adiponectin and its receptors (AdipoR1 and AdipoR2) in chicken ovary: potential role in ovarian steroidogenesis

Adiponectin and its receptors (AdipoR1 and AdipoR2) mRNAs are expressed in various chicken tissues including ovary. However, the cellular expression and the role of adiponectin system have never been investigated in chicken ovary. Here, we have shown that the level of adiponectin mRNA is about 10- to 30-fold higher (p < 0.001) in theca cells than in granulosa cells from each hierarchical yellow follicle studied (F4–F1). In contrast, the level of AdipoR1 mRNA expression was about two-fold lower in theca cells than in granulosa cells (p < 0.05) whereas those of AdipoR2 was similar in both ovarian cells. Whereas expression of adiponectin mRNA increased with follicular differentiation in theca cells, it decreased in granulosa cells. In contrast, mRNA expression of AdipoR1 and AdipoR2 in both theca and granulosa cells remained stable during yellow follicle development. To determine whether adiponectin is involved in the ovarian steroidogenesis, LH (100 ng/ml)-, FSH (100 ng/ml)- and IGF-1 (100 ng/ml)-induced progesterone production was measured in absence or presence of human recombinant adiponectin (10 μg/ml) for 36 h in cultured granulosa cells from F1, F2 and mixed F3 and F4 follicles. In absence of LH, FSH and IGF-1, adiponectin treatment had no effects on progesterone production whatever vitollegenic follicle studied. However, it increased by about two-fold IGF-1-induced progesterone secretion in F2 and F3/4 follicles whereas it halved progesterone production in response to gonadotropins (LH and FSH) in F3/4 follicles. Thus, in chicken, adiponectin, mainly expressed in theca cells, could exert paracrine or autocrine effect on the ovarian steroidogenesis.     

鹌鹑卵泡发育过程中颗粒细胞黄体生成素受体mRNA的表达

用Ｎorthern杂交的方法研究了鹌鹑排卵泡内颗粒细胞黄体生成素受体（ＬＨＲ）mＲＮＡ的表达。在预计排卵前２０h和３h分别取出最大的３个卵泡（Ｆ１、Ｆ３卵泡）以及小黄卵泡（ＳＹＦ）,剥离颗粒层提取出总ＲＮＡ,并经变性凝胶电泳后将ＲＮＡ转移到滤膜上。杂交所用的探针是用特异的引物经反转录一多聚栈链式反应（ＲＴ－ＰＣＲ）扩增出编码ＬＨＲcＤＮＡ的细胞外区（ＥＣ）和跨膜区（ＴＭ）cＤＮＡ。结果表明,颗粒细胞ＬＨＲmＲ     

Androgen receptor mRNA expression in the bovine ovary

Previous studies have shown that androgen receptor (AR) is expressed in granulosa cells of healthy, growing ovarian follicles in rats and primates. However, AR expression in the bovine ovary has not been examined. Therefore, a 346-base pair segment of the bovine AR was cloned and sequenced. Using a ribonuclease protection assay, AR expression was detected in total RNA from bovine ovarian cortex. Expression (absence or presence) of AR mRNA was detected by in situ hybridization in bovine ovarian cortex. Follicles (n = 32) were classified as follows: type 1 (1 layer of flattened granulosa cells), type 2 (1-1.5 layers of cuboidal granulosa cells), type 3 (2-3 layers of granulosa cells), type 4 (4-6 layers of cuboidal granulosa cells and formation of thecal layer), and type 5 (>6 layers of cuboidal granulosa cells, defined theca layer, and antrum formation). Frequency of AR mRNA expression increased (P < 0.001) as follicles entered the growing pool. Expression of AR mRNA was absent in type 1 follicles (n = 8), but present in the granulosa cells of 41% of type 2 follicles (n = 12). In types 3-5 follicles, AR mRNA expression was present in granulosa cells of 100% of follicles examined (n = 4, 4, and 4, respectively) and was greater than type 1 follicles (P = 0.002). These data provide evidence of AR mRNA expression in bovine follicles and suggest that AR mRNA increases during early follicle development.     

Follicle-stimulating hormone (FSH) stimulates the expression of Pin1, a peptidyl-prolyl isomerase, in the bovine granulosa cells

A peptidyl-prolyl isomerase, Pin 1, has been shown to play a role in the regulation of cell cycle progression, both in vitro and in vivo. However, the involvement of Pin 1 during follicular development is not well understood. The aim of this study was first to investigate the expression of Pin 1 mRNA in the granulosa and theca cells of the follicle at different developmental stages of follicles in the bovine ovary, and second, to examine the effects of follicle-stimulating hormone (FSH) and estradiol (E2) on the expression of Pin 1 in the cultured bovine granulosa cells. Follicles were classified into four groups based on the diameter (dominant follicles >8.5mm in diameter, subordinate follicles <8.5mm in diameter) and the relative levels of E2 and progesterone (P4) (E2:P4>1, estrogen active; E2:P4<1, estrogen inactive): i.e. preovulatory dominant follicles (POFs); E2 active dominant follicles (EADs); E2 inactive dominant follicles (EIDs); small follicles (SFs). The expression of the Pin 1 gene was significantly increased in the granulosa cells of EADs as compared with those of other follicles, whereas its expression in theca cells did not differ among follicles at different developmental stages. The concentration of 5 ng/ml FSH alone and the combination of 1 ng/ml E2 and 5 ng/ml FSH stimulated the expression of the Pin 1 gene in bovine granulosa cells. Our data provide the first evidence that Pin 1 expression in the granulosa cells but not the theca cells changes during follicular development, and that FSH stimulate the expression of the Pin 1 gene. These results suggest that Pin 1 regulates the timing of cell proliferation and may act as an intracellular signal responder in the granulosa cells during bovine follicle development.     

Expression of messenger ribonucleic acid encoding for steroidogenic acute regulatory protein and enzymes, and luteinizing hormone receptor during the spring transitional season in equine follicles

The period of spring transition, from the anovulatory to the ovulatory season, is characterized in many mares by cyclical growth and regression of large dominant follicles. These follicles produce only low concentrations of estradiol and it is thought that acquisition of steroidogenic competence by large follicles during spring transition is prerequisite in stimulating LH prior to first ovulation. In situ hybridization was used to localize and quantify expression of factors that play a key role in follicular steroidogenesis: StAR, P450scc (CYP11A1), P450c17 (CYP17), P450arom (CYP19), and LH receptor (LHr). One ovary was obtained from mares on the day after detection of an actively growing 30 mm transitional anovulatory follicle (defined as the transitional follicle), and the remaining ovary was removed at the third estrus of the breeding season on the day after the preovulatory follicle reached 30 mm in diameter (defined as the preovulatory follicle). Messenger RNAs encoding StAR, CYP11A1, and CYP17 were detected only in theca cells and CYP19 mRNA was confined to the granulosa layer. There was significantly lower expression of mRNAs for the steroidogenic enzymes, StAR (P<0.001) and LHr (P<0.05) in transitional follicles than in preovulatory follicles. In conclusion, large equine follicles during spring transition have low levels of mRNA encoding steroidogenic enzymes, StAR and LHr which will contribute to the steroidogenic incompetence of dominant follicles during spring transition and their subsequent regression.     

Comparison of cadherin and integrin localization in bovine cystic and healthy follicles

As stage progresses in the cystic follicle, granulosa cells are lost. We hypothesized that the granulosa and theca interna layers are detached in association with weakened expression of cell adhesion molecules such as cadherin (cell–cell adhesion) and integrin (cell–extracellular matrix adhesion) in cystic follicles. To elucidate this hypothesis, we immunolocalized these molecules in the granulosa and theca interna and compared them between cystic and small healthy follicles. Sections were immunostained with cadherin and integrin β₁ antibodies and their localizations were compared. Cadherin‐positive reaction was seen in the cytoplasma of all granulosa cells. No increase in the frequency of cadherin‐positive area in the granulosa layers and the intensity of cadherin immunoreaction in the theca interna was detected in cystic follicles compared with healthy ones. A dense immunoreaction product of integrin β₁ was detected in the theca interna in both cystic and healthy follicles. Intensity of integrin β₁‐immuno reaction in the granulosa layers and integrin β₁‐positive area in the theca interna was significantly lower in the cystic follicle than in the healthy follicles. These results suggest that granulosa and theca interna cells are detached while maintaining the cell–cell adhesion, resulting in the consequent loss of these layers from the cystic follicle.     

Expression of Vascular Endothelial Growth Factor Receptors in Bovine Cystic Follicles

Cystic follicles have excess fluid derived from blood flow in the theca interna of the follicle; therefore, the vasculature network is related to cystic follicle formation. Vascular endothelial growth factor (VEGF) is a potent stimulator of blood vessel permeability and angiogenesis. The aim of this study was to examine the expression of VEGF receptors proteins and mRNA in cystic follicles to elucidate the VEGF system in cystic follicles. The expression of protein for VEGF receptors; fms‐like‐tyrosine kinase‐1 (Flt‐1) and foetal liver kinase‐1 (Flk‐1) was detected by the immunohistochemical method. The mRNA expression of Flt‐1 and Flk‐1 in cystic follicles was determined by RT‐PCR. Concentration of oestradiol‐17β and progesterone in the follicular fluid of cystic follicles was determined using ELISA. Flt‐1‐ and Flk‐1 proteins were localized in granulosa and theca interna cells and endothelial cells of theca layers. The intensity of Flt‐1 and Flk‐1 immunoreaction was similar among cystic follicles with various ratios of oestradiol‐17β/progesterone concentrations. The expression of Flt‐1 and Flk‐1 mRNA was similar, regardless of the ratio of oestradiol‐17β to progesterone in follicular fluid. These results demonstrate that cystic follicles have both VEGF receptors in the granulosa and theca interna layers, which may be responsible for the increased permeability of microvessels, causing the accumulation of follicular fluid in cystic follicles.     

Immunohistochemical localization of 3beta-hydroxysteroid dehydrogenase in the granulosa and theca interna layers of bovine cystic follicles

The aim of the present study was to determine whether the alteration of population of cells containing 3beta-hydroxysteroid dehydrogenase (3beta-HSD) is responsible for the formation of cystic follicles. Paraffin sections of healthy (2 to 5 mm in diameter), atretic (2 to 5 mm) and cystic follicles (more than 25 mm) were immunohistochemically stained with rabbit polyclonal antibody to bovine 3beta-HSD. The 3beta-HSD-positive cells were counted in 4 different regions of the follicles from the apical to the basal side. The frequencies of 3beta-HSD-positive granulosa cells in cystic follicles were significantly higher than those in the healthy follicles (P<0.05), although the number of 3beta-HSD-positive granulosa cells in the cystic follicle were fewer than half the cells (30 to 40%) and was much smaller than that in preovulatory follicles (Conley et al., 1995). The frequencies of 3beta-HSD-positive cells were higher in the granulosa layer and lower in the theca interna layer of the cystic follicles than the atretic follicles. These results suggest that the differentiation of granulosa cells to express 3beta-HSD might be insufficient in cystic follicles and accordingly they fail to ovulate. The differences of frequencies of 3beta-HSD-positive cells in the granulosa and theca interna layers between cystic and atretic follicles may be one of the reasons why regression is delayed in cystic follicles.     

Deficient proliferation and apoptosis in the granulosa and theca interna cells of the bovine cystic follicle

The aim of the present study was to examine the frequencies of cell proliferation and death of granulosa and theca interna layers during development of cystic follicles in order to understand the mechanisms of cystic follicle formation. Paraffin sections of cystic follicles were immunostained with antibodies against proliferating cell nuclear antigen (PCNA) and cleaved caspase-3 in order to observe proliferating and apoptotic cells, respectively. The concentrations of estradiol-17beta and progesterone in the follicular fluid of these follicles were measured by ELISA. The granulosa and theca interna layers contained both PCNA- and caspase-3-positive cells, although their numbers were limited. There was significant negative correlation between the estradiol-17beta and progesterone concentrations in the follicular fluid. Regression analysis revealed no significant correlation, except for that between the PCNA-positive cells in the theca interna and the caspase-3-positive cells in the granulosa layer. These results indicate that the granulosa and theca interna cells of the cystic follicle show weak proliferative activity and low apoptotic frequency; this implies that the cystic follicle grows slowly and then maintains a static condition without degeneration, which leads to long-term persistence of the follicle.     

In situ hybridization analysis of ovarian prostaglandin endoperoxide synthase mRNA throughout the periovulatory period of the ewe.

Cellular alterations in level of expression of mRNA encoding for prostaglandin endoperoxide synthase were quantified within ovarian tissues of sheep obtained before, during and after induction of the preovulatory surge of LH and ovulation with LHRH. This was accomplished by isotopic in situ hybridization using a selective cRNA probe to ovine prostaglandin endoperoxide synthase mRNA. A significant elevation in mRNA was detected within the theca interna of the preovulatory follicle at 8, 16 and 24 hr following administration of LHRH. Very close to the time of ovulation (ie., at 24 hr post-LHRH) a marked rise in mRNA was observed in association with epithelial cells covering the apical surface of the follicle. Ovarian cyclooxygenase metabolites of arachidonic acid produced during the ovulatory process in the ewe originate within the thecal layer and germinal epithelium of the follicle destined to ovulate.     

Lysophosphatidic acid expression in theca cells depends on the type of bovine ovarian follicle

Lysophosphatidic acid (LPA) exerts various actions on the mammalian reproductive system. In cows, LPA stimulates the synthesis and secretion of luteotropic factors in the ovary, which affects the growth and development of ovarian follicles. The role of LPA in granulosa cells, oocyte and oocyte‐cumulus complex (COC) has previously been investigated; but its role in the theca layer, which is an important structural and functional component of the ovarian follicle, is still unclear. The goal of this study was to investigate the expression of LPA in theca cells originating from different bovine ovarian follicle types. Theca cells were separated from healthy, transitional and atretic ovarian follicles, based on intrafollicular estradiol: progesterone ratios. LPA concentration in the follicular fluid (FF) in different follicle types was measured, and expression of the enzymes responsible for LPA synthesis (autotaxin [AX], phospholipase A2 [PLA2]) and receptors for LPA (LPAR1‐4) were determined. The obtained results confirmed the follicle‐type dependent presence of LPA in the FF of the bovine ovarian follicles. The highest concentration of LPA was detected in follicles classified as healthy and dominant. LPAR1‐4, PLA2 and AX expression in theca cells in all of the types of follicles examined were detected at mRNA and protein level. These results suggest that theca cells can be a source of LPA synthesis other than granulosa cells and COCs, as well as the target for its action in the bovine ovarian follicle, with PLA2 and LPAR4 playing major roles in LPA synthesis and action.     

Expression analysis of melatonin receptor subtypes in the ovary of domestic chicken

Melatonin (N-acetyl-5-methoxytryptamine), an indole hormone, regulates various biological functions through three different receptor subtypes (Mel-1a, Mel-1b, and Mel-1c). However, the distribution of different melatonin receptor subtypes in chicken reproductive tissues was not known. In the present investigation, the partial sequences of ovarian melatonin receptor subtypes (Mel-1a, Mel-1b, and Mel-1c) were characterized. Further, the expression profile of melatonin receptor subtypes in the granulosa and theca layers of different preovulatory and postovulatory follicles (POF) were studied by semi-quantitative RT-PCR. The expression of all three subtypes of melatonin receptors were observed in the ovary of domestic chicken. Analysis of partial sequences of ovarian melatonin receptors revealed that the melatonin subtypes were identical to the brain receptors. In small white ovary follicles, we observed only the expression of mel-1b receptors, but not mel-1a or mel-1c receptors. In yellow follicles, all the three subtypes of receptors expression were noticed. Interestingly, we observed the expression of mel-1a receptor only in thecal layer, but not in granulosa layer. In contrast, mel-1b and -1c receptors were expressed in both granulosa and thecal layer. During the regression of POF, we observed significant upregulation of melatonin receptors (mel-1a and 1c) expression, that downregulated in the later stages of regression. We assume that the expression of melatonin receptors might have been influenced by the atresia or apoptosis of different follicular layers in POF. Our findings suggest that the differential distribution of melatonin receptor subtypes might have distinct downstream cellular functions in the ovarian tissues.     

Ghrelin对鸡等级前卵泡发育的影响

旨在研究Ghrelin对产蛋鸡等级前卵泡发育的调节作用.通过RT-PCR方法检测卵泡Ghrelin受体(GH-SR)的表达情况,采用组织学和免疫组化方法探讨Ghrelin和促卵泡素(FSH)单独或联合处理对鸡等级前卵泡形态学的变化以及层粘连蛋白(LN)和缝隙连接蛋白43(Cx43)的标记率.结果表明,GHSR在颗粒层和膜层上均有表达,其表达量随着卵泡的发育分别至大白卵泡(LWF)和小黄卵泡(SYF)达到最高,随后逐渐降低.悬浮培养的卵泡经Ghrelin和FSH处理24 h后,LWF和SYF的颗粒层和膜层厚度及颗粒细胞密度均显著增加(P＜0.05).同时,颗粒细胞中LN和Cx43的标记率显著提高(P＜0.05),且以Ghrelin与FSH联合处理组最为显著.结果提示,Ghrelin可通过提高LN和Cx43的表达以增强颗粒细胞的连接功能,促进鸡等级前卵泡颗粒细胞的增殖,并可协同FSH调节卵泡的发育.     

Tumor necrosis factor-alpha (TNF alpha) inhibits progesterone and estradiol-17beta production from cultured granulosa cells: presence of TNFalpha receptors in bovine granulosa and theca cells

The aim of this study was to investigate whether functional tumor necrosis factor-alpha (TNFalpha) receptors are present in the granulosa cells and the cells of theca interna (theca cells), obtained from bovine follicles classified into one of three groups. Each group was defined as either small vesicular ovarian follicles (small follicles; 3-5 mm in diameter), preovulatory mature ovarian follicles (preovulatory follicles) or atretic follicles (12-18 mm) according to gross examination of the corpus luteum in the epsilateral or contralateral ovary and the uterus (size, color, consistency and mucus), and the ratio of progesterone (P(4)) and estradiol-17beta (E(2)) concentrations in follicular fluid. A Scatchard analysis showed the presence of a high-affinity binding site on both granulosa and theca cells from all follicles examined (dissociation constant: 4.7 +/- 0.15 to 6.9 +/- 1.40 nM). Moreover, TNFalpha receptor concentrations in granulosa and theca cells obtained from atretic follicles were significantly higher than those in the cells from preovulatory follicles (P<0.05). Exposure of cultured granulosa cells from small antral follicles to recombinant human TNFalpha (rhTNFalpha; 0.06-6 nM) inhibited E(2) secretion in a dose-dependent fashion (P<0.01), but did not affect P(4) secretion. In addition, rhTNFalpha inhibited follicle stimulating hormone-, forskolin- or dibutylyl cyclic AMP-induced P(4) and E(2) secretion by the cells (P<0.01). These results indicate the presence of functional TNFalpha receptors in bovine granulosa and theca cells in small, preovulatory and atretic follicles, and suggest that TNFalpha plays a role in regulating their secretory function.