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.     

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.     

Microvascular distribution and vascular endothelial growth factor expression in bovine cystic follicles

The aim of this study was to examine the distribution of microvessels in the theca and the expression of vascular endothelial growth factor (VEGF) in the theca and granulosa of cystic follicles. Paraffin sections of cystic follicles were stained with Bandeiraea simplicifolia-I (BS-I) to visualize the endothelial cells of microvessels. The other sections were immunostained with anti-VEGF antibody. The mRNA expression of VEGF in the theca interna of cystic and healthy follicle was determined by RT-PCR. In the theca interna, cystic follicles with granulosa cells had significantly greater microvessel number density (the number of microvessels per given field) and area (area occupied by microvessels per given area) than healthy follicles in various sizes (<3, 4–8, >9 mm). Loss of granulosa cells from cystic follicles resulted in a similar number density, but significantly smaller area of microvessels in the theca interna. There was no significant difference in the microvessel number density and area of the theca externa between the types of follicle. VEGF protein was expressed in the granulosa and theca interna of healthy and cystic follicles. These results demonstrate that cystic follicles have a highly developed vasculature network in the theca interna, especially in cystic follicles containing granulosa cells. It is also suggested that VEGF is highly expressed in the cystic follicle as well as healthy follicle, which may be associated with advanced vasculature and the accumulation of follicular fluid in cystic follicles.     

Cell Proliferation in the Atretic Follicles of Buffalo and Cattle Ovary

The present study was carried out to describe the proliferative activity of granulosa and theca cells in healthy antral and atretic follicles of Philippine buffaloes (BU) and Holstein-Friesian (HF) cows. Paraffin sections of ovary were immunostained with mouse monoclonal antibody to proliferating cell nuclear antigen (PCNA). Then the follicles were classified into healthy and various stages of atretic follicles. The granulosa layer of healthy follicles had a significantly higher frequency of PCNA-positive cells than the early and advanced atretic follicles in both breeds. In the theca interna, significantly reduced populations of the PCNA-positive cells were found in both breeds as atresia progressed. Moreover, HF had significantly higher PCNA-positive cells in the theca interna of healthy, early atretic and advanced atretic follicles than BU. A reduction of PCNA-positive cells during atresia was also noted in the theca externa in both animals although differences were not significant. The results of the present work suggest that the proliferative activity of granulosa and theca cells decreases in association with follicular atresia in the BU similar to HF. Furthermore, a significantly deficient cell proliferative activity of theca interna was found in BU compared with HF.     

Sex steroids level in blood plasma and ovarian follicles of the chimeric chicken

The study was performed to determine the hormonal status of mature germline chimeras obtained by blastodermal cell transfer from chicken embryos of a donor breed [Green-legged Partridgelike breed (GP) x Araucana (AR)] to those of a recipient breed [White Leghorn (WL)] being at the same stage of embryonic development. The egg-laying chimeras and WL hens (control) of the same age were used in the experiment. At first, blood samples were taken from each bird at 0.5, 5, 12.5 and 18.5 h following oviposition. Subsequently, the chimeras and the WL hens were decapitated 1-2 h after ovulation. A stroma and the following follicles were isolated from the ovary: white normal (1-4, 4-6 and 6-8 mm), white atretic and yellow preovulatory follicles (F4-F1). Sex hormones, progesterone (P4), testosterone (T) and oestradiol (E2) in blood plasma and ovarian follicles were determined radioimmunologically. The activity of the 3beta-hydroxysteroid dehydrogenase (3beta-HSD) in the granulosa and theca layers of the follicles was analysed histochemically. In chimeric chickens, a higher level of T in blood plasma during the ovulatory cycle was noticed. However, in the stroma, white prehierarchical and medium-size preovulatory ovarian follicles the level of T was significantly lower. With respect to E2, its elevated levels were found both in blood and in the ovarian follicles. There were no significant differences in P4 concentrations in blood plasma while in ovarian follicles a higher level was observed only in white 6-8 mm follicles. 3beta-HSD activity in granulosa and theca layers of the ovarian follicles in chimeras was not different from that in the WL hens. In conclusion, the results obtained indicate that germline chimeras exhibit significant alterations in sex hormone levels in the ovary and blood plasma, which in turn may affect their reproductive abilities.     

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.     

Immunolocalization of steroidogenic cells in small follicles of the chicken ovary: Anatomical arrangement and location of steroidogenic cells change during follicular development

Previous anatomical and histochemical studies suggested that interstitial cells were the only steroidogenic cells in the theca layer of small follicles of the chicken ovary. However, the precise cellular site of steroid production in the small follicles is not certain. Therefore, our goal was to identify steroidogenic cells in small follicles (< 10 mm in diameter) of the chicken ovary which have not entered the follicular hierarchy by localizing steroidogenic enzymes with immunocytochemistry. Polyclonal antisera used were anti-cholesterol side-chain-cleavage cytochrome P450 (P450scc), anti-17-hydroxylase cytochrome P450 (P450c17), and anti-aromatase cytochrome P450 (P450arom) for pregnenolone-, androgen-, and estrogen-producing cells, respectively. Ovaries were collected 2 hr after oviposition and embedded in Paraplast after fixation with 4% paraformaldehyde, 10% formaldehyde, or Bouin's solution. Tissues were sectioned (4–6 μm) and sections were mounted on poly-L-lysine coated slides. Sections were incubated overnight at room temperature with each specific antiserum raised in rabbits against cytochrome P450 steroidogenic enzymes or normal rabbit serum as a control and were immunostained with an avidin-biotin-peroxidase complex. Immunoreactivity for the P450 enzymes was absent in the granulosa layer but was present in the theca layer of the small follicles (< 10 mm in diameter). Interstitial cells in the single theca layer of cortical follicles embedded in the ovarian cortex (less than 1 mm in diameter) contained P450scc and P450c17. Cells which contained P450arom, identified as aromatase cells, surrounded the interstitial cells in the theca layer. In small white follicles (approximately 1 mm in diameter), large white follicles (approximately 2–4 mm in diameter), and small yellow follicles (approximately 5–10 mm in diameter) which protruded from the surface of the ovary, the theca layer is divided into the theca interna and the theca externa. P450scc and P450c17 were localized in interstitial cells in the theca interna and externa whereas P450arom was localized in aromatase cells of the theca externa. With follicular development, more interstitial cells staining for P450scc and P450c17 appeared in the theca interna than in the theca externa whereas aromatase cells staining for P450arom were localized only in the theca externa. The distance between interstitial cells and aromatase cells within the theca layer increased as the follicles matured, resulting in a change in the anatomical relationship of steroidogenic cells. Our results of immunolocalization of cytochrome P450 steroidogenic enzymes in developing small follicles suggest that: 1) granulosa cells in small follicles are steroidogenically inactive; 2) steroids are produced in two distinct cell populations in the theca layer of small follicles, namely interstitial cells and aromatase cells; and 3) the anatomical relationship and location of interstitial cells and aromatase cells in the theca layer change with follicular maturation (a two-cell model for steroidogenesis in small follicles during follicular development).     

Immunolocalization of von Willebrand factor and vascular endothelial growth factor during follicular atresia in the swamp buffalo ovary

The aim of this study was to investigate the distribution pattern of von Willebrand factor (vWF) and vascular endothelial growth factor (VEGF) in the healthy antral and atretic follicles of Philippine swamp buffaloes (SB) in comparison with Holstein-Friesian cows (HF). Paraffin sections of healthy follicles and atretic follicles at various stages were immunostained with vWF antibody and VEGF antibody. The density of vWF-positive capillary vessels in the theca interna significantly increased as atresia progressed in SB, whereas the density significantly decreased in late atretic follicles compared with advanced ones in HF. On the other hand, the area of vWF-positive capillary vessels in the theca interna significantly increased as atresia progressed in both SB and HF. Immunoreactions of VEGF in the granulosa cells (in all follicle types) were observed in both SB and HF. In the granulosa layer, a reduction in the VEGF immunoreaction was noted as follicles progressed from healthy to advanced atretic follicles in both animals. Granulosa cells (in both SB and HF) showed a higher immunopositive staining than theca cells. In the theca interna, VEGF immunostaining diminished as follicles progressed to the late atretic follicles in both animals. These results indicate that during atresia, changes of vWF expression are the opposite of VEGF expression in SB. Both vWF and VEGF are suggested to be associated with follicular atresia in SB.     

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.     

Messenger ribonucleic acid expressions of hepatocyte growth factor, angiopoietins and their receptors during follicular development in gilts

Angiogenic factors are associated with angiogenesis during follicular development in the mammalian ovary. The aim of the present study was to determine the relationships between the vascular network and mRNA expressions of angiopoietins (Ang)-1, Ang-2 and hepatocyte growth factor (HGF), and their receptors in follicles at different developmental stages during follicular development. Ovaries in gilts were collected 72 h after equine chorionic gonadotropin (eCG, 1250 IU) treatment for histological observation of the capillary network. Granulosa cells and thecal tissues in small (<4 mm), medium (4-5 mm) or large (>5 mm) individual follicles were collected for detection of mRNA expression of HGF, Ang-1 and Ang-2 in granulosa cells, and HGF receptor (HGF-R) and Tie-2 in the theca cells by semi-quantitative RT-PCR. The number of capillaries in the thecal cell layer increased significantly in healthy follicles at all developmental stages in the eCG group compared with those in controls. The expression of Ang-1 mRNA declined in granulosa cells of medium and large follicles and the level of Ang-2 mRNA increased in granulosa cells of small follicles after eCG treatment. The ratio of Ang-2/Ang-1 increased in small, medium and large follicles from ovaries after eCG treatment, but Tie-2 mRNA expression in the theca cells did not change. The level of HGF mRNA increased in granulosa cells of small follicles after eCG treatment but HGF-R in theca cells was not increased by eCG. These data suggested that the angiopoietins might be associated with thecal angiogenesis during follicular development in eCG-treated gilts.     

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.     

Heat Shock Protein 70 and Sex Steroid Receptors in the Follicular Structures of Induced Ovarian Cysts

The purpose of this study was to estimate the expression and relative amounts of estrogen (ER) and progesterone receptors (PR) and their isoforms as well as heat shock protein 70 (HSP70) in ovaries of rats with induced cystic ovarian disease (COD). Primary, secondary, tertiary, atretic and cystic follicles were evaluated by immunohistochemistry and total ovarian proteins were analyzed by Western blot. In the granulosa layer, growing and cystic follicles in the treated group have a higher expression of ERα than growing follicles of control individuals. In the theca interna layer, tertiary follicles presented a significantly higher expression of ERα in the treated group. An increase in total ERα protein was detected in the treated group. Granulosa cells of all growing, atretic and cystic follicles show a lower expression of ERβ in animals with COD, and the total protein expression of ERβ was lower in this group. The expression of PR was lower in the granulosa cell layer of tertiary and cystic follicles in treated animals, and theca interna layer had less intense immunostaining in this group. Although there were no differences in the expression of PR-B by Western blotting, the expression of PR-A was higher and the expression of PR-C was smaller in the treated group. An intense HSP70 immunostaining was observed in the cells of cystic follicles. By Western blotting, higher protein expression of HSP70 was detected in the ovarian samples of the control group than those of the treated ones. Ovaries of animals with COD exhibited an altered steroid receptor expression and subtype balance as compared with control animals, and an increase in HSP70 immunoexpression.     

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.     

Apoptosis in the Antral Follicles of Swamp Buffalo and Cattle Ovary: TUNEL and Caspase-3 Histochemistry

The present study was carried out to investigate the pattern of apoptosis in the healthy antral and atretic follicles of Philippine swamp buffaloes (BU) in comparison with Holstein-Friesian (HF) cows. Paraffin sections of healthy follicles and various stages of atretic follicles were stained using the terminal deoxynucleotidyl transferase (TdT)-mediated biotinylated deoxyuridine triphosphates (dUTP) nick end-labelling (TUNEL) method to detect DNA fragmentation and cleaved caspase-3 antibody to detect cells committed to undergo apoptosis. Five equidistant areas of a follicle were counted for the presence of TUNEL- and caspase-3-positive cells. Healthy follicles of BU and HF contained no TUNEL-positive cells in the granulosa and theca layer but showed some caspase-3 positivity. The granulosa layer of advanced atretic follicles showed a significantly higher frequency of caspase-3 positivity than the healthy and early atretic follicles in both breeds. The frequency of caspase-3-positive cells of BU was significantly higher than HF in the granulosa layer of healthy, early atretic and advanced atretic follicles. In the theca interna layer, BU and HF showed a significantly lower and higher frequency of TUNEL-positive cells in the late atretic follicles compared with advanced atretic follicle, respectively. However, the frequency of caspase-3-positive cells of both BU and HF in the late atretic follicles was significantly higher than the advanced atretic follicles in the theca interna layer. These results indicate that caspase-3 and DNA fragmentation is involved in the buffalo ovarian apoptotic process.     

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.     

Leptin Immunohistochemical Staining in the Porcine Ovary

This study aimed to investigate leptin immuno‐staining of the porcine ovary in different reproductive stages. Ovaries from 21 gilts were collected from slaughterhouses. The ovarian tissue sections were incubated with a polyclonal anti‐leptin as a primary antibody. The immuno‐staining in ovarian tissue compartments was calculated using imaging software. Leptin immuno‐staining was found in primordial, primary, preantral and antral follicles. Leptin immuno‐staining was expressed in the oocyte and granulosa and theca interna layers in both preantral and antral follicles. In the corpora lutea, leptin immuno‐staining was found in the cytoplasm of the luteal cells. The leptin immuno‐staining in the granulosa cell layer of preantral follicles did not differ compared to antral follicles (90.7 and 91.3%, respectively, P > 0.05). However, the leptin immuno‐staining in the theca interna layer of preantral follicles was lower than antral follicles (49.4 and 74.3%, respectively, P < 0.001). There was no difference in leptin immuno‐staining in the granulosa cell layer between follicular and luteal phases (92.4 and 89.7%, respectively, P > 0.05). However, the leptin immuno‐staining in the theca interna layer of follicular phase was greater than that in the luteal phase (72.7 and 51.0%, respectively, P < 0.001). These findings indicated that leptin exists in different compartments of the porcine ovary, including the oocyte, granulosa cells, theca interna cells, corpus luteum, blood vessel and smooth muscles. Therefore, this morphological study confirmed a close relationship between leptin and ovarian function in the pig.     

Distribution of Cytochrome P450-side Chain Cleavage in the Theca Interna Layers of Bovine Small Antral and Cystic Follicles

Cystic follicle is anovulatory follicular structure that is caused by an endocrine imbalance. The activity of cytochrome P450‐side chain cleavage (P450scc) is essential for the initiation of steroidogenesis in the follicle. The present study was designed to compare the frequency of cells containing P450scc between healthy and atretic small antral follicles, and among several types (I, II and III, classified based on the presence of granulosa layer) of cystic follicles. Paraffin sections of healthy (2–5 mm in diameter), atretic (2–5 mm) and cystic follicles (>25 mm) were immunohistochemically stained with rabbit polyclonal antibody to bovine P450scc. The P450scc‐positive cells were counted in four different regions of the follicles from the apical to the basal side. In small antral follicles and cystic follicles, P450scc‐positive cells were localized in the theca interna layers but not granulosa layers. The P450scc‐positive cell populations decreased in the late atretic follicles compared with the early and advanced atretic follicles at all the regions of follicle. Type III cystic follicles showed significantly lower frequencies of P450scc‐positive cells than those in the types I and II cystic follicles. These results suggest that in both small and cystic follicles in cows, total loss of granulosa cells may be associated with the reduction of frequency of P450scc‐positive cells in theca interna layer.     

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.