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.     

The endothelial nitric oxide synthase/nitric oxide system is involved in the defective quality of bovine oocytes from low mid-antral follicle count ovaries

In a previous survey concerning cows of reproductive age, we demonstrated that oocytes isolated from ovaries with <10 medium antral follicles of 2 to 6 mm in diameter (low ovaries; Lo) show less developmental competence than oocytes collected from ovaries with >10 medium antral follicles (high ovaries; Hi). The aim of the present study was to evaluate whether a defective endothelial nitric oxide synthase/nitric oxide (eNOS/NO) system and vasculature in healthy medium antral follicles is likely to reduce oocyte competence from Lo ovaries. Thus, experiments were conducted to 1) immunolocalize eNOS protein during folliculogenesis; 2) quantify eNOS protein/vasculature in the follicle wall; and 3) verify if NO donor, S-nitroso acetyl penicillamine (SNAP) administration during in vitro maturation affects developmental competence of oocytes isolated from Lo ovaries. Endothelial nitric oxide synthase protein was detected in granulosa and theca cells, as well as in blood vessels from primordial to antral follicles. Quantitative analysis indicated that in medium antral follicles from Lo ovaries, eNOS protein expression and vasculature were reduced (P < 0.05). The addition of SNAP improved blastocyst and hatching rates of oocytes from Lo ovaries, promoting a percentage similar to oocytes from Hi ovaries, and reduced the percentage of apoptotic nuclei in in vitro-produced blastocysts (P < 0.05). Results from our study suggest that in bovine ovaries with small mid antral follicle number, a defective eNOS/NO system is related to a reduced follicle vasculature and may affect oocyte quality, thus inducing a premature decline of fertility.     

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.     

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.     

Ovarian localization of 11β-hydroxysteroid dehydrogenase (11βHSD): effects of ACTH stimulation and its relationship with bovine cystic ovarian disease

Cystic ovarian disease (COD) is an important cause of infertility in cattle, and ACTH has been involved in regulatory mechanisms related to ovarian function associated with ovulation, steroidogenesis, and luteal function. Here, we examined the localization of 11β-hydroxysteroid dehydrogenase type 1 (11βHSD1) and 11βHSD2 proteins in the ovary of healthy cows and animals with spontaneous and ACTH-induced COD and the in vitro response of the follicular wall exposed to ACTH. After stimulation by ACTH, we documented changes in 11βHSD expression and cortisol secretion by the follicular wall of large antral and follicular cysts. Follicular cysts showed a higher constitutive expression of both enzymes, whereas ACTH induced an increase in 11βHSD1 in tertiary follicles and follicular cysts and a decrease in 11βHSD2 in follicular cysts. Moderate expression of 11βHSD1 was observed by immunohistochemistry in granulosa of control animals, with an increase (P < 0.05) from primary to secondary, tertiary, and atretic follicles. The level of immunostaining in theca interna was lower than that in granulosa. The expression of 11βHSD2 was lower in the granulosa of primary follicles than in that of secondary, tertiary, and atretic follicles and was lower in the theca interna than in the granulosa. In ACTH-induced and spontaneously occurring follicular cysts, differences from controls were observed only in the expression of 11βHSD1 in the granulosa, being higher (P < 0.05) than in tertiary follicles. These findings indicate that follicular cysts may be exposed to high local concentrations of active glucocorticoids and indicate a local role for cortisol in COD pathogenesis and in regulatory mechanisms of ovarian function.     

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.     

Expression of gap junction protein connexin 43 during follicular atresia in the ovary of swamp buffaloes

The present study was performed to detect the presence of gap junction protein connexin 43 (Cx43) and describe the changes in its expression during ovarian follicular atresia in the swamp buffalo in comparison with cattle. Ovaries of Philippine swamp buffaloes (Bubalus bubalis; SB) and Holstein-Friesian cows (Bos taurus; HF) were collected from slaughterhouses, fixed in 10% formalin in PBS and embedded in paraffin. Sections of healthy follicles and at various follicular stages of atresia were immunostained with anti-Cx43 antibody. Cx43 appeared as punctate staining between granulosa cells (healthy to advanced atretic follicles), indicating assembled gap junctions, but was absent in the theca interna. In SB as well as in HF, granulosa cells showed a dense, moderate, and sparse immunoreactivity to Cx43 in healthy, early atretic, and advanced atretic follicles, respectively. Cumulus cells (in the advanced atretic follicle) surrounding oocytes and adjacent granulosa layers retain the Cx43 protein, although there was only a sparse expression of Cx43 observed in the granulosa layers distant from oocytes in the same follicles. The results indicate that gap junction protein Cx43 decreases in association with atresia and supports the concept that a loss of gap junctional communication plays a coordinating role in the process of atresia. Furthermore, the schema of Cx43 immunoreactivity in SB granulosa cells is similar to that of HF.     

Changes in the thecal vasculature during follicular atresia in the ovary of swamp buffalo

This study aimed to describe the changes in the thecal vasculature during ovarian follicular atresia in the swamp buffalo. Ovaries of Philippine swamp buffalo (Bubalus bubalis; SB), crossbred (SB x Murrah buffalo; CB) and Holstein-Friesian cow (Bos taurus; HF) were collected from slaughterhouses, fixed in 10% formalin in PBS and embedded in paraffin. Sections of healthy follicle and various follicular stages of atresia were stained with Bandeiraea simplicifolia-I lectin (BSL-I) to visualize the endothelial cells of blood vessels. In the theca interna, healthy follicles in SB had a significantly lower number of capillary vessels than other breeds and other atretic stages of follicle. From healthy to early atretic follicle, theca interna in all breeds showed a significant decrease in the area of capillary vessel. Capillary vessel area significantly increased (but was smaller than in healthy follicle) in the middle stage of atresia and declined again in the late atretic follicle (greater than in early atresia but smaller than in healthy follicle) in SB only. No significant change in the capillary vessel area of theca interna was noted in both CB and HF from early to late atretic follicles. There was no significant difference in the capillary vessel number and area of theca externa among the different breeds and atretic stages of follicle. These results suggest that there are dynamic changes occurring in the thecal vasculature of SB but not CB during follicular atresia which differs among cattle.     

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.     

Colocalization of DNA fragmentation and caspase-3 activation during atresia in pig antral follicles

Apoptosis is the cellular mechanism of ovarian follicular atresia. The major downstream effector of this phenomenon in many tissues is caspase-3 but little is known about its role in pig ovarian apoptosis. In the present study, we detected the localization of caspase-3 in parallel with nuclear fragmentation (TUNEL) on healthy and early atretic antral follicles. In healthy antral follicles caspase-3 and TUNEL positivity were occasionally recorded within theca layer. The incidence of DNA fragmentation, as indicated also by the biochemical detection, increased mainly in the granulosa layer of early atretic follicles. Quantitative analysis revealed, besides, that atresia was accompanied by a higher incidence of caspase-3 (57.20 +/- 20.05 versus 3.64 +/- 0.61 positive cells in atretic versus healthy follicles, respectively; P < 0.05), of TUNEL positivity (20.13 +/- 9.33 versus 0.42 +/- 0.12; P < 0.05) and simultaneous immunostaining for caspase-3 and TUNEL (15.02 +/- 6.95 versus 0.31 +/- 0.05; P < 0.05) in the granulosa layer. In detached granulosa cells isolated from the follicular fluid of early atretic follicles a further significantly increase was recorded in the percentage of TUNEL positivity and in the incidence of cells that showed colocalization of caspase-3 activity and DNA fragmentation. Granulosa cells of early atretic follicles exhibited a higher positivity for caspase-3 localized in the cytoplasm and occasionally in the nucleus area of granulosa cells. These results indicate that capsase-3 was involved and precociously activated during the process of atresia. Finally, the progressively higher incidence of TUNEL positivity and of double immunostaining in atretic cells collected within the follicular fluid seems to indicate that proteases activity leads only tardily in a detectable DNA fragmentation.     

Comparison of Antral and Preantral Ovarian Follicle Populations Between Bos indicus and Bos indicus‐taurus Cows with High or Low Antral Follicles Counts

The objective was to compare populations of antral and pre‐antral ovarian follicles in Bos indicus and Bos indicus‐taurus cows with high and low antral follicle counts. Nelore (Bos indicus, n = 20) and Nelore X Angus (1/2 Bos indicus‐taurus, n = 20) cows were subjected to follicular aspiration without regard to the stage of their oestrous cycle (day of aspiration = D0) to remove all follicles ≥3 mm and induce growth of a new follicular wave. Ovaries were examined by ultrasonography on D4, D19, D34, D49 and D64, and antral follicles ≥3 mm were counted. Thereafter, cows were assigned to one of two groups: high or low antral follicular count (AFC, ≥30 and ≤15 antral follicles, respectively). After D64, ovaries were collected after slaughter and processed for histological evaluation. There was high repeatability in the numbers of antral follicles for all groups (range 0.77–0.96). The mean (±SD) numbers of antral follicles were 35 ± 9 (Bos indicus) and 38 ± 6 (Bos indicus‐taurus) for the high AFC group and 10 ± 3 (Bos indicus) and 12 ± 2 (Bos indicus‐taurus) follicles for the low AFC. The mean number of preantral follicles in the ovaries of Bos indicus‐taurus cows with high AFC (116 226 ± 83 156 follicles) was greater (p < 0.05) than that of Bos indicus cows (63 032 ± 58 705 follicles) with high AFC. However, there was no significant correlation between numbers of antral and preantral follicles.     

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.     

Regulation of secondary follicle growth by theca cells and insulin-like growth factor 1

Ovaries contain follicles at various stages of development, including primordial, primary, secondary, antral and Graafian follicles. Although the growth of these follicles is controlled to maintain regular ovulation, the mechanism through which this occurs remains unclear. In our study, we found that the growth rate of cultured secondary follicles separated from mice ovaries differed between follicles. After 4 days of culture, the size of some secondary follicles was markedly increased, while that of others had either slightly increased, remained unchanged or shrunk. We compared the expression levels of growth factors between these secondary follicles and found that the growth rate of cultured secondary follicles correlated with the expression level of insulin-like growth factor 1 (Igf1) mRNA. Igf1 mRNA expression level in secondary follicles containing theca cells was higher than that in secondary follicles without theca cells, and the granulosa cell proliferation around follicles containing theca cells was increased. Furthermore, an IGF1 inhibitor also inhibited the granulosa cell proliferation, and administration of IGF1 to secondary follicles without growth promoted granulosa cell proliferation. These results indicated that the theca cells of secondary follicles induced the expression of IGF1 and promoted the follicle growth.     

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.     

Gene expression and immunolocalization of fibroblast growth factor 2 in the ovary and its effect on the in vitro culture of caprine preantral ovarian follicles

This study quantified Fibroblast growth factor 2 (FGF-2) mRNA and localized FGF-2 protein in different categories of follicles isolated from goat ovaries. In addition, we verified the effects of this factor on the in vitro culture of preantral follicles isolated from goats. For mRNA quantification, we performed real-time PCR using primordial, primary and secondary follicles, as well as cumulus-oocyte complexes (COCs) and mural granulosa and theca cells of small and large antral follicles. For FGF-2 protein localization, the ovaries were subjected to conventional immunohistochemical procedures. Preantral follicles were isolated and cultured in vitro for 12 days in either control (basic) or supplemented with FGF-2 medium. The expression of FGF-2 mRNA was detected in all categories of follicles and there was no difference in preantral follicles and COCs or granulosa/theca cells from small and large antral follicles. However, in large antral follicles, COCs showed expression levels significantly lower than in granulosa/theca cells (p < 0.05). We observed moderate expression of FGF-2 protein in preantral follicles but not in granulosa cells of primordial follicles and theca cells of secondary follicles. In both small and large antral follicles, strong, moderate and weak staining was observed in oocytes, granulosa and theca cells, respectively. The addition of FGF-2 caused a significant increase in the daily follicular growth rate compared to the control group. We conclude that FGF-2 mRNA is expressed throughout follicular development and that its protein can be found in different patterns in preantral and antral follicles. Furthermore, FGF-2 increases the follicular growth rate in vitro.     

Expression of gap junctional connexin proteins in ovine fetal ovaries: effects of maternal diet

Gap junctions have been implicated in the regulation of cellular metabolism and the coordination of cellular functions during growth and differentiation of organs and tissues, and gap junctions play a major role in direct cell-cell communication. Gap junctional channels and connexin (Cx) proteins have been detected in adult ovaries in several species. Furthermore, it has been shown that several environmental factors, including maternal diet, may affect fetal organ growth and function. To determine whether maternal diet affects expression of Cx26, Cx32, Cx37, and Cx43 in fetal ovaries, sheep were fed a maintenance (M) diet with adequate (A) selenium (Se) or high (H) Se levels from 21 d before breeding to day 132 of pregnancy. From day 50 to 132 of pregnancy (tissue collection day), a portion of the ewes from the ASe and HSe groups was fed a restricted (R; 60% of M) diet. Sections of fetal ovaries were immunostained for the presence of Cxs followed by image analysis. All four Cxs were detected, but the distribution pattern differed. Cx26 was immunolocalized in the oocytes from primordial, primary, secondary, and antral follicles; in granulosa and theca layers of secondary and antral follicles; stroma; and blood vessels. Cx32 was in oocytes, granulosa, and theca cells in a portion of antral follicles; Cx37 was on the borders between oocyte and granulosa/cumulus cells of primordial to antral follicles and in endothelium; and Cx43 was on cellular borders in granulosa and theca layers and between oocyte and granulosa/cumulus cells of primordial to antral follicles. Maternal diet affected Cx26 and Cx43 expression, Cx26 in granulosa layer of antral follicles was decreased (P < 0.01) by HSe in the M and R diets, and Cx43 in granulosa layer of primary and granulosa and theca of antral follicles was increased (P < 0.05) by the M diet with HSe. Thus, Cxs may be differentially involved in regulation of fetal ovarian function in sheep. These data emphasize the importance of maternal diet in fetal growth and development.     

Does Apoptosis Occur during Follicular Atresia in the Follicular Walls of the Porcine Ovary?

Contents In this experiment, the possibility that the follicular-wall cells' death during ovarian follicular atresia occurs as a result of apoptosis was examined. Programmed cell death or apoptosis is a process whereby cells die in a controlled fashion, triggered by changes in levels of specific physiological stimuli. Morphological transformations of the cells are preceded by endo- nuclease-mediated genomic-DNA cleavage. The analysis of DNA from the theca and granulosa layers of follicles indicated that internucleosomal fragmentation of DNA occurred in atretic granulosa cells but not in atretic theca cells. The healthy granulosa and theca cells in all classes of follicles showed no apoptosis. This paper demonstrates that the death of porcine ovarian-follicle walls can be caused by different processes and, contrary to granulosa cells' apoptosis, either does not or only partly concerns the internal theca layer.     

Distribution of immunoreactive von Willebrand factor in the microvascular network of bovine cystic follicles

The present study was undertaken to examine whether the von Willebrand factor (vWF) expression in the follicular microvasculature in the cystic follicles differs from that in the atretic follicles. Paraffin sections of healthy, atretic and cystic follicles were immunostained with rabbit monoclonal antibody to vWF. The vWF-positive cells were counted in four different regions of a follicle from the apical to the basal side. In all types of follicles, immunoreactions for vWF were observed in the endothelial cells of capillaries as well as veins and arteries in the theca interna and externa. In the theca interna, vWF-positive areas were significantly lower in the Type A and B cystic follicles compared to advanced and late atretic follicles. In the theca externa, the vWF-positive blood vessels and vWF-positive area were significantly smaller in all types of cystic follicles than in the healthy or atretic follicles. From these results, it is suggested that in the cystic follicles the induction of vWF in the follicular microvasculature system is reduced, which may suppress the degeneration of vascular system. Continuation of stability in vasculature may be one of the factors that delays the tissue regression in the cystic follicles, and also contributes to the accumulation of follicular fluid that originates from the serum.     

An immunohistochemical study of bovine antral follicles,with special attention to non‐atretic follicles with and without atypical granulosa cells

Summary

Inhibin and oxytocin were immunohistochemically demonstrated in all non‐atretic and light‐atretic follicles >2 mm from untreated and pregnant mare's serum gonadotrophin (PMSG)‐treated heifers and cows. Immunostaining for luteinizing hormone (LH) and oestradiol was observed in all non‐atretic follicles >4 mm, but only in follicles from PMSG‐treated cows. Inhibin and oestradiol immunoreactivity was restricted to the granulosa. Oxytocin and LH immunoreactiviity was visualized in both the theca interna and the granulosa. Within the granulosa, LH immunoreactivity was mainly present in cells that were located near the basement membrane. Normal granulosa cells differed from atypical granulosa cells (AGCs) with respect to their ability to bind LH and oestradid It is concluded that immunostaining for α‐inhibin, oxytocin, oestradiol and LH cannot be used as a marker of follicle quality to discriminate between non‐atretic follicles with AGCs and non‐atretic follicles without AGCs in mid‐luteal bovine ovaries.