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.     

A morphological and immunohistochemical study of healthy and atretic follicles in the ovary of the sexually immature ostrich (Struthio camelus)

The morphology of healthy and atretic follicles in the ovary of the sexually immature ostrich was described in the present study. In addition, the distribution of the intermediate filaments desmin, vimentin and smooth muscle actin, in these ovarian follicles, was demonstrated. Healthy and atretic primordial, pre-vitellogenic and vitellogenic follicles were present in the ovaries of the sexually immature ostrich. Atresia occurred during all stages of follicular development. Atretic primordial and pre-vitellogenic follicles were characterized by the presence of a shrunken oocyte surrounded by a multilayered granulosa cell layer. Two forms of atresia (types 1 and 2) were identified in vitellogenic follicles. In the advanced stages of type 1 atresia the follicle was dominated by a hyalinized mass. In contrast, in type 2 atresia the granulosa and theca interna cells differentiated into interstitial gland cells. Positive immunostaining for desmin was observed in the granulosa cells of only healthy primordial and pre-vitellogenic follicles. Atretic primordial and pre-vitellogenic follicles were immunonegative for desmin. Vimentin immunoreactivity was demonstrated in the granulosa cells of all follicles except the vitellogenic atretic follicles. The results of the present study indicate that ovarian follicles in the sexually immature ostrich undergo a cycle of growth and regression, which is similar to that reported in other avian species. Furthermore, based on the results of the immunohistochemical study, it would appear that the distribution and immunostaining of intermediate filaments changes during follicular development and atresia.     

Expression of Mitochondria‐Associated Genes (PPARGC1A,NRF‐1, BCL‐2 and BAX) in Follicular Development and Atresia of Goat Ovaries

Most follicles undergo atresia during the developmental process. Follicular atresia is predominantly regulated by apoptosis of granulosa cells, but the mechanism underlying apoptosis via the mitochondria‐dependent apoptotic pathway is unclear. We aimed to investigate whether the mitochondria‐associated genes peroxisome proliferator‐activated receptor‐gamma, coactivator1‐alpha (PPARGC1A), nuclear respiratory factor‐1 (NRF‐1), B‐cell CLL/lymphoma 2 (BCL‐2) and BCL2‐associated X protein (BAX) played a role in follicular atresia through this pathway. The four mitochondria‐associated proteins (PGC‐1α, which are encoded by the PPARGC1A gene, NRF‐1, BCL‐2 and BAX) mainly expressed in granulosa cells. The mRNA and protein levels of PPARGC1A/PGC‐1α and NRF‐1 in granulosa cells increased with the follicular development. These results showed that these genes may play a role in the regulation of the follicular development. In addition, compared with healthy follicles, the granulosa cell in atretic follicles had a reduced expression of NRF‐1, increased BAX expression and increased ratio of BAX to BCL‐2 expression. These results suggested that changes of the mitochondria‐associated gene expression patterns in granulosa cells may lead to follicular atresia during goat follicle development.     

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.     

Expression of ski in the granulosa cells of atretic follicles in the rat ovary

The aim of the present study was to locate Ski protein, a product of cellular protooncogene c-ski, in rat ovaries in order to predict the possible involvement of Ski in follicular development and atresia. First, expression of c-ski mRNA in the ovaries of adult female rats was confirmed by RT-PCR. Then, ovaries obtained on the day of estrus were subjected to immunohistochemical analysis for Ski and proliferating cell nuclear antigen (PCNA) in combination with terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL). Ski was expressed in granulosa cells that were positive for TUNEL, but negative for PCNA, regardless of the size of follicles. Expression of Ski in TUNEL-positive granulosa cells, but not in PCNA-positive granulosa cells, was also verified in immature hypophysectomized rats having a single generation of developing and atretic follicles by treatment with equine chorionic gonadotropin. These results indicate that Ski is profoundly expressed in the granulosa cells of atretic follicles, but not in growing follicles, and suggests that Ski plays a role in apoptosis of granulosa cells during follicular atresia.     

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.     

Detection of Follicular Apoptosis in Water Buffalo (Bubalus bubalis) Ovary by Histology and Nick End Labelling Technique

The objective of this experiment was to assess the features and extent of follicular apoptosis in the water buffalo (Bubalus bubalis) ovary using classical histology and nick end labelling technique. Ovaries (n = 40) procured from the slaughterhouse were used for the study. The sections (5 μm) were used for detection of terminal deoxynucleotidyl transferase‐mediated dUTP‐biotin nick end labelling (TUNEL) and classical histology (H&E). Those follicles showing ≥ 5% TUNEL positivity (TUNEL assay) and pyknotic nuclei (histology) in granulosa cells were classified as atretic. Based on histology, the atretic primary and secondary follicles (%) were 93.82 and 95.62 respectively. The histology study reveals that the rates (%) of atresia in <1, 1–3, 3–5 mm and >5 mm were 36.90, 40.50, 62.84 and 74.5 respectively. Further the atretic tertiary follicles (%) were significantly lower than the primary and secondary classes of follicles. TUNEL assay reveals that the atretic rate (%) of tertiary follicles in <1, 1–3, 3–5 and ≥ 5 mm class follicles were 50.88, 53.84, 81.81 and 36.36 respectively. The percentage of atresia in >5 mm diameter follicles were significantly lower in TUNEL than histology. Percentages of granulosa and thecal cells positive for atresia by TUNEL were 30.7 ± 0.53 and 13.82 ± 0.18 respectively per follicle. The initial structural changes in atretic follicles were seen primarily in the granulosa cells. In severely atretic follicles TUNEL positive granulosa cells along with theca cells have to be considered in assessing the rate and extent of atresia.     

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.     

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.     

The regulation of ovarian granulosa cell death by pro- and anti-apoptotic molecules

In the mammalian ovary, follicular development and atresia are closely regulated by cell death and survival-promoting factors, including hormones (gonadotropins) and intraovarian regulators (gonadal steroids, cytokines, and intracellular proteins). Several hundred thousand primordial follicles are present in the mammalian ovary; however, only a limited number of primordial follicles develop to the preovulatory stage and ovulate. The others, more than 99% of follicles, will be eliminated via a degenerative process known as "atresia". The endocrinological regulatory mechanisms involved in follicular development and atresia have been characterized to a large extent, but the precise temporal and molecular mechanisms involved in the regulation of these events have remained largely unknown. Recent studies suggest that the apoptosis of ovarian granulosa cells plays a major role in follicular atresia. In this review, we provide an overview of development and atresia of follicles, and apoptosis of granulosa cells in mammals.     

Insulin-like growth factor binding proteins in granulosa and thecal cells from bovine ovarian follicles at different stages of development

Because IGFBP inhibit IGF-stimulated cellular proliferation and differentiation, it is hypothesized that variations among IGFBP in individual follicles might contribute to the regulation of recruitment, selection, dominance, and turnover of ovarian follicles. Sources of IGFBP in fluid of bovine follicles are not well established; thus, objectives of this study were to determine levels of IGFBP binding activities and messenger RNA (mRNA) in granulosa and theca interna cells at different stages of follicular development (small [< 6 mm], medium [6 to < 8 mm], and large [> or = 8 mm]) and to characterize associations of these levels measured in the cells with levels of IGFBP and steroids in follicular fluid. Thecal and granulosa cells from large healthy follicles contained two- to twentyfold less (P < 0.05) IGFBP-2, -3, and -5 than cells from small, medium, and large atretic follicles. Thecal cells from small, medium, and large atretic follicles contained more (P < 0.05) IGFBP-3 and -4 than granulosa cells from these follicles, whereas granulosa cells from these follicles contained more IGFBP-2 activity than thecal cells. Differences in IGF binding activity were paralleled by differences in levels of mRNA for the respective IGFBP. Developmental differences in IGFBP activity in follicular fluid were positively associated with activity in granulosa and/or thecal cells, with the exception of IGFBP-4, which was low in fluid from large healthy follicles but markedly increased (mRNA and binding activity) in granulosa cells from these follicles. It is concluded that developmental changes in follicular fluid IGFBP-2 and -5 binding activities seem to be controlled in part by alterations in synthesis of these IGFBP by granulosa and thecal cells, whereas diminished IGFBP-4 in fluid from large healthy follicles occurs concomitantly with increased levels of IGFBP-4 mRNA and activity in granulosa cells, implicating posttranslational regulation by specific proteases.     

Expression of Connexin 43 in the Porcine Foetal Gonads During Development

This study was designed to reveal connexin 43 (Cx43) mRNA and protein expression in porcine foetal gonads using RT‐PCR, immunohistochemistry and Western blot analysis. Expression of Cx43 was investigated in porcine foetal ovaries and testes on days 50, 70 and 90 post coitum (p.c.). RT‐PCR results indicated that Cx43 mRNA was expressed in both foetal ovaries and testes at all gestational ages examined. Cx43 protein was found in the foetal ovary but its distribution varied across ovarian compartments and changed during development. In foetal ovaries, Cx43 was localized between the interstitial cells surrounding egg nests on all investigated days of prenatal period. Moreover, Cx43 expression was observed between germ cells on day 50 p.c. as well as between pre‐granulosa and granulosa cells of primordial and primary follicles on days 70 and 90 p.c. In the foetal testes, Cx43 protein was detected between neighbouring Leydig cells on all examined days of prenatal period and between adjacent Sertoli cells exclusively on day 90 p.c. The presence of Cx43 protein in all investigated foetal gonads was confirmed by Western blot analysis. Cx43 protein detection between pre‐granulosa cells of primordial follicles suggests its role in regulation of the initial stages of follicle development. The Cx43 immunoexpression between neighbouring Leydig and between Sertoli cells indicates its involvement in controlling their functions. We propose that Cx43‐mediated gap junctional communication is involved in the regulation of porcine foetal gonadal development.     

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.