Autophagy and apoptosis interplay during follicular atresia in fish ovary: a morphological and immunocytochemical study

Follicular atresia in fish ovary provides an interesting model for studying autophagy and apoptosis. In order to improve knowledge of the mechanisms regulating ovarian regression, we investigated the immunolocalisation of various proteins involved in the complex network of autophagy and apoptosis. Females of three species of freshwater fish maintained in captivity were sampled after the reproductive period and the main events of follicular atresia were assessed by histology: splits in the zona radiata, yolk degradation and reabsorption, hypertrophy of the follicular cells, accumulation of autophagic vacuoles, closing of the follicular lumen and thickening of the theca. The interplay of apoptosis and autophagy was analysed by TUNEL in situ and by immunocytochemistry for caspase-3, bax, bcl-2, beclin-1 and cathepsin-D. During early and advanced stages of follicular regression, the actin cytoskeleton was well developed and labelling for bcl-2 and cathepsin-D were pronounced in the follicular cells at a stage when they were intensively involved in yolk phagocytosis. Immunofluorescence for beclin-1 was prevalent in the follicular cells, punctate labelling often surrounding autophagic vacuoles during the advanced stage of follicular regression, a critical step towards cell death. TUNEL-positive reaction and immunostaining for bax and caspase-3 demonstrated the participation of apoptosis in late follicular regression. Overall, this study provides evidence that autophagic and apoptotic proteins are activated in a coordinated fashion depending on the stage of follicular regression, with interplay between autophagy and apoptosis being essential in determining the fate of the cell during follicular atresia in fish ovary.     

Regulation of prohibitin expression during follicular development and atresia in the mammalian ovary

Prohibitin is a ubiquitous and highly conserved protein implicated as an important regulator in cell survival. Prohibitin content is inversely associated with cell proliferation, but it increases during granulosa cell differentiation as well as in earlier events of apoptosis in a temperature-sensitive granulosa cell line. In the present study, we have characterized the spatial expression patterns for prohibitin using established in vivo models for the induction of follicular development and atresia in the mammalian ovary. Comparative Western blot analyses of granulosa cell lysates from control ovaries and from ovaries primed with eCG or treated with eCG plus anti-eCG (gonadotropin withdrawal) were conducted. Prohibitin was immunolocalized in rat ovarian sections probed with antibodies against either proliferating cell nuclear antigen (PCNA) or cholesterol side-chain cleavage cytochrome P450 (P450(scc)) or in terminal deoxynucleotidyl transferase-mediated dUTP nick end labeled sections. Additionally, porcine oocytes, zygotes, and blastocyts were also immunolocalized with prohibitin antibody. Immunolocalization revealed the presence of prohibitin in granulosa cells, theca-interstitial cells, and the oocyte. The results indicate that prohibitin protein expression in the gonadotropin-treated cells was upregulated. Immunoreactivity of prohibitin was inversely related to PCNA expression during follicular maturation and colocalized with P450(scc). Prohibitin appeared to be translocated from the cytoplasm to the nucleus in atretic follicles, germinal vesicle-stage oocytes, zygotes, and blastocysts. These results suggest that prohibitin has several functional regulatory roles in granulosa and theca-interstitial cells and in the ovum during follicular maturation and atresia. It is likely that prohibitin may play an important role in determining the fate of these cells and eventual follicular destiny.     

Ultrastructure of follicular atresia in the rat

Observations were made on the sequence of morphologic changes in atresia of medium-sized preantral follicles in the rat. Ultrastructural studies indicated that in both control and hormonally treated animals granulosa cell changes, including nuclear condensation and alterations in cytoplasmic organelles, occurred prior to effects on the oocyte. In more advanced stages of atresia, extensive disruption of granulosa cell cytoplasm was associated with loss of microvilli and cytoplasmic vacuolization in oocytes. The findings are consistent with the view that follicular atresia begins with alterations in granulosa cells, effects on the oocyte occurring later in the atretic process.     

Theca interna: the other side of bovine follicular atresia

Currently, histological classifications of ovarian follicular atresia are almost exclusively based on the morphology of the membrana granulosa without reference to the theca interna. Atresia in the bovine small antral ovarian follicle has been redefined into antral or basal atresia where cell death commences initially within antral or basal regions of the membrana granulosa, respectively. To examine cell death in the theca interna in the two types of atretic follicles, bovine ovaries were collected and processed for immunohistochemistry and light microscopy. Follicles were classified as healthy, antral atretic, or basal atretic. Follicle diameter was recorded and sections stained with lectin from Bandeiraea simplicifolia to identify endothelial cells or with an antibody to cytochrome P450 cholesterol side-chain cleavage to identify steroidogenic cells and combined with TUNEL labeling to identify dead cells. The numerical density of steroidogenic cells within the theca interna was significantly reduced (P < 0.001) in basal atretic follicles in comparison with other follicles. Cell death was greater in both endothelial cells (P < 0.05) and steroidogenic cells (P < 0.01) of the theca interna of basal atretic follicles compared with healthy and antral atretic follicles. Thus, we conclude that the theca interna is susceptible to cell death early in atresia, particularly in basal atretic follicles.     

In vitro development of individually matured bovine oocytes in relation to follicular wall atresia

Morphologically good-quality cumulus oocyte complexes (COCs) can originate from slightly atretic follicles. Biochemical and ultrastructural investigations reveal that a very high percentage of bovine antral follicles express some degree of atresia. The aim of the present study was to determine the developmental competence of good quality COCs in relation to their biochemically estimated follicular wall apoptosis. For experimental design a single oocyte maturation system was established, followed by group culture processing oocytes together according to their level of follicular wall atresia estimated by an ELISA for apoptotic cell death. Single oocyte culture during maturation reduced the developmental capacity of oocytes significantly (P < 0.01), with 5% blastocysts versus 25% after common group culture. Blastocyst formation for single oocyte maturation was found exclusively in oocytes isolated from luteal stage ovaries with low degree of apoptosis. The level of follicular wall apoptosis in luteal stage follicles (0.79 +/- 0.05 units/mg protein, n = 198) was lower than in follicular stage follicles (1.14 +/- 0.05 units/mg protein, n = 208). This was caused by significant higher levels in small (< 3.5 mm diameter) and large (> 5.5 mm diameter) follicles of the latter group. In conclusion, despite reduced developmental capacity after single oocyte maturation, we were able to reveal some functional relationship between oocyte origin and quality. It was shown that morphologically good quality COCs isolated from follicles with higher degree of apoptosis lose their developmental capacity.     

miR-26b promotes granulosa cell apoptosis by targeting ATM during follicular atresia in porcine ovary

More than 99% of ovarian follicles undergo atresia in mammals, but the mechanism of follicular atresia remains to be elucidated. In this study, we explored microRNA (miRNA) regulation of follicular atresia in porcine ovary. A miRNA expression profile was constructed for healthy, early atretic, and progressively atretic follicles, and the differentially expressed miRNAs were selected and analyzed. We found that miR-26b, which was upregulated during follicular atresia, increased the number of DNA breaks and promoted granulosa cell apoptosis by targeting the ataxia telangiectasia mutated gene directly in vitro.     

Mechanism of granulosa cell death during follicular atresia depends on follicular size

Changes in granulosa cell lysosomal and mitochondrial functions in relation to follicular size and to the stage of atresia were studied by fluorescent emission spectra and intensity using flow cytometry. Antral follicles were grouped by size in two groups: small, 3-6 mm and large, >6mm in diameter, and classified into three stages of atresia: non-atretic, initially atretic and advanced atretic. Differences in Rhodamine 123 (Rh123) and Acridine Orange (AO) fluorescent intensity indicated that changes in mitochondrial function are the primary mechanism of granulosa cell death in atretic follicles 3-6 mm in diameter, while its role in granulosa cell death in >6 mm atretic follicles seemed to be less important. However, modifications in lysosomal function (shown by a decrease in fluorometric intensity of AO incubated granulosa cells) were mainly associated with cell death in large atretic follicles. Our results support the hypothesis that the pathway of granulosa cell death during follicular atresia depends on the state of energy metabolism or on the production of hypoxic conditions related to follicular size. Changes in mitochondrial membrane potential and production of permeability transition pores were the main changes found in small follicles, while lysosomal function destabilization seemed to be the major cause of granulosa cell death during atresia in large follicles.     

Effects of gonadotropins on follicular development, ovulation, and atresia in the mature guinea pig

The induction of follicular growth, ovulation, and atresia by heterologous gonadotropic preparations was studied late in the reproductive cycle of the adult female guinea pig. Human chorionic gonadotropin (HCG) administration (10 IU) 12 days following the first signs of opening of the vaginal membrane was found to stimulate ovulation within 24 h in all animals studied, as evidenced by recovery of ova from their oviducts as well as the presence of postovulatory follicles in their ovaries. Histologically, ovaries of animals receiving HCG exhibited atretic changes in most of the follicles smaller than 999 micrometer in diameter. Pregnant mares serum gonadotropin (PMSG, 10 IU) administered on days 9 and 10 of the cycle was not sufficient to stimulate ovulation in this species although histological changes in the follicular complement were observed. Administration of PMSG prior to the HCG appeared to have an inhibitory effect on ovulation induction. Follicles luteinizing with entrapped ova were seen in all groups receiving exogenous gonadotropin, although they were most prevalent in the animals receiving the maximum total gonadotropin doses (i.e. PMSG + HCG).     

Chemotactic detection of incipient ovarian follicular atresia.

Chemotactic activity was measured in the follicular fluid collected from normal and atretic Graafian follicles isolated from the rat ovaries. The atresia of Graafian follicles was induced by pentobarbitone injections for 3 days beginning the day of proestrous. The chemotactic activity, as measured by direct morphological evaluation of cellular locomotion of individual cells and Boyden leading front assay, was significantly higher in follicular fluid from atretic follicles and it showed a progressive increase from day 1 to day 3 of blockade of ovulation. In vitro exposure of blocked follicles to PMSG and hCG on day 1, 2 and 3 failed to alter the chemotactic response of leukocytes towards follicular fluid of atretic follicles. Increased chemotaxis in the follicular fluid after 24 hr of blockade of ovulation appears to form an important criterion to identify atretic follicles well in advance, before the morphological symptoms of degeneration become apparent and the incipient change once induced in follicles is not reversed by gonadotropins.     

Alterations in follicular fluid steroids and follicular hCG and FSH binding during atresia in hamster

Preovulatory follicles from hamsters treated on proestrus for 1-3 days with phenobarbital sodium exhibited early signs of atresia after 2-3 days of ovulatory delay. A significant increase in follicular fluid progesterone was evident by Day 1 of delay. Concentrations of androstenedione in follicular fluid were unaffected by ovulatory delay. Follicular fluid levels of estradiol in delayed follicles were either higher than proestrous values after 1 day of delay or lower after 2 and 3 days of delay. hCG binding was slightly higher than proestrous controls after ovulatory delay whereas FSH binding was significantly lower than controls after 2 and 3 days of ovulatory delay. These results indicate that in the barbiturate-treated hamster the elevated follicular fluid levels of progesterone precede by 1-2 days the previously reported increase in steroidogenic capability of delayed follicles to produce progesterone in vitro; this correlated with an increase in the ratio of hCG:FSH binding and this was mostly due to a decrease in FSH binding to whole follicles.     

Ovarian follicular atresia is mediated by heterophagy, autophagy, and apoptosis in Prochilodus argenteus and Leporinus taeniatus (Teleostei: Characiformes)

We investigated apoptosis, cell proliferation antigen (PCNA), and heat shock protein (HSP70) during ovarian follicular atresia in two freshwater teleost species from the São Francisco River basin, Brazil: curimatã-pacu, Prochilodus argenteus and piau-jejo, Leporinus taeniatus. Fishes were maintained in captivity after the reproductive period and ovarian regression was assessed by gonadosomatic index for three stages: early, advanced, and late regression. Follicular atresia was analysed by light and transmission electron microscopy, as well as by TUNEL and immunohistochemistry for HSP70 and PCNA. During early regression, atretic follicles exhibited zona pellucida breakdown, yolk degeneration, and hypertrophied follicular cells (e.g. granulosa in mammals). Intense heterophagy to engulf the yolk, and autophagy were detected in the follicular cells during advanced and late atresia. The TUNEL assay detected DNA fragmentation, mainly in late follicular atresia. The apoptosis rate of the follicular cells increased up to 10% during follicular atresia in both species and was negatively correlated with follicular area. Immunohistochemistry reaction for HSP70 stained the follicular cells strongly during advanced atresia, when they are intensively involved in yolk engulfment, whereas the reaction for PCNA labelled theca cells. We inferred that heterophagy, autophagy, and apoptosis contributed to follicular atresia in teleost ovaries, thereby achieving a more efficient removal of the degenerating oocyte and dying follicular cells. Additionally, HSP70 may protect the follicular cells before apoptosis when they are involved in yolk engulfment, and cell proliferation in the theca contributed to ovarian remodelling.     

Expression and function of Fas antigen vary in bovine granulosa and theca cells during ovarian follicular development and atresia

Fas antigen is a receptor that triggers apoptosis when bound by Fas ligand (FasL). A role for Fas antigen in follicular atresia was studied in follicles obtained during the first wave of follicular development during the bovine estrous cycle (estrus is Day 0). Granulosa and theca cells were isolated from healthy dominant follicles and the two largest atretic subordinate follicles on Day 5, atretic dominant follicles on Days 10-12, and preovulatory follicles on Day 1. Fas antigen mRNA levels were highest in granulosa cells from subordinate as compared to other follicles, and lowest in theca cells from healthy Day 5 dominant as compared to other follicles. FasL alone had no effect on viability of granulosa or theca cells but became cytotoxic in the presence of interferon-gamma (IFN). IFN has been shown to induce responsiveness to Fas antigen-mediated apoptosis in other cell types. In the presence of IFN, killing of granulosa cells by FasL was greater in subordinate compared to healthy dominant follicles on Day 5, did not differ between healthy and atretic dominant follicles, and was similar in theca among all follicles. Granulosa cells from preovulatory follicles, which had been exposed to the LH surge in vivo, were completely resistant to FasL-induced killing. In summary, Fas antigen expression, and responsiveness to Fas antigen-mediated apoptosis, vary during follicular development.     

Ovarian follicular atresia in two teleost species: a histological and ultrastructural study

Follicular atresia is a common phenomenon in vertebrate ovaries involving the oocyte and the follicular wall degeneration. Female Astyanax bimaculatus lacustris and Leporinus reinhardti were kept in aquaculture cages inside tanks from February 1994 to January 1995 for the study of the characteristics of different stages of follicular atresia. Histological and ultrastructural analysis demonstrated similarities in the degenerative events and in the resorption of oocytes in both species. Degradation of organelles, such as mitochondria, cortical alveoli, and annulate lamellae occurred in the peripheral ooplasm during the initial stage of the process. Follicle cells showed marked phagocytic activity with digestive vacuoles, myelin figures, and lipofuscin granules during the intermediate and advanced stages of follicular atresia. Granulocytes were in activity during the final stage of follicle resorption. The duration of follicular atresia was 4 months in Leporinus reinhardti and 7 months in Astyanax bimaculatus lacustris. When submitted to induced reproduction in December 1995, 50-60% of the females of both species responded to induced spawning, indicating the recovery of gonadal activity. It is suggested that, in captive conditions, follicular atresia is shorter in total-spawning fishes when compared to those showing partial spawning, and that it has no apparent deleterious effects on induced reproduction in the subsequent cycle.     

血管紧张素Ⅱ在小鼠卵泡闭锁中的作用

应用幼年小鼠经孕马血清促性腺激素（pregnant mares serum gonadotropin,PMSG)处理的动物模型,研究了卵泡从发育到闭锁动态变化过程中血管紧张素Ⅱ（AngⅡ）的作用。结果表明：（1）24日龄小鼠给予PMSG（10IU/只）后6d时,卵巢中出现大量闭锁卵泡,颗粒细胞DNA琼脂糖电泳显示了梯形条带;（2）随卵泡闭锁发生,卵巢AngⅡ含量增加;（3）AngⅡ显著拮抗FSH刺激颗粒细胞雌二醇生成的作用。我们认为,AngⅡ参与了对小鼠卵泡闭锁的调节。     

The effects of the environmental antiandrogen vinclozolin on the induction of granulosa cell apoptosis during follicular atresia in pigs

The aim of this study was to investigate whether the androgens testosterone and dihydrotestosterone (DHT) and the antiandrogenic fungicide vinclozolin (Vnz) exert proapoptotic effects on porcine granulosa cells (GCs), and to examine the roles of these compounds in follicular atresia. Granulosa cells isolated from pig follicles were cultured for 24 hours, and then exposed to 0.1 μM testosterone, 0.1 μM DHT, 14 μM Vnz, or the equivalent concentrations of testosterone and Vnz or DHT and Vnz for a further 24 hours. Apoptosis and necrosis of the GCs were determined via Hoechst staining and flow cytometry analyses of annexin V-stained cells. Whole porcine follicles were also exposed to the same compounds and combinations of compounds for 24 hours. The sections were stained with hematoxylin and eosin for morphologic assessments, and a Terminal deoxynucleotidyl Transferase Biotyn-dUTP Nick-End Labeling (TUNEL) assay was performed to determine the number of apoptotic cells. The progesterone and estradiol concentrations secreted into the culture media by isolated GCs and follicles were also measured. Exposure to the androgens resulted in an increased number of apoptotic GCs both in vitro and in the organotypic model. Vinclozolin exposure increased and decreased the number of necrotic and apoptotic GCs, respectively. Furthermore, compared with control follicles, those exposed to testosterone, DHT, or Vnz displayed enhanced atresia, and coadministration of Vnz attenuated the promotive effect of these androgens on atresia. Estradiol secretion was stimulated by the combination of testosterone and Vnz, whereas exposure to Vnz alone reduced it. Progesterone production declined after the combined addition of androgens and the antiandrogen. In summary, Vnz caused massive necrosis of GCs in vitro and induced apoptosis of GCs in whole follicles. The androgens testosterone and DHT enhanced these effects. The results presented here suggest that selective destruction of porcine follicles is a serious consequence of exposure to Vnz, and may lead to premature ovarian failure in affected animals.