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In recent years, numerous studies conducted on teleost fish have highlighted the contribution of transcriptomic studies in elucidating the physiological mechanisms underlying the molecular events of oogenesis and follicular atresia, enabling the identification of potential genes and molecular networks that participate in both the reproductive cycle and the process of follicular atresia. Atresia can affect the reproductive potential of females by reducing the healthy eggs that a female can spawn in both aquaculture and wild populations. The substantial diversity of reproductive strategies exhibited by teleost fish has contributed to the difficulty in identifying common genes between species, but a set of core genes has emerged as potential markers for atresia in relation to apoptosis/autophagy, lipid metabolism, oxidative metabolism and other physiological processes similar to those identified in other vertebrates, even mammals. We review the current status of the genes that have been identified in ovaries with atretic oocytes. Our primary goal is to review the current status regarding gene expression during gonadal development and follicular atresia. This information will enable us to understand the factors and expression patterns involved in the follicular atresia of teleost fish., (Copyright © 2019 Elsevier B.V. All rights reserved.)

In mammals, more than 99% of ovarian follicles undergo a degenerative process known as atresia. The molecular events involved in atresia initiation remain incompletely understood. The objective of this study was to analyze differential gene expression profiles of medium antral ovarian follicles during early atresia in pig. The transcriptome evaluation was performed on cDNA microarrays using healthy and early atretic follicle samples and was validated by quantitative PCR. Annotation analysis applying current database ( Sus scrofa 11.1) revealed 450 significantly differential expressed genes between healthy and early atretic follicles. Among them, 142 were significantly upregulated in early atretic with respect to healthy group and 308 were downregulated. Similar expression trends were observed between microarray data and quantitative RT-PCR confirmation, which indicated the reliability of the microarray analysis. Further analysis of the differential expressed genes revealed the most significantly affected biological functions during early atresia including blood vessel development, regulation of DNA-templated transcription in response to stress and negative regulation of cell adhesion. The pathway and interaction analysis suggested that atresia initiation associates with (1) a crosstalk of cell apoptosis, autophagy and ferroptosis rather than change of typical apoptosis markers, (2) dramatic shift of steroidogenic enzymes, (3) deficient glutathione metabolism and (4) vascular degeneration. The novel gene candidates and pathways identified in the current study will lead to a comprehensive view of the molecular regulation of ovarian follicular atresia and a new understanding of atresia initiation., (© 2018 Society for Reproduction and Fertility.)

Most follicles in the mammalian ovary undergo atresia. Granulosa cell apoptosis is a hallmark of follicle atresia. Our previous study using a microRNA (miRNA) microarray showed that the let-7 microRNA family was differentially expressed during follicular atresia. However, whether the let-7 miRNA family members are related to porcine (Sus scrofa) ovary follicular apoptosis is unclear. In the current study, real-time quantitative polymerase chain reaction showed that the expression levels of let-7 family members in follicles and granulosa cells were similar to our microarray data, in which miRNAs let-7a, let-7b, let-7c, and let-7i were significantly decreased in early atretic and progressively atretic porcine ovary follicles compared with healthy follicles, while let-7g was highly expressed during follicle atresia. Furthermore, flow cytometric analysis and Hoechst33342 staining demonstrated that let-7g increased the apoptotic rate of cultured granulosa cells. In addition, let-7 target genes were predicted and annotated by TargetScan, PicTar, gene ontology and Kyoto encyclopedia of genes and genomes pathways. Our data provide new insight into the association between the let-7 miRNA family in granulosa cell programmed death.

Follicular atresia, a key phenomenon in follicle development, eliminates most of the follicles in mammalian ovaries. To investigate the molecular mechanism of follicular atresia in porcine ovaries, we investigated the mRNA expression of three important cell death ligand-receptor systems and Fox O1 in follicles with a diameter of 3-5 mm. The phosphorylation and subcellular localization of Fox O1 during granulosa cell apoptosis was also determined. TRAIL and Fas L played an important role in follicular atresia at this stage. Fox O1 expression was upregulated during atresia, and was confined to the nucleus of granulosa cells; however, phosphorylated Fox O1 was localized to the cytoplasm. These results suggest Fox O1 involvement in the regulation of TRAIL and Fas L expression during follicular atresia in pigs.

The ovary receives a finite pool of follicles during fetal life. Atresia remains the major form of follicular expenditure at all stages since development of ovary. The follicular reserve, however, declines at an exponential rate leading to accelerated rate of decay during the years preceding menopause. We examined if diminished follicle reserve that characterizes ovarian aging impacts the attrition rate. Premature ovarian aging was induced in rats by intra-embryonic injection of galactosyltransferase-antibody on embryonic day 10. On post-natal day 35 of the female litters, either a wedge of fat (sham control) or a wild type ovary collected from 25-day old control rats, was transplanted under the ovarian bursa in both sides. Follicular growth and atresia, and ovarian microenvironment were evaluated in the follicle-deficient host ovary and transplanted ovary by real time RT-PCR analysis of growth differentiation factor-9, bone morphogenetic protein 15, and kit ligand, biochemical evaluation of ovarian lipid peroxidation, superoxide dismutase (SOD) and catalase activity, and western blot analysis of ovarian pro- and anti-apoptotic factors including p53, bax, bcl2, and caspase 3. Results demonstrated that the rate of follicular atresia, which was highly preponderant in the follicle-deficient ovary of the sham-operated group, was significantly prevented in the presence of the transplanted ovary. As against the follicle-deficient ovary of the sham-operated group, the follicle-deficient host ovary as well as the transplanted ovary in the ovary-transplanted group exhibited stimulated follicle growth with increased expression of anti-apoptotic factors and down regulation of pro-apoptotic factors. Both the host and transplanted ovaries also had significantly lower rate of lipid peroxidation with increased SOD and catalase activity. We conclude that the declining follicular reserve is perhaps the immediate thrust that increases the rate of follicle depletion during the final phase of ovarian life when the follicle reserve wanes below certain threshold size.

More than 99 % of follicles in mammalian ovaries undergo a degenerative process known as atresia, and thus only a limited number of ovarian follicles actually ovulate after full growth and development. The endocrinological regulatory mechanisms involved in follicular development have been studied extensively, but the precise and systematic molecular mechanisms of steroidogenesis enzymes involved in atresia are unclear. In the present study, we examined whether and how the steroidogenesis enzymes are involved in porcine ovary follicular atresia. Expression of steroidogenic acute regulatory protein, CYP11, CYP17, 3β-hydroxysteroid dehydrogenase (3β-HSD), CYP19, as well as related pituitary and ovarian hormone receptors were quantified in ovaries. During porcine follicular atresia, expressions of P450 cholesterol side chain cleavage enzyme, progesterone and androgen receptors increased significantly during the late atretic stage, while the expression of aromatase and follicle-stimulating hormone receptors decreased significantly in the early stage. These data suggested that the regulation of aromatase by follicle-stimulating hormone might induce follicular atresia, and that progesterone and androgen production further promoted follicular atresia. Additionally, a correlation analysis indicated a large and complex interactive network among these genes and the endocrinological microenvironment of the follicles. Significant correlations were observed between expression of steroidogenic enzymes and their receptors, and also between progesterone and 17β-estradiol (E2) levels in follicular fluid. Taken together, these results suggest that CYP19 plays a role during early atresia by regulating the production of E2, whereas CYP11 and 3β-HSD increase atresia progression by increasing progesterone levels.

The physiological significance of androgens in female reproduction was unclear until female mice with global knockout of androgen receptor (AR) expression were found to have reduced fertility with abnormal ovarian function. However, because ARs are expressed in a myriad of reproductive tissues, including the hypothalamus, pituitary, and various ovarian cells, the role of tissue-specific ARs in regulating female fertility remained unknown. To examine the importance of ovarian ARs in female reproduction, we generated granulosa cell (GC)- and oocyte-specific AR-knockout (ARKO) mice by crossing AR-flox mice with MisRIIcre (GC-specific) or growth differentiation factor growth differentiation factor-9cre (oocyte-specific) mice. Relative to heterozygous and wild-type mice, GC-specific ARKO mice had premature ovarian failure and were subfertile, with longer estrous cycles and fewer ovulated oocytes. In addition, ovaries from GC-specific knockout mice contained more preantral and atretic follicles, with fewer antral follicles and corpus lutea. Finally, in vitro growth of follicles from GC-specific AR-null mice was slower than follicles from wild-type animals. In contrast to GC-specific AR-null mice, fertility, estrous cycles, and ovarian morphology of oocyte-specific ARKO mice were normal, although androgens no longer promoted oocyte maturation in these animals. Together, our data indicate that nearly all reproductive phenotypes observed in global ARKO mice can be explained by the lack of AR expression in GCs. These GC-specific ARs appear to promote preantral follicle growth and prevent follicular atresia; thus they are essential for normal follicular development and fertility.

CD44 on macrophages is recognized as a phagocytic receptor involved in the phagocytosis of apoptotic cells. Recently, we detected CD44 on macrophages in atretic follicles during atresia. In this study, we evaluated the distribution of the principal CD44 ligand hyaluronan (HA) and the expressions of HA synthases (HAS: HAS1, HAS2, and HAS3) during atresia in pig ovaries. We determined the 2139-bp sequence of Sus scrofa HAS1 and raised an anti-HAS1 polyclonal antibody. The S. scrofa HAS1 sequence contained six putative HA-binding motifs and conserved amino acid residues crucial for GlcNac transferase activity. HAS1 mRNA expression was upregulated during atresia; however, HAS2 and HAS3 mRNA expression levels were low and very low to undetectable, respectively. Western blotting showed that HAS1 was markedly upregulated during atresia. Immunohistochemical analyses revealed HAS1 distribution in theca cells of healthy and early atretic (stages I and II) follicles and in progressing atretic (stage III) follicles. Hyaluronan was visualized with the HA-binding protein; it accumulated in the theca layer during all stages and in stage III follicles. Hyaluronan assay showed a significantly increased HA concentration in follicular fluid at stage III. Flow cytometry showed HAS1 expression in 55.7% of SIRPA-positive macrophages in stage III follicles. Our results suggest that the HA concentration in follicular fluids increased during atresia and that HAS1 may be the dominant HAS protein in theca cells to produce HA in pig ovaries.

Thrombospondin (TSP)-1 is an antiangiogenic extracellular matrix glycoprotein that modulates several aspects of cellular function. The aim of this study was to determine the pattern of TSP-1 mRNA and protein expression as well as expression of its receptor CD36 in the marmoset ovary and to investigate the effects of inhibition of gonadotropins or VEGF activity on TSP-1 and CD36 expression in vivo. GnRH antagonist or VEGF Trap, a soluble decoy receptor, was administered on d 0 of the follicular phase of the cycle, and ovaries were collected at the end of the follicular phase (d 10). TSP-1 mRNA and protein were present in granulosa cells of preantral and antral follicles, with the highest staining at the late secondary and tertiary stages. Moreover, expression of TSP-1 mRNA and protein was significantly increased in tertiary follicles undergoing atresia. CD36 protein was detected in granulosa cells of preantral and antral follicles as well as in endothelial cells of large vessels. Inhibition of gonadotropin secretion or VEGF activity had no effect on TSP-1 expression; however, expression of CD36 protein was inhibited by the VEGF Trap. In conclusion, TSP-1 may be involved in the cessation of angiogenesis in follicles undergoing atresia; alternatively, TSP-1 may act on granulosa and/or endothelial cells to promote follicular atresia in the ovary. Angiogenesis is likely to involve a balance between pro- and antiangiogenic factors. Our results suggest that loss of VEGF activity does not regulate TSP-1 expression directly but may influence TSP-1 activity via down-regulation of the CD36 receptor.

Follicular selection is performed in mammalian ovaries, as most follicles undergo atresia during follicular development and growth. Follicular regression is indicated to begin with granulosa cell apoptosis. To reveal the molecular mechanisms of the selection, we examined the changes in the levels of cellular-Flice like inhibitory protein (cFLIP) expression in porcine granulosa cells. cFLIP is the homologue of intracellular apoptosis inducer (procaspase-8/Flice), and has two alternative splicing isoforms: cFLIP short form (cFLIP(S)) and long form (cFLIP(L)). By competing with caspase-8, cFLIP inhibits apoptosis initiated by death receptors. The changes in the levels of cFLIP(S) and cFLIP(L) mRNA and protein expression in granulosa cells were determined by RT-PCR and Western blotting, respectively. cFLIP(L) mRNA and protein were highly expressed in granulosa cells of healthy follicles and decreased during atresia. cFLIP(S) mRNA levels in granulosa cells were low and showed no change among the stages of follicular development, and its protein level was extremely low. We examined the changes in the localization of cFLIP mRNAs in pig ovaries by in situ hybridization and found that cFLIP(L) is abundant in granulosa cells of healthy follicles in comparison with those of atretic follicles. Immunohistochemical analyses demonstrated that the cFLIP protein is highly expressed in the granulosa cell of healthy follicles but weakly expressed in that of atretic follicles. We presumed that cFLIP, especially cFLIP(L), plays an anti-apoptotic role in the granulosa cells of healthy follicles of pig ovaries, and that cFLIP could be a major survival factor that determines whether growth or atresia occurs in porcine follicles., ((c) 2005 Wiley-Liss, Inc.)

To reveal the molecular mechanism of selective follicular atresia in porcine ovaries, we investigated the changes in the expression of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and its receptor (DR4) proteins and TRAIL mRNA in granulosa cells during follicular atresia. Immunohistochemical, Western immunoblotting and reverse transcription-polymerase chain reaction analyses (RT-PCR) revealed that significant increases in TRAIL protein and mRNA levels but not DR4 protein were changed during atresia. The RT-PCR product was confirmed to be porcine TRAIL by the cDNA sequence determination. An in vitro apoptosis inducing assay using cultured granulosa cells prepared from healthy follicles showed that TRAIL could activate caspase-3 and induce apoptotic cell death in the cells. The present findings confirm that TRAIL induces apoptosis in granulosa cells during atresia in porcine ovaries.

We have investigated which follicular compartment and stage of follicular development are associated with endothelin-1 (ET-1) gene expression in the porcine ovary. The localization of mature ET-1 peptide and of its mRNA was determined by immunohistochemistry and by in situ hybridization. Stage of follicular development associated with ET-1 expression was investigated in terms of follicular class and occurrence of atresia. The latter was investigated by determining the occurrence of DNA fragmentation in apoptotic cells on adjacent sections to those used for ET-1 gene expression. Fifteen ovaries from 10 prepubertal pigs stimulated with gonadotropin were collected; a total of 1050 follicles were examined. Specific ET-1 immunoreactivity was restricted to the ovarian vasculature and to the granulosa cell compartment of antral follicles. The pattern of ET-1 mRNA expression was similar to that found for ET-1 immunoreactivity. Primordial, primary, and most secondary follicles did not express ET-1. The theca cell layer did not express ET-1 regardless of follicle developmental stage. ET-1 expression occurred with a significantly greater probability (P < 0.001 by the likelihood ratio test) in the granulosa cell compartment of antral follicles than in any other follicle class. Furthermore, in antral follicles, ET-1 expression occurred with a greater likelihood in large antral follicles than in small antral follicles (P < 0.001 by the likelihood ratio test). In small antral follicles, only 16.8% expressed ET-1; in contrast, 66.7% of large antral follicles exhibited ET-1 expression. It is interesting that in follicles in which ovulation had already occurred, intense ET-1 expression was found only in the prominent developing vasculature, the other cells present in the luteinized follicle did not display any ET-1 expression. The pattern of ET-1 gene expression observed in this study would be in agreement with our previous suggestion of a plausible physiological role for ET-1 in preventing premature progesterone production by granulosa cells of an antral follicle. The occurrence of atresia and expression of ET-1 in the same follicle was rare. Small and large antral follicles constituted 5.1% and 5.6%, respectively, of the examined follicles in this category. The majority of atretic follicles did not express ET-1 and, conversely, follicles that expressed ET-1 were not atretic. To the best of our knowledge, this is the first report in which large, nonatretic follicles are clearly identified as the population of follicles expressing ET-1. The results of this study delineate the follicular developmental stage and the compartment of when and where ET-1 may be physiologically meaningful.

We detected that Fas receptor (Fas) was expressed at transcriptional levels in oocytes/eggs and some granulosa cells of murine ovary, whereas, Fas ligand (FasL) was found to be strongly expressed in granulosa cells by means of in situ hybridisation. These results were supported by an indirect immunofluorescence (IIF) test with anti-Fas monoclonal antibody (mAb)/FasL Ab. The lysates from granulosa cells were precipitated by anti-FasL Ab, exhibiting a specific band at 40 kDa. When zona pellucida (ZP)-free eggs were incubated with granulosa cells in vitro, apoptosis (DNA fragmentation) was induced in the eggs, as detected by the terminal deoxynucleotide transferase mediated dUTP-nick end labelling (TUNEL) method. To examine whether FasL-bearing cells can induce apoptosis in eggs expressing Fas through molecular interaction between FasL and Fas, we employed a baculovirus expression system to generate FasL on the surface of Spodoptera frugiperda (Sf9) cells. The co-incubation of eggs with Autographa californica nuclear polyhedrosis virus (AcNPV) and FasL transfected Sf9 (Sf9-FasL) cells in vitro was performed and resulted in the induction of apoptosis in eggs as detected by the TUNEL method. Apoptosis was absent in eggs co-incubated with AcNPV-1393 transfected Sf9 (Sf9-1393) cells. Thus, ovarian atresia was suggested to be induced by molecular interaction between FasL on granulosa cells and Fas on oocytes/eggs during oogenesis.

We investigated apoptosis in 37 human ovaries from normally cycling women to determine the mechanism of the various stages of follicular atresia and the regression of corpus luteum in human. Apoptosis was examined by 3'-hydroxy nick end-labeling, by immunostaining of an apoptosis-related antigen, Le(y) and by cell death-related genes, bcl-2. Immunoreactivity for Le(y) and nick end-labeling reactive cells were not observed in the follicular and luteal phases, except for scattered cells in the degenerating corpus luteum. On the other hand, immunoreactivity for bcl-2 was observed in the granulosa cells of antral follicles. These results indicated that apoptosis is not obvious through follicular maturation and luteal regression. This may be due to the relatively long process of human follicular growth and atresia.

Physiological cell death mechanisms (termed apoptosis, programmed cell death, active cell death, and biological cell death) play a fundamental role in the cyclic function of the ovary as it strives in each cycle to ovulate a viable egg for fertilization. Healthy ovarian follicles and corpora lutea are also required for the secretion of steroids to prepare the female reproductive tract for embryo implantation and gestation. Using molecular biological approaches combined with classic histological examinations, several recent studies have confirmed the occurrence of apoptosis in female germ cells (oogonia and oocytes) during fetal ovarian development, granulosa cells during follicular atresia, and cells of the corpus luteum during luteolysis. Additionally, new light has been shed on the potential gene products which function to precisely coordinate the balance of life and death in the ovary. It is the purpose of this review to discuss the concepts of physiological cell death as they relate to ovarian function, and to offer testable hypotheses concerning the intracellular effector pathways responsible for directing ovarian cell fate in response to changes in hormonal stimuli.