Immunohistochemical and ultrastructural visualization of different routes of oocyte elimination in adult rats

Cell death is a process for maintaining homeostasis in tissues and organs. In the ovary, apoptotic cell death has been implicated in follicular atresia; in the elimination of the follicles that are not ovulated during adult life. Recent studies indicate that apoptosis and autophagy are two programmed processes of cell death. Apoptosis is performed by proteases called caspases and leads to such morphological traits as DNA fragmentation. Autophagy, in turn, is characterized by the exacerbated formation of autophagosomes; a process in which the amount of the LC3 and Lamp 1 proteins increases. In this study, oocytes from all stages of the estrous cycle of Wistar rats were analyzed. The apoptosis process was identified by immunodetecting active Caspase-3 and locating DNA fragmentation using the TUNEL technique. Autophagy was evaluated through immunodetection of the LC3 and Lamp 1 proteins, and by ultrastructural localization of autophagic vesicle formation. All techniques were conducted using the same oocytes. Results show that all phases of the estrous cycle contain dying oocytes that test positive simultaneously for apoptosis and autophagy markers. The highest level of apoptosis was found during estrus; while the proestrous stage had the highest level of autophagy. The diestrous and metestrous phases were characterized by a high frequency of the presence of markers of apoptosis and autophagy in the same oocyte. Our results demonstrate that during oocyte elimination in adult rats the proteins involved in both processes, apoptosis and autophagy, are present in the same cell at the same time.     

Autophagy and Cathepsin D mediated apoptosis contributing to ovarian follicular atresia in the Nile tilapia

Follicular atresia is a hormonally controlled degenerative process involving apoptosis of the somatic and germ cells. Since different signaling pathways can induce cell death, the aim of the present study was to investigate cell death signaling and crosstalk between autophagic, apoptotic, and lysosomal proteins during follicular atresia in Nile tilapia. For this, females were kept in controlled conditions for 21 days, and ovary samples were collected weekly. The atretic follicles (AF) were analyzed in three regression phases: Early, advanced, and late. Under electron microscopy, the follicular cells exhibited numerous protein synthesis organelles in the early AF. Immunoreactivity for Bcl2, Beclin1, Lc3, and Cathepsin D increased significantly in advanced AF (p < .001), when follicular cells were in intense yolk phagocytosis. In this phase, autophagosomes and autolysosomes were frequently observed. In the late AF, follicular cells had a markedly electron‐lucid cytoplasm and immunoreactivity for Bax and TUNEL assay indicated an elevated apoptosis rate. Colocalisation of Lamp1/Cathepsin D and Lc3/Caspase‐3 suggests dynamic crosstalk between the autophagy, apoptosis, and lysosome pathways. Taken together, the data indicate that autophagy plays a role in the homeostasis and clearance of the follicular cells preceding Cathepsin D mediated apoptosis during follicular atresia in Nile tilapia.     

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.     

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.     

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.     

家禽卵泡闭锁机制的研究进展

家禽卵泡闭锁是一种受多种机制共同调控卵泡退化的过程,与家禽的繁殖性能密切相关。近年来,随着分子技术的快速发展,针对家禽卵泡细胞的凋亡和自噬引起卵泡闭锁的研究取得巨大进展。综述了家禽卵泡内细胞凋亡与自噬对家禽卵泡闭锁调控机制,讨论了卵泡细胞凋亡与自噬引起卵泡闭锁的意义及存在的问题。通过对家禽卵泡闭锁机制的研究,以期为家禽产蛋性能的充分利用和挖掘提供理论依据。     

Analysis of different cell death processes of prepubertal rat oocytes in vitro

The processes of cell death were studied in vitro in populations of oocytes isolated from prepubertal rats. In order to identify apoptosis, the externalized phosphatidylserine was recognized with Annexin-V coupled to FITC and the fragmentation of DNA was demonstrated by means of electrophoresis. Oocytes were tested for autophagy by means of the incorporation of monodansylcadaverine and monitoring Lc3-I/Lc3-II by western blot. The expression of mRNA marker genes of autophagy and of apoptosis was studied by means of RT–PCR in pure populations of oocytes. Some oocytes expressed at least one of the following markers: caspase-3, lamp1 and Lc3. Some oocytes were positive to Annexin-V or to monodansylcadaverine. However, most of them were simultaneously positive to both markers. The relative frequency of oocytes simultaneously positive to markers of apoptosis and autophagy did not change in the different ages studied. The transformation of Lc3-I in Lc3-II was present in all populations of oocytes studied. The mRNAs for caspase-3, lamp1 and Lc3 were present in all populations of oocytes analyzed. Our results demonstrate that oocytes of rats from new born to prepubertal age are eliminated by means of three different cell death processes: apoptosis, autophagy and a mixed event in which both routes to cell death participate in the same cell.     

Differential Expression of Programmed Cell Death on the Follicular Development in Normal and Miniature Pig Ovary

Follicles are important in oocyte maturation. Successful estrous cycle requires remodeling of follicular cells, and proper execution of programmed cell death is crucial for normal follicular development. The objectives of the present study were to understand programmed cell death during follicle development, to analyze the differential follicle development patterns, and to assess the patterns of apoptosis and autophagy expression during follicle development in normal and miniature pigs. Through the analysis of differential patterns of programmed cell death during follicular development in porcine, MAP1LC3A, B and other autophagy-associated genes (ATG5, mTOR, Beclin-1) were found to increase in normal pigs, while it decreased in miniature pigs. However, for the apoptosis-associated genes, progression of genes during follicular development increased in miniature pigs, while it decreased in normal pigs. Thus, results show that normal and miniature pigs showed distinct patterns of follicular remodeling manifesting that programmed cell death largely depends on the types of pathway during follicular development (Type II or autophagy for normal pigs and Type I or apoptosis for miniature pigs).     

Ovarian follicle apoptosis at the onset of standing estrus in virgin and repeat-breeder dairy heifers

There is evidence that repeat breeding in dairy cattle can be caused by both extrinsic, environmental factors and intrinsic, animal factors. In repeat-breeder heifers (RBH), disturbed endocrine patterns and estrous events result in a subsequent decreased fertility associated with delayed ovulation. Whether infertility is also due to the presence of an unsuitable follicular environment impairing normal fertilization, remains to be determined. At the onset of standing estrus, ovaries were obtained from 7 strictly defined RBH and 5 virgin heifers (VH) of the Swedish Red and White breed. Detection of apoptosis in the preovulatory and three subordinate follicle walls was done by using the TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling) technique at light microscopy level. The follicles were histologically assessed for degree of atresia. The ultrastructure of the follicle wall and recovered oocytes was studied using transmission electron microscopy. The overall degree of apoptosis in membrana granulosa and theca interna of preovulatory and subordinate follicles did not differ between RBH and VH, but the numbers of TUNEL-positive cells differed significantly between preovulatory and subordinate follicles in both RBH and VH. There was a strong relationship between density of apoptotic cells and degree of atresia. No differences in follicle wall apoptosis nor morphology were detectable, suggesting that repeat breeder heifers enter standing estrus with the same morphological prerequisites as normal animals, considering follicular structure.     

Combined apoptosis and autophagy,the process that eliminates the oocytes of atretic follicles in immature rats

We studied the alterations of dying oocytes in 1–28 days old rats using TUNEL method, immunolocalizations of active caspase 3, lamp1, localization of acid phosphatase, and DAPI staining. All procedures were performed in adjacent sections of each oocyte. In most dying oocytes exist simultaneously features of apoptosis as active caspase 3 and DNA breaks, and a large increase of lamp1 and acid phosphatase characteristic of autophagy. Large clumps of compact chromatin and membrane blebbing were absent. Electron microscope observations demonstrated the presence of small clear vesicles and autophagolysosomes. All these features indicate that a large number of oocytes are eliminated by a process sharing features of apoptosis and autophagy. In dying oocytes of new born rats the markers of apoptosis predominate over those of autophagy. However, fragmentation and apoptotic bodies were not found. These features suggest that in different cytophysiological conditions the processes of cell death may be differently modulated.     

Follicular populations, recruitment and atresia in the ovaries of different strains of mice

Follicular atresia is a key event in the selection of the ovulatory follicles and occurs during all developmental stages. The aims of the study were to evaluate the follicular population as well as the rates of follicular recruitment and atresia in different strains of mice. Ovaries were obtained from four strains of mice: G1/ Swiss, G2/ F1 Swiss×C57BL/6, G3/ inbred strain C57BL/6, and G4/ F1 C57BL/6×Swiss. All mice used in the study were 60 days old. Ovaries collected from the mice were fixed and processed for histological analysis. The G2 ovaries were also used to examine immunolocalization of active caspase-3. The pimordial follicle population was smaller in G3 mice than in G1, G2 and G4 groups (7 565±1 845 vs. 17 180±3 159, 14 785±3 319 and 13 325±2 685, respectively; p<0.05). The rate of follicular recruitment in G3, however, was higher than in the other groups (29.2% vs. 18.2%, 17.3% and 13.0% in G1, G2 and G4, respectively; p<0.05), resulting in a similar (p>0.05) number of antral follicles among groups. The small follicular pool in G3 mice was also associated with a lower rate of follicular atresia (11.4% vs. 17.2%, 16.7% and 13.6% for G3, G1, G2 and G4, respectively; p<0.05). The number of follicles stained with active caspase-3 was higher (p<0.05) during the final stage of preantral folliculogenesis than in other stages of follicular development suggesting that apoptosis in mice occurs earlier in comparison to large animals. Thus, it was concluded that differences in follicle reservoir among mice strains are compensated by an increased rate of follicular recruitment and a decreased rate of follicular atresia; and atresia occurs in mice mainly at the end of the preantral stage of folliculogenesis.     

Relationship between low-molecular-weight insulin-like growth factor-binding proteins, caspase-3 activity, and oocyte quality

Bovine follicular atresia is associated with the apoptosis of granulosa cells and the subsequent loss of oocyte competence through the reduction of cellular contact (e.g., gap junctions). Several components of the insulin-like growth factor (IGF) system are thought to affect follicular atresia. Whereas the IGF-binding proteins (IGFBPs) are present in varying quantities throughout follicular development, IGFBP-5 appears to be present only during atresia, in parallel with its regulation in other tissue remodeling systems. However, to our knowledge, no connection has yet been made between atresia, low-molecular-weight IGFBP content, and oocyte quality in the bovine ovary. Caspases are actively involved in ovarian follicular atresia, and apoptosis in antral follicles is caspase-3-dependent. Hence, the aim of the present study was to investigate the use of these factors in the assessment of oocyte quality and developmental potential. Oocytes were aspirated, morphologically classified, and individually matured in vitro. The follicular fluid and granulosa cells of these follicles were analyzed for IGFBP profile and caspase-3 activity, respectively. A significant correlation was found between the presence of low-molecular-weight IGFBPs in bovine follicular fluid and caspase-3 activity of granulosa cells isolated from individual follicles. The highest percentage of development to the blastocyst stage was observed in oocytes from slightly atretic follicles. This group of oocytes contained an equal proportion of oocytes at grades 1-3. These data demonstrate that low-molecular-weight IGFBP profile is a more reliable method than the traditional morphological assessment of oocytes and can be used as an effective marker of developmentally competent oocytes. Importantly, these results have implications for the use of noninvasive follicular fluid markers in the selection of competent oocytes to improve outcomes of in vitro fertilization.     

Growth differentiation factor 9 is antiapoptotic during follicular development from preantral to early antral stage

Ovarian follicular atresia represents a selection process that ensures the release of only healthy and viable oocytes during ovulation. The transition from preantral to early antral stage is the penultimate stage of development in terms of gonadotropin dependence and follicle destiny (survival/growth vs. atresia). We have examined whether and how oocyte-derived growth differentiation factor 9 (GDF-9) and FSH regulate follicular development and atresia during the preantral to early antral transition, by a novel combination of in vitro gene manipulation (i.e. intraoocyte injection of GDF-9 antisense oligos) and preantral follicle culture. Injection of GDF-9 antisense suppressed basal and FSH-induced preantral follicle growth in vitro, whereas addition of GDF-9 enhanced basal and FSH-induced follicular development. GDF-9 antisense activated caspase-3 and induced apoptosis in cultured preantral follicles, a response attenuated by exogenous GDF-9. GDF-9 increased phospho-Akt content in granulosa cells of early antral follicles. Although granulosa cell apoptosis induced by ceramide was attenuated by the presence of GDF-9, this protective effect of GDF-9 was prevented by the phosphatidylinositol 3-kinase inhibitor LY294002 and a dominant negative form of Akt. Injection of GDF-9 antisense decreased FSH receptor mRNA levels in cultured follicles, a response preventable by the presence of exogenous GDF-9. The data suggest that GDF-9 is antiapoptotic in preantral follicles and protects granulosa cells from undergoing apoptosis via activation of the phosphatidylinositol 3-kinase/Akt pathway. An adequate level of GDF-9 is required for follicular FSH receptor mRNA expression. GDF-9 promotes follicular survival and growth during the preantral to early antral transition by suppressing granulosa cell apoptosis and follicular atresia.     

Expression and Preliminary Functional Profiling of the let-7 Family during Porcine Ovary Follicle Atresia

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.     

Apoptosis and ovarian function: novel perspectives from the teleosts

Apoptosis is a fundamental mechanism in follicular atresia and postovulatory regression in mammals, but its role in teleost ovarian function is currently unknown. This study tested the hypotheses that apoptosis mediates follicular atresia in teleosts and is inducible in vitro by incubation in serum-free conditions. Vitellogenic follicles from rainbow trout (Oncorhynchus mykiss) and goldfish (Carassius auratus) were incubated overnight in serum-free medium and examined for apoptosis by 3'-end-labeling and/or TUNEL analysis. Primary, postovulatory, and oocytectomized vitellogenic trout follicles and atretic goldfish follicles were evaluated in similar fashion. Overall, goldfish follicles had lower levels of DNA fragmentation than trout follicles. The DNA fragmentation in atretic goldfish follicles was similar to that measured in healthy vitellogenic and prematurational follicles; DNA fragmentation did not change after incubation. In the trout, postovulatory and oocytectomized vitellogenic follicles showed significantly greater in vitro susceptibility to apoptosis than intact vitellogenic follicles, whereas primary follicles were least susceptible. The TUNEL analyses revealed that in trout vitellogenic follicles, more thecal/epithelial cells than granulosa cells showed fragmented DNA in vivo, but incubation (24 h) did not result in increased apoptosis in cells of either type. These results indicate that apoptosis is involved in normal ovarian growth and postovulatory regression in teleosts, but that it does not appear to be an early event in teleost follicular atresia.     

Distribution of laminin β2, collagen type IV, fibronectin and MMP-9 in ovaries of the teleost fish

Extracellular matrix in the ovarian follicle has been characterised for several mammalian species but there are no reports that describe the immunolocalisation of the extracellular matrix elements, matrix metalloproteinases, and its relation to plasma 17β estradiol levels and follicular apoptosis during the teleost’s reproductive cycle. The present study used immunohistochemistry to characterise the distribution of laminin β2, collagen type IV, fibronectin and matrix metalloproteinases-9 (MMP-9). The TUNEL in situ technique was used to quantify apoptosis and indirect immunofluorimetric to determine plasma 17β estradiol levels. The TUNEL-positive reaction associated with morphological features exhibited follicular apoptosis. During postovulatory follicle involution, the drop in plasma 17β estradiol levels after spawning contributed to the intense apoptosis observed. By immunohistochemical analysis, laminin β2 and collagen type IV were identified as the major constituents of the basement membrane. The loss of integrity of the basement membrane occurred due to lyses of the major constituents, and coincides with increased follicular apoptosis. The integrity of the basement membrane is important for the survival of follicular cells. Furthermore, the MMP-9 results suggest that this enzyme is involved in final oocyte maturation and regression of postovulatory follicles. Fibronectin was observed on the surface of follicular cells of the postovulatory follicle in P. argenteus, this being important for maintaining normal cell adhesion to extracellular matrix. In conclusion, our results suggest that the structure and composition of the extracellular matrix, and plasma 17β estradiol levels related to apoptosis, play an important role during the follicular development and post-spawning involution in teleost fishes.     

卵泡颗粒细胞凋亡相关蛋白的研究进展

哺乳动物的卵巢中存在大量卵泡。大多数卵泡在发育过程中发生闭锁而消失,只有少数可以发育到成熟而排卵。卵泡是由卵母细胞与其周围的颗粒细胞构成的。卵泡颗粒细胞的凋亡是卵泡闭锁的主要原因。颗粒细胞凋亡相关蛋白通过参与凋亡通路及凋亡信号转导调节凋亡。本文就哺乳动物卵泡颗粒细胞凋亡相关蛋白的研究进展进行综述。