| For mammals, only a few ovarian follicles can develop to maturity and ovulation and the fate of the vast majority of follicles is atresia. In fact, follicular atresia is happened from the early stage of follicular development until ovulation. There were many reports aimed to interpret this phenomenon, however, the mechanism controlling follicular development and atresia remains unclear.In order to study the porcine oocyte nest breakdown, primordial follicle formation and development characteristics, according to the previous study of our laboratory, the ovaries of 90 day postcoitum(dpc) pigs,1,25,70 and 150 day(s) postpartum (dpp) were selected, the application of hematoxylin-eosin (HE) staining was used for histological studies. The results showed that germ cells were mainly oocyte nests in the embryo at 90 days, when the nest was breakdown and primordial follicle formed; sows at 1dpp the ovarian germ cells existed mainly in primordial follicles, and oocyte nests were still existed; primary follicle formation could be observed. Ratios of the follicles at different stages were analyzed according to HE stainings. In short, oocyte nests were gradually breakdown and the primordial follicles formated between 90 dpc to 1 dpp, therefore, this period was critical for porcine oocyte nest breakdown and primordial follicles formation. The 1dpp is the critical time for porcine primary follicle formation. Therefore, our data indicated that the initial recruitment of primordial follicles started from ldpp to 25dpp and 70dpp, which was accompanied with oocyte nest breakdown.Even inside of the body, cells are still subject to various stress, results of this effect is breakage of DNA strands and cell death. Poly (ADP-ribosylation), which occurs rapidly in cells following DNA damage and is regulated by poly (ADP-ribose) polymerase-1 (PARP-1), is a post-translational modification of proteins playing a crucial role in many processes, including DNA repair and cell death. Although PARP-1 has recently been implicated in a variety of physiological and pathological processes, its role in the process of follicular development and atresia is not yet completely defined. The present study was designed to investigate the cellular expression pattern and immunolocalization of PARP-1, cleaved PARP-1, caspase-3 and cleaved caspase-3 in fetal, neonatal and adult porcine ovaries. Our results showed that PARP-1 cleavage is involved in the process of oocyte nest breakdown, primordial follicle formation and transition to primary follicles in fetal and neonatal pigs. The results of immunohistochemistry indicated that PARP-1 cleavage was involved in the process of follicular development and atresia, similar to our previous study. However, it was noted that cleaved caspase-3 was mainly localized in and around the nucleus of apoptotic granulosa cells, whereas cleaved PARP-1 was mainly localized in the nucleus of the apoptotic granulosa cells. Radioimmunoassay (RIA) data showed that serum concentrations of progesterone (P4) and estradiol (E2) increased with age after birth. Collectively, our findings suggested that the PARP-1 signaling pathway was involved in oocyte nest breakdown and primordial follicle formation in fetal and neonatal porcine ovaries, but was different from follicular atresia in adult porcine ovarie.In production of livestock or medicine, in normal circumstances or under stress situations the ovarian fillicles are likely to be atretic. Ovarian follicle atresia is a common phenomenon in vertebrate ovaries and this process is characterized by follicular wall degeneration. The molecular mechanism underlying follicle atresia is apoptotic granulusa cell death; however, the exact signaling pathway is still unclear. PARP-1, the founding member of the poly (ADP-ribose) polymerase (PARP) family, plays an important role in a large variety of physiological processes. Although its cleavage has recently been implicated in a variety of physiological and pathological processes, its role in the process of follicular atresia is not yet completely defined. We identified the cleavage of PARP-1 involved in the process of follicle degeneration, which is known as "follicular atresia", both from in vivo models and cell culture data. The results from immunohistochemistry (IHC) showed that cleaved PARP-1 was mainly located in apoptotic granulosa cells (GCs); and the expression of PARP-1 and caspase-3 were decreased in apoptotic granulosa cells (GCs). The results from western blotting showed that PARP-1 expressions were significantly decreased in atretic follicles compared with healthy (H) follicles, and the cleavage of caspase-3 (17 kDa) significantly increased in atretic follicles. Along with the cleavage of caspase-3, the expression of cleaved PARP-1 (24 kDa) product was significantly increased, which confirmed caspase-3 activation. Serum starvation led to a reduction in PARP-1 and an increase in cleaved PARP-1 (24 kDa) and caspase-3 (17 kDa), suggesting that caspase-3 was activated under the stress of withdrawal of growth factors, in accordance with the in vivo study. In the present study, the concentration of estradiol (E2) and progesterone (P4) as well as the P4/E2 (P/E) ratio were combined with morphological features to determine follicular classification. In summary, the present study demonstrated that cleavage of PARP-1 by caspase-3 was involved in the process of granulosa cell apoptosis. PARP-1 might through its cleavage act as a critical regulator in the process of porcine follicular atresia. Our results identified that cleavage of PARP-1 by activated (cleaved) caspase-3 might play a key role in controlling follicular atresia through granulosa cell degeneration. These findings were helpful in understanding the regulatory mechanisms controlling follicular development and atresia.As well as PARP-1, Sirtuins is also NAD+-dependent enzymes, however, the intracellular NAD+pool is limited. When investigate the role of PARP-1, it is necessary to study the changes of Sirtuins. SIRT-1 (The Silent Information Regulatorl) and SIRT-2 (The Silent Information Regulator2) is a histone deacetylase (Histone Deacetylase, HDAC) third class members, belonging to the family members deacetylase Sirtuins, wide expressed in cells, which play a role in the gene silencing, cell cycle regulation, fatty acid metabolism and many other cellular activities. It was found that SIRT-1 and SIRT-2 play an important regulatory role in apoptosis, however, for their roles on follicular development and atresia is unclear. My current study aimed to investigate the role of SIRT-1 and SIRT-2 in the ovary of fetus, newborn and adult pigs. The data showed that SIRT-1 and SIRT-2 might be involved in oocyte nest breakdown, primordial follicle formation and transformation process to primary follicles. Immunohistochemistry data showed that SIRT-1 and SIRT-2 were involved in the process of follicular development and atresia. However, the mechanisms of SIRT-1 and SIRT-2 in oocyte nest breakdown, primordial follicle formation and development as well as the process of follicular atresia remain to be further investigated.Many intracellular proteins can be modificated by acetylation and deacetylation, which are associated protein activity. The most studied acetylase is CBP/p300.CREB (CREB binding protein) CBP/p300 coactivator binding protein transcription factor family is composed of two closely related transcriptional co-activator protein compositions. It is found that CBP/p300 play an important regulatory role in apoptosis, however, for their roles on follicular development and atresia is unclear. This study aimed to investigate the roles of CBP/p300 in the ovaries of fetus, newborn and adult pigs. The data showed that CBP/p300 might be involved in oocyte nest breakdown, primordial follicle formation and transformation process to primary follicles. Immunohistochemistry data showed that CBP/p300 were involved in the process of follicular development and atresia. However, the mechanisms of CBP/p300 in oocyte nest breakdown, primordial follicle formation and development as well as the process of follicular atresia remain to be further investigated.It is found that deacetylase CBP/p300 is capable of histone H3 acetylation, and SIRT-1 and SIRT-2 can perform deacetylation modification on histone H3. The dynamic equilibrium exists between the two kinds of modifications. Therefore, histone acetyltransferase (HATs) and deacetylases (HDACs) have the effect of maintaining homeostasis. In general, histone acetylation promotes transcription, while deacetylation inhibits transcription. Histone H3 in eukaryotic cells chromatin structure is one of the five major histones. Histone H3 modifications including acetylation and deacetylationare catalyzed by the histone acetyltransferase (HATs) and de-acetyltransferase (HDACs). The modification of histone H3 is believed to play a role in the gene silencing, cell cycle regulation, fatty acid metabolism and many other cellular activities. Histone H3 acetylation and deacetylation was found to play an important regulatory role in apoptosis, however, for their roles on follicular development and atresia was unclear. This study aimed to investigate the roles of histone H3 acetylation in the ovaries of fetus, newborn and adult pigs. The data showed that histone H3 acetylation might be involved in oocyte nest breakdown, primordial follicle formation and transformation process to primary follicles. Immunohistochemistry data showed that histone H3 acetylation were involved in the process of follicular development and atresia. However, the mechanisms of histone H3 acetylation in oocyte nest breakdown, primordial follicle formation and development as well as the process of follicular atresia remain to be further investigated. |
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