Study Of Mechanism On Porcine Follicular Cysts

Ovarian cyst disease is one of the most common causes of reproductive failure in females, often classified into two groups:follicular cysts and luteal cysts, the former is much more common than the latter. Follicular cysts, one of major infertility diseases in sows, are characterized by anovulation of follicular structures and disturbance of estrous cycle. Numerous studies have been published on cystic ovarian disease since first describe in 1831. Up to now, this disease has been reported in bovine, ovine, porcine, human and other species. The incidences of follicular cysts have been reported to be 10%of the reproductive failure in sows, from 10 to 13%in cows, from 2.8 to 4.2%in water buffalo. This disease greatly decreases the reproductive performance of females, and results in severe economic loss to the breeding farm. The incidence of PCOS in human was 5%and accounted for 70%of the anovulatory infertility in women. Therefore, we have to know more about this disease urgently, and then control them effectively.The mechanisms leading to follicular cysts have been reported in many aspects. It is widely accepted that the endocrine imbalance is an important factor in the etiology of follicular cysts. The abnormal regulation of the hypothalamic-pituitary-gonadal axis may be responsible for the disorder of endocrine and estrous cycle, and anovulation of cystic follicles. Ovaries as effector for many hormones, play an important role in maintaining relatively stability of the endocrine system. Studies have reported that intraovarian mechanism exists in the interruption of preovulatory LH surge. Therefore, the local factors have an essential role in the regulation of the cystic formation process. Follicular fluid (FF) could reflect the function of endocrine, paracrine and autocrine system. The compositions of FF could reflect the physiology and pathology of hormone synthesis, cellular metabalism, oocyte maturation, ovulation, luternization, et al. Today, follicular fluid is becoming the focus of the researching in ovarian disease.The proteins in follicular fluid have an important role in the follicular development and oocyte maturation. Increasing knowledge with respect to protein compositions, protein concentrations and particular proteins function of follicular fluid has greatly contributed to a more insight to the physiological processes related to follicular growth and oocyte maturity. Numerous studies have reported on protein profiling of FF, including cows, rat and human, but none in porcine. On the other hand, atresia is the process of physiological degradation. Apoptosis plays a crucial role in the regression process of atretic follicles. The latest reports show that delay of folliclular regression following ovulatory failure may be an alternative cause of cysts, low frequency of apoptosis in cystic follicles may be responsible for the persistence of cystic follicles.In the present study, we analyzed the proteomes of FF from normal and cystic follicles by comparative proteomic approach. Our aim was to explore the key proteins associated with pathogenesis of follicular cysts, some of which may be candidate biomarkers for the condition; as well as we examined the apoptosis and proliferation of cystic follicles. Our findings will contribute to further insight into the pathogenesis of follicular cysts. The principal results were described as follows:1. Histology and hormone assayThe morphological and hisological structures of cystic follicles were both altered. We found that the weight and volume of ovary and diameter of the large follicles in cystic ovaries were significantly larger than those in normal ovaries. In cystic follicles, the granulosa cells were exfoliated and less layered; the follicular basal lamina had disappeared in part. Concentrations of FSH, estradiol and testosterone in serum were lower, while those of LH and progesterone were higher in the cystic group than in normal groups. Progesterone concentrations were significantly higher in cystic follicles (p<0.01) than those in normal follicles. These findings show that the cystic follicles were in the later stages of development.2. Comparative proteomic analysis of follicular fluidWe have established the 2-DE map for porcine follicular fluid by comparative proteomic analysis. About three hundred spots were reproducibly detected on 2-DE gels of samples of each group. We analyzed FF proteins that are differentially expressed in small, medium, large and cystic follicles. There were twenty-three spots corresponding to differentially expressed proteins in the small and medium follicles. Medium and large follicles differed with respect to the expression of nineteen proteins. Between large and cystic follicles, forty spots were differentially expressed. Of these, thirty-two specific proteins of interest were identified by HDMS, the identified protein spots corresponded to only twenty-eight different proteins. Seventy five percent of the identified proteins were detected in sow FF for the first time. The proteins identified as being of potential significance can be classified according to their function, as follows:proteins whose main roles relate to cellular metabolism; signal transduction:nucleic acid metabolism; cytoskeleton; immunity and apoptosis. RBP-4 and ApoA-I might be candidate biomarkers for porcine follicular cvsts.For further investigation on the source and function of the identified protein, we cultured the granulosa cells and oocytes in vitro. Using methods of RT-PCR and ELISA.we found that RBP-4 and transferrin in FF may also originate from the granulosa cells aparting from blood. Addition of different concentration of tansferrin (1 ng/ml.10 ng/ml,100 ng/ml) into the maturation media of oocytes did not significantly affect the cumulus expansion and PBI exclusion.3. Apoptosis and proliferation of cystic folliclesWe evaluated the frequency of apoptotic cells in normal, atretic and cystic follicles by the terminal deoxynucleotidyl transferase-mediated biotinylated deoxyuridine triphosphate nick end-labeling (TUNEL) method. We found that the apoptotic cells were both detected in follicles of all categories, the frequency of apoptosis in cystic follicles were lower than that in atretic follicles. We examined the expression of apoptotic related factors (XIAP, bax, bcl-2, caspase-3) by Immunohistochemistry as well. We found that immunostaining for caspase-3 was observed in the cellular nucleus and cytoplasm. The most intense immunostaining was determined in the atretic follicles, and weak immunostaining in cystic follicles, similar to those in the normal follicles. In granulosa and theca layers. The greater immunostaining for bcl-2 was observed in normal and cystic follicles, and lesser intensity in atretic follicles. By contrast, immunostaining for bax in granulosa and theca layers was greater in atretic follicles. Immunostaining for XIAP was observed in cellular nucleus, and strongly expressed in granulosa layer of normal and cystic follicles, but weakly expressed in atretic follicles. With the use of Western blotting and real-time PCR, high levels of bcl-2 and XIAP protein and mRNA expressions were examined in cystic follicles compared with those in normal follicles, but expressions of bax and caspase-3 protein and mRNA were low. The results indicate that low frequency of apoptosis might be associated with decreased amounts of apoptotic-related factors (bax and caspase-3). and increased amounts of anti-apoptotic factors (XIAP and bcl-2) in cystic follicles.On the other hand, we evaluated the cell proliferation by the expression of PCNA. together with proliferation index of in vitro cultural granulosa and theca cells by ELISAs in cystic follicles. We found that the PCNA-positive cells were strongly expressed in granulosa and theca layer of normal follicles, but the atretic and cystic follicles showed minor proliferation by immunohistochemistry. Low levels of PCNA protein and mRNA expression in cystic follicles were examined by western blotting and real-time PCR. The cell proliferation activities of granulosa and thecal cells from cystic follicles were significantly lower (P<0.01) by ELISAs. These results indicate that the program cell death and cell proliferation system were altered in cystic follicles. Low apoptotic frequency and weak proliferative activity may be one of the new feature of porcine cystic follicles.In summary, we have established the 2-DE map for porcine FF. seventy five percent of the identified proteins were detected in porcine FF for the first time. The present study enriches our understanding of the proteins of FF. RBP-4 and ApoA-I might be candidate biomarkers for porcine follicular cysts. By further investigation, we found that RBP-4 and transferrin may also originate from the granulosa cells aparting from the blood. These data suggest that changes in FF protein concentrations may reflect the changes of both the synthetic ability and metabolism of follicular cells and the structure of follicular wall in various pathological states. Our results indicate that the programed cell death and cell proliferation system were altered in cystic follicles. The disorder between apoptosis and proliferation was responsible for maintaining a static condition without degeneration, which leads to the long-term persistence of follicles. These findings provide important novel insights into the pathogenesis of follicular cysts in sows.