| The development of ovarian follicles in mammals is a physiological process regulated by many factors.During the development of follicles,most of the follicles are blocked and degraded and only a few of primordial follicles can ovulate successfully.Studies have shown that changes in granulosa cells play a crucial role in the development and atresia of follicles.In recent years,studies have shown that Smad4 plays an important role in granulosa cell apoptosis and follicular atresia.In this study,pig ovary follicles were used as research objects.Hematoxylin-eosin(HE)staining,radioimmunoassay(RIA),transmission electron microscopy(TEM),immunohistochemistry(IHC)and Western blotting(WB)were used to study the morphological characteritics of granulosa cells during follicular atresia in pigs.At the same time,the expression and localization of Smad4 in porcine ovary were explored,and the phosphorylation of Smad4 in follicular granulosa cells in different healthy status was further studied.The research results will enrich the content of porcine reproductive biology and provide basis for the involvement of Smad4 in the regulation of follicle development and atresia.1.Morphological study of granulosa cells in follicular atresia of ovary in Suhuai pigs150-day-old porcine ovaries were harvested to investigate the internal characteristics of follicles during follicular atresia in Suhuai pigs.And the healthy follicles,the early atresia follicles and the late atresia follicles were peeled with a diameter of 5-8mm.The ovarian histology and granular.cell morphology were detected by HE technique.The levels of hormones in three follicles were detected by RIA and the subcellular structure of granulosa cells was detected by TEM.The results of HE showed that 150 days old Suhuai pig ovary had primordial follicles,primary follicles,secondary follicles,mature follicles and atresia follicles.In healthy follicles,the granulosa cell layer was tightly distributed on the inner side of the membrane cells and follicular cavity was normal.Granular cells lived in normal state.In early occluded follicles,some granulosa cells in the granulosa cell layer began to fall off and apoptotic bodies appeared.There were apoptotic granule cells in the follicular cavity.In late atresia follicles,the granular cell layer was no longer present.Granular cells were scattered in the follicular cavity.Nuclear chromatin was marginalized,and the color was dark.Many apoptotic cells aggregated into apoptotic bodies.RIA results showed that the concentration of E2 in the three follicular fluids decreased significantly with follicular atresia(P<0.01);there was no significant difference in the concentration of P4 in the follicular fluid of healthy follicles and early atresia follicles,and the concentration of P4 in the follicular fluid of the late atresia follicles was significantly increased(P<0,05).With the follicular atresia,the P/E value increased significantly(P<0.01).TEM results showed that the membrane of granulosa cells in healthy follicles were intact,their organelles were normal,and the nuclear membrane was intact.The mitochondria in the granule cells in early atresia follicles have begun to swell and the chromatin has begun to shrink.The granular mitochondria in the late atresia follicles have been completely swollen and transparent,the chromatin was completely condensed,and most of the cell membrane and nuclear membrane have been broken.These results indicate that the granulosa cell structure changes during the process of follicular atresia which provides a theoretical basis for studying the involvement of granulosa cells in the regulation of follicular development and atresia.2.Expression and localization of Smad4 in follicular development and atresia of ovary in Suhuai pigsOvaries of 25,70 and 150 days old pigs were collected to investigate the expression and localization of Smad4 in follicular development and atresia of ovary in Suhuai pigs.The localization of Smad4 protein was studied by immunohistochemistry.Western blotting was used to study the expression of Smad4 and p-Smad4 in ovarian healthy,early atresia and late atresia follicles of 150-day-old pigs.It was found that in the ovaries of 25-day-old sows,the presence of nests of oocytes was observed,and primordial follicles,primary follicles and secondary follicles were observed at the same time.No antral follicles were found.Smad4 was expressed with different degrees in granulosa cells and oocytes.However,with the development of follicles,the expression of Smad4 in oocytes gradually weakened,and the expression of Smad4 in granulosa cells gradually increased.In a 70-day-old sow ovary,the oocyte nest was no longer present.Primary primordial follicles,primary follicles,and secondary follicles were observed.Antral follicles have not yet been found,Smad4 was mainly expressed on granulosa cells but not in oocytes.In the 150-day-old sow's ovary,primordial follicles,primary follicles,secondary follicles,and antral follicles(including atresia follicles)were also observed.Smad4 was mainly expressed in granulosa cells.With the development of follicles,expression in granulosa cells was getting more and more enhanced.When the follicles began to infiltrate,the granule cell layer disintegrated and the apoptosis of granulosa cells was scattered in the follicular fluid.The expression of Smad4 in granulosa cells also gradually decreased,and the Smad4 was also expressed on membrane cells of early atresia follicles.Granule cells in late atresia follicles had no Smad4 expression.Western Blot results showed that the expression of Smad4 was highest in healthy follicles,and the expression of Smad4 protein gradually decreased with follicular atresia.Further examination of the phosphorylation level of Smad4 in follicular granulosa cells in different healthy states revealedr-that p-Smad4 in the three follicular granulosa cells also showed a decreasing trend.However,there was no significant difference between healthy follicles and early atresia follicles.These results provide scientific basis for the further study of the mechanism of Smad4 and its phosphorylation modification involved in the regulation of porcine follicular development and atresia. |
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