| Follicular formation and development is a complex procedure, which includes follicular initiation, recruitment, selection, dominance, ovulation, and luteinization. This procedure can be regulated by a lot of factors, such as hormones and growth factors. It is well known that only a few follicles can reach into dominant follicles and ovulation, more than 99% follicles go atresia in mammals. Erhualian pig is one of the seven local class groups of Taihu pigs which is one of the four famous local pigs in China. Erhualian pig is well known for its reproductive performance, good maternity and good maternality. Although there are some reports about the reproductive characteristics of Erhualian pigs, knowledge of reproductive physiology in this species is extremely limited.In female mammals, the reproductve period and fertility are much determined by the number of primordial follicle pool and oocytes in the ovary. The primordial follicle formation is known as follicle assembly. In this process, the primordial follicles formed after the oocyte completed mitosis and entered into the process of meiosis. In rodents, the follicular formation occurred during the first few days after birth, while this process occurred during the fetal life before birth in the human, cattle, sheep and pig. Recent studies have shown that steroids, PI3K/Akt and NO/sGC/cGMP signaling pathways play important roles in regulating the formation and development of ovarian follicles in rats, mice, cattle, etc. However, all of these above-mentioned pathways of early stage of follicular formation and development in pigs are limited. Therefore, it is immediately required to carry out the further study on the ovarian cell biology and regulatory mechanisms of the signaling pathway involved in the follicular development in the female pig.The current study is focused on histological observation on follicular development in fetal and neonatal Erhualian pigs first, and then examines the period of the primordial follicles formation and stages of the early development of the follicles. The ovarian concentrations of estrogen and progesterone in fetal and neonatal pigs during the follicular formation and development are studied by using the method of immunohistochemistry and chemical luminescenc, and the PTEN/PKB/FOXO signaling pathway is investigated by using immunohistochemistry and western blotting in the early formation and development of ovarian follicles. At the same time, the role of the proteins involved in the NOS signaling pathway are also examined during these stages.1. The early stages of follicular formation and development in Erhualian pigsIn order to study the pattern of follicular development, the ovaries of day 50,70 and 90 post coitum (dpc), days 1,20,70 and 120 postpartum (dpp) of Erhualian pigs were sampled for histological analysis. The results indicated that the germ cells were existed as egg nests in the early stage of the ovary, primordial follicles were first discovered in the 70-dpc ovary and corpus luteum was found on 120 dpp. Stat3 as a marker for labelling germ cells were used to check single follicles and egg nests, the results showed that the structure of egg nest reduced significantly and the proportion of single follicle increased in the fetal aged from 70dpc to 90dpc, respectively. In addition, antral follicles appeared in the ovary on 70 dpp, and the Corpus luteum was observed on 120 dpp. PCNA were used as a marker to examine the proliferation of germ cells, the results showed that proliferation of oocytes was the most important during the stage of follicular formation and development. All of these results suggested that the primordial follicles formed in 70 dpc, the puberty ranged from 70 dpp to 120 dpp and the predominant growth before follicular activation was the oocyte.2. Concentrations of estrodiol and progesterone during stage of ovarian follicular development and localization of ovarian estradiol receptor (ER) and progesterone receptor (PR) in the fetal and neonatal pigIn order to investigate the relationship between the ovarian concentration of estrogen and progesterone and the follicular formation and development, five ovaries of the 70dpc, 90dpc, and ldpp were used to do localization of ER and PR by immunohistochemistry, respectively. The results showed that in the early stage of pig ovary, ERs expressed mainly in in the egg nests, cytoplasma of oocytes and granulosa cells of follicles ranging from primordial to secondary stages. The pattern of immunostaining of PR was similar to the ERs. Simultaneously, the levels of estrogen and progesterone of ovarian tissue and blood serum were determined by chemical luminescence method in experimental pigs, and the findings implied that with gestational age increased, ovarian levels of estrodiol and progesterone reduced as the number of primordial follicles increased. Together, our morphological observation on the stage and quantity of follicular formation, the ovarian concentration of estrodiol and progesterone suggested that estrodiol and progesterone might play a potential role in follicular formation and development in fetal and neonatal pigs.3. Immunolocalization of PTEN, PKB, FoxO3a and FoxO1 proteins in the ovary of Erhualian pigsTo study the function of PTEN PKB/FOXO/signaling pathways in the early stage of follicular development, localization of PTEN, PKB, FoxO1 and Fox03a proteins in porcine ovary were carried by using immunochemistry. The results showed that PTEN, PKB, FoxO1 and Fox03a proteins were mainly expressed in the oogonia in egg nest, the cytoplasm of oocytes in primodial and primary follicles, and vascular endothelial cells in the fetal and neonatal pigs. In postnatal pigs, PKB, FoxO1 PTEN and Fox03a were expressed mainly in the granulosa cell layer of the preantral and antral follicles, theca cell layer, corpus luteum cells and cytoplasm of oocytes. Furthermore, the intensive immunostaining for FoxOl in granulosa cell layers was the most intensive. The result of western blotting also showed that PTEN, PKB and FoxO3a proteins were located in the ovary of fetal pigs. All of these results suggested PKB/FOXO PTEN/signaling pathways might play an important role in the early stage of follicular formation, development and luteinization.4. Expression of PTEN and FoxO3a proteins in the granulosa cells of different diameter folliclesTo investigate the effect of FoxO3a and PTEN on pGCs, the GCs were isolated from small (1.5-3 mm) and large (larger than 5 mm) antral follicles in different diameters. Then, the percent of the health follicles was counted, granulosa cells isolated and follicular fluid collected. The granulosa cell survival rate was tested by the injection experiment with Taiwan phenol; the expression levels of FoxO3a, pAKT and PTEN were detected with western blotting, respectively; and and the concentrations of E2 in pFF were detected. The results showed that the survival rate of pGC from large follicles was the higher (P<0.05); with the growth of follicles, the rate of the healthy follicles, the expression of FoxO3a and PTEN increased; concentrations of E2 in porcine follicular fluid increased with the enlargement of antral follicles. These results indicated that the expression of FoxO3a and PTEN proteins in in granulosa cells may be involved in the survival and apoptosis of pGC.5. Immunolocalization of nitric oxide synthase (NOS) isoforms and soluble guanylyl cyclase (sGC) al andβ1 subunits in the ovaries of Erhualian pigs.In order to investigate the roles of NO/sGC/cGMP pathway in follicular formation and development, localization of three different NOS isoforms (eNOS, iNOS, nNOS) and sGC subunits proteins in porcine ovary were carried by using immunochemistry. The results showed that three NOS were all expressed in the oocytes cell nest, the oocytes of the follicle at all levels, ovarian epithelial cells, vascular endothelial cells and corpus lutea. These proteins were also expressed in the the granulosa and the membrane cell layer of the growing and antral follicles. Furthermore, in granulosa cells and membrane cells, the expression levels of the three NOS strengthened gradually with the follicular development. In addition, sGCal and sGCβ1 were expressed in the oocytes cell nests, primordial follicles, and granulosa cells in the small developing follicles, and vascular endothelial cells.In the ovary of 90-dpc-old and 1-dpp-old, the expression intensity were very high in the granulosa cells of the primodial follicle and primary follicle, however, which gradually decreased with the growth of the follicles, theca cell of antral follicle and corpus luteum cell also had moderate expression.Furthermore, the bioactivity of total NOS, eNOS, iNOS and nNOS in these ovaries were also examined. The results showed that ovarian total NOS activities were significantly higher in fetal and neonatal stage compared to postnatal stages. Taken together, our findings suggest that NO/sGC/cGMP pathway involved in the follicular development.In conclusion, Follicular formation and development is a complex procedure, which can be regulated by a lot of factors. The results showed that steroids, PI3K/Akt and NO/sGC/cGMP signaling pathways play potential roles in regulating the formation and development of ovarian follicles in pigs. The regulatory mechanism involved in these pathways required further investigation. |
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