Study On Endocrine And Molecular Cytological Alterations Of Porcine Antral Follicles During Development And Atresia

In mammalian ovaries, there are huge numbers of follicles at various stage and only a few follicles could develop to mature and ovulation during follicular development. More than 99% of follicles will undergo a degeneration process called atresia. Atresia is a normal physiological phenomenon during follicular development, this process is essential for the maintainence of ovarian homeostasis in mammals. The regulation of follicular development and atresia is a complex process and involves in many cell death factors and survival-promoting ones, and the regulation of follicular development and atresia have been characterized to a large extent. However, the precise temporal and molecular mechanisms involved in the regulation of these events have remained to be unclear. There are many difficulties for elucidating these molecular mechanisms, one of the most important reasons is lackage of an elegant system for such a study. Recent studies suggest that the apoptosis of ovarian granulosa cells plays a major role in follicular atresia, but very little was known for the molecular mechanisms that control apoptotic cell death in granulosa cells during follicle selection. It is of importance theoretically and practically to elucidate the mechanisms on follicular atresia for a better understanding to reveal the mysteries in reproductive control, to increase the utilization rate of ovaries and IVM rate of oocytes and to improve the efficiency of livestock breeding. In the present study, the paraffin section preparation for single intact antral follicle which isolated from porcine ovaries has been reported, then we observed the morphological characteristic of porcine antral follicles during development and atresia using the HE staining technique; through isolated and cultured porcine antral follicles in vitro, we established a culture model for porcine antral follicles, and analyzed the relationship of the morphology of follicles and the apoptotic granulosa cells or the levels of steroids of porcine follicle during development and atresia; and quantified the expression of FasL and Fas mRNA in granulosa cells by real time quantitative PCR; or investigated the effect of FSH on porcine different size follicles and studyed on the relationship between follicular cells apoptosis and the developmental competence of oocytes, so as to supply experimental data for further revealing of the regulatory mechanism of follicular development and atresia. This study contained the following 6 parts.Experiment 1. The morphological observation of porcine antral follicles during development and atresiaThe paraffin section was prepared for porcine intact antral follicle which isolated from different developmental stage, and the morphological characteristic of porcine antral follicles during development and atresia was observed using HE staining. There is abundant follicular fluid in antral follicle, so it is difficult to obtain the paraffin section of natural and integrated structures by the routine paraffin section method. In this experiment, isolated follicles were fixed into the Bouin's stationary liquid for 72 h, before the process of anhydration used the mixed liquor of xylene and ethanol (1:1) as a transition approach, besides lengthen the time make sure that the paraffin literally penetrate into ovarian follicles, a better section of a single integrity follicle was obtained.. Only limited numbers of the apoptotic cells have been observation around the follicular antrum of healthy antral follicles. Different degree of separation in the basilemma and granulosa cells layer have been observation in early atretic follicles, the junction among granulosa cells was loose and began to fall off into the follicular antrum, the cumulus-oocyte complexes (COC) of individual small follicles (<3mm) fall off into the follicle antrum, the apoptotic cumulus cells or oocytes were not observed. In progress atretic follicles, the theca layer deformed and loose, the theca cells near the outer region of theca layer were desquamate, limited number of the theca interna cells have hypertrophied. Cell structure is not clear, the boundary between the granulosa and theca cells became indistinguishable, the basilemma disappeared, the follicular antrum contained numerous apoptotic granulosa cells or filled with remnants of aptotic bodies. In the process of follicular atresia, there are no significant difference in the morphological characteristics among the large (>5mm), medium (3-5mm) and small follicles. Morphology research shows that the acuracy of classification by morphometric criteria of healthy follicles, early atretic follicles and progress atretic follicles is 76%-92% in this experiments, especially in healthy follicles, providing the basis for the eye examination of follicles and follicular classificationExperiment 2. Establishment of a culture model for isolated porcine antral folliclesIn this experiment, isolated porcine antral follicles are firstly used to culture in vitro, in order to establishment of a investigative model for follicular development and atresia in vitro. Porcine small (<3 mm), medium (3～5 mm) and large (>5 mm) healthy follicles were cultured in serum-free TCM199, respectively. After culture for 8 h, the morphology of follicles was no visible differences compared to the follicles before culture. When cultured for 16 h, the vessels of the follicle wall are not visible, the follicular fluid of medium and small follicles become slightly turbid, the follicle becomes opaque, but the oocytes from various sizes of follicles possessed 5 or more layers of compact cumulus cells with a uniform ooplasm. Although the morphological structure of various sizes of follicles were complete at 24 h, but the cells of the surface of follicles began to fall off and follicular fluid became turbid, however 22.2% of oocytes from small follicles had no more than 5 layers of cumulus cells and contained occasional pyknotic oocytes. When cultured for 48 h, the large and medium follicles collapse and relaxation were observed,83.8% of oocytes isolated from large follicles and 88.3% of oocytes from medium follicles only had 1～2 layers of cumulus cells. The follicles collapsed and the follicular wall of small follicles began to dissolve, and granulosa cells floated free in the follicular fluid, almost all oocytes from small follicles were denuded and degenerated. This experiment showed that the serum-free culture system can effectively induce further atresia of porcine antral follicles, which set a appropriate research model for further research on the function of gonadotropin, growth factors and cytokines on ovarian follicular development and steroid hormone.Experiment 3. The analysis of the granulosa cells apoptosis and the changes of hormone during porcine follicular development and atresiaIn this experiment, intact antral follicles were separated from porcine ovaries, which are divided into 3 categories according to the quality: healthy follicles, early atretic follicles and progress atretic follicles. Then divided into 3 groups according to the follicular diameter: large follicles group (>5 mm), medium follicles group (3～5 mm) and small follicles group (<3 mm). Selection of the healthy follicles used for culture in vitro, after culture for 0 (control group), 8,16 and 24 h, analyzed for apoptosis in granulosa cells via AnnexinⅤand propidium iodide (PI) staining, or examined the concentrations of oestradiol (E2) and progesterone (P4) in follicular fluid of cultured above different size follicles using radioimmunoassay. The results showed that there are limited numbers of apoptotic granulosa cells in the healthy follicles of different sizes, and the percentage of the apoptotic granulosa cells was significantly increased during follicular atresia, especially in small follicles. About 60% of granulosa cell became apoptosis in late atresia small follicles. In healthy large follicles, the concentrations of E2 of follicular fluid was significantly higher than that in the medium and small follicles, the concentration of P4 was no difference with the medium follicles but significantly higher than that in the small follicles. Ratio of P4/E2 is variability between different size follicles when they are in different health state. Ratio of P4/E2 is less than 5 in follicular fluid of healthy medium and small follicles, but the ratio of P4/E2 is less than 1 in follicular fluid of healthy large follicles. When follicular atresia occurs, the concentrations of E2 significant decline and progesterone concentrations significantly increased among follicle size groups. Ratio of P4/E2 is more than 1 in follicular fluid of early atretic large follicles. Ratio of P4/E2 is between 5 to 15 in early atretic medium and small follicles, when the ratio past 15, then the follicles become the progress atretic state, however, the ratio of P4/E2 in follicular fluid of small and medium progress atretic follicle was significantly higher than those in large follicles. It showed that the total apoptotic rate of granulosa cells (early apoptotic cells+progress apoptotic cells) derived from cultured follicles could reach over 70% at 8 h after culture and be 81.1%-94.6% at 24 h after culture. Concentrations of E2 and P4 decreased significantly with time in culture, concentrations of E2 and P4 of follicular fluid in small follicles were higher than those of medium and large follicles, but the difference not significant. The experiments showed that the follicular culture system without serum used could effectively induce the apoptosis of follicular granulose cells, cell apoptosis is the main cause of follicular atresia, the degree of which varied with the size of follicles, small follicles seemed to be easier atretic than medium and large follicles. The concentrations ratio of P4/E2 of follicular fluid can be determined as a standard of the quality of antral follicles.Experiment 4. The expression of the Fas/FasL mRNA of granulosa cells derived from porcine antral folliclesTo study the relationships between Fas/FasL mRNAand granulosa cells apoptosis at different developmental stages of the porcine antral follicles, and investigate the regularity and molecular mechanism of follicular atresia. In this experiment, intact antral follicles were separated from porcine ovaries, which are divided into 3 categories according to the quality: healthy follicles, early atretic follicles and progress atretic follicles. Then divided into 3 groups according to the follicular diameter:large, medium and small follicles group. Selection of the healthy follicles used for culture in vitro, after culture for 0,8,16,24,48 and 72 h, quantified the expression of FasL and Fas mRNA in granulosa cells by real time quantitative PCR SYBRgreen method. Fas/FasL mRNA expressed in granulosa cells of various developmental stages of procine antral follicles. When follicular atresia happened, the expression of FasL mRNA significantly increased, while the expression of Fas mRNA was no significant difference compared with healthy follicles. In the early atretic follicles group, the expression of FasL mRNA in granulosa cells of small follicles is significantly higher than those of the large and medium follicles group. In vitro culture showed that the levels of FasL mRNA in the granulosa cells of different size of follicles has a significant increase with time in culture and reach the maximum at 24 h (P＜0.05); the level of FasL mRNA in granulosa cells of small follicle is higher than those of large and medium follicles group. The relative expression of Fas mRNA in the granulosa cells among different sizes of follicles were not significant difference before cultured (0 h), then significantly increase at 8 h, reach the maximum at 48 h. The results show that, Fas/FasL system plays an important regulatory role in the granulosa cells apoptosis during the different developmental stages of porcine antral follicular atresia. There are differences of the factors that regulate the expression of Fas mRNA between in vivo and in vitro. The high expression of FasL mRNA in vivo may is the main inducement that initiate Fas/FasL apoptosis pathway.Experiment 5. The effect of FSH on porcine isolated antral follicles cultured in vitroThrough the established serum-free culture system of porcine antral follicles to approach the effection of FSH on porcine antral follicles in vitro. In this experiment, intact healthy antral follicles were separated from porcine ovaries, which are divided into 3 groups according to the follicular diameter: large, medium and small follicle groups. Then those follicles cultured in serum-free TCM199 with 1,3 and 5IU/ml FSH, respectively. After culture for 0,8,16,24,48 and 72 h, analyzed for apoptosis in granulosa cells via Annexin V and propidium iodide (PI) staining, or quantified the expression of FasL and Fas mRNA in granulosa cells by real time quantitative PCR. Additionally, the concentrations of E2 and P4 in follicular fluid of cultured above different size follicles were examined using radioimmunoassay. The results showed that,1 IU/ml FSH could not significantly inhibit granulosa cells apoptosis of different size of follicles, the level of Fas and FasL mRNA were no difference within each culture time compared with those of control group.3-5 IU/ml FSH with a dose dependent manner significantly inhibited granulosa cells apoptosis of different size follicles, especially for granulosa cells of large follicles. Supplementation with 5 IU/ml FSH seemed to decrease FasL and Fas mRNA expression in granulosa cells regardless of follicle size, especially at 24 h. And 3 IU/ml FSH has no significant inhibition on the level of Fas mRNA. Treatment with FSH increased the concentrations of either E2 or P4, but no significance level was observed.Experiment 6. Apoptosis within follicular cells during porcine follicular development and its effect on the developmental competence of oocytesTo study the effect of follicular cell apoptosis on the developmental competence of oocytes, as well as the important role of follicular integrity to support and promote the porcine oocyte maturation. The changes of oocytes diameter of porcine antral follicles at different developmental stages were measured in this experiment. The healthy antral follicles (3-5 mm) and cumulus-oocyte complexes (COC) collected from the same size follicles were choosen for serum-free culture in vitro. After culture for 8 h, granulosa cells and cumulus cells of antral follicle group or the cumulus cells of COC group were collected to analyze the apoptotic rate using Annexin V and propidium iodide (PI) staining. The FDA staining was used for the analysis of survival of the oocytes and the in vitro fertilization was used for the detection of cleavage rate. The results showed that oocytes diameter significantly increased with the growth of antral follicles, when the follicular diameter up to 5 mm or above, oocyte diameters could reach the size at matural. Follicular development is significantly suppressed once the medium and small follicular atresia. There is no difference between early atretic medium follicles compared with the healthy medium follicles in the oocytes diameter, then a small amount (5%) of oocytes is occasionally death. In progress atretic follicles, the oocyte survival rate or diameter of medium follicles significantly decrease, but significantly higher than those of small follicles. However, the oocyte survival rate or diameter in the early atretic large follicles is no difference compared with those in healthy large follicles. The COC morphology of small follicles is more effected by follicular than that of large and middle follicles, but still have more than 80% of the oocytes possessed 3 or more layers of cumulus cells in progress atretic small follicles. The percentage of apoptotic cumulus cells of intact follicle groups was significantly lower than that of COC group cultured in vitro, and the oocyte survival rate was no difference between the two groups, the cleavage rate was significantly higher than that of COC group (24.55% VS 0%, P< 0.05), but were significantly lower than control group. These results showed that the structural integrity of follicles seemed to have protective effect for the cumulus cell aopotosis and the developmental capacity of oocytes. The integrated follicular structure and the interaction between oocyte and follicular cells inside was inducing to delay the inhibition to the oocytes due to the follicular atresia.