| Mammall ovaries contain a large number of oocytes at different developmental stages, most of them existing in the no cavity follicles. However, most follicles atresia and degraded in no cavity (preantral follicle) stage, are not be given the appropriate use, all that is undoubtedly a great waste of animal genetic resources and breeding. Therefore, the development of preantral follicles is of an effective and significance way to solve this problem. Culture technology of preantral follicles in vitro has made tremendous development and established a variety of culture systems, but different culture system has its own advantages and disadvantages, researchers have been exploring for a suitable culture model of the situation in order to find a way to simulate the follicular development of environmental conditions and pattern in vivo. There are many factors affect preantral follicle culture in vitro, summing up successful experience of predecessors, in this study, several areas were explored for discussing, which we just want to explore a suitable mouse preantral follicle separation and culture system.1. Using paraffin section-HE staining technique to study follicles' distribution and development pattern at the different stages of ovarian, selecting the appropriate developmental stages of mice, and laying a solid basement for the efficient separation of preantral follicles, as well as an identification standard during the follicular development in vitro. The results showed that:with the development of mouse, ovary develops from non-cortex and medulla assigned in the initia to can be seen apparently. Follicles developed from the streak were gathered so as to develop into primordial follicles, primary follicles, secondary follicles, until cavity formation, judging from the numbers, primordial follicles decreased in the initial stage follicles gradually, primary follicles and secondary follicles increased gradually, the number of antral follicles from scratch to less than mature follicles relatively. Ovarian follicle development from the initial scratch to many non-sizes at different developmental stages of follicles, the majority cavity-like structure. preantral follicles most in mouse that 10 days to 15 days after birth, and at this time,there were less ovarian connective tissue, adipose tissue, yellow body and was easy to get hold of the completed and a large number of isolated preantral follicles. In addition, the distribution of follicles in the ovaries is regional, there were a large number of primordial follicles in the outermost layer of ovarian cortex with the higher density and primary follicle was followed, secondary follicles were mostly located in the most inner layer with the large junction distribution in the skin marrow relatively. Third follicles and mature follicle will be highlighted in the epidermis. The experimental results showed that it is more ideal that the ovaries of mouse 15 days after birth target as a preantral follicles separation object in vitro.2. By using separation time, the number of normal rate as well as recovery rate as evaluation indexes to compare the two methods of enzyme-mechanical combination methods and micro separation methods separation to find a suitable and an effective preantral follicles separation method that separated from 15days postnatal mice ovarian and make a first vital step for successfully-cultured mouse preantral follicles in vitro. The Results showed that:Enzyme mechanical ion and recovery of the number of preantral follicles that separated by enzyme-mechanical combination methods obtained significantly more than the simple mechanical separation, and 0.1% collagenase selected could obtain common follicles more than the normal. Micro-separation time was longer, less number, but high survival rates of follicles after 6days cultured in vitro. Considering those various factors, in this study, Micro-dissection method was used for separating preantral follicles witch from 15days after birth in mice in vitro.3. Through establishing a serum-free culture system that was suitable for development of mouse preantral follicles in vitro to explore the additive FSH, LH, VC to mouse follicle/oocyte growth and development, to explore a suitable culture in vitro system for mouse preantral follicles growth and development. The results showed that:with time going, preantral follicles became more and more bigger, the diameter of follicle and its acolyte were increased as well, but the acolyte growth was not faster than follicle growth. And the concentration of every group culture in 4 days, the diameter of follicle and acolyte growth rate were the fastest. And follicular growth appears a significant differences between the first 4 days and the following 2,6,8 days, (P<0.05). Serum-free basic medium were added to FSH400+LH 200, VC50ug/ml showed a greater degree of follicles and acolytes in promoting growth and improving the survival rate of follicles and cavity rate, In the eighth day, rHCG and EGF were added for culturing 24h, the occurrence of GVBD and PB1 emission have a higher rate.4. Through Using DNA agarose gel electrophoresis and RT-PCR technologys to detect the above selected serum-free culture system for preantral follicles at different times of apoptosis and Bcl-2/Bax in mRNA levels of gene expression situation from molecular biology,In order to further verify the adequacy of the serum-free culture system. Thus, to explore a suitable mouse preantral follicle growth culture system in vitro. The results showed that:In the follicle culture 0 days and 2, 6 days, regardless of the basis of medium or above complete culture medium were added, bax and bcl-2 are expressed, and the expression growing, but in the training of junior,the expression of bax and bcl-2 remains quite. Only in the sixth day, the control group showed a slightly stronger expression. In the view of the training results, the experimental group and control group was compared in the same culture, Bax, Bcl-2 gene expression are stronger in the experimental group than the control group. DNA agarose gel electrophoresis for follicle apoptosis showed that:there are no typical features of apoptosis no matter the experimental group or control group, It showed that the basic medium were added to FSH400 mIU/mL, LH 200 mIU/mL and VC50ug/ml for preantral follicles cultivation in vitro we used in the present study, is feasible5. In this study, bovine preantral ovarian follicle and antral follicle's distribution and morphological characteristics were also studied, while the ovaries of bovine preantral follicles were isolated and cultured in vitro.The results showed that:bovine follicle's distribution was obvious regional, and the same with the distribution of mouse ovaries follicle's. Mature oocytes in vitro maturation experiment had been released the first polar body. |
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