| Buffalo ovary contains about tens of thousands of primordial follicles at birth, but only about two hundred follicles can develop to reach maturation and ovulation during the life cycle, about99.9%of follicles undergo atresia and degradation during their growth. How to excavate the reproductive potential of female Buffalo, develop and applicate ovarian preantral follicles, is an important subject in the research of reproductive physiology of Buffalo. The aim of this study was to detecte expression of the candidate genes in the different period of preantral follicles, summarize the characteristics and differences of expression, it can lay a foundation for ascertaining the molecular mechanism of buffalo follicular development and optimizing the culture system of buffalo preantral follicles. Now the relevant research results are summarized as follows.Firstly, expression and localization research of PCNA and VASA prot-eins in buffalo ovarian tissue. The localization of PCNA and VASA proteins were demonstrated in different periods follicles of buffalo ovarian tissue through the method of paraffin sections and immunohistochemistry. The results showed that,(1) in primordial follicles, no remarkable staining for PCNA and VASA in granulosa cells was observed, but staining in the oocytes.(2) In primary follicles, weak immunoreactivity of PCNA and VASA were localized in oocytes and some granulosa cells.(3) In secondary follicles, positive staining of PCNA and VASA in oocytes and in most granulosa cells were detected, but no remarkable staining for VASA was detected in nuclei of oocyte.(4) In antral follicles, extensive PCNA labeling was expressed in the layers of granulosa cells, theca cells and the cumulus cells encircling the oocyte, but no remarkable VASA staining was found in all of these cells. With the growth of follicles, the expression of PCNA had a significant upward trend, and the expression of VASA in different periods of the follicles had a significant difference.Secondly, the mRNA expression level of PCNA, FSHR, IGFR-1, IGF-Ⅱ, IGFR-2, FGF-2and FGFR-2were researched in different periods preantral follicles of buffalo. Primordial follicles, primary follicles and seconda-ry follicles of buffalo were isolated, and detected their expression changes of th-ese genes through method of QRT-PCR. The results showed that,(1) PCNA mRNA were present in all preantral follicles, and showed a upward trend, the mRNA expression level of PCNA was significantly higher in primary follicles than primordial follicles (1.63±0.09vs1±0.01, P<0.05), the mRNA expression level in secondary follicles was about43times as much as primordial follicles, and highly significantly higher than primary follicles (43.50±0.14vs1.63±0.09 vs1±0.01, P<0.01), the result of QRT-PCR is accordant with the results of the immunohistochemistry.(2) FSHR wasn’t present in primordial follicles, and the mRNA expression level of FSHR was significantly higner in secondary follicles than primary follicles (1.64±0.09vs1±0.10, P<0.05).(3) The expression of IGFR-1were detected in all preantral follicles, and showed a upward trend, the mRNA expression level of IGFR-1was significantly higher in primary follicles than primordial follicles (1.93±0.004vs1±0.07, P<0.05), and it was highly significantly higner in secondary follicles than primordial follicles and primary follicles, and was about12times as much as in primordial follicles (12.07±0.02vs1.93±0.004vs1±0.07, P<0.01).(4) IGF-Ⅱ were present in all preantral follicles, and showed a upward trend, the mRNA expression level of IGF-Ⅱ in secondary follicles and primary follicles were significantly higner than primord-ial follicles (5.59±0.46vs3.60±0.35vs1±0.40, P<0.05), but there were no significantly difference between secondary and primary follicles; The expression of IGFR-2were detected in all preantral follicles, and showed a upward trend, the mRNA expression level of IGFR-2was highly significantly higner in seconddary follicles than primary follicles and primordial follicles (10.74±0.01vs1.24±0.07vs1±0.05, P<0.01), there were no significantly differentce betwe-en primary follicles and primordial follicles.(5) FGF-2were present in all preantral follicles, and showed a downward trend, the mRNA expression level of FGF-2was significantly higner in primordial follicles than primary follicles and seconddary follicles (1±0.16vs0.60±0.18vs0.51±0.09, P<0.05), there were no significantly difference between primary follicles and seconddary folic-les; The expression of FGFR-2were detected in all preantral follicles, and showed a upward trend, there were significant difference between primordial follicles and primary folicles, and the mRNA expression level of FSHR was significantly higner in secondary follicles than primary follicles(2.74±0.16vs1.47±0.09vs1±0.05, P<0.05). The results above showed that, with the growth of follicles, the mRNA expression level of FSHR、IGFR-1、IGF-2、IGFR-2and FGFR-2in primordial follicles, primary folicles and seconddary follicles had a upward trend; but the mRNA expression level of FGF-2had a downward trend, this may be associated with endocrine physiology environment of follicle development. The results of this study provided a molecular basis for optimizing the culture system of buffalo preantral follicles. |
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