الفهرس 
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Abstract
The better understanding of molecular aspect in follicular growth may be a step forward to improve the response to MOET, in order to maximize exploitation of buffalo cows during their life. Hence, the current experiments were designed. Experiment 1, aimed to (I) study the expression profile of selected genes in GC during follicular and luteal phases, (II) evaluate the correlations between GC gene expression, and steroid concentrations as estradiol (E2) & progesterone (P4) in follicular fluid (FF), and (III) to determine the effect of ovarian status on follicular population as well as follicular size frequency. At slaughterhouse, ovaries were collected in pairs from buffalo cows (n=178). Ovaries were classified into; follicular and luteal phases, where ovaries in luteal stage were subdivided into; hemorrhagic, developing, mature and albicans. Moreover, the antral follicles from luteal groups were classified only into: small (<4mm), and large (9-20 mm). Additionally, the antral follicles from follicular groups were classified into three subgroups: small (<4mm), medium (5-8 mm), and large (9-20 mm).The FF and GC were collected for steroids measurement as well as analysis gene expression, respectively. The LHCGR, and CYP19 mRNAs in small size follicles significantly (P<0.001) decreased compared to medium size follicles. However, large size follicle showed significant (P<0.001) increase in LHCGR and CYP19 genes compared to medium size follicles. Additionally, FSHR was clearly (P<0.001) decreased in large size follicles compared to medium size ones. The PCNA was significantly increased in small (P<0.001), and large (P<0.05) sizes follicles. Meanwhile, AMH and PLA2G3 were significantly (P<0.001) decreased in small and large size follicles. The different stages of luteal phase had a profound impact on follicular size& number, and GC gene expression, represented by up-regulation of FSHR and CYP19, as well as down-regulation of LHCGR, PCNA, CASP3, and AMH, mainly at mature stage of CL. The follicular size and phase of estrous cycle had a profound effect on the concentrations of E2 and P4 in FF. We found strong (positive and/or negative) correlations between expressed genes in GC and steroids hormones in FF. Collectively, the precise regulation of GC is necessary to maintain the proper growth and development of follicles and CL. The highly orchestrated pattern of LHCGR, FSHR, CYP19, PCNA, CASP3, and AMH in GC during luteal phase, may suggest that the proper time for applying superovulation protocols is 8-11th day, during mid-cycle.
Experiment 2, aimed to (I) characterize cultured GCs from different follicles sizes morphologically and molecularly, and (II) select the suitable model according to the follicular size, which maintained GCs function during culture. Ovaries of buffalo were collected from slaughterhouse, and follicles were classified morphologically into: 1st group ≤ 4, 2nd group 5-8, 3rd group 9-15 and 4th group 16-20 mm diameter. The GCs pellet was divided into two portions; 1st portion served as control fresh pellet, and 2ndwas used for GCs culture for a week. Total RNA was isolated, and qRT-PCR was performed for FSHR, CYP19, LHCGR, PCNA, CASP3, AMH, and PLA2G3 mRNAs. Estradiol (E2) and progesterone (P4) levels were measured in the culture supernatant, and in follicular fluids, using ELISA. Basic DMEM-F12 maintained the morphological appearance of cultured GCs. The relative abundance of FSHR, CYP19, and LHCGR mRNAs was (0.001 ≤P≤ 0.01) decreased at the end of culture, compared to fresh pellet. There was a fine balance between expression pattern of proliferative gene (PCNA), and proapoptotic marker gene (CASP3). The AMH mRNA was significantly increased (P< 0.001) in cultured GCs from small follicles, while the cultured GCs from other three categories; 5-8, 9-15 and 16- 20 mm showed a clear reduction (P< 0.001). Interestingly, the relative abundance of PLA2G3 mRNA was significantly (P< 0.001) increased in all cultured GCs. The concentrations of E2 and P4 were significantly (P< 0.001) decreased in all cultured groups. The primary cultured GCs from small follicle could be a good model for better understanding of follicular development in Egyptian buffaloes.