Dynamic patterns of expressed genes in granulosa cells during follicular and luteal stages in Egyptian buffaloes.

A better understanding of the molecular mechanisms in granulosa cells (GC) is warranted, during different follicular and luteal developmental stages in buffalo cows. We aimed to (I) study the expression of selected genes in GC during follicular and luteal phases, (II) evaluate correlations between GC gene expression and steroid concentrations {17-beta estradiol (E2) and progesterone (P4)} in follicular fluid (FF), and (III) study effect of ovarian status on follicular population as well as follicular size frequency. Ovaries were collected in pairs from buffaloes (n = 178). Ovaries bearing corpus luteum (CL) were subdivided into hemorrhagic, developing, mature, and albicans. Follicles from luteal groups were classified only into small (< 4 mm) and large (9-20 mm), while follicles from follicular groups were classified into three subgroups: small (< 4 mm), medium (5-8 mm), and large (9-20 mm). The FF and GC were collected for steroid concentrations measurement and gene expression, respectively. In the follicular phase, luteinizing hormone/choriogonadotropin receptor (LHCGR) and cytochrome P450 aromatase (CYP19) in small follicles decreased compared to medium ones. Large follicle showed an increase in LHCGR and CYP19 compared to medium ones. Follicle-stimulating hormone receptor (FSHR) decreased in large compared to medium size follicles. Proliferating cell nuclear antigen (PCNA) increased in small and large follicles. Meanwhile, anti-Mullerian hormone (AMH) and phospholipase A2 group III (PLA2G3) decreased in small and large follicles. The different stages of luteal phase had a profound impact on GC gene expression. There were strong (positive and/or negative) correlations between gene expression and steroid hormones. The different scenarios between expressed genes in GC and steroid concentrations are required for the proper growth and development of follicles and CL.

Expression profiling of primary cultured buffalo granulosa cells from different follicular size in comparison with their in vivo counterpart.

This study aimed to: (i) characterize cultured granulosa cells (GCs) from different follicle sizes morphologically and molecularly; and (ii) select a suitable model according to follicular size that maintained GC function during culture. Buffalo ovaries were collected from a slaughterhouse and follicles were classified morphologically into: first group ≤ 4 mm, second group 5-8 mm, third group 9-15 mm and fourth group 16-20 mm diameter. GC pellets were divided into two portions. The first portion served as the control fresh pellet, and the secondwas used for 1 week for GC culture. Total RNA was isolated, and qRT-PCR was performed to test for follicle-stimulating hormone receptor (FSHR), cytochrome P450 19 (CYP19), luteinizing hormone/choriogonadotropin receptor (LHCGR), proliferating cell nuclear antigen (PCNA), apoptosis-related cysteine peptidase (CASP3), anti-Müllerian hormone (AMH), and phospholipase A2 group III (PLA2G3) mRNAs. Estradiol (E2) and progesterone (P4) levels in the culture supernatant and in follicular fluids were measured using enzyme-linked immunosorbent assay (ELISA). Basic DMEM-F12 medium maintained the morphological appearance of cultured GCs. The relative abundance of FSHR, CYP19, and LHCGR mRNAs was 0.001 ≤ P ≤ 0.01 and decreased at the end of culture compared with the fresh pellet. There was a fine balance between expression patterns of the proliferation marker gene (PCNA) and the proapoptotic marker gene (CASP3). AMH mRNA was significantly increased (P < 0.001) in cultured GCs from small follicles, while cultured GCs from other three categories (5-8 mm, 9-15 mm 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. E2 and P4 concentrations were significantly (P < 0.001) decreased in all cultured groups. Primary cultured GCs from small follicles could be a good model for better understanding follicular development in Egyptian buffaloes.

Establishment a protocol for total RNA isolation from buffalo fresh and frozen semen for molecular applications.

To date, there is no an established protocol for total RNA isolation in Egyptian buffalo spermatozoa. The present study aimed (I) to establish a defined protocol for total RNA isolation from fresh and frozen spermatozoa, (II) to evaluate RNA quality and quantity from different extraction methods and studying gene expression. Warm and standard room temperature modified QIAzol Lysis Reagents were used for total RNA extraction. The quality and quantity of extracted RNA were checked, and subsequently qRT-PCR was performed using androgen receptor-like and three reference gene primers (GAPDH, ACTB and 18S). The warm modified QIAzol Lysis Reagents resulted higher yield of good quality RNA from fresh (569.54 ± 18.83 ng/µl) and frozen spermatozoa (110.59 ± 4.43 ng/µl), compared to standard room temperature modified QIAzol (421.26 ± 7.18 ng/µl) and (29.07 ± 5.25 ng/µl), for fresh and frozen semen samples respectively. The 260/280 ratio was 1.90 and 1.89 for fresh and frozen isolated semen by warm method respectively. The integrity of RNA was good and appeared as a sharp band on 2% agarose gel. The most stable reference gene was 18S. Reliable extraction method of high quality RNA yield could be a step forward for understanding mechanisms of spermatogenesis for improving male fertility.