116 Differential expression of connexin 43 and 37 mRNA transcripts during the estrous cycle in canines

Abstract

Gap junctions are intercellular channels that mediate cell-to-cell communication, allowing the passage of small signalling molecules. In the ovary, connexin 43 (Cx43) and connexin 37 (Cx37) are important gap junctional proteins expressed in the granulosa and cumulus cells or oocytes of several species. Gap junctions and connexins are required for the regulation of the oocytes meiotic resumption in preovulatory follicles after the surge of LH. However, unlike other species, canine oocytes do not resume meiosis before ovulation, which could be related to expression patterns of Cx43 and Cx37 during oocyte development and ovulation. Therefore, this study aimed to address the canine Cx37 and Cx43 gene expressions throughout the oestrous cycle, including the preovulatory period. The ovaries were obtained from bitches 1-6 years old (n = 72) following ovariohysterectomy. The stage of the oestrous cycle was assessed according the ovarian structures and by measurements of serum progesterone (P4) levels obtained from blood samples on the day of surgery. Anestrus was <0.1 ng/mL P4 and absence of follicles or corpus luteum in the ovarian surface; proestrus was 0.2-2 ng/mL P4 and growing small to medium follicles on the surface of the ovaries; oestrus was 2-19 mg/mL P4 and large follicles on the surface of the ovaries; and diestrus was >20 ng/mL P4 and mainly predominant corpus luteum on the ovaries. For Cx43 analysis, follicular cells (granulosa and theca) were mechanically recovered from follicles (n = 620) distributed into 4 types: prenatal (1 layer of granulosa cells up to the onset of antrum formation), small antral (~0.2-0.39 mm), medium antral (~0.4-5.9 mm), and large antral (~6-10 mm). For Cx37 study, the cumulus-oocytes complexes (COC) from the same follicles were used. Total RNA extraction was performed, and the evaluation of gene expression levels was achieved by relative quantification quantitative PCR analysis in follicular cells and COC. The data from at least 3 independent experiments for each gene were evaluated by ANOVA. The gene expression of both Connexins were observed in all stages of follicular development; however, the mRNA levels varied over the oestrous cycle. Both Cx43 and Cx37 transcripts showed the highest (P < 0.05) levels at anestrus when compared to other phases. The mRNA levels of both genes remained without changes in large follicles at oestrus stage, suggesting that, in contrast to other mammals where LH down-regulates connexins expression leading to the subsequent loss of intercellular coupling, the communication between the oocyte and follicular cells was maintained in canines. In conclusion, these 2 connexin genes were differentially expressed in canine follicular cells and COC during the follicular development. The maintenance of the gene expression of these connexins at the final follicular growth may be involved in the prolonged meiotic arrest in this species.

Supported by Ga grant from FONDECYT (1171670).