Vascular endothelial growth factor A isoforms modulate follicle development in peripubertal heifers independent of diet through diverse signal transduction pathways.

Follicular progression during peripuberty is affected by diet. Vascular endothelial growth factor A (VEGFA) induces follicle progression in many species; however, there are limited studies to determine if diet may alter the effects of angiogenic VEGFA165-stimulated follicle progression or antiangiogenic VEGFA165b follicle arrest. We hypothesized that diet affects the magnitude of angiogenic and antiangiogenic VEGFA isoform actions on follicular development through diverse signal transduction pathways. To test this hypothesis, beef heifers in our first trial received Stair-Step (restricted and refeeding) or control diets from 8 to 13 months of age. Ovaries were collected to determine follicle stages, measure vascular gene expression and conduct ovarian cortical cultures. Ovarian cortical cultures were treated with phosphate-buffered saline (control), 50 ng/ml VEGFA165, VEGFA165b, or VEGFA165 + VEGFA165b. The Stair-Step heifers had more primordial follicles (P < 0.0001), greater messenger RNA abundance of vascular markers VE-cadherin (P < 0.0001) and NRP-1 (P < 0.0051) than controls at 13 months of age prior to culture. After culture, VEGFA isoforms had similar effects, independent of diet, where VEGFA165 stimulated and VEGFA165b inhibited VEGFA165-stimulated follicle progression from early primary to antral follicle stages. In vitro cultures were treated with VEGFA isoforms and signal transduction array plates were evaluated. VEGFA165 stimulated expression of genes related to cell cycle, cell proliferation, and growth while VEGFA165b inhibited expression of those genes. Thus, VEGFA isoforms can act independently of diet to alter follicle progression or arrest. Furthermore, follicle progression can be stimulated by VEGFA165 and inhibited by VEGFA165b through diverse signal transduction pathways.

Determination of testicular estrogen receptor alpha expression of male chickens (Gallus domesticus) with age.

BACKGROUND AND AIM: Estrogen activity, a central component of reproductive growth, is regulated by the receptor proteins, estrogen receptor alpha (ERα), and ER beta (ERß) in chickens as in many other species. ERα expresses predominantly in gonads. Although the expression of ERα in embryonic gonads has been studied in detail, the expression of ERα in post-hatching male gonads has not been studied adequately. Therefore, the current research was conducted to determine the post-hatching changes in the expression of ERα in the left gonads of male chickens with age. MATERIALS AND METHODS: Shaver Brown male chickens were raised and cared for according to the management guide and sacrificed at the intervals of 1, 4, and 8 weeks of age. The total RNA was extracted from the left gonads using the Trizol method and reverse transcribed using a pair of gene-specific primers. Following polymerase chain reaction amplification, the expression of ERα was quantified relative to the expression of the reference gene GAPDH. RESULTS: The results showed that ERα expression significantly increases with age at p=0.0032. However, the increment of ERα expression from week 1 to week 4 was 2.04-fold and from week 4 to week 8 was 1.39-fold, with the later age reflecting a diminishing pattern in the increment. CONCLUSION: These results differentiate the post-hatching ERα expression of the left gonads of male chickens increase with age but with a diminishing gradient that may support their reproductive functions in later stages of life.

Testosterone induces activation of porcine primordial follicles in vitro.

Purpose: The mechanism underlying primordial follicle activation is poorly understood. In this study, in-vitro culture and subsequent xenotransplantation were conducted to determine whether testosterone promotes the activation of porcine primordial follicles. Methods: Prepubertal porcine ovarian cortical strips containing primordial follicles were cultured in the presence of testosterone for 7 days, and subsequently transplanted to immunodeficient mice for 2 months. After culture and transplantation, development of follicles was examined histologically. The presence of androgen receptors in oocytes was assessed by use of western blot and immunohistochemical analyses. Results: Testosterone at 10-6 m induced the primordial follicle transition to the intermediate (19 ± 4%) and primary (3 ± 1%) stages after 7-day culture, while 56 ± 5% of primordial follicles remained in the initial pool. Higher concentrations, above 10-5 m, or lower concentrations, below 10-6 m, did not induce follicle transition to the primary stage. After 7-day culture with 10-6 m testosterone, ovarian cortical strips were transplanted to immunodeficient mice. Some of the follicles developed to the secondary (15 ± 3%) and antral (10 ± 3%) stages, whereas 44 ± 7% of primordial follicles remained in the initial pool. In the culture experiment, estradiol-17ß (10-7-10-5 m) had no significant effect on follicle activation. The androgen receptor antagonist, cyproterone acetate, inhibited the stimulatory effect of testosterone on primordial follicle activation, suggesting an androgen receptor-mediated action of testosterone. Western blot and immunohistochemical analyses revealed that androgen receptors were present in the oocytes of primordial follicles. Conclusions: These results suggest that testosterone at 10-6 m promotes the activation of porcine primordial follicles in vitro through the androgen receptors in the oocytes.