Changes on corpus luteum structure and progesterone synthesis pathway after hCG or GnRH treatment during the early luteal phase in sheep.

This study investigated the effect of hCG or GnRH on structural changes of the corpora lutea (CL) and the regulation of the expression of steroidogenic enzymes involved in P4 secretion in post-ovulatory (po-CL) and accessory CL (acc-CL). Sixty-four ewes were assigned to three groups receiving: 300 IU of hCG (hCG) or 4 µg Buserelin (GnRH) or 1 mL of saline solution (Control) on Day (d) 4 post artificial insemination (FTAI). Laparoscopic ovarian were performed on d 4, 14 and, 21 post-FTAI to determine the numbers of CL. Blood samples were collected for serum LH and P4 analysis. On d 14 post-FTAI, both CL were removed from the ovary to determine large luteal cell (LLC) number and to evaluate the expression of steroidogenic enzymes (HSD3B1, STAR, CYP11A1). Only hCG and GnRH treated ewes generated acc-CL. The LLC in both po- and acc-CL were significantly greater in the hCG group compared to GnRH and Control groups (P<0.05). Overall, hCG group showed the greatest immunodetection of HSD3B1and STAR in both po- and acc-CL (P<0.05). rnRNA expression of HSD3B1, STAR and CYP11A1 in the acc-CL tended to be greater in hCG group than in GnRH group (P<0.1). The LH concentration was increased in GnRH group (P<0.05) and P4 concentration was greater in hCG group compared to the other groups (P<0.05). In conclusion, administration of hCG has a notably impact on acc-CL development and the expression of steroidogenic enzymes compared to GnRH treatment in ewes. This leads to elevated P4 concentration and improved luteal function.

Altered expression of angiogenic factors in dominant preovulatory follicles of dairy cattle treated with ACTH.

Studies in cows have reported that ovulation, steroidogenesis and angiogenesis are affected by stress and consequently fertility decreases. The purpose of this study was to evaluate the effects of ACTH administration during the preovulatory period on the expression of growth factors (CD-31, PDGF-A, PDGF-B, VEGFA-164, VEGFA-164b, VEGF-R1 and VEGF-R2) associated with the angiogenic process by immunohistochemistry in cows (n = 14). Results evidenced the expression of these growth factors in theca and granulosa cells from antral, atretic and dominant preovulatory follicles of ACTH-treated cows, suggesting that, under stress conditions, their expression continues to be required. VEGFA-164, VEGF-R1 and VEGF-R2 expression was greater in theca cells of dominant preovulatory follicles of the ACTH-treated group than in those of the control group. CD-31 protein expression was lower in the dominant preovulatory follicles of the ACTH-treated group than in those of the control group. PDGF-A and PDGF-B expression did not differ between groups, either in granulosa or in theca cells. These results suggest that VEGFA-164, its receptors and CD-31 are actors in the normal cycle of the ovaries and could have greater pathophysiological importance in the altered angiogenic process and other events that occur during anovulation and stress conditions. This dysregulation reinforces the importance of the angiogenic process in the pathophysiology of cystic ovarian disease in cows. This is the first report on the expression and localization of components of the VEGF and PDGF systems and CD-31 in cells from dominant preovulatory follicles after ACTH administration.

An altered expression of components of the IGF system could contribute to follicular persistence in Holstein cows.

In dairy cows, reproductive diseases such as cystic ovarian disease (COD) represent a major problem that impacts on dairy production. It has been postulated that the insulin-like growth factor (IGF) system may contribute to follicular persistence and development of COD. Thus, the aim of the present study was to analyze relevant members of the IGF system in a critical period immediately after the expected time of ovulation, to obtain information about their role in follicular persistence in dairy cows. Proteins of the IGF system were evaluated at 0 (expected day of ovulation), 5, 10 and 15 days of follicular persistence to determine whether the changes previously detected in cows with COD occur early in COD pathogenesis. The serum concentration of IGF1 was higher in cows with 10 and 15 days of follicular persistence than in control cows. IGF1 expression in granulosa cells was similar in the follicles analyzed. In contrast, in theca cells, persistent follicles of days 5 and 10 showed the lowest IGF1 expression. IGF binding protein (IGFBP) 2 and 3 expression was lower in persistent follicles than in dominant follicles of the control group. Although IGF receptor (IGFR) 1 expression was similar in the groups analyzed, p-IGFR1 expression was significantly higher in dominant follicles of the control group than in persistent follicles. These data suggest alterations in the IGF system at the early stages of follicular persistence. The evidences obtained allow supporting that the IGF system could plays a key role in dairy cattle reproduction.

Alterations in the insulin signaling pathway in bovine ovaries with experimentally induced follicular persistence.

Reproductive diseases in dairy cows as cystic ovarian disease (COD) represent a major problem that impacts on dairy production. COD is characterized by anovulation, persistence of the dominant follicle, and interruption of normal estrous cycles. Anovulation is attributable to a failure in the LH surge, due to endocrine imbalances and alterations in local factors, such as the insulin signaling pathway. Thus, the aim of this study was to examine the expression of critical nodes of the insulin pathway, including insulin receptor (IR), IR substrate (IRS), phosphatidylinositol-3-kinase (PI3K), and protein kinase B (also known as Akt or pan-Akt), in ovarian follicular structures of cows during the development of follicular persistence induced by long-term progesterone administration. Immunoexpression of IR, p-IR, IRS1, p-IRS1, PI3K, pan-Akt and p-pan-Akt was evaluated in situ by immunohistochemistry and the concentration of insulin in serum and follicular fluid was determined by radioimmunoassay. p-IR, p-IRS1, PI3K and p-pan-Akt expression was decreased in follicles at different times of persistence in relation to the control dominant follicles, in both granulosa and theca cells, whereas IR and IRS1 immunoexpression was decreased in persistent follicles at 5 and 15 days of persistence in granulosa cells. Serum and follicular fluid insulin concentration was higher in cows with persistent follicles than in control cows. These results show that decreased expression and/or activation of the receptors and other intermediates of the insulin signaling pathway in persistent follicles indicates that reduced response/resistance to insulin rather than the concentration of insulin per se may be one of the important molecular mechanisms in the development of persistent follicles in dairy cows.

Developmental Programming: Does Prenatal Steroid Excess Disrupt the Ovarian VEGF System in Sheep?

Prenatal testosterone (T), but not dihydrotestosterone (DHT), excess disrupts ovarian cyclicity and increases follicular recruitment and persistence. We hypothesized that the disruption in the vascular endothelial growth factor (VEGF) system contributes to the enhancement of follicular recruitment and persistence in prenatal T-treated sheep. The impact of T/DHT treatments from Days 30 to 90 of gestation on VEGFA, VEGFB, and their receptor (VEGFR-1 [FLT1], VEGFR-2 [KDR], and VEGFR-3 [FLT4]) protein expression was examined by immunohistochemistry on Fetal Days 90 and 140, 22 wk, 10 mo (postpubertal), and 21 mo (adult) of age. Arterial morphometry was performed in Fetal Day 140 and postpubertal ovaries. VEGFA and VEGFB expression were found in granulosa cells at all stages of follicular development with increased expression in antral follicles. VEGFA was present in theca interna, while VEGFB was present in theca interna/externa and stromal cells. All three receptors were expressed in the granulosa, theca, and stromal cells during all stages of follicular development. VEGFR-3 increased with follicular differentiation with the highest level seen in the granulosa cells of antral follicles. None of the members of the VEGF family or their receptor expression were altered by age or prenatal T/DHT treatments. At Fetal Day 140, area, wall thickness, and wall area of arteries from the ovarian hilum were larger in prenatal T- and DHT-treated females, suggestive of early androgenic programming of arterial differentiation. This may facilitate increased delivery of endocrine factors and thus indirectly contribute to the development of the multifollicular phenotype.