The Journal of Reproduction and Development Supplement

Improving pregnancy rates associated with the use of cryopreserved human oocytes would be an important advance in human assisted reproductive technology. The use of oocyte in vitro maturation (IVM) should reduce the need for gonadotrophin-induced ovarian hyperstimulation and side effects. Nowadays the proposed system for human IVM is not consistent in results and produces low developmental competence oocytes. For the present study, bovine oocytes were aspirated from follicles of 3 to 8 mm in diameter from ovaries obtained from a slaughterhouse. Bovine cumulus-oocyte complexes (COCs) were matured in TCM199 + 10% FBS as control. The treatment group was supplemented with cAMP modulators (FSK) and phosphodiesterase inhibitor (hypoxantine) during IVM, increasing COC’s cAMP levels. The aim was to increase cAMP which supports the citoplasmatic maturation to improve the developmental competence of the oocytes. These effects on COC had consequences for oocyte developmental potential increasing the quality of the embryos and blastocysts rate (25% to 43%) compared to conventional IVM and IVF oocytes. This approach of IVM mimics some characteristics of oocyte maturation in vivo and substantially improves oocyte developmental outcomes. Adaption of this system developed in bovine for clinical application should have significant implications for infertility management and bring important benefits to patients.

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Anterior pituitary (AP) is the complex organ collecting signals from both hypothalamus and various periphery tissues, and secreting various hormones to control critical events, including ovulation. To gain a better understanding of gene expressions in AP of Japanese Black (JB) before and after ovulation, this study has utilized deep sequencing of transcriptomes by next generation sequencing. The JB heifers were slaughtered on the day of estrus or three days after ovulation. APs were collected, frozen in liquid nitrogen. For the analysis of gene expression, total RNA was extracted, and RNA quality and quantity were verified by Bioanalyzer. Libraries were prepared from the samples to sequence. High-quality sequences were retained and aligned to the bovine genome sequence. Genes that differentially expressed were interpreted in the context of biological processes and functions and by their networks pathways. About 13,000 genes were identified in the bovine AP based on a definition that the reads per kilobase per million mapped reads (RPKM) was greater than 1 in at least one stage. The total RPKM value of all transcriptomes in AP was about 700,000, and about half were for the AP hormones. Especially, RPKM value of prolactin was highest among all genes. The RPKM value of TSHB decreased after ovulation, whereas the RPKM value of FSHB, GH1, LHB, and CGA increased after ovulation. AP expressed about 260 receptor genes, including ESR1 which was increased after ovulation. This study detected only small difference in expressions of hypothalamic hormone receptors between pre- and post-ovulation. Therefore, this study discovered dynamic changes of gene expressions from pre-ovulation to post-ovulation.

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Picomolar concentrations of estradiol produce the rapid suppression of GnRH-induced LH secretion from the bovine anterior pituitary (AP) via GPR30 as non-genomic manner. However, no research has investigated the cytoplasmic mechanism for rapid estradiol suppression of LH secretion from AP cells. ERK1/2/5 pathway and PKA pathway were reported as important pathways for genomic effect of estradiol in lactotroph. However, it is not clear whether PKA pathway and ERK1/2/5 pathway are the pathways in the downstream of GPR30 to suppress LH secretion in non-genomic mechanism. Therefore, this study was conducted to test the hypothesis that ERK1/2/5 or PKA pathways were the important pathway for estradiol mediated by GPR30 to suppress GnRH-induced LH release from bovine AP through rapid, non-genomic manner. The bovine AP cells (n = 8) were cultured for 3 days in steroid-free conditions. The AP cells were treated with either 1 µM of ERK1/2/5 inhibitor (U1026) or 5 µM of PKA inhibitor (H89) or both inhibitors for 30 min, then, also with 0.01 nM estradiol or GPR30-specific agonist, G1, for 5 min before GnRH stimulation. Pre-treatment with 0.01 nM estradiol or G1 in the absence of inhibitors treatment suppressed GnRH-stimulated LH secretion significantly (P<0.01). In contrast, pre-treatment with 0.01 nM estradiol in the presence of U0126 alone, H89 alone, or both inhibitors had no suppressive effect on GnRH-stimulated LH secretion. Therefore, both ERK1/2/5 pathway and PKA pathway are important for the rapid non-genomic suppression of estradiol on the GnRH-induced LH secretion from bovine AP cells.

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The study was undertaken in pubertal beef bulls to: (1) determine the temporal relationship of pulsatile secretion among LH, insulin-like peptide 3 (INSL3) and testosterone (T); and (2) monitor acute regulation of INSL3 secretion by LH using GnRH and hCG. Blood samples were taken at 15-min interval for 8 h from the intact Japanese Black bulls (n=6). The bulls were also treated with GnRH analogue and hCG and blood samples were taken after the treatments for 6 h and 12 days, respectively. Plasma concentrations of LH, INSL3 and T were measured by EIAs. INSL3, LH and T secretion in plasma were pulsatile and the frequency of LH, INSL3 and T pulses per 8 h was 4.7, 3.8 and 1.0, respectively. The increasing rate (peak/basal) of INSL3 pulses was lower (P<0.001) than that of T pulses. After GnRH treatment, LH concentrations increased at 1 h and remained high until 5 h (P<0.01), and a higher INSL3 concentration was observed at 1 h, 2 h and 6 h post-treatment (P<0.05). T concentrations increased at 1 h after GnRH treatment and remained high during 6 h (P<0.01). After hCG treatment, an increase of INSL3 concentration was observed at 2 h, 4 h, day 2, day 4 and day 8 of treatment (P<0.05), and T concentrations remained high till 8 day post-treatment (P<0.01). The increasing rate (maximum/pre-treatment) of INSL3 was much lower (P<0.001) than that of T after GnRH and hCG treatments. Results demonstrate that INSL3 secretion in blood is pulsatile in bulls. Endogenous and exogenous LH can stimulate INSL3 secretion soon after the treatment, suggesting the acute regulation of INSL3 by LH. Moreover, INSL3 can be used as a less-fluctuating marker than T to evaluate functions of testicular Leydig cells in bulls.

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