The Ovulatory Signal Precipitates LRH-1 Transcriptional Switching Mediated by Differential Chromatin Accessibility.

In the ovary, follicular growth and maturation are complicated processes that involve a series of morphological and physiological changes in oocytes and somatic cells leading to ovulation and luteinization, essential processes for fertility. Given the complexity of ovulation, characterization of genome-wide regulatory elements is essential to understand the mechanisms governing the expression of specific genes in the rapidly differentiating follicle. We therefore employed a systems biology approach to determine global transcriptional mechanisms during the early stages of the ovulatory process. We demonstrate that, following the hormonal signal that initiates ovulation, granulosa cells undergo major modification of distal regulatory elements, which coincides with cistrome reprogramming of the indispensable orphan nuclear receptor liver receptor homolog-1 (LRH-1). This cistromic reorganization correlates with the extensive changes in gene expression in granulosa cells leading to ovulation. Together, our study yields a highly detailed transcriptional map delineating ovarian cell differentiation during the initiation of ovulation.

Granulosa cells of prepubertal cattle respond to gonadotropin signaling and upregulate genes that promote follicular growth and prevent cell apoptosis.

Oocytes collected from prepubertal animals are known to be less developmentally competent than those from adult animals. There is evidence suggesting that acquisition of developmental competence in bovine oocytes may be linked to the expression profile of genes in the granulosa cells (GCs). Cumulus-oocyte complexes (COC) and GCs were collected from 12 Holstein heifers between 2 and 6 months of age (nine follicle-stimulating hormone [FSH] treated and three untreated) and eight FSH-treated cows. The COCs from prepubertal animals were matured, fertilized, and cultured in vitro to assess development to the blastocyst stage. The relative messenger RNA (mRNA) abundance of FSHR, StAR, CYP19A1, HSD3B1, CX43, FOXO1, and XIAP in GCs were quantified by real-time quantitative polymerase chain reaction. Results from this study revealed that GCs of prepubertal animals respond to FSH treatment by increasing mRNA levels of genes promoting estradiol synthesis and follicular growth ( FSHR and CYP19A1), and preventing cell apoptosis ( XIAP), and by decreasing mRNA levels of genes promoting progesterone production ( StAR and HSD3B1). This study also revealed that the relative mRNA abundance of FOXO1 in GCs is associated with oocyte competence to support embryo development to the blastocyst stage in prepubertal Holstein heifers.

Interval of gonadotropin administration for in vitro embryo production from oocytes collected from Holstein calves between 2 and 6 months of age by repeated laparoscopy.

Laparoscopic Ovum Pick-Up (LOPU) in calves followed by in vitro embryo production (IVEP) and transfer (ET) into adult recipients has great potential for accelerated genetic gain through shortening of the generation interval. In this study, 11 Holstein calves were subjected to up to six LOPU procedures between the ages of 2-6 months at 2-3 weeks interval. In all cases, the animals received a CIDR 5 days prior to LOPU and were gonadotropin-stimulated starting at 72 h before LOPU using one of three protocols that were rotated twice among the animals during the study. Calves were injected with FSH every 12 h (FSH12h), or every 8 h (FSH8h) or every 8 h until -36 h from LOPU at which point the FSH was replaced with a single dose of 400 IU eCG (FSH8h-eCG). No statistical differences were observed among the 3 treatments in terms of mean follicles available for aspiration (35.7 ±â€¯16 vs. 38.5 ±â€¯25 vs. 31.1 ±â€¯22), mean oocytes recovered (26.5 ±â€¯14 vs. 21.6 ±â€¯10 vs. 19.4 ±â€¯14) and cleavage rate (66.0 ±â€¯14 vs. 61.1 ±â€¯11 vs. 72.2 ±â€¯8), for FSH12h, FSH8h and FSH8h-eCG, respectively. However, FSH8h-eCG resulted in a significantly higher rate of transferable embryos (17.5 ±â€¯8%) compared with FSH12h (8.9 ±â€¯5%, P < 0.05). Oocytes from follicles of ≥5 mm in diameter yielded a higher rate (P < 0.05) of development to the blastocyst stage (13.8%) than those collected from <5 mm follicles (6.8%). Animal age, by comparing animals at <100, 101 to 130 and > 130 days of age, did not affect the mean number of follicles (34.2 ±â€¯15 vs. 39.3 ±â€¯26 vs. 31.6 ±â€¯25), the mean number of oocytes recovered (21.2 ±â€¯10 vs. 24.5 ±â€¯15 vs. 22.6 ±â€¯17), and the cleavage rate (68.6 ±â€¯11 vs. 61.7 ±â€¯12 vs. 70.7 ±â€¯10%), respectively. However, animals in the older age range had significantly higher development to the blastocyst stage (19.9 ±â€¯6 vs. 9.5 ±â€¯8%, P < 0.01) and better embryo quality, as evidenced by higher average cell numbers (119.1 ±â€¯47 vs. 91.5 ±â€¯25, P < 0.05) compared with those in the lower age. Finally, we tested the benefits of relieving endoplasmic reticulum stress by supplementing the culture medium with 50 µM tauroursodeoxycholic acid (TUDCA) and found a numerically higher rate of development to the blastocyst stage (21.1 ±â€¯8 vs. 18.6 ±â€¯4%), but not statistically different, compared with control culture. Overall, our findings indicate that a significant number of transferable embryos (range 10-30) can be produced from Holstein calves before they reach 6 months of age.

The effect of age and length of gonadotropin stimulation on the in vitro embryo development of Holstein calf oocytes.

The use of oocytes recovered from prepubertal donors for in vitro embryo production has great potential for accelerating the rate of genetic gain in the dairy industry. However, these oocytes are known to be less developmentally competent than those from adult donors. In this study, we investigated the effect of age and gonadotropin stimulation in Holstein heifers subjected to oocyte collection every two weeks between 2 and 6 months of age. In order to assess the effect of gonadotropin stimulation, animals were subjected to one of three treatments, namely Short (ST; 36-42 h), Long (LT; ≥72 h) and No Treatment (NT) prior to laparoscopic ovum pick-up (LOPU). Our results show that the LT significantly improved the proportion of large follicles (>5 mm diameter) present in the ovary (LT 34.0% vs. ST 11.2% vs. NT 2.4%, P < 0.05), as well as the percentage of good-quality cumulus oocyte complexes (COCs) recovered (LT 95.3 ± 18% vs. ST 85.4 ± 22% vs. NT 82.2 ± 14%, P < 0.05) and blastocyst rate (LT 36.7 ± 26% vs. ST 18.3 ± 15% vs. NT 16.7 ± 9%, P < 0.05). Recovery rate was affected by treatment (LT 70.4 ± 25 vs. ST 85.4 ± 29 vs. NT 72.7 ± 23, P < 0.05). To assess the impact of age, data was grouped into <100 days (A), 100-130 days (B) and >130 days (C) of age at LOPU. We found that as animals got older, although the average number of COCs per donor per LOPU declined (A: 17.5 ± 11 vs. B: 14.7 ± 7 vs. C: 11.9 ± 8), the blastocyst rate increased (A: 12.8 ± 20% vs. B: 17.1 ± 21% vs. C: 21.8 ± 25%, P < 0.05). We also evaluated the incidence of polyspermy and confirmed it is a critical limitation for IVF in calf oocytes. The incidence of polyspermy was unaffected by gonadotropin treatment, but significantly decreased with age. The capacity for full development to term of in vitro produced embryos from calf oocytes was tested by embryo transfer into 21 synchronized adult recipients, which resulted in 13 pregnancies (62%), full development to term and healthy calves born. Finally, the study allowed evaluating the safety of the procedure since, on average, each animal was subjected to 8 LOPU procedures over a period of 4 months. Our results showed that the procedure is safe (no incidents during laparoscopy), and was not harmful for the reproductive future of the animals, as those that were bred became pregnant after reaching sexual maturity.

Comparative analysis of granulosa cell gene expression in association with oocyte competence in FSH-stimulated Holstein cows.

Ovarian stimulation with exogenous FSH followed by FSH withdrawal or 'coasting' is an effective means of increasing the number of oocytes obtainable for the in vitro production of cattle embryos. However, the quality of the oocytes thus obtained varies considerably from one cow to the next. The aim of the present study was to gain a better understanding of the follicular conditions associated with low oocyte developmental competence. Granulosa cells from 94 Holstein cows in a commercial embryo production facility were collected following ovarian stimulation and coasting. Microarray analysis showed 120 genes expressed with a differential of at least 1.5 when comparing donors of mostly competent with donors of mostly incompetent oocytes. Using ingenuity pathway analysis, we revealed the main biological functions and potential upstream regulators that distinguish donors of mostly incompetent oocytes. These are involved in cell proliferation, apoptosis, lipid metabolism, retinol availability and insulin signalling. In summary, we demonstrated that differences in follicle maturity at collection could explain differences in oocyte competence associated with individual animals. We also revealed deficiencies in lipid metabolism and retinol signalling in granulosa cells from donors of mostly incompetent oocytes.

Effect of cow age on the in vitro developmental competence of oocytes obtained after FSH stimulation and coasting treatments.

The use of oocytes obtained from younger donors for IVF followed by embryo transfer represents an opportunity to accelerate genetic gain by reducing generation time. In this study, we investigated the relationship between donor age and the in vitro developmental competence of oocytes obtained from Holstein females (aged 5-18 months) after FSH stimulation and coasting. The follicle size patterns showed a significantly higher total number of small follicles (5-6 mm) from donors aged 5 to 10 months and a higher total number of medium-sized follicles (7-10 mm) in donors aged 6 to 7 months. Our analysis also revealed that the total number of follicles was significantly higher (P < 0.05) in donors aged 5 to 8 months and tended to be higher (P = 0.053) in nine-month-old donors. However, oocytes obtained from donors aged 5 to 10 months yielded fewer embryos reaching the morula and blastocyst stages. In summary, our results demonstrate that a higher number of oocytes can be obtained from younger animals but lower developmental competence negates this gain.