MicroRNA expression profile in bovine cumulus–oocyte complexes: Possible role of let-7 and miR-106a in the development of bovine oocytes

The objectives of this study included: (1) identify the expression of miRNAs specific to bovine cumulus–oocyte complexes (COCs) during late oogenesis, (2) characterize the expression of candidate miRNAs as well as some miRNA processing genes, and (3) computationally identify and characterize the expression of target mRNAs for candidate miRNAs. Small RNAs in the 16–27 bp range were isolated from pooled COCs aspirated from 1- to 10-mm follicles of beef cattle ovaries and used to construct a cDNA library. A total 1798 putative miRNA sequences from the cDNA library of small RNA were compared to known miRNAs. Sixty-four miRNA clusters matched previously reported sequences in the miRBase database and 5 miRNA clusters had not been reported. TaqMan miRNA assays were used to confirm the expression of let-7b, let-7i, and miR-106a from independent collections of COCs. Real-time PCR assays were used to characterize expression of miRNA processing genes and target mRNAs (MYC and WEE1A) for the candidate miRNAs from independent collections of COCs. Expression data were analyzed using general linear model procedures for analysis of variance. The expression of let-7b and let-7i were not different between the cellular populations from various sized follicles. However, miR-106a expression was greater (P \u3c 0.01) in oocytes compared with COCs and granulosa cells. Furthermore, all the miRNA processing genes have greater expression (P \u3c 0.001) in oocytes compared with COCs and granulosa cells. The expression of potential target mRNAs for let-7 and let-7i (i.e., MYC), and miR-106a (i.e., WEE1A) were decreased (P \u3c 0.05) in oocytes compared with COCs and granulosa cells. These results demonstrate specific miRNAs within bovine COCs during late oogenesis and provide some evidence that miRNAs may play a role regulating maternal mRNAs in bovine oocytes

Localization of Period 1 mRNA in the ruminant oocyte and investigations of its role in ovarian function

The clock gene Period 1 (Per1) may be a prolificacy gene, because it localized to the mouse oocyte and Per1-null drosophila shed fewer eggs. Because Per1 mapped to a region of mouse chromosome 11 syntenic to bovine chromosome 19 where a quantitative trait loci (QTL) for ovulation rate existed, we hypothesized that Per1 influenced folliculogenesis and ovulation rate in ruminants. Ovarian cortex was collected at slaughter on days 5, 12, 15, 17, and 20 after estrus for real-time RT-PCR evaluation of Per1 mRNA expression in Dorset (n = 18), Romanov (n = 10), Romanov/Dorset (n = 21), and Composite (n= 22) ewes. Ovarian cortexwas also collected from cows selected for increased ovulation rate (n = 37) or unselected controls (n = 28) on days 4, 5, and 6 of the estrous cycle for in situ hybridization and real-time RT-PCR. To examine the role of Per1 in early follicular development, ovarian cortex from neonatal calves (n= 5) was cultured for 10 days and Per1 mRNA levels were measured on day 0 and on day 10 of culture. The primers generated a 483 bp amplicon with 100% sequence homology to bovine RIGUI-like protein (Per1). In silico mapping of this sequence placed Per1 on bovine chromosome 19; however, it was 20 cM from the QTL. Per1 mRNA expression was unaffected by prolificacy, day of the cycle, or pregnancy status in ewes or cows. The riboprobe hybridized to oocytes of bovine preantral and antral follicles. In bovine ovarian cortical cultures on day 0, the tissue contained mostly primordial follicles (5.6±0.6 follicles/section); however, after 10 days in culture, the number of primordial follicles per section decreased (0.5 follicles/section) and the number of primary follicles increased as follicles activated (day 0 = 0.5±0.6 versus day 10 = 10.4±0.6 primary follicles/section; P \u3c 0.001). Per1 mRNA did not change over time in culture.We conclude that Per1 mRNA is expressed by ruminant oocytes in preantral and antral follicles; however, its physiological role in mammalian ovarian function remains to be elucidated. Published by Elsevier B.V

TWINNING IN CATTLE: II. GENETIC AND ENVIRONMENTAL EFFECTS ON OVULATION RATE IN PUBERAL HEIFERS AND POSTPARTUM COWS AND THE EFFECTS OF OVULATION RATE ON EMBRYONIC SURVIVAL

The potential of ovulation rate before 18 to 21 mo of age in puberal heifers as an indirect selection criterion for twinning rate was considered. Heritability (h2) was .07 ± .03 for single observations and .34 ± .18 for the mean of 7.9 estrous cycles per heifer. Estimated repeatability (r) of ovulation rate was ≤h2, indicating negligible permanent environmental effects. Expected h2 for mean ovulation rate (assuming h2 = r = .07 for single observations) for increasing numbers of estrous cycles would be as follows: 4, .23; 6, .31; 8, .38; and 10, .43. About 50% of the heifers produced no multiple ovulations, but 27% produced multiple ovulations in more than 15% of their estrous cycles. Ovulation rate varied seasonally and increased about .01 per month of age (P \u3c .05). Genetic correlation of mean ovulation rate with adjusted 368-d weight was low (.08 ± .32). Ovulation rate in postpartum cows was higher (P \u3c .05) in fall than in spring (1.15 vs 1.08). In postpartum cows, estimated h2 = .24 ± .13 and r = .17. Mean ovulation rate for postpartum cows was 1.12 vs 1.09 in puberal heifers, accounting in part for the higher h2. Pregnancy rate was higher (P \u3c .O5) in multiple- than in single-ovulating cows. Effects of ovulation rate on embryonic survival were small (P \u3e .05). Unilateral and bilateral multiple ovulations were not diffemt in embryonic survival. Accuracy of ovulation rate determination by palpation per rectum was lower in multiple- than in single-ovulating postpartum cows, because some unilateral multiple corpora lutea, especially, were recorded as singles. Results suggest that use of ovulation rate in puberal heifers should permit effective indirect selection for twinning rate among yearling heifers based on individual performance and among young sires based on ovulation rate of sibs and daughters