S. E. Echternkamp

The ovarian insulin and insulin-like growth factor system with an emphasis on domestic animals☆

Insulin and insulin-like growth factors (IGFs) have direct effects on cultured ovarian cells. These effects include stimulation of granulosa cell mitogenesis, granulosa and luteal cell progesterone production, and thecal cell androgen production and appear similar among species. However, species differences exist with regard to insulin and IGF-I effects on granulosa cell estradiol production. In addition to endocrine effects of insulin and IGFs, IGFs are produced by granulosa, thecal, and luteal cells, allowing for an intraovarian autocrine and paracrine system. Granulosa, thecal, and luteal cells contain receptors for insulin and IGFs, and these receptors appear to mediate the effects of insulin and IGFs. Adding to the complexity of the regulatory role of IGFs is the presence of IGF-binding proteins (IGFBPs) within the ovary. These IGFBPs are produced by granulosa, thecal, and luteal cells, and their production is hormonally regulated. Evidence for a coherent mechanism by which insulin, IGFs, and IGFBPs interact and regulate ovarian function in vivo has yet to be found.

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<0.01) in oocytes compared with COCs and granulosa cells. Furthermore, all the miRNA processing genes have greater expression (P<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<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.

Initial results of genomic scans for ovulation rate in a cattle population selected for increased twinning rate.

Genomic scans were conducted with 273 markers on 181 sires from a cattle population selected for increased twinning rate to identify chromosomal regions containing genes that influence ovulation rate. Criteria used for selecting markers were number of alleles, ease of scoring, and relative position within linkage group. Markers were multiplexed or multiple-loaded on the gels to reduce the costs and labor required to obtain genotypic data. This approach reduced the number of gels by 45% when compared with running each marker independently. Male animals selected for the genomic scan sired the majority of the population. A modified interval analysis was used in a granddaughter design to compare effects of each allele within sire for 10 different sire families. The midparent deviation of the sons estimated breeding value for ovulation rate was used as the phenotype. Forty-one potential peaks were identified with a nominal significance level < or = 0.05. The 10 peaks with the highest significance levels (P < 0.02) were selected for further analysis. Markers were genotyped across daughters of the sire where nominal significance was found for each of the 10 peaks. One peak (BTA5, relative position 40 cM) was found to be nominally significant in the daughters. The nominal significance levels were P = 0.01 for the sons (n = 32) and P = 0.02 for the daughters (n = 94) of sire 784403. A combined genomewide significance value (P = 0.07) was calculated that accounted for the 10 analyses with sons and the 10 analyses with daughters. These results strongly suggest that this region contains a gene(s) that is involved in the follicular recruitment and development process.

Size of ovulatory follicles in cattle expressing multiple ovulations naturally and its influence on corpus luteum development and fertility.

Long-term genetic selection of cattle for fraternal twins has increased the frequency of twin and triplet ovulations. In contrast, the ratio of fetal numbers to ovulation sites in pregnant females with twin (0.83) or triplet (0.73) ovulations is <1.0 and the number of calves per parturition is 1.6 and 2.0, respectively. Failure of individual twin or triplet ovulations to yield a conceptus in fertile females indicates a significant contribution of ovulation or oocyte anomalies to increased fertilization failure or early embryonic mortality. The present objective was to identify physiological traits affecting conception in cyclic cattle expressing multiple ovulations naturally, including the effect of ovulation rate on follicle or corpus luteum (CL) size, and their relationship to conception. Diameter of the individual ovulatory follicles was measured by transrectal ultrasonography at AI and ranged from 8 to 30 mm, with a trend for diameter of the individual follicles, and associated CL, to decrease with increasing ovulation rate. Independent of ovulation rate, ovulatory follicles were smaller (P < 0.05) for nulliparous heifers (1.5 yr) compared with parous cows (> or =2.5 yr). Pregnancy and fetal status were diagnosed by transrectal ultrasonography between 42 and 72 d after AI. Fertility was reduced (P < 0.01) for small twin or triplet ovulatory follicles (8 to 8.9 mm vs. 10 to 17.9 mm diam.), whereas fertility in monovular females was reduced (P < 0.01) for large ovulatory follicles (> or =22 vs. 14 to 17.9 mm). Plasma progesterone concentrations increased with ovulation rate and were correlated positively with total CL or ovulatory follicle volume per female, indicating that CL size and function were influenced by the size of the follicle of origin. Progesterone was greater (P < 0.05) in the blood of nulliparous heifers compared with parous cows. The increased proportion of small ovulatory follicles associated with twin and triplet ovulations indicates that some ovulatory follicles were either selected to ovulate at a lesser stage of maturity or rescued while undergoing atresia, thus compromising oocyte competency or ovulation. Of greatest importance for reduced fertility was the greater incidence of pregnancy losses occurring in the middle of gestation in females gestating 2 or more fetuses as an apparent effect of uterine crowding, especially when 2 or more fetuses were contained within 1 uterine horn.

Confirmation of quantitative trait loci using a low-density single nucleotide polymorphism map for twinning and ovulation rate on bovine chromosome 5.

Traditional genetic selection in cattle for traits with low heritability, such as reproduction, has had very little success. With the addition of DNA technologies to the genetic selection toolbox for livestock, the opportunity may exist to improve reproductive efficiency more rapidly in cattle. The US Meat Animal Research Center Production Efficiency Population has 9,186 twinning and 29,571 ovulation rate records for multiple generations of animals, but a significant number of these animals do not have tissue samples available for DNA genotyping. The objectives of this study were to confirm QTL for twinning and ovulation rate previously found on BTA5 and to evaluate the ability of GenoProb to predict genotypic information in a pedigree containing 16,035 animals when using genotypes for 24 SNP from 3 data sets containing 48, 724, or 2,900 animals. Marker data for 21 microsatellites on BTA5 with 297 to 3,395 animals per marker were used in conjunction with each data set of genotyped animals. Genotypic probabilities for females were used to calculate independent variables for regressions of additive, dominance, and imprinting effects. Genotypic regressions were fitted as fixed effects in a 2-trait mixed model analysis by using multiple-trait derivative-free REML. Each SNP was analyzed individually, followed by backward selection fitting all individually significant SNP simultaneously and then removing the least significant SNP until only significant SNP were left. Five significant SNP associations were detected for twinning rate and 3 were detected for ovulation rate. Two of these SNP, 1 for each trait, were significant for imprinting. Additional modeling of paternal and maternal allelic effects confirmed the initial results of imprinting done by contrasting heterozygotes. These results are supported by comparative mapping of mouse and human imprinted genes to this region of bovine chromosome 5.

Effect of postweaning diet on ovarian development and fertility in replacement beef heifers.

Programs for developing replacement heifers are designed for heifers to calve at 2 yr of age and to extend their stayability in the herd and minimize feed cost. The experimental objective was to determine whether developing prepubertal heifers on less dietary energy and to a BW of 55% rather than 65% of mature BW at 14 mo of age would compromise ovarian development and reduce fertility. In a 3-yr study, 8-mo-old Angus (n = 60/yr) and composite MARC II (n = 60/yr) heifers were assigned equally by age, BW, and breed to receive either a low (LG) or high (HG) BW gain diet fed to achieve an ADG of either 0.45 or 0.8 kg/d from 8 to 15 mo of age, including the first 21 d of breeding, and then transferred to pasture. At 14 mo, heifers were housed with fertile bulls for 47 d. Estrus was monitored for 21 d. Within 12 h after detection of estrus, ovarian length and height, preovulatory follicle diam., and antral follicle count (AFC) were measured by transrectal ultrasonography. Corpus luteum (CL) volume and plasma progesterone concentration were measured 5 to 15 d after estrus. Data were analyzed by ANOVA with treatment, breed, and year and their 2-way interactions as independent variables. At breeding, HG heifers were heavier than LG heifers (419.9 vs. 361.8 ± 7.5 kg; P < 0.01); ADG for the treatment period was 0.79 vs. 0.47 ± 0.04 kg/d (P < 0.01), respectively. In 2010 and 2011, 97.2% of heifers were cyclic by 21 d of breeding. Size of the ovary, preovulatory follicle, CL, and AFC did not differ between HG and LG, but preovulatory follicle diam. and ovarian length were greater (P ≤ 0.05) for MARC II vs. Angus heifers. Progesterone concentrations were less for LG vs. HG heifers (P ≤ 0.02), whereas CL volume was not affected by treatment or breed but was correlated positively with preovulatory follicle size (P < 0.01). Total AFC ranged from 5 to 49 and was correlated positively with ovarian volume but was not associated with fertility. A greater proportion of HG vs. LG heifers conceived within the first 21 d of the breeding period (64.4% vs. 49.2% ± 3.8%, respectively; P < 0.01), but overall pregnancy rate was not affected by treatment (83.0% vs. 77.7% ± 3.1%, respectively; P > 0.10). Pregnancy rate was 10% less (P < 0.01) for Angus vs. MARC II heifers. Developing beef heifers at a lesser ADG to a lighter BW (55% vs. 64% of mature BW) at breeding did not influence postweaning ovarian development or AFC or compromise pregnancy rate during the 47-d breeding period.

Identification of an ionotropic glutamate receptor AMPA1/GRIA1 polymorphism in crossbred beef cows differing in fertility

A proposed functional polymorphism in the ionotropic glutamate receptor AMPA1 (GRIA1) has been reported to influence antral follicle numbers and fertility in cows. Repeat breeder cows that fail to produce a calf in multiple seasons have been reported to have reduced numbers of small (1 to 3 mm) antral follicles in their ovaries. Therefore, we tested the hypothesis that this GRIA1 polymorphism was affecting antral follicle numbers in repeat breeder cows. Repeat breeder cows (n = 64) and control cows (n = 72) that had always produced a calf were housed in a dry lot and observed twice daily for behavioral estrus. Blood samples were collected, and cows were genotyped for this GRIA1 polymorphism and for a polymorphism in the GnRH receptor (GnRHR) that was proposed to influence age at puberty. On d 3 to 8 after estrus cows were slaughtered, and reproductive organs were collected to determine antral follicle count, ovary size, and uterine horn diameter. Repeat breeder cows were older at first calving than control cows (P = 0.006). The length (P = 0.03) and height (P = 0.02) of the ovary contralateral to the corpus luteum (CL) were greater in control cows than repeat breeder cows. The endometrial diameter in the horn ipsilateral to the CL was greater in the control cows than the repeat breeder cows. Repeat breeder cows had fewer small (1 to 5 mm) antral follicles than control cows (P = 0.003); however, there was no association between GRIA1 genotype and antral follicle number. The GnRHR polymorphism was associated with age at first calving because cows that were homozygous for the C allele had a greater age at first calving than heterozygous cows or cows that were homozygous for the T allele (P = 0.01). In the granulosa cells from small (1 to 5 mm) antral follicles, mRNA abundances of 2 markers of oocyte quality, anti-Müllerian hormone and pentraxin 3, did not differ between fertility groups (P ≥ 0.12). We conclude that this GRIA1 polymorphism exists in beef cows but that it does not influence antral follicle numbers. The association between GnRHR genotype and age at first calving is likely not causal as this polymorphism is not functional. The utility of this polymorphism as a genetic marker for early conception in heifers will require further validation. Screening postpartum cows by ultrasonography to determine antral follicle numbers may aid in making culling decisions.

The Hedgehog System in Ovarian Follicles of Cattle Selected for Twin Ovulations and Births: Evidence of a Link Between the IGF and Hedgehog Systems

ABSTRACT Hedgehog signaling is involved in regulation of ovarian function in Drosophila, but its role in regulating mammalian ovarian folliculogenesis is less clear. Therefore, gene expression of Indian hedgehog (IHH) and its type 1 receptor, patched 1 (PTCH1), were quantified in bovine granulosa (GC) or theca (TC) cells of small (1–5 mm) antral follicles by in situ hybridization and of larger (5–17 mm) antral follicles by real-time RT-PCR from ovaries of cyclic cows genetically selected (Twinner) or not selected (control) for twin ovulations. Expression of IHH mRNA was localized to GC and cumulus cells, whereas PTCH1 mRNA was greater in TC than in GC. Estrogen-active (E-A; follicular fluid concentration of estradiol > progesterone) versus estrogen-inactive follicles had a greater abundance of mRNA for IHH in GC and PTCH1 in TC. Abundance of IHH mRNA in GC was not affected by cow genotype, whereas TC PTCH1 mRNA was less in large E-A follicles of Twinners than in controls. In vitro, estradiol and wingless-type (WNT) 3A increased IHH mRNA in IGF1-treated GC. IGF1 and BMP4 treatments decreased PTCH1 mRNA in small TC. Estradiol and LH increased PTCH1 mRNA in IGF1-treated TC from large and small follicles, respectively. In summary, functional status of ovarian follicles was associated with differences in hedgehog signaling in GC and TC. We hypothesize that as follicles grow and develop, increased free IGF1 may suppress expression of IHH mRNA by GC and PTCH1 mRNA by TC, and these effects are regulated in a paracrine way by estradiol and other intra- and extragonadal factors.

Increased abundance of aromatase and follicle stimulating hormone receptor mRNA and decreased insulin-like growth factor-2 receptor mRNA in small ovarian follicles of cattle selected for twin births.

Cattle genetically selected for twin ovulations and births (Twinner) exhibit increased ovarian follicular development, increased ovulation rate, and greater blood and follicular fluid IGF-1 concentrations compared with contemporary cattle not selected for twins (Control). Experimental objectives were to 1) assess relationships among aromatase (CYP19A1), IGF-1 (IGF1), IGF-2 receptor (IGF2R), and FSH receptor (FSHR) mRNA expression in small (≤5 mm) antral follicles and 2) determine their association with increased numbers of developing follicles in ovaries of Twinner females. Ovaries were collected from mature, cyclic (d 3 to 6) Twinner (n = 11), and Control (n = 12) cows at slaughter and pieces of cortical tissue were fixed and embedded in paraffin. Expression of mRNA was evaluated by in situ hybridization using (35)S-UTP-labeled antisense and sense probes for CYP19A1, FSHR, IGF1, and IGF2R mRNA. Silver grain density was quantified within the granulosa and theca cells of individual follicles (2 to 7 follicles/cow) by Bioquant image analysis. Follicles of Twinners tended to be smaller in diameter than Controls (1.9 ± 0.1 vs. 2.3 ± 0.1 mm; P = 0.08), but thickness of granulosa layer did not differ (P > 0.1) by genotype. Relative abundance of CYP19A1 (P < 0.01) and FSHR (P < 0.05) mRNA was greater in granulosa cells of Twinners vs. Controls, respectively, whereas IGF2R mRNA expression was less in both granulosa (P < 0.01) and theca (P < 0.05) cells in follicles of Twinners vs. Controls, respectively. Abundance of CYP19A1 mRNA in granulosa cells was correlated negatively with IGF2R mRNA expression in both granulosa (r = -0.33; P < 0.01) and theca (r = -0.21; P = 0.05) cells. Expression of IGF1 mRNA was primarily in granulosa cells, including cumulus cells, and its expression did not differ between Twinners vs. Controls (P > 0.10). Detected increases in CYP19A1 and FSHR, but not IGF1, mRNA expression along with decreases in IGF2R mRNA expression in individual follicles of Twinners support the hypothesis that increased follicular development and steroidogenesis in Twinner females result from increased extra-ovarian IGF-1 production. Furthermore, a reduction in follicular IGF2R mRNA expression accompanied by a reduction in receptor numbers would increase availability of free IGF-2 and its stimulation of follicular development in Twinners.

Possible role of IGF2 receptors in regulating selection of 2 dominant follicles in cattle selected for twin ovulations and births

Abundance of IGF-2 receptor (IGF2R), FSH receptor (FSHR), and LH receptor (LHCGR) mRNA in granulosa cells (GCs) or theca cells (TCs) or both cells as well as estradiol (E2), progesterone (P4), and androstenedione concentrations in follicular fluid were compared in cows genetically selected (Twinner) or not selected (control) for multiple ovulations and twin births. Cows were slaughtered at day 3 to 4 (day 3) and day 5 to 6 (day 5) of an estrous cycle, and ovaries, follicular fluid, GCs, and TCs were collected. The two largest (F1 and F2) E2-active (EA) and E2-inactive (EI) follicles were selected according to their E2-to-P4 ratio and diameter. Androstenedione levels in EA F1 and F2 follicles were 5-fold greater (P < 0.05) in Twinner cows than in control cows on day 3 but did not differ on day 5. Twinner cows also had greater (P < 0.05) E2 and P4 concentrations, whereas steroid levels in EI follicles did not differ (P > 0.10) between genotypes. In EA F2 follicles, IGF2R levels in GCs were greater (P < 0.05) in control cows than in Twinner cows on day 3 and day 5, whereas IGF2R mRNA in TCs did not differ (P > 0.10). On day 3, FSHR mRNA levels were greater (P < 0.05) in GCs of EA F1 and EI F2 follicles of control cows than of Twinner cows. LH receptor mRNA expression was less in GCs and greater in TCs of EA F2 follicles in control cows than in Twinner cows (P < 0.05). We hypothesize that reduced GC IGF2R expression in F2 follicles of Twinner cows may play a role in the development of 2 or more dominant follicles.