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Excessive FSH doses during ovarian stimulation in the small ovarian reserve heifer (SORH) cause premature cumulus expansion and follicular hyperstimulation dysgenesis (FHD) in nearly all ovulatory-size follicles with predicted disruptions in cell-signaling pathways in cumulus cells and oocytes (before ovulatory hCG stimulation). These observations support the hypothesis that excessive FSH dysregulates cumulus cell function and oocyte maturation. To test this hypothesis, we determined whether excessive FSH-induced differentially expressed genes (DEGs) in cumulus cells identified in our previously published transcriptome analysis were altered independent of extreme phenotypic differences observed amongst ovulatory-size follicles, and assessed predicted roles of these DEGs in cumulus and oocyte biology. We also determined if excessive FSH alters cumulus cell morphology, and oocyte nuclear maturation before (premature) or after an ovulatory hCG stimulus or during IVM. Excessive FSH doses increased expression of 17 cumulus DEGs with known roles in cumulus cell and oocyte functions (responsiveness to gonadotrophins, survival, expansion, and oocyte maturation). Excessive FSH also induced premature cumulus expansion and oocyte maturation but inhibited cumulus expansion and oocyte maturation post-hCG and diminished the ability of oocytes with prematurely expanded cumulus cells to undergo IVF or nuclear maturation during IVM. Ovarian stimulation with excessive FSH is concluded to disrupt cumulus cell and oocyte functions by inducing premature cumulus expansion and dysregulating oocyte maturation without an ovulatory hCG stimulus yielding poor-quality cumulus-oocyte complexes that may be incorrectly judged morphologically as suitable for IVF during ART., (© The Author(s) 2023. Published by Oxford University Press on behalf of European Society of Human Reproduction and Embryology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

High follicle-stimulating hormone (FSH) doses during ovarian stimulation protocols for assisted reproductive technologies (ART) are detrimental to ovulatory follicle function and oocyte quality. However, the mechanisms are unclear. In a small ovarian reserve heifer model, excessive FSH doses lead to phenotypic heterogeneity of ovulatory size follicles, with most follicles displaying signs of premature luteinization and a range in severity of abnormalities. By performing whole transcriptome analyses of granulosa cells, cumulus cells, and oocytes from individual follicles of animals given standard or excessive FSH doses, we identified progressive changes in the transcriptomes of the 3 cell types, with increasing severity of follicular abnormality with the excessive doses. The granulosa and cumulus cells each diverged progressively from their normal phenotypes and became highly similar to each other in the more severely affected follicles. Pathway analysis indicates a possible dysregulation of the final stages of folliculogenesis, with processes characteristic of ovulation and luteinization occurring concurrently rather than sequentially in the most severely affected follicles. These changes were associated with disruptions in key pathways in granulosa and cumulus cells, which may account for previously reported reduced estradiol production, enhanced progesterone and oxytocin production and diminished ovulation rates. Predicted deficiencies in oocyte survival, stress response, and fertilization suggest likely reductions in oocyte health, which could further compromise oocyte quality and ART outcomes., (© The Author(s) 2022. Published by Oxford University Press on behalf of the Endocrine Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

High follicle-stimulating hormone (FSH) doses during ovarian stimulation are detrimental to ovulatory follicle function and decrease live birth rate in cattle and women. However, the mechanism whereby excessive FSH causes ovarian dysfunction is unknown. This study tested the hypothesis that excessive FSH during ovarian stimulation induces premature luteinization of ovulatory-size follicles. Small ovarian reserve heifers were injected twice daily for 4 days with 70 IU (N = 7 heifers) or 210 IU (N = 6 heifers) Folltropin-V [commercial FSH-enriched preparation of porcine pituitary glands with minor (<1%) luteinizing hormone (LH) contamination, cpFSH]. Ovulatory-size (≥10 mm) follicles were excised from ovaries after the last cpFSH injection and hormone concentrations in follicular fluid (FF) were determined using ELISA. Luteinization was monitored by assessing cumulus cell-oocyte complex (COC) morphology and measuring concentrations of estradiol (E), progesterone (P), and oxytocin (O) in FF. COCs were classified as having compact (cCOC) or expanded (eCOC) cumulus cell layers, and as estrogen-active (E:P in FF ≥1), estrogen-inactive (EI, E:P in FF ≤1 > 0.1), or extreme-estrogen-inactive (EEI, E:P in FF ≤0.1). A high proportion (72%) of ovulatory-size follicles in 210 IU, but not 70 IU, dose heifers displayed eCOCs. The high doses also produced higher proportions of EI or EEI follicles which had lower E:P ratio and/or E but higher P and/or O concentrations compared with the 70 IU dose heifers. In conclusion, excessive cpFSH doses during ovarian stimulation may induce premature luteinization of most ovulatory-size follicles in heifers with small ovarian reserves., (Published by Oxford University Press on behalf of Society for the Study of Reproduction 2022.)

Increased knowledge of reproduction and health of domesticated animals is integral to sustain and improve global competitiveness of U.S. animal agriculture, understand and resolve complex animal and human diseases, and advance fundamental research in sciences that are critical to understanding mechanisms of action and identifying future targets for interventions. Historically, federal and state budgets have dwindled and funding for the United States Department of Agriculture (USDA) National Institute of Food and Agriculture (NIFA) competitive grants programs remained relatively stagnant from 1985 through 2010. This shortage in critical financial support for basic and applied research, coupled with the underappreciated knowledge of the utility of non-rodent species for biomedical research, hindered funding opportunities for research involving livestock and limited improvements in both animal agriculture and animal and human health. In 2010, the National Institutes of Health and USDA NIFA established an interagency partnership to promote the use of agriculturally important animal species in basic and translational research relevant to both biomedicine and agriculture. This interagency program supported 61 grants totaling over $107 million with 23 awards to new or early-stage investigators. This article will review the success of the 9-year Dual Purpose effort and highlight opportunities for utilizing domesticated agricultural animals in research., (Published by Oxford University Press on behalf of Society for the Study of Reproduction 2022.)

High FSH doses during superovulation of heifers with a small ovarian reserve increase the number of dysfunctional ovulatory-size follicles that do not ovulate in response to human chorionic gonadotropin (hCG). Thus, anti-Müllerian hormone (AMH) and antral follicle count (AFC), two well-established biomarkers of responsiveness of individuals to superovulation, are hypothesized to be positively linked to number of dysfunctional ovulatory-size follicles developing in response to superovulation with high FSH doses. To test this hypothesis, heifers with a small ovarian reserve were stimulated beginning on Day 1 of the estrous cycle with twice daily treatments for 4 days with each of four Folltropin-V (FSH) doses (35 IU, 70 IU (industry standard), 140 IU, or 210 IU) followed by prostaglandin F 2α to regress corpora lutea (CL) from the previous estrous cycle and hCG to induce ovulation. Ovulatory-size follicles were classified as functional or dysfunctional based on whether they ovulated and formed CL in response to hCG. FSH dose did not impact the relationship between AMH, AFC and the number of functional or dysfunctional ovulatory-size follicles developing in response to superovulation. Thus, data from the four superovulations were averaged for each heifer. AMH and AFC were positively associated with the subsequent number of functional and dysfunctional ovulatory-size follicles and the proportion of ovulatory-size follicles that are dysfunctional after superovulation. Because measurements of AMH concentration and AFC predict the number but not functionality of ovulatory-size follicles, which may also impact oocyte quality, these ovarian reserve biomarkers are concluded to be unlikely useful to improve IVF or embryo transfer outcomes in heifers with a small ovarian reserve., Competing Interests: Declaration of competing interest None., (Published by Elsevier Inc.)

Purpose: To evaluate whether total FSH dose was negatively correlated with number of oocytes retrieved in a large data set where previously, a negative correlation between FSH dose and live birth rate was identified., Methods: Data from 650,637 fresh autologous in vitro fertilization (IVF) cycles reported to the Society for Assisted Reproductive Technology between 2004 and 2012 were included. Logistic regression analysis was performed to determine if the relationship between total FSH dose used during ART with number of oocytes retrieved was impacted by the patient's health prognosis, age, BMI, ovarian stimulation protocol, or infertility diagnosis., Results: The number of oocytes retrieved was negatively correlated with FSH dose (P < 0.0001). Regardless of patient prognosis, age, BMI, ovarian stimulation protocol, and infertility diagnosis, the highest number of oocytes retrieved was in the 1001-2000 IU FSH group, and was 36-51% lower in the > 5000 IU compared with the optimal, 1001-2000 IU, FSH groups. Overall, ~80% of patients received FSH doses outside of the optimal FSH dose. Moreover, 61% of good prognosis patients (excludes individuals likely prescribed higher FSH doses) received doses exceeding the optimal dose range., Conclusion: The inverse relationship between FSH dose and the number of oocytes retrieved independent of patient age or health implies that excessive FSH doses during ART may be detrimental to oocyte retrieval., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

When women with small ovarian reserves are subjected to assisted reproductive technologies, high doses of gonadotropins are linked to high oocyte and embryo wastage and low live birth rates. We hypothesized that excessive follicle-stimulating hormone (FSH) doses during superovulation are detrimental to ovulatory follicle function in individuals with a small ovarian reserve. To test this hypothesis, heifers with small ovarian reserves were injected twice daily for 4 days, beginning on Day 1 of the estrous cycle with 35, 70, 140, or 210 IU doses of Folltropin-V (FSH). Each heifer (n = 8) was superovulated using a Williams Latin Square Design. During each superovulation regimen, three prostaglandin F2α injections were given at 12-h interval, starting at the seventh FSH injection to regress the newly formed corpus luteum (CL). Human chorionic gonadotropin was injected 12 h after the last (8th) FSH injection to induce ovulation. Daily ultrasonography and blood sampling were used to determine the number and size of follicles and corpora lutea, uterine thickness, and circulating concentrations of estradiol, progesterone, and anti-Müllerian hormone (AMH). The highest doses of FSH did not increase AMH, progesterone, number of ovulatory-size follicles, uterine thickness, or number of CL. However, estradiol production and ovulation rate were lower for heifers given high FSH doses compared to lower doses, indicating detrimental effects on ovulatory follicle function., (© The Author(s) 2020. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

The objectives of this study were to establish if exposure of pregnant dairy cows to high environmental temperatures and humidity during the first trimester of pregnancy impairs the establishment of the ovarian reserve (total number of healthy follicles and oocytes in ovaries) and fertility in their offspring. Serum anti-Müllerian hormone (AMH) concentrations and number of follicles ≥3 mm (antral follicle count; AFC) were assessed on a random day of the estrous cycle in 310 sixteen-month-old dairy heifers. Based on season of their conception and early fetal life, heifers were separated into 2 groups: summer (mean monthly temperature-humidity index = 69.33 ± 2.6) and winter (temperature-humidity index = 54.91 ± 1.08). The AMH and AFC were lower in summer (419.27 ± 22.81 pg/mL and 9.32 ± 0.42 follicles, respectively) compared with winter heifers (634.91 ± 47.60 pg/mL and 11.84 ± 0.46 follicles, respectively) and were not influenced by farm and age at sampling. Heifers born to dams that were not being milked during gestation had lower AMH and AFC compared with offspring of cows on their first lactation, whereas no difference was detected between offspring of cows on their first and subsequent lactations. Summer and winter heifers had similar age at first service and at first calving, and similar number of services per conception. Regardless of season in early fetal life, heifers were classified into 3 groups based on AMH and AFC (low = 20%, intermediate = 60%, high = 20%). Heifers with the lowest AMH were older at first service compared with herd mates with intermediate AMH, but age at first calving and number of services per conception were similar among AMH categories. No difference was detected in any of the fertility measures among AFC categories. Heifers born to mothers exposed to high environmental temperatures in early gestation had smaller ovarian reserves compared with herd mates conceived in winter, but no association between season of early fetal life and fertility at first conception was established. Season of conception and maternal lactation status affect the size of the ovarian reserve, but not fertility, at first conception in the progeny., (© 2020, The Authors. Published by Elsevier Inc. and Fass Inc. on behalf of the American Dairy Science Association®. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).)

Maternal nutritional status programs the development of several systems in female offspring, with effects that depend on the severity, duration, and window of development when the nutritional perturbation is imposed. On the basis of the developmental origins of health and disease concept, we hypothesize that gestational low caloric intake may induce maternal subclinical hyperandrogenism during early pregnancy and compromise cardiovascular health and fertility in the female offspring. To examine this possibility, a literature search for human and animal studies was conducted using two electronic databases, PubMed and Cochrane until April 2019 to address the following questions: (a) Do androgens have a developmental role in cardiovascular and ovarian development? (b) Is excess maternal testosterone linked to cardiovascular disease and infertility? and (c) Could early pregnancy undernutrition enhance maternal androgen production and compromise health and fertility in female offspring? The observations reviewed, establish a potential causative link between maternal undernutrition and subclinical hyperandrogenism with hypertension and reduced ovarian reserve in the progeny. Further studies in appropriate models are needed to better understand whether low energy intake and subclinical maternal hyperandrogenism during early pregnancy can negatively affect the health of the female offspring., (© 2019 Wiley Periodicals, Inc.)

This review summarizes studies we conducted to test the hypothesis that size of the ovarian reserve (number of healthy follicles and oocytes in ovaries) positively impacts ovarian function and fertility in cattle. Key results, primarily in Bos taurus dairy cattle, show that antral follicle count (AFC) during follicular waves is highly variable between individuals, but very highly repeatable within individuals. Cycling heifers with low (≤15 follicles ≥3 mm, ~20% of a herd) vs. a high AFC (≥25, ~20% of a herd) have a smaller ovarian reserve, higher FSH but lower anti-Müllerian hormone (AMH), androstenedione, estradiol, and progesterone concentrations. Moreover, cattle with low AFC have a thinner endometrium, decreased response of granulosal, thecal, or luteal cells to FSH or LH and a poorer response to superovulation compared to cattle with high AFC. Interestingly, cows with a very high AFC as heifers have reduced fertility, fewer lactations, and shorter herd longevity, whereas cows with a low vs. intermediate AFC have reduced fertility, fewer lactations, and shorter herd longevity. Anti-Müllerian hormone concentrations are static within individuals but highly positively correlated with AFC, but fertility is not correlated with circulating AMH concentration in heifers and dairy cows with low vs. a higher AMH as heifers have reduced fertility and a shorter herd longevity. Anti-Müllerian hormone concentrations in dairy heifers are a moderately heritable trait (36%), and negatively impacted by inadequate maternal nutrition during early pregnancy or high maternal somatic cell count. We conclude that genetic or environmental manipulations of AMH could enhance size of the ovarian reserve and ovarian function, thereby improving fertility, response to superovulation, and longevity in dairy cows., (© The Author(s) 2019. Published by Oxford University Press on behalf of the American Society of Animal Science. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Anti-Müllerian hormone (AMH) is an ovarian growth factor that plays an important role in regulation of ovarian follicle growth. The objectives of this study were to estimate the genomic heritability of AMH and identify genomic regions associated with AMH production in a genome-wide association (GWA) analysis. Concentrations of AMH were determined in 2,905 dairy Holstein heifers genotyped using the Zoetis medium density panel (Zoetis Inclusions, Kalamazoo, MI) with 54,519 single nucleotide polymorphism (SNP) markers remaining after standard genotype quality control edits. A linear mixed model was used to model the random effects of sampling day and genomics on the logarithm of AMH. The genomic heritability (± standard error of the mean) of AMH was estimated to be 0.36 ± 0.03. Our GWA analysis inferred significant associations between AMH and 11 SNP markers on chromosome 11 and 1 SNP marker on chromosome 20. Annotated genes with significant associations were identified using the Ensembl genome database (version 88) of the cow genome (version UMD 3.1; https://www.ensembl.org/biomart). Gene set enrichment analysis revealed that 2 gene ontology (GO) terms were significantly enriched in the list of candidate genes: G-protein coupled receptor signaling pathway (GO:0007186) and the detection of chemical stimulus involved in sensory perception (GO:0050907). The estimated high heritability and previously established associations between AMH and ovarian follicular reserve, fertility, longevity, and superovulatory response in cattle implies that AMH could be used as a biomarker for genetic improvement of reproductive potential., (Copyright © 2018 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

Development of a bovine model without ovarian follicular waves (waveless) and transient increases in gonadotropin secretion during estrous cycles may lead to new methods to more consistently regulate ovulatory follicle growth thereby improving efficiency of embryo transfer. We hypothesized that the GnRH antagonist acyline would inhibit gonadotropin secretion thereby blocking follicular waves, ovarian function and ovulation during estrous cycles of cattle. To test this hypothesis, beef heifers (n = 5 per group) were treated twice daily with vehicle (control) or 25 or 50 μg/kg acyline beginning 12 h after GnRH-induced ovulation and ending 21 days later. Each animal was subjected to ovarian ultrasonography for 25 days to monitor number and growth of follicles ≥3 mm in diameter and growth of the corpus luteum (CL). Blood samples were taken at various intervals to determine circulating concentrations of FSH, LH, anti-Müllerian hormone (AMH, marker of small follicle growth) and progesterone. Results show that acyline treatment decreased or blocked: circulating concentrations of LH, transient FSH spikes associated with emergence of follicular waves, preovulatory FSH/LH surges, follicular waves, dominant follicle development, CL growth, and progesterone production. In contrast, the largest acyline dose increased AMH concentrations. In conclusion, long-term acyline treatment blocks follicular waves but not growth of preantral and small antral follicles (≤ 3 mm). Future studies will determine if the waveless bovine model, which has enhanced development of preantral and small antral follicles, can be used to develop new methods to improve predictability of response of cattle to superovulation., (Copyright © 2018 Elsevier B.V. All rights reserved.)

A reliable, easy to assess marker for fertility in agricultural species would be highly desirable and Anti-Müllerian Hormone (AMH) is a promising candidate. This review summarizes recent findings concerning AMH and its role in fertility management, mainly in cattle. It focuses on (1) alterations in circulating AMH concentrations from birth to puberty and during estrous cycles; (2) correlation of circulating AMH concentrations with ovarian follicle numbers and ovarian reserve; (3) factors that impact circulating AMH concentrations; (4) use of AMH as a predictor of fertility. Circulating AMH concentrations can be easily and reliably measured with a single blood sample in adult cattle because AMH varies minimally during the estrous cycle and is repeatable across multiple cycles. Circulating AMH concentrations are positively associated with several measures of fertility. Dairy heifers with low compared with higher AMH concentrations subsequently had lower pregnancy rates, higher probability of being culled after birth of their first calf and shorter herd longevity. Also, AMH is predictive of response to superovulation in cattle and sheep. Several factors contribute to the variability in AMH concentrations among individuals; for example, beef cattle have higher AMH than dairy cattle. Nutritional imbalances, disease and endocrine disruptors during fetal life may negatively program the size of the ovarian reserve and consequently serum AMH concentrations and potential fertility in adulthood. We conclude that AMH may be a predictor of fertility and herd longevity in cattle, whereas in sheep and other farm species, the potential association between AMH and reproductive performance remains largely unexplored.Free Italian abstract: An Italian translation of this abstract is freely available at http://www.reproduction-online.org/content/154/1/R1/suppl/DC1., (© 2017 Society for Reproduction and Fertility.)

Fertility and productive herd life (time in herd after birth of first calf) are inferior in dairy cows with relatively low compared with intermediate but not high numbers of follicles growing during ovarian follicular waves. The present study, therefore, tested the hypothesis that fertility and productive herd life are lower in dairy heifers with high follicle numbers compared with age-matched herdmates with fewer follicles. To test this hypothesis, 11 to 15 mo old Holstein heifers were subjected to a single ultrasound measurement of the number of follicles ≥3 mm in diameter. Heifers were classified into a high- (≥25 follicles), mid- (16-24), or low-range (≤15) follicle number group (FNG). All heifers not removed from the herd before first calving (n = 408) had the opportunity to start their fifth or sixth lactation after birth of their first calf. During this time, performance and health parameters for each individual were recorded daily by herd managers. Results showed that heifers in the high-range FNG had a 180-d shorter productive herd life, reduced survival rate, and greater probability of being culled after birth of the first calf, as well as fewer lactations compared with heifers in the low-range FNG. Cows in the high-compared with the mid- or low-range FNG also had greater involuntary culling rates, days open, and services per conception, and lower pregnancy rates during the first, second, or third lactations. We concluded that dairy heifers with ≥25 follicles ≥3 mm in diameter have suboptimal fertility and a shorter productive herd life compared with herdmates with fewer follicles., (Copyright © 2017 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

Objective: To evaluate the correlation between total gonadotropin dose and live birth rate., Design: Retrospective analysis., Setting: Not applicable., Patient(s): A total of 658,519 fresh autologous cycles of in vitro fertilization (IVF) reported to the Society for Assisted Reproductive Technology from 2004 to 2012., Intervention(s): None., Main Outcome Measure(s): Logistic regression models were fitted to live birth rates with the use of categorized values for total FSH dose and number of oocytes retrieved as the primary predictor variables. To reduce the effect of the most significant confounders that may lead physicians to prescribe higher doses of FSH, additional analyses were performed limited to good-prognosis patients (<35 years of age, body mass index <30 kg/m(2), and no diagnosis of diminished ovarian reserve, endometriosis, or ovulatory disorder) and including duration of gonadotropin treatment., Result(s): Live birth rate significantly decreased with increasing FSH dose, regardless of the number of oocytes retrieved. The statistically significant decrease in live birth rate with increasing FSH dose remained in patients with good prognosis, and regardless of female age, except for women aged ≥ 35 years with 1-5 oocytes retrieved., Conclusion(s): This analysis suggests that physicians may wish to avoid prescribing a high dose of FSH. However, the results of this study do not justify the use of minimal-stimulation or natural-cycle IVF., (Copyright © 2015 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

Five isoforms of follistatin (FST) (Mr 31, 33, 35, 37, and 41  kDa) were purified from bovine follicular fluid (bFF). Comparison of their activin and heparan sulphate proteoglycan (HSP) binding properties and biopotencies in the neutralisation of activin A action in vitro revealed that all five isoforms bound activin A, but they did so with different affinities. Only the 31  kDa isoform (FST-288) bound to HSP. FST-288 also showed the greatest biopotency, and the 35 and 41  kDa isoforms were the least potent. To determine whether bovine follicle development is associated with changing intrafollicular FST and activin profiles, we analysed bFF from dominant follicles (DFs) and subordinate follicles (SF) collected at strategic times during a synchronised oestrous cycle. Total FST, activin A and activin AB were measured by immunoassay, whereas individual FST isoforms were quantified by immunoblotting. Follicle diameter was positively correlated with oestrogen:progesterone ratio (r=0.56) in bFF but negatively correlated with activin A (r=-0.34), activin AB (r=-0.80) and 'total' FST (r=-0.70) levels. Follicle diameter was positively correlated with the abundance of the 41  kDa isoform (r=0.59) but negatively correlated with the abundance of the 33 and 31  kDa isoforms (r=-0.56 and r=-0.41 respectively). Both follicle statuses (DF and SF) and cycle stage affected total FST, activin A and activin B levels, whereas follicle status, but not cycle stage, affected the abundance of the 41, 37, 33 and 31  kDa FST isoforms. Collectively, these findings indicate that intrafollicular FST isoforms, which differ in their ability to bind and neutralise activins and to associate with cell-surface proteoglycans, show divergent changes during follicle development. Enhanced FST production may play an important negative role, either directly or via the inhibition of the positive effects of activins, on follicle growth and function during follicular waves., (© 2015 Society for Reproduction and Fertility.)

Reliable biomarkers predictive of productive herd life (time in herd after birth of first calf) have heretofore not been discovered in dairy cattle. However, circulating concentrations of anti-Müllerian hormone (AMH) are positively associated with number of follicles or antral follicle count (AFC), ovarian function, and fertility, and approximately 25% of cows have a relatively low AFC and low AMH concentrations. The present study tested the hypothesis that heifers with the lowest AMH concentrations have suboptimal fertility and are removed from a herd for poor reproductive performance at a greater rate, and therefore have a shorter productive herd life compared with age-matched herdmates with higher AMH. To test this hypothesis, 11- to 15-mo-old Holstein heifers (n=281) were subjected to a single measurement of AMH. All heifers not removed from the herd had the opportunity to complete 2 lactations and start their third lactation after calving. During this time, performance and health parameters for each individual were recorded daily by herd managers. Results showed that the quartile of heifers with the lowest AMH concentration also had, on average, a shorter productive herd life (by 196 d), a reduced survival rate after birth of the first calf, the lowest level of milk production (first lactation), the lowest total percentage of cows pregnant (across all lactations), the highest culling rates (first and second lactations and overall), and the highest culling rate for poor reproduction (first lactation) compared with age-matched herdmates with higher AMH. We concluded that a single determination of AMH concentration in young adult dairy heifers may be a simple diagnostic method to predict herd longevity, and AMH may be a useful phenotypic marker to improve longevity of dairy cows., (Copyright © 2015 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

Previous studies have documented that ovarian antral follicle count (AFC) is positively correlated with number of healthy follicles and oocytes in ovaries (ovarian reserve), as well as ovarian function and fertility in cattle. However, environmental factors (e.g., nutrition, steroids) during pregnancy in cattle and sheep can reduce AFC in offspring. The role that genetic and environmental factors play in influencing the variability in AFC and, correspondingly, the size of the ovarian reserve, ovarian function, and fertility, are, however, poorly understood. The present study tests the hypothesis that variability in AFC in offspring is influenced not only by genetic merit but also by the dam age and lactation status (lactating cows vs. nonlactating heifers) and milk production during pregnancy. Antral follicle count was assessed by ultrasonography in 445 Irish Holstein-Friesian dairy cows and 522 US Holstein-Friesian dairy heifers. Heritability estimates for AFC (± standard error) were 0.31 ± 0.14 and 0.25 ± 0.13 in dairy cows and heifers, respectively. Association analysis between both genotypic sire data and phenotypic dam data with AFC in their daughters was performed using regression and generalized linear models. Antral follicle count was negatively associated with genetic merit for milk fat concentration. Also, AFC was greater in offspring of dams that were lactating (n=255) compared with nonlactating dams (n=89) during pregnancy and was positively associated with dam milk fat concentration and milk fat-to-protein ratio. In conclusion, AFC in dairy cattle is a moderately heritable genetic trait affected by age or lactation status and milk quality but not by level of dam's milk production during pregnancy., (Copyright © 2014 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

Follicular development occurs in wave like patterns in monotocous species such as cattle and humans and is regulated by a complex interaction of gonadotropins with local intrafollicular regulatory molecules. To further elucidate potential mechanisms controlling dominant follicle selection, granulosa cell RNA harvested from F1 (largest) and F2 (second largest) follicles isolated at predeviation (PD) and onset of diameter deviation (OD) stages of the first follicular wave was subjected to preliminary RNA transcriptome analysis. Expression of numerous WNT system components was observed. Hence experiments were performed to test the hypothesis that WNT signaling modulates FSH action on granulosa cells during follicular waves. Abundance of mRNA for WNT pathway members was evaluated in granulosa cells harvested from follicles at emergence (EM), PD, OD and early dominance (ED) stages of the first follicular wave. In F1 follicles, abundance of CTNNB1 and DVL1 mRNAs was higher and AXIN2 mRNA was lower at ED versus EM stages and DVL1 and FZD6 mRNAs were higher and AXIN2 mRNA was lower in F1 versus F2 follicle at the ED stage. Bovine granulosa cells were treated in vitro with increasing doses of the WNT inhibitor IWR-1+/- maximal stimulatory dose of FSH. IWR-1 treatment blocked the FSH-induced increase in granulosa cell numbers and reduced the FSH-induced increase in estradiol. Granulosa cells were also cultured in the presence or absence of FSH +/- IWR-1 and hormonal regulation of mRNA for WNT pathway members and known FSH targets determined. FSH treatment increased CYP19A1, CCND2, CTNNB1, AXIN2 and FZD6 mRNAs and the stimulatory effect on CYP19A1 mRNA was reduced by IWR-1. In contrast, FSH reduced CARTPT mRNA and IWR-1 partially reversed the inhibitory effect of FSH. Results support temporal and hormonal regulation and a potential role for WNT signaling in potentiating FSH action during dominant follicle selection.

We previously established a potential role for cocaine and amphetamine regulated transcript (CARTPT) in dominant follicle selection in cattle. CARTPT expression is elevated in subordinate versus dominant follicles, and treatment with the mature form of the CARTPT peptide (CART) decreases follicle-stimulating hormone (FSH)-stimulated granulosa cell estradiol production in vitro and follicular fluid estradiol and granulosa cell CYP19A1 mRNA in vivo. However, mechanisms that regulate granulosa cell CART responsiveness are not understood. In this study, we investigated hormonal regulation of granulosa cell CART-binding sites in vitro and temporal regulation of granulosa cell CART-binding sites in bovine follicles collected at specific stages of a follicular wave. We also determined the effect of inhibition of CART receptor signaling in vivo on estradiol production in future subordinate follicles. Granulosa cell CART binding in vitro was increased by FSH, and this induction was blocked by estrogen receptor antagonist treatment. In follicles collected in vivo at specific stages of a follicular wave, granulosa cell CART binding in the F2 (second largest), future subordinate follicle increased during dominant follicle selection. Injection into the F2 follicle (at onset of diameter deviation) of an inhibitor of the o/i subclass of G proteins (previously shown to block CART actions in vitro) resulted in increased follicular fluid estradiol concentrations in vivo. Collectively, results demonstrate hormonal regulation of granulosa cell CART binding in vitro and temporal regulation of CART binding in subordinate follicles during dominant follicle selection. Results also suggest that CART signaling may help suppress estradiol-producing capacity of the F2 (subordinate) follicle during this time period.

Severe prenatal undernutrition is usually associated with low birth weights in offspring and disorders including hypertension, obesity, and diabetes. Whether alterations in maternal nutrition insufficient to impair birth weight or prenatal growth impact the cardiovascular, stress, or metabolic systems is unknown. In addition, little is known about the effects of maternal dietary restriction on development of the reproductive system in mammals. Here, we use the bovine model, which has a gestational length and birth rate similar to humans, to show that offspring from nutritionally restricted dams (during the first trimester) were born with identical birth weights and had similar postnatal growth rates (to 95 wk of age), puberty, glucose metabolism, and responses to stress compared to offspring from control mothers. However, an increase in maternal testosterone concentrations was detected during dietary restriction, and these dams had offspring with a diminished ovarian reserve (as assessed by a reduction in antral follicle count, reduced concentrations of anti-Müllerian hormone, and increased follicle-stimulating hormone concentrations), enlarged aorta, and increased arterial blood pressure compared with controls. Our study links transient maternal undernutrition and enhanced maternal androgen production with a diminished ovarian reserve as well as potential suboptimal fertility, enlarged aortic trunk size, and enhanced blood pressure independent of alterations in birth weight, postnatal growth, or stress response and glucose tolerance. The implications are that relatively mild transient reductions in maternal nutrition during the first trimester of pregnancy (even those that do not affect gross development) should be avoided to ensure healthy development of reproductive and cardiovascular systems in offspring.

Members of the bone morphogenetic protein (BMP) family regulate follicular development and granulosa cell function. However, changes in expression of BMP2 and its receptors during follicular waves in cattle and ability of BMP2 to modulate bovine granulosa cell estradiol production are not well understood. The objectives of this study were to determine temporal regulation of mRNA for BMP2 and its type I and II receptors (BMPR1A and BMPR2) in bovine follicles collected at specific stages of a follicular wave (predeviation, early dominance, mid dominance, preovulatory), ability of BMP2 to modulate bovine granulosa cell steroidogenesis, and whether effects of BMP2 on granulosa cell estradiol production are influenced by cotreatment with cocaine- and amphetamine-regulated transcript (CART), an intrafollicular regulatory peptide shown to inhibit estradiol production in response to other trophic hormones (FSH and IGF1). Relative abundance of mRNAs for Bmp2 and Bmpr2 was elevated at the mid dominance stage relative to earlier stages of the follicular wave and further increased at the preovulatory stage. Abundance of mRNA for Bmpr1a was lowest at early dominance stage and highest at preovulatory stage relative to other stages of the follicular wave examined. Treatment of bovine granulosa cells in vitro with BMP2 increased estradiol but decreased progesterone concentrations. Co-incubation with CART reduced the BMP2-stimulated increase in granulosa cell estradiol production. Results suggest that BMP2 may play a regulatory role in development of bovine follicles to the preovulatory stage and that CART can inhibit granulosa cell estradiol production in response to multiple hormones/growth factors, including BMP2., (Copyright © 2013 Elsevier Inc. All rights reserved.)

The total number of ovarian follicles ≥ 3mm in diameter (antral follicle count, AFC) during follicular waves varies among cattle of similar age, but AFC is highly repeatable within individuals. We hypothesized that lower AFC could be associated with reduced fertility in cattle. The AFC was assessed by ultrasonography for 2 d consecutively during the first wave of follicular growth of the estrous cycle, 4.6±1.43 d (mean ± SD) after estrus, in 306 Holstein-Friesian dairy cows approximately 70 d postpartum. Cows were classified into 3 groups based on AFC: low (AFC ≤15), intermediate (AFC=16 to 24), and high (AFC ≥25). During the cycle in which AFC was assessed and in subsequent cycles, cows were artificially inseminated (AI) following detection of estrus, and pregnancy status was assessed using ultrasonography. Cows with high AFC had 3.34 times greater odds of being pregnant at the end of the breeding season compared with cows with low AFC; the odds of a successful pregnancy at first service were 1.75 times greater in the intermediate compared with the low group. The predicted probability of a successful pregnancy by the end of the breeding period (length of breeding season was 86±16.3 d) was 94, 88, and 84% for the high, intermediate, and low AFC groups, respectively. No difference was evident among groups in 21-d submission rate (proportion of all cows detected in estrus and submitted for AI in the first 21 d of the breeding season), but the interval from calving to conception was shorter in the high (109.5±5.1 d) versus low (117.1±4 d) group, and animals with intermediate AFC received fewer services during the breeding season (2.3±0.1) compared with animals with low AFC (2.7±0.1). Lactating cows with ≤15 ovarian follicles have lower reproductive performance compared with cows with higher numbers of follicles, but the existence of a positive association between high numbers of ovarian follicles and fertility is yet to be established., (Copyright © 2012 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

The reason ovarian function and fertility are diminished in women with a low antral follicle count (AFC), despite significant numbers of follicles remaining in ovaries, is unknown. The bovine model is unique to address this question because cattle and women with a low AFC exhibit similar phenotypic characteristics including a diminished ovarian reserve, reduced circulating concentrations of anti-Müllerian hormone (AMH) but heightened FSH secretion during reproductive cycles. Because women and cattle with a low AFC respond minimally to gonadotropin stimulation during IVF cycles or superovulation, granulosa cells in individuals with a low AFC are hypothesised to be refractory to FSH. The present study evaluates this hypothesis by testing whether capacity of granulosa cells to respond to FSH differs between cattle with a low and a high AFC. Granulosa cells from cattle with a low (≤15 follicles ≥3 mm in diameter) or a high (≥25 follicles) AFC were cultured with different doses of FSH. Treatments were evaluated by measurement of oestradiol (E), progesterone (P) and AMH in media and abundance of mRNAs for aromatase (CYP19A1), AMH, FSH receptor (FSHR) and oxytocin (OXT). Progesterone and OXT mRNA are well-established markers of granulosa cell luteinisation. Although high doses of FSH induced granulosa cell luteinisation, basal and FSH-induced increases in E and AMH production and expression of mRNAs for CYP19A1, FSHR and AMH in granulosa cells were much lower, while P production and OXT mRNA expression were higher in non-luteinised and luteinised granulosa cells from the low than the high AFC group. Granulosa cells in cattle with a low AFC are refractory to FSH action, which could explain why ovarian function, responsiveness to gonadotropin stimulation and fertility are diminished in individuals with a low versus a high AFC.

The objective of this study was to identify prostaglandin F(2α) (PG)-induced changes in the transcriptome of bovine corpora lutea (CL) that are specific to mature, PG-responsive (day 11) CL vs. developing (day 4) CL, which do not undergo luteolysis in response to PG administration. CL were collected at 0, 4, and 24 h after PG injection on days 4 and 11 of the estrous cycle (n = 5 per day and time point), and microarray analysis was performed with GeneChip Bovine Genome Arrays. Data normalization was performed with affy package and significance testing with maanova from Bioconductor. Significance (relative to 0 h time point) was declared at fold change >2.0 or <0.5 and false discovery rate of <5%. At 4 and 24 h after PG, 221 (day 4) and 661 (day 11) and 248 (day 4) and 1,421 (day 11) regulated genes, respectively, were identified. The accentuated gene expression response in day 11 CL was accompanied by specific enrichment of PG-regulated genes in distinctive gene ontology categories (immune related and other), particularly at 24 h after injection. Specificity in putative transcription factor binding sites was observed among PG-regulated genes on day 11 vs. day 4, including a potential association of ETS transcription factors with acute PG-induced gene expression specific to day 11 CL. Temporal and PG-induced regulation of abundance of mRNA for ETS transcription factor family members linked to the stage-specific response to PG was not observed. Increased abundance of protein and/or mRNA for six PG-regulated putative ETS-responsive genes was noted in day 11 but not day 4 CL. Results reveal insight into stage-specific gene expression in bovine CL in response to PG and potential transcriptional mediators of luteolysis.

The mechanism whereby the inherently high variation in ovary size and the total number of high-quality oocytes in ovaries (ovarian reserve) impact on ovarian function and fertility, diagnostics to measure the size of the ovarian reserve and the factors that cause variation in the ovarian reserve are unknown. Our results show that cattle can be phenotyped reliably based on the number of antral follicles growing during follicular waves (antral follicle count, AFC). Young adult cattle with a consistently low v. a high AFC have smaller gonads, a markedly diminished ovarian reserve and many other phenotypic characteristics usually associated with ovarian aging and infertility. A powerful new approach based on a single measurement of serum concentration of anti-Müllerian hormone (AMH) is described to test the longstanding hypothesis that the size of the ovarian reserve is positively associated with fertility. Also, new evidence shows that maternal environment has a critical role in regulation of the high variation in the ovarian reserve and perhaps fertility in offspring. These results support the conclusion that the inherently high variation in the ovarian reserve, potentially caused by alterations in the maternal environment, has a negative impact on ovarian function that may result in suboptimal fertility in young adult cattle, and a single AMH measurement can be used reliably in future studies to determine if fertility is suboptimal in young adult cattle with low circulating AMH concentrations and a correspondingly diminished ovarian reserve.

Androgens have an important role in ovarian follicular growth and function, but circulating androgen concentrations are also associated with ovarian dysfunction, cardiovascular disease, and metabolic disorders in women. The extent and causes of the variation in androgen production in individuals, however, are unknown. Because thecal cells of follicles synthesize androstenedione and testosterone, variation in production of these androgens is hypothesized to be directly related to the inherently high variation in number of healthy growing follicles in ovaries of individuals. To test this hypothesis, we determined whether thecal CYP17A1 mRNA (codes for a cytochrome P450 enzyme involved in androgen synthesis), LH-induced thecal androstenedione production, androstenedione concentrations in follicular fluid, and circulating testosterone concentrations were lower in cattle with relatively low versus high number of follicles growing during follicular waves and whether ovariectomy reduced serum testosterone concentrations. Results demonstrated that cattle with a low follicle number had lower (P<0.05) abundance of CYP17A1 mRNA in thecal cells, reduced (P<0.01) capacity of thecal cells to produce androstenedione in response to LH, lower (P<0.01) androstenedione concentrations in ovulatory follicles, and lower (P<0.02) circulating testosterone concentrations during estrous cycles compared with animals with high follicle number. Also, serum testosterone in cattle with low or high follicle number was reduced by 63 and 70%, respectively, following ovariectomy. In conclusion, circulating androgen concentrations are lower in cattle with low versus high number of follicles growing during follicular waves, possibly because of a reduced responsiveness of thecal cells to LH.

In cattle we have noted that the antral follicle count (AFC, follicles > or = 3 mm in diameter) varies greatly among animals (from 5 to 50), is repeatable within animals, and is highly correlated with the total number of healthy follicles in ovaries. Also, animals with low AFC have higher serum concentrations of FSH and LH, but lower concentrations of Anti-Mullerian Hormone, progesterone and androgens than animals with high AFC. We have investigated the effect of maternal environment during gestation on their offspring AFC by restricting maternal nutrition to 60% of maintenance requirements (compared with 100% in controls) during the first third of gestation. Calves born to nutritionally restricted mothers had 60% lower AFC compared with calves born to mothers fed control diets. In other studies we have evidence to indicate that fertility may be compromised in animals with low AFC due to effects on oocytes, progesterone and the endometrium compared with animals with high AFC. To examine this directly we assessed AFC in post-partum dairy cows and found that cows with a high AFC had higher pregnancy rates, shorter calving to conception intervals and received fewer services during the breeding season compared with cows with a low AFC. In addition, the high variation in follicle numbers in adults may not only be reflective of reproductive disorders and suboptimal fertility, but there is evidence to indicate that it may be associated with alterations in the function of other non-reproductive systems (e.g. cardiovascular) that may have profound effects on the animal's health and welfare.

We hypothesised that higher serum FSH concentrations in cattle with low v. high follicle numbers during follicular waves are caused by a different capacity of the pituitary gland to produce gonadotropins. Dairy cows with high (> or = 30; n = 5) and low (< or = 15; n = 5) follicle numbers were selected and serum concentrations of oestradiol and FSH during an oestrous cycle were measured. Cows were ovariectomised at oestrus and bled frequently up to 8 days after ovariectomy. After 33 days, cows were injected with gonadotropin-releasing hormone (GnRH) and bled intensively up to 8 h after GnRH injection. One day later, animals were injected with follicular fluid (FF) from bovine follicles and were bled intensively up to 2 days after the first injection. Serum concentrations of FSH and LH were measured. After 2 days, cows were killed and their pituitary glands collected. Prior to ovariectomy, serum oestradiol concentrations were similar between groups, whereas FSH concentrations were higher in cattle with low v. high numbers of follicles. No differences were detected in serum gonadotropin concentrations after ovariectomy, GnRH injection or FF challenge between groups. The results indicate that the inherent capacity of the pituitary gland to secrete gonadotropins does not differ between cattle with high v. low numbers of follicles during follicular waves.

The mechanisms regulating development of a single (dominant) follicle capable of ovulation during each follicular wave in cattle and atresia of remaining follicles (dominant follicle selection) are not well understood. FSH and IGF1 are known regulators of follicle growth and granulosa cell estradiol production during follicular waves. Recent evidence indicates cocaine and amphetamine regulated transcript (CARTPT), with intraovarian expression only in single-ovulating species, is a novel regulator of follicular development. The mature CARTPT peptide (CART) is a potent negative regulator of FSH and IGF1 action on granulosa cells in vitro and can inhibit follicular estradiol production in vivo. Follicular fluid CART concentrations in healthy follicles decrease after dominant follicle selection and CARTPT mRNA is lower in healthy versus atretic follicles collected prior to and early after initiation of follicle dominance, suggestive of a regulatory role in the selection process. The inhibitory actions of CART on FSH signaling and estradiol production are dependent on the G(o/i)-subclass of inhibitory G proteins and linked to multiple components of the FSH signal transduction pathway resulting in reduced CYP19A1 mRNA and estradiol production. Evidence to date supports a potential important functional role for CART in regulation of dominant follicle selection and the species-specific ovulatory quota in monotocous species.

We demonstrated previously a negative association of granulosa cell cocaine- and amphetamine-regulated transcript (CARTPT) expression with follicle health status and inhibitory effects of the mature CARTPT peptide (CART) on follicle-stimulating hormone (FSH) signal transduction in vitro, resulting in reduced bovine granulosa cell CYP19A1 mRNA and estradiol production. The objectives of this study were to investigate temporal regulation of granulosa cell CARTPT expression (granulosa cell mRNA and follicular fluid CART peptide concentrations) during follicular waves, CART regulation of androstenedione production (precursor for estradiol biosynthesis) by thecal tissue collected at specific stages of a follicular wave, FSH regulation of granulosa cell CARTPT mRNA expression, and the ability of CART to inhibit granulosa cell estradiol production and CYP19A1 mRNA expression when administered in vivo. CART concentrations in healthy, estrogen-active follicles (estradiol greater than progesterone in follicular fluid) decreased after dominant follicle selection, and CARTPT mRNA was lower in healthy, estrogen-active versus estrogen-inactive atretic follicles (progesterone greater than estradiol) collected at the predeviation and early dominance stages. CART treatment reduced luteinizing hormone-induced androstenedione production by thecal tissue collected at predeviation and early dominance stages but not at later stages of a follicular wave. The FSH or insulin-like growth factor 1 treatment in vitro reduced granulosa cell CARTPT mRNA in a dose-dependent fashion. Administration of CART in vivo into follicles at the early dominance stage reduced follicular fluid estradiol concentrations and granulosa cell CYP19A1 mRNA. Collectively, results support a potential stage-specific regulatory role for CART in negative regulation of estradiol production associated with selection of the dominant follicle.

Low progesterone concentrations and diminished ovarian reserves (total number of healthy oocytes) during reproductive cycles are linked to infertility in single-ovulating species like cattle. However, the extent and mechanisms whereby the inherently high variation in ovarian reserves may negatively affect progesterone production are unknown. Cattle were chosen to address these questions because the size of their ovarian reserves can be predicted based on an antral follicle count (AFC) during follicular waves. The present study determined if progesterone concentrations, differentiation and function of the corpus luteum (CL), and endometrial thickness differed during reproductive cycles of age-matched healthy young adult cattle with low versus high AFC during follicular waves. The results showed that, despite enhanced LH secretion, progesterone concentrations were lower during estrous cycles for animals with low versus high AFC. Animals with low versus high AFC also had a decreased basal, LH-, and 25-hydroxycholesterol-induced capacity of luteal and granulosal cells to produce progesterone, reduced amounts of STAR and mRNAs for STAR and LH receptor in the CL, and no change in endometrial thickness during estrous cycles. Taken together, these results 1) supported the conclusion that high variation in ovarian reserves of young adults is associated with alterations in differentiation and function of the CL and 2) provided insight into the potential factors that may cause suboptimal luteal function (e.g., heightened LH secretion and desensitization of the LH receptor, diminished LH responsiveness, diminished STAR, inherent deficiency in capacity of granulosal cells to undergo luteinization) and infertility (e.g., low progesterone, poor endometrial growth) in individuals with diminished ovarian reserves.

Previous studies established a positive relationship between oocyte competence and follistatin mRNA abundance. Herein, we used the bovine model to test the hypothesis that follistatin plays a functional role in regulation of early embryogenesis. Treatment of early embryos with follistatin during in vitro culture (before embryonic genome activation) resulted in a dose-dependent decrease in time to first cleavage, increased numbers of blastocysts, and increased blastocyst total and trophectoderm cell numbers. To determine the requirement of endogenous follistatin for early embryogenesis, follistatin ablation/replacement studies were performed. Microinjection of follistatin small interfering RNA into zygotes reduced follistatin mRNA and protein and was accompanied by a reduction in number of embryos developing to eight- to 16-cell and blastocyst stages and reduced blastocyst total and trophectoderm cell numbers. Effects of follistatin ablation were rescued by culture of follistatin small interfering RNA-injected embryos in the presence of exogenous follistatin. To investigate whether follistatin regulation of early embryogenesis is potentially mediated via inhibition of endogenous activin activity, the effects of treatment of embryos with exogenous activin, SB-431542 (inhibitor of activin, TGF-beta, and nodal type I receptor signaling) and follistatin plus SB-431542 were investigated. Activin treatment mimicked positive effects of follistatin on time to first cleavage and blastocyst development, whereas negative effects of SB-431542 treatment were observed. Stimulatory effects of follistatin on embryogenesis were not blocked by SB-431542 treatment. Results support a functional role for oocyte-derived follistatin in bovine early embryogenesis and suggest that observed effects of follistatin are likely not mediated by classical inhibition of activin activity.

The mechanisms whereby the high variation in numbers of morphologically healthy oocytes and follicles in ovaries (ovarian reserve) may have an impact onovarian function, oocyte quality, and fertility are poorly understood. The objective was to determine whether previously validated biomarkers for follicular differentiation and function, as well as oocyte quality differed between cattle with low versus a high antral follicle count (AFC). Ovaries were removed (n = 5 per group) near the beginning of the nonovulatory follicular wave, before follicles could be identified via ultrasonography as being dominant, from heifers with high versus a low AFC. The F1, F2, and F3 follicles were dissected and diameters determined. Follicular fluid and thecal, granulosal, and cumulus cells and the oocyte were isolated and subjected to biomarker analyses. Although the size and numerous biomarkers of differentiation, such as mRNAs for the gonadotropin receptors, were similar, intrafollicular concentrations of estradiol and the abundance of mRNAs for CYP19A1 in granulosal cells and ESR1, ESR2, and CTSB in cumulus cells were greater, whereas mRNAs for AMH in granulosal cells and TBC1D1 in thecal cells were lower for animals with low versus a high AFC during follicle waves. Hence, variation in the ovarian reserve may have an impact on follicular function and oocyte quality via alterations in intrafollicular estradiol production and expression of key genes involved in follicle-stimulating hormone action (AMH) and estradiol (CYP19A1) production by granulosal cells, function and survival of thecal cells (TBC1D1), responsiveness of cumulus cells to estradiol (ESR1, ESR2), and cumulus cell determinants of oocyte quality (CTSB).

The molecular mechanisms of ovulation and luteinization have not been well established, partially due to lack of a comprehensive understanding of functionally significant genes up-regulated in response to an ovulatory stimulus and the signaling pathways involved. In the present study, transcripts increased in bovine preovulatory follicles following a GnRH-induced LH surge were identified using microarray technology. Increased expression of 368 and 878 genes was detected at 12 (368 genes) and 20 h (878 genes) following GnRH injection. The temporal, cell specific and prostanoid-dependent regulation of selected genes (ADAM10, DBI, CD36, MTSS1, TFG, and RABGAP1) identified from microarray studies and related genes (ADAM17 and AREG) of potential significance were also investigated. Expression of mRNA for DBI and CD36 was simultaneously up-regulated in theca and granulosa cells (GC) following the LH surge, whereas temporal regulation of ADAM10, MTSS1, TFG, and RABGAP1 was distinct in the two cell compartments and increased granulosa TFG and RABGAP1 mRNA were prostanoid dependent. AREG mRNA was increased in theca and GCs at 12 and 24 h following GnRH injection. ADAM17 mRNA was increased in theca, but reduced in GCs 24 h following GnRH injection. The increased ADAM17 and AREG mRNA were prostanoid dependent. ADAM10 and ADAM17 protein were increased specifically in the apex but not the base of preovulatory follicles and the increase in ADAM17 was prostanoid dependent. Results reveal novel information on the regulation of preovulatory gene expression and suggest a potential functional role for ADAM10 and ADAM17 proteins in the region of follicle rupture.

Pleiotropic actions of cocaine- and amphetamine-regulated transcript (CART) are well described in the central nervous system and periphery, but the intracellular mechanisms mediating biological actions of CART are poorly understood. Although CART is not expressed in mouse ovaries, we have previously established CART as a novel intracellular regulator of estradiol production in bovine granulosa cells. We demonstrated that inhibitory actions of CART on estradiol production are mediated through inhibition of FSH-induced cAMP accumulation, Ca(2+) influx, and aromatase mRNA expression via a G(o/i)-dependent pathway. We also reported that FSH-induced estradiol production is dependent on Erk1/2 and Akt signaling, and CART may regulate other signaling proteins downstream of cAMP essential for estradiol production. Here, we demonstrate that CART is a potent inhibitor of FSH-stimulated Erk1/2 and Akt signaling and the mechanisms involved. Transient CART stimulation of bovine granulosa cells shortens the duration of FSH-induced Erk1/2 and Akt signaling whereas a prolonged (24 h) CART treatment blocks Erk1/2 and Akt activation in response to FSH. This CART-induced accelerated termination of Erk1/2 and Akt signaling is mediated both by induced expression and impaired ubiquitin-mediated proteasome degradation of dual specific phosphatase 5 (DUSP5) and protein phosphatase 2A. Results also support existence of a negative feedback loop in which CART via a G(o/i)-MAPK kinase dependent pathway activates Erk1/2, and the latter induces DUSP5 expression. Moreover, small interfering RNA mediated ablation of DUSP5 and/or protein phosphatase 2A prevents the CART-induced early termination of Erk1/2 and Akt signaling. Results provide novel insight into the intracellular mechanism of action of CART in regulation of FSH-induced MAPK signaling.

Methods to predict numbers of healthy oocytes in the ovaries of young adults could have important diagnostic relevance in family planning and animal agriculture. We have observed that peak antral follicle count (AFC) determined by serial ovarian ultrasonography during follicular waves is very highly reproducible within individual young adult cattle, despite 7-fold variation among animals. Herein, we tested the hypothesis that AFC is positively associated with the number of morphologically healthy oocytes and follicles in ovaries and with serum concentrations of anti-Müllerian hormone (AMH), an indirect marker for number of healthy follicles and oocytes in ovaries. In the present study, age-matched young adult cattle (12-18 mo old) were subjected to serial ultrasonography to identify animals with a consistently high (> or =25 follicles that were > or =3 mm in diameter) or low (< or =15 follicles) AFC during follicular waves. Differences in serum AMH concentrations, ovary weight, and number of morphologically healthy and atretic follicles and oocytes were determined. The phenotypic classifications of cattle based on AFC during follicular waves or AMH concentrations both predict reliably the relative number of morphologically healthy follicles and oocytes in ovaries of age-matched young adult cattle.