J.L.H. Ireland

Numbers of Antral Follicles During Follicular Waves in Cattle: Evidence for High Variation Among Animals, Very High Repeatability in Individuals, and an Inverse Association with Serum Follicle-Stimulating Hormone Concentrations

Abstract The extent, causes, and physiological significance of the variation in number of follicles growing during ovarian follicular waves in human beings and cattle are unknown. Therefore, the present study examined the variability and repeatability in numbers of follicles 3 mm or greater in diameter during the follicular waves in bovine estrous cycles, and we determined if the variation in number of follicles during waves was associated with alterations in secretion of FSH, estradiol, inhibin, and insulin-like growth factor I (IGF-I). Dairy cattle were subjected to twice-daily ultrasound analysis to count total number of antral follicles 3 mm or greater in diameter throughout 138 different follicular waves. In another study, blood samples were taken at frequent intervals from cows that consistently had low or very high numbers of follicles during waves and were subjected to immunoassays. Results indicate the following: First, despite an approximately sevenfold variation in number of follicles during waves among animals and marked differences in age, stage of lactation, and season of the year, a very highly repeatable (0.95) number of follicles 3 mm or greater in diameter is maintained during the ovulatory and nonovulatory follicular waves of individuals. Second, variation in number of follicles 3 mm or greater in diameter during waves and the inverse association of number of follicles during waves with FSH are not directly explained by alterations in the patterns of secretion of estradiol, inhibin, or IGF-I. Third, ovarian ultrasound analysis can be used reliably by investigators to identify cattle that consistently have low or high numbers of follicles during waves, thus providing a novel experimental model to determine the causes and physiological significance of the high variation in antral follicle number during follicular waves among single-ovulating species, such as cattle or humans.

Antral Follicle Count Reliably Predicts Number of Morphologically Healthy Oocytes and Follicles in Ovaries of Young Adult Cattle

Abstract 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 (≥25 follicles that were ≥3 mm in diameter) or low (≤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.

Alterations in Intrafollicular Regulatory Factors and Apoptosis During Selection of Follicles in the First Follicular Wave of the Bovine Estrous Cycle

Abstract Changes in follicular fluid (FF) concentrations of estradiol, inhibin forms, and insulin-like growth factor binding proteins (IGFBPs), percentage of apoptotic granulosa cells (%A), and follicular size for individual follicles in a growing cohort were determined throughout the first wave of follicular development during the bovine estrous cycle and related to FSH decline. Four groups of heifers (n = 31) were ovariectomized between Days 1.5 and 4.5 of the estrous cycle at 5 ± 1, 33 ± 2, 53 ± 1, and 84 ± 2 h after the periovulatory peak in FSH concentrations. Follicles ≥2.5 mm were dissected, measured, and FF aspirated. The five largest follicles were ranked based on their diameter (F1 to F5). Diameters of F1 to F5 were positively correlated with interval from FSH peak (r ≥ 0.6, P < 0.05). Five hours after the FSH peak, follicular diameter and FF concentrations of estradiol, inhibins, and IGFBPs were similar for F1 to F5. From 5 to 33 h, amounts of the six precursor inhibin forms (≥48 kDa) increased (P < 0.05) in F1 follicles. The IGFBPs in F1 follicles remained low at all time periods. At 33 h, amounts of IGFBP-4 and -5 were higher (P < 0.05) in F4 and F5 compared with F1 follicles. At 84 h, IGFBP-2, -4, and -5 were increased (P < 0.05) in F3, F4, and F5 compared with F1. At 5, 33, or 53 h, %A was not different between follicles in any size class. At 84 h %A was increased (P < 0.05) in follicles <6 mm in diameter. However, at that time, %A did not differ between the selected DF and the largest subordinate follicle. For individual heifers, the selected DF at 84 h was largest in size, highest in estradiol, and lowest in IGFBP-2 and -4. The F1 follicle had highest estradiol in 23 of 27 heifers irrespective of stage of the wave and lowest IGFBP-4 in 19 of 21 heifers from 33 h. We concluded that the earliest intrafollicular changes that differentiate a dominant-like follicle from the growing cohort are enhanced capacity to produce estradiol and maintenance of low levels of IGFBPs.

Identification of Potential Intrafollicular Factors Involved in Selection of Dominant Follicles in Heifers

Abstract A surgical procedure to aspirate follicular fluid concurrently from individual follicles from the same heifer was validated and used to determine if intrafollicular amounts of estradiol, progesterone, inhibins, activin-A, follistatins, and insulin-like growth factor binding proteins (IGFBP) differed for the future dominant compared with subordinate follicles during selection of the first wave dominant follicle. Heifers were subjected to surgery and aspiration of follicular fluid from the two or three largest follicles on Day 3 of the estrous cycle (∼1.5 days after emergence). Ultrasound was used to determine the fate of each aspirated follicle after surgery. At aspiration, diameter of the future dominant and largest subordinate follicle was similar in heifers. However, estradiol was higher, whereas IGFBP-4 was lower in the future dominant compared with the largest or next largest subordinate follicles. Also, the future dominant follicle in most cohorts had the highest estradiol and lowest IGFBP-4 compared with future subordinate follicles. We concluded that: IGFBP-4 and estradiol may have key roles in determining the physiological fate of follicles during selection of the first wave dominant follicle in heifers, and that both are reliable markers to predict which follicle in a growing cohort of 5- to 8.5-mm follicles becomes dominant.

Identification of Genes Involved in Apoptosis and Dominant Follicle Development During Follicular Waves in Cattle

Abstract We hypothesize that granulosa and theca cells from growing dominant follicles, with relatively high intrafollicular concentrations of estradiol, have a greater expression of genes involved in inhibiting apoptosis pathways and lower expression of genes involved in apoptosis pathways than growing subordinate follicles with lower estradiol concentrations. Using the well-characterized bovine dominant follicle model, we collected granulosa and theca cells from individual dominant and the largest subordinate follicle 3 days after initiation of a follicular wave in four animals. Based on ultrasound analysis, both follicle types were in the growth phase at the time of ovariectomy. However, dominant follicles were larger (9.8 ± 1.0 versus 7.6 ± 0.6 mm in diameter, P < 0.05) and had greater intrafollicular concentrations of estradiol (132.2 ±3 8.5 versus 24.1 ± 12.1 ng/ml, P < 0.05), compared with the largest subordinate follicles. We used bovine cDNA microarrays, which contained a total of 1400 genes, including a subset of 53 genes known to be involved in apoptosis pathways, to determine which apoptosis and marker genes from each of the four dominant versus subordinate follicles were potentially differentially expressed. Using a low stringency-screening criterion, 22 genes were identified. Quantitative real-time polymerase chain reaction confirmed that 16 of these genes were differentially expressed. Our novel results demonstrate that the high intrafollicular concentrations of estradiol in growing dominant follicles were positively associated with enhanced expression of mRNAs in granulosa cells for aromatase, LH receptor, estradiol receptor β, DICE-1, and MCL-1, compared with granulosa cells from subordinate follicles (all survival-associated genes). In contrast, the relatively low intrafollicular concentrations of estradiol in growing subordinate follicles were positively associated with enhanced expression of mRNAs in granulosa cells for β glycan, cyclo-oxygenase-1, tumor necrosis factor α, caspase-activated DNase, and DRAK-2, and in theca cells for β glycan, caspase 13, P58(IPK), Apaf-1, BTG-3, and TS-BCLL, compared with granulosa or theca cells from dominant follicles (genes that are all associated with cell death and/or apoptosis). We suggest that that these genes may be candidate estradiol target genes and that they may be early markers for the final stages of follicle differentiation or initiation of apoptosis and thus selection of dominant follicles during follicular waves.

Molecular Evidence That Growth of Dominant Follicles Involves a Reduction in Follicle-Stimulating Hormone Dependence and an Increase in Luteinizing Hormone Dependence in Cattle

Abstract The bovine dominant follicle (DF) model was used to identify molecular mechanisms potentially involved in initial growth of DF during the low FSH milieu of ovarian follicular waves. Follicular fluid and RNA from granulosa and theca cells were harvested from 10 individual DF obtained between 2 and 5.5 days after emergence of the first follicular wave of the estrous cycle. Follicular fluid was subjected to RIA to determine estradiol (E) and progesterone (P) concentrations and RNA to cDNA microarray analysis and (or) quantitative real-time PCR. Results showed that DF growth was associated with a decrease in intrafollicular E:P ratio and in mRNA for the FSH receptor, estrogen receptor 2 (ER beta), inhibin alpha, activin A receptor type I, and a proliferation (cyclin D2) and two proapoptotic factors (apoptosis regulatory protein Siva, Fas [TNFRSF6]-associated via death domain) in granulosa cells. In contrast, mRNAs for the LH receptor in granulosa cells and for two antiapoptotic factors (TGFB1-induced antiapoptotic factor 1, LAG1 longevity assurance homolog 4 [Saccharomyces cerevisiae]) and one proapoptotic factor (tumor necrosis factor [ligand] superfamily, member 8) were increased in theca cells. We conclude that the bovine DF provides a unique model to identify novel genes potentially involved in survival and apoptosis of follicular cells and, importantly, to determine the FSH-, estradiol-, and LH-target genes regulating its growth and function. Results provide new molecular evidence for the hypothesis that DF experience a reduction in FSH dependence but acquire increased LH dependence as they grow during the low FSH milieu of follicular waves.

Evidence That High Variation in Ovarian Reserves of Healthy Young Adults Has a Negative Impact on the Corpus Luteum and Endometrium During Estrous Cycles in Cattle

Abstract 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.

Maternal Undernutrition in Cows Impairs Ovarian and Cardiovascular Systems in Their Offspring

ABSTRACT 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.

Effects of Maternal Environment During Gestation on Ovarian Folliculogenesis and Consequences for Fertility in Bovine Offspring

Mammals such as cattle, swine, sheep and humans are born with a highly variable number of ovarian follicles and oocytes in the ovaries that dwindle during ageing and are never replenished. This variation in the ovarian reserve is reflected in the numbers of antral follicles in the ovaries at all ages after birth. As numbers of follicles in ovaries are determined during gestation, the role of maternal nutrition and health during gestation (at time of ovarian development in their foetuses) has been investigated as factors that may impact oogonia proliferation and thus follicle numbers post-natally. These studies have found that both nutrition and health impact numbers of follicles in their offspring. The idea that numbers of follicles and oocytes in ovaries impact fertility is a long-held belief in reproductive biology. This has recently been tested in cattle, and it has been shown that cows with a relatively high number of antral follicles in ovaries have higher pregnancy rates, shorter calving to conception intervals and fewer artificial inseminations during the breeding season compared with cows with a lower number of follicles, and similarly, heifers with many follicles had higher pregnancy rates than those with fewer follicles. Studies summarized in this review highlight the importance of the maternal environment during gestation in determining the size of the ovarian reserve in their offspring and also the contribution of the ovarian reserve to subsequent fertility in cattle.

Evidence that high variation in antral follicle count during follicular waves is linked to alterations in ovarian androgen production in cattle

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.