Exposure of dairy cows to high environmental temperatures and their lactation status impairs establishment of the ovarian reserve in their offspring.

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.

Genomic heritability and genome-wide association analysis of anti-Müllerian hormone in Holstein dairy heifers.

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.

Anti-Müllerian Hormone (AMH) and fertility management in agricultural species.

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.

A single ultrasound determination of ≥25 follicles ≥3 mm in diameter in dairy heifers is predictive of a reduced productive herd life.

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.

Concentration of anti-Müllerian hormone in dairy heifers is positively associated with productive herd life.

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.

Heritability and impact of environmental effects during pregnancy on antral follicle count in cattle.

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.

Low numbers of ovarian follicles ≥3 mm in diameter are associated with low fertility in dairy cows.

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.

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.

Does size matter in females? An overview of the impact of the high variation in the ovarian reserve on ovarian function and fertility, utility of anti-Müllerian hormone as a diagnostic marker for fertility and causes of variation in the ovarian reserve in cattle.

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.

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.

Inherent capacity of the pituitary gland to produce gonadotropins is not influenced by the number of ovarian follicles > or = 3 mm in diameter in cattle.

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.

Differential expression of signal transduction factors in ovarian follicle development: a functional role for betaglycan and FIBP in granulosa cells in cattle.

Ovarian follicles develop in groups yet individual follicles follow different growth trajectories. This growth and development are regulated by endocrine and locally produced growth factors that use a myriad of receptors and signal transduction pathways to exert their effects on theca and granulosa cells. We hypothesize that differential growth may be due to differences in hormonal responsiveness that is partially mediated by differences in expression of genes involved in signal transduction. We used the bovine dominant follicle model, microarrays, quantitative real-time PCR and RNA interference to examine this. We identified 83 genes coding for signal transduction molecules and validated a subset of them associated with different stages of the follicle wave. We suggest important roles for CAM kinase-1 and EphA4 in theca cells and BCAR1 in granulosa cells for the development of dominant follicles and for betaglycan and FIBP in granulosa cells of regressing subordinate follicles. Inhibition of genes for betaglycan and FIBP in granulosa cells in vitro suggests that they inhibit estradiol production in regressing subordinate follicles.

Differential expression of genes for transcription factors in theca and granulosa cells following selection of a dominant follicle in cattle.

Transcription factors inhibit or assist RNA polymerases in the initiation and maintenance of transcription; however, the cell specific expression and roles of transcription factors within bovine ovarian follicles during development are unknown. The aim of present study was to determine if the expression of transcription factors in theca and granulosa cells differ between the dominant and the largest subordinate follicles at different stages of the follicle wave. We used a bovine cDNA microarray to screen granulosa and theca cells from dominant and subordinate follicles for differential expression of genes coding for transcription factors. Expression was confirmed using reverse transcription polymerase chain reaction and differences in mRNA abundance further examined at Emergence, Selection and Dominance stages of the follicle wave. We have identified five genes encoding for transcription factors that have not been previously described in developing follicles with greater mRNA abundance in subordinate compared to dominant follicles. The genes (and their putative roles) are CEBP-beta (responsible for luteinization), SRF (cell survival), FKHRL1 (stimulates apoptosis), NCOR1 (modulation of the actions of the oestradiol receptor) and Midnolin (control of development via regulation of mRNA transport in cells).

Development of the "waveless" bovine model.

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.

Development of nonovulatory antral follicles in heifers: changes in steroids in follicular fluid and receptors for gonadotropins.

The objective of this study was to examine changes in steroid concentrations in follicular fluid (FF) and in gonadotropin receptors in nonovulatory follicles that develop after ovulation in heifers. Six groups of six to seven heifers were ovariectomized on days 3, 5, 7, 9, 11, and 13 of an estrous cycle. Follicles 6 mm or more in diameter were separated into two classes, estrogen-active (E-A) and estrogen-inactive (E-I). E-A follicles had higher concentrations of estradiol than progesterone and androgens in FF and a low incidence of atresia. E-I follicles had higher concentrations of progesterone or androgens than estradiol in FF and a high incidence of atresia. Diameters, numbers of granulosa cells, and capacities of granulosa cells and thecal tissue to bind [125I]iodo-hCG increased in E-A follicles from days 3-7. During this time, diameters, numbers of granulosa cells, concentrations of estradiol, and capacities of granulosa cells and thecal tissue to bind gonadotropins were greater in E-A than E-I follicles. On days 9, 11, and 13, each heifer had only a single E-I follicle. Compared with E-A follicles on day 7, E-I follicles on days 9, 11, and 13 were smaller and had less granulosa cells, less estradiol in FF, and a lower capacity to bind [125I]iodo-hCG. After day 7, E-A follicles were not observed until day 13. We conclude that at least two periods of growth and atresia of follicles occur between days 3-13 of an estrous cycle in heifers. One is during days 3-7 when a single E-A follicle 6 mm or more in diameter develops and all other E-I follicles regress. The other is between days 7 and 13 when the E-A follicle from day 7 becomes an E-I follicle and regresses, and another E-A follicle develops.

Development of antral follicles in cattle after prostaglandin-induced luteolysis: changes in serum hormones, steroids in follicular fluid, and gonadotropin receptors.

Changes in growth, concentrations of steroids in follicular fluid, and numbers of gonadotropin receptors in follicles 6 mm or more in diameter were examined 12, 24, 36, 48, 60, and 72 h after prostaglandin (PG)-induced luteolysis in heifers. Follicles with higher concentrations of estradiol than progesterone and androgens in follicular fluid (FF) were classified as estrogen active (E-A). Follicles with higher concentrations of progesterone or androgens than estradiol in follicular fluid were classified as estrogen inactive (E-I). Although E-A and E-I follicles were similar in size 12-72 h after PG treatment, before the preovulatory surge of LH, numbers of granulosa cells, estradiol concentrations, and capacities of follicles to specifically bind gonadotropins were markedly lower in E-I follicles. Most E-I follicles had histological signs of atresia, whereas E-A follicles did not. Sizes, concentrations of steroids in FF, and specific binding of gonadotropins to follicle cells did not change in E-A or E-I follicles 12-72 h after PG treatment but before the preovulatory surge of LH. However, diameters of E-A follicles were highly correlated with concentrations of estradiol and progesterone in FF and with capacities of granulosa and thecal cells to specifically bind [125I] iodo-hCG. In contrast, diameters of E-A follicles were highly negatively correlated with concentrations of testosterone in FF and with capacities of granulosa cells to specifically bind [125I] iodobovine FSH. We conclude that E-I follicles are atretic and E-A follicles are potentially ovulatory follicles. In addition, after a single injection of PG during an estrous cycle in heifers, the growth the differentiation of ovulatory follicles were not synchronized.

The differential effect of progesterone on concentrations of luteinizing hormone and follicle-stimulating hormone in heifers.

The effects of progesterone on LH and FSH concentrations in cattle are not clearly understood. The objective of this experiment was to determine the effect of various concentrations of progesterone on serum concentrations of LH and FSH in heifers. After estrous synchrony, animals were assigned to four different treatment groups (n = 10/group). Heifers received vaginal coils containing either 2% or 6.75% progesterone between days 8-10 (luteal phase) or days 17-18 (follicular phase) after estrus. Coils remained in place for 7 days. One day before removal of coils, all animals received an injection of prostaglandin-F2 alpha to cause regression of existing corpora lutea. Both the stage of the cycle and the percentage of progesterone in the coil affected serum concentrations of progesterone while the coils were in the vagina. Concentrations of FSH during progesterone treatment were not different for animals on different treatments. While coils were in the vagina, there was a significant increase in serum LH concentrations in heifers in the follicular phase given 2% progesterone coils, but no increase in LH was observed in the other three treatment groups. After removal of the progesterone coils, but before the preovulatory gonadotropin surges, LH increased but not FSH. The LH and FSH surges occurred synchronously. After the surges, FSH concentrations increased at a faster rate than LH. These data suggest that differential feedback mechanisms exist for LH and FSH in heifers. (Endocrinology 108: 568, 1981)

CI628 inhibits follicle-stimulating hormone (FSH)-induced increases in FSH receptors of the rat ovary: requirement of estradiol for FSH action.

Although estradiol (E2) alone does not increase receptors for FSH in granulosa cells, E2 priming before administration of FSH increases numbers of FSH receptors significantly compared with FSH alone. We hypothesized that if E2 is required for FSH to increase its own receptor, blocking estrogen action should prevent FSH-induced increases in FSH receptors. Five groups of hypophysectomized rats were injected sc with: saline at 0 h; the antiestrogen CI628 (1 mg) at -6 h; human FSH (hFSH, 2 micrograms) at 0 h; CI628 at -6 h, then hFSH at 0 h; and CI628 plus E2 (2 mg) at -6 h, then FSH at 0 h. Animals were decapitated at 0, 6, 12, or 24 h, and granulosa membrane receptors for FSH, LH, and nuclear receptors for E2 were measured. LH receptor levels increased only after administration of E2 before hFSH. Treatment with hFSH for 6 h increased numbers of FSH receptors 3-fold (P less than 0.01) without any increase in numbers of E2 receptors. At 12 and 24 h, hFSH increased numbers of FSH and E2 receptors 6- and 7-fold (P less than 0.01) over controls. CI628 prevented the hFSH-induced increases in FSH receptors at 6, 12, and 24 h. Administration of E2 concomitant with CI628 before hFSH significantly reversed the inhibitory effects of CI628 on hFSH-induced increases in FSH receptors. There were no changes in affinity of FSH or E2 receptors from 0 to 24 h. To determine whether E2 was acting on the adenylate cyclase system, the ability of hFSH to increase the content of cAMP in granulosa cells in each treatment group was determined. After an iv injection of hFSH, cAMP levels were similar in CI628- and saline-treated rats but had increased 6-fold (P less than 0.01) in hFSH or CI628 plus hFSH-treated animals. Thus, blocking hFSH-induced increases in FSH and E2 receptor appeared to have no effect on FSH stimulation of cAMP. In conclusion E2 appears to be required for FSH action, perhaps by acting within granulosa cells distal to the cAMP-adenylate cyclase system.

Changes in inhibin-like bioactivity in ovulatory and atretic follicles and utero-ovarian venous blood after prostaglandin-induced luteolysis in heifers.

The objectives of this study were to develop a bioassay for measuring inhibin bioactivity in untreated samples of bovine follicular fluid (BFF) and then examine changes in inhibin bioactivity in ovulatory and atretic follicles and utero-ovarian venous blood during the periovulatory period in heifers. A rat pituitary cell culture system was used to bioassay inhibin-like activity. Addition of 0.005 to 1 microliter untreated (whole), unfiltered charcoal-stripped, or filtered whole BFF to pituitary cultures caused a linear suppression of LHRH-induced FSH release but had no effect on LH secretion. Steroids in BFF did not suppress FSH secretion, since removal of steroids from BFF with charcoal did not remove the FSH-suppressive activity in BFF. Addition of ether extracts of BFF caused a slight but nonparallel suppression of FSH secretion; however, heating these extracts removed most of this suppressive activity. Removal of BFF from pituitary cultures completely restored the capacity of pituitary cultures to respond to LHRH. It was concluded that the inhibin bioassay was specific for detecting inhibin-like activity in fluids from individual follicles without interference of steroids. Within 12 h after a prostaglandin (PG) injection during the luteal phase of heifers, LH levels in serum increased 2- to 4-fold and remained at this level until the occurrence of the preovulatory gonadotropin surge. In contrast, FSH did not change before the gonadotropin surge. Inhibin bioactivity was measured in all follicles (greater than or equal to 6 mm) 0, 12, 24, 36, 48, 60, and 72 h after and in utero-ovarian venous serum 0, 24, and 36 h after PG-induced luteolysis. From 0-36 h after PG administration, inhibin-like activity increased linearly in presumed ovulatory follicles and utero-ovarian venous serum. Then, from 48-72 h after PG treatment, before the preovulatory LH surge, inhibin activity decreased in ovulatory follicles. After the surge but before ovulation, inhibin-like activity increased in ovulatory follicles. Inhibin-like activity in atretic follicles did not change after PG treatment and was lower in atretic than ovulatory follicles. Since a single hypothalamic releasing factor, LHRH, may control the secretion of LH and FSH, increased secretion of inhibin from preovulatory follicles before the preovulatory LH and FSH surges could account for the absence of a presurge rise in FSH in blood, as was observed for LH during this time in heifers. Diminished follicular production of inhibin during the gonadotropin surge could explain the preovulatory release of FSH along with LH during this time.

Evidence for autocrine or paracrine roles of alpha2-macroglobulin in regulation of estradiol production by granulosa cells and development of dominant follicles.

alpha(2)-Macroglobulin (alpha(2)-M) inhibits proteinases and modulates the actions of growth factors and cytokines. Despite the key roles proteinases, growth factors, and cytokines have in folliculogenesis, the role of alpha(2)-M in follicular development is unknown. Our objectives were to: 1) determine whether granulosa cells produce alpha(2)-M and have alpha(2)-M receptors, 2) examine the effect of alpha(2)-M on estradiol production by granulosa cells, 3) establish whether amounts of alpha(2)-M and alpha(2)-M receptors were altered during dominant nonovulatory follicle development, and 4) examine alpha(2)-M's mechanism of action. The results demonstrated that bovine granulosa cells contain 5.2- and 15-kb mRNAs and 720- and 500-kDa proteins that correspond, respectively, to sizes of mRNAs and proteins for alpha(2)-M and the alpha(2)-M receptor. Treatment of granulosa cells with alpha(2)-M resulted in a specific dose-responsive increase in estradiol production. Cell viability, cell number, and the amount of aromatase in granulosa cells were not altered by alpha(2)-M. Treatment of granulosa cells with factors that bind alpha(2)-M or its receptor did not mimic alpha(2)-M action. Although intrafollicular amounts of alpha(2)-M remained unchanged, amounts of alpha(2)-M receptor in granulosa cells were strongly inversely associated with concentrations of estradiol in dominant and subordinate follicles. Based on these results, we concluded that alpha(2)-M may have autocrine or paracrine roles in granulosa cells potentially important for regulation of estradiol production and development of dominant follicles.