Activin B is produced early in antral follicular development and suppresses thecal androgen production

Little is known about the role of activin B during folliculogenesis. This study investigated the expression levels of activin/inhibin subunits (βA, βB, and α), steroid enzyme, and gonadotrophin receptors in theca (TC) and granulosa cells (GC) by QPCR and activin A and B and inhibin A protein levels in follicular fluid (FF) of developing sheep follicles during estrus and anestrus. The effect of activin B on androgen production from primary TC cultures in vitro was also assessed. During folliculogenesis, in anestrus and estrus, FF activin B concentrations and thecal and GC activin βB mRNA levels decreased as follicle diameter increased from 1-3 to >6 mm regardless of estrogenic status. Estrogenic preovulatory follicles had reduced concentrations of FF activins B and A, and TC and GCs expressed higher levels of activin βA mRNA at 3-4 mm, and TCs more inhibin α mRNA at >4 mm stages of development compared with nonestrogenic follicles. Activin B decreased androstenedione production from primary TCs in vitro, an effect blocked by inhibin A. Thus, sheep follicles 1-3 mm in diameter contained high FF levels of activin B, which decreased as the follicle size increased, and, like activin A, suppressed thecal androgen production in vitro, an effect blocked by inhibin. Furthermore, the theca of large estrogenic follicles expressed high levels of inhibin α and activin βA mRNA suggesting local thecal derived inhibin A production. This would inhibit the negative effects of thecal activins B and A ensuring maximum androgen production for enhanced estradiol production by the preovulatory follicle(s).     

Dietary unsaturated fatty acids influence preovulatory follicle characteristics in dairy cows

Dietary unsaturated fatty acids (UFAs) have been implicated in several reproductive processes in dairy cows through a variety of mechanisms. This study examined the effects of periparturient supplementation of rumen bypass fats low or high in proportion of UFAs (oleic and linoleic) on preovulatory follicle characteristics. Forty-two 256-day pregnant dairy cows were divided into three groups and were fed a control diet (n=14) or supplemented with fats either low (LUFA; n=14) or high (HUFA; n=14) in UFAs. At 14-15 days following behavior estrus, the cows received a prostaglandin F(2)(alpha) injection and 48 h later >7 mm follicles were aspirated. Progesterone (P(4)), androstenedione (A(4)), and estradiol (E(2)) were determined in the follicular fluid. Out of 75 follicles, 37 follicles that were aspirated between 55 and 70 days post partum were regarded as E(2)-active follicles (E(2)/P(4) ratio >1) and subjected for further analysis. The diameter of preovulatory follicles was greater in cows fed HUFA than in those fed control or LUFA. The concentrations and content of A(4) and E(2) in follicles and E(2)/P(4) ratio were higher in the HUFA group than in the other two groups. The P450 aromatase mRNA expression in granulosa cells that were collected from the aspirated preovulatory follicles was also higher in the HUFA cows than in the other groups. A significant correlation was observed between E(2) concentrations in preovulatory follicles and E(2) concentrations in plasma at aspiration. In conclusion, dietary UFA increased the size of and elevated steroid hormones in preovulatory follicles, which may be beneficial to consequent ovarian function.     

Induction of persistent ovarian follicular structures following administration of progesterone near the onset of estrus in dairy cattle

The objective of this study was to determine if progesterone administered near the onset of estrus in dairy cows would block the preovulatory surge of LH and result in subsequent persistence of ovarian follicular structures. Following synchronization of estrus with prostaglandin F2 alpha, 20 multiparous, non-lactating Holstein cows were randomly assigned into three groups: 1 ml ethanol administered i.m. at 12-h intervals for 24 h (n = 6; group 1); 1 mg progesterone administered i.m. at 12-h intervals for 24 h (n = 7; group 2); 2.5 mg progesterone administered i.v. at the onset of standing estrus (n = 7; group 3). Ovarian structures were palpated per rectum on the day of estrus and twice weekly for 14 d. Blood was collected every 2 h from onset of standing estrus for 30 h, and concentrations of LH and progesterone were determined. Numbers of cows diagnosed with persistent follicles 10 d after estrus were 1 of 6 (group 1), 2 of 7 (group 2), and 5 of 7 (group 3). The preovulatory surge of LH did not occur during the sampling period (30 h) for 1 of 6, 7 of 7 and 5 of 7 cows, for groups 1, 2, and 3, respectively, and mean serum concentrations of LH were higher in group 1 than in groups 2 and 3. Serum concentration of progesterone (ng/ml) was higher in group 1 (1.9 +/- .4) than in groups 2 (.9 +/- .4) and 3 (.9 +/- .4) 10 d following estrus. Blocking the preovulatory surge of LH with exogenous progesterone resulted in persistence of ovarian follicles.     

Effect of calcium soaps of fatty acids and administration of somatotropin on milk production, preovulatory follicular development, and plasma and follicular fluid lipid composition in high yielding dairy cows

The effect of fat and bovine somatotropin (bST) on preovulatory follicular hormones and lipids was evaluated by feeding cows for 150 d from parturition a control diet, a control diet plus 0.55 kg/d of calcium soaps of fatty acids, or a control diet with 500 mg of bST injected every 14 d. Fourteen days after a synchronized or natural estrus, cows were injected with a PGF2 alpha analogue; 48 h later, follicular fluid from all ovarian follicles > 8 mm was aspirated. Cows fed fat or injected with bST produced more milk and milk solids than did control cows, and cows on the bST treatment lost more body condition after calving than did cows on the other treatments. Both treatments changed the proportion of estradiol-active follicles (> 400 ng of estradiol/ml of follicular fluid) and the correlation between follicular fluid estradiol concentration and the total number large follicles per cow. In follicles aspirated between 60 and 90 DIM the percentage of estradiol-active follicles was 67, 40, and 0 for cows on the control, calcium soaps of fatty acids, and bST treatments, respectively. After 90 DIM, no differences existed between treatments in the percentage of estradiol-active follicles. Estradiol concentration in follicular fluid was correlated with DIM at follicle aspiration (r = 0.51). The proportion of oleic acid in free fatty acids in plasma at 50 DIM was lower in control cows and was lower in follicular fluid of estradiol-active follicles. Both calcium soaps of fatty acids and bST had a considerable effect on follicular development and activity and the composition of fatty acids in follicles.     

Follicular development after administration of adrenocorticotropin to nonlactating Holstein cows

The effect of chronic administration of adrenocorticotropin on ovarian follicular development was studied. Twelve nonlactating Holstein cows received either 100 IU adrenocorticotropin (n = 6) or saline (n = 6) at 12-h intervals, commencing d 16 and continuing until d 23 of an induced estrous cycle (estrus = d 0). Cows were slaughtered on d 24, ovaries collected, and number of visible antral follicles recorded. Estradiol-17 beta, androstenedione, and testosterone in follicular fluid, and luteinizing hormone and follicle-stimulating hormone receptors in follicular tissue of the largest follicles were determined. Largest follicles were classified as ovulatory or nonovulatory based on the estrogen to androgen ratio. One cow treated with adrenocorticotropin, but none treated with saline, had ovulated by slaughter. The numbers of small, medium, and large antral follicles were 0, 1, and 5 for cows treated with adrenocorticotropin and 0, 1, and 6 for cows treated with saline. Follicular diameter (15.0 +/- 1.0 versus 14.0 +/- 2.0 mm) and follicular fluid volume (2.9 +/- .8 versus 2.2 +/- .5 ml) of the largest follicle in cows treated with adrenocorticotropin or saline were not different. No differences were found in the number of luteinizing hormone and follicle stimulating hormone receptors nor in the proportion of ovulatory versus nonovulatory follicles between treatments. We conclude that adrenocorticotropin administered at 100 IU every 12 h during the follicular phase does not significantly alter follicular development in the nonlactating dairy cow.     

Relationships between FSH patterns and follicular dynamics and the temporal associations among hormones in natural and GnRH-induced gonadotropin surges in heifers

Preovulatory LH and FSH surges and the subsequent periovulatory FSH surge were studied in heifers treated with a single injection of GnRH (100 microg, n = 6) or saline (n = 7). Blood samples were collected every hour from 6 h before treatment until 12 h after the largest follicle reached > or =8.5 mm (expected beginning of follicular deviation). The GnRH-induced preovulatory LH and FSH surges were higher at the peak and shorter in duration than in controls, but the area under the curve was not different between groups. The profiles of the preovulatory LH and FSH surges were similar within each treatment group, suggesting that the two surges involved a common GnRH-dependent mechanism. Concentrations of FSH in controls at the nadir before the preovulatory surge and at the beginning and end of the periovulatory surge were not significantly different among the three nadirs. A relationship between variability in the periovulatory FSH surge and number of 5.0 mm follicles was shown by lower FSH concentrations during 12-48 h after the beginning of the surge in heifers with more follicles (11.0 +/- 1.0 follicles (mean+/-s.e.m.) n = 7) than in heifers with fewer follicles (5.7 +/- 0.4, n = 6). This result was attributed to increased FSH suppression from increased numbers of follicles reaching 5.0 mm. Grouping of heifers into those with longer vs shorter intervals from a 4.5 mm to an 8.5 mm largest follicle did not disclose any relationship between length of the interval and FSH characteristics (e.g. profile of surge, area under curve, FSH concentrations at specific events). The hypothesis of a relationship between variation in the periovulatory FSH surge and variation in follicular dynamics was supported for the number of 5.0 mm follicles but not for the hour the largest follicle reached 8.5 mm. Thus, the expected time of follicle deviation was not altered by the extensive variation in the wave-stimulating FSH surge.     

E- and N-cadherin expression and distribution during luteinization in the rat ovary

Cadherins, a family of Ca(2+)-dependent cell adhesion molecules, play an important role in ovarian tissue remodelling processes. The aim of this study was to examine the expression pattern of E- and N-cadherin in rat preovulatory follicles, luteinizing follicles and corpora lutea. Immature female rats were treated with equine chorionic gonadotrophin (eCG) to promote preovulatory follicle development. At 48 h after eCG treatment, the rats were injected with an ovulatory dose of hCG. Ovaries were analysed by western blot analysis and immunofluorescence for E- and N-cadherin expression at 48 h after eCG injection, and at 24 and 72 h after hCG injection. Ovaries of cyclic adult rats were examined to assess whether the changes in the expression pattern of cadherin were in agreement with those of the gonadotrophin-treated rats. Finally, expression of E-cadherin in luteinizing granulosa cells in vitro was assessed by RT-PCR and western blot analysis. Immunofluorescence results indicate that E-cadherin is expressed in the theca-interstial cells surrounding preovulatory follicles. N-cadherin expression is prominent in the membrana granulosa of these follicles. The initiation of luteinization with hCG leads to a decreased expression of N-cadherin in the membrana granulosa, whereas expression of E-cadherin starts within the luteinizing follicle. Both cadherins are prominently expressed in the fully formed corpus luteum at 72 h after hCG treatment. Immunofluorescence results revealed that the patterns of E- and N-cadherin expression in the gonadotrophin-treated rats were similar to those of the cyclic adult rats. Western blot analysis reflected similar changes for N-cadherin in the ovaries of both the cyclic adults and gonadotrophin-treated rats; however, they were different in E-cadherin expression. The expression of E-cadherin mRNA and protein was induced in vitro in luteinized granulosa cells. These results support the hypothesis that modulation of cadherin expression is an integral component of remodelling processes, including corpus luteum formation, in the ovary. The results also indicate that expression of E- and N-cadherin in granulosa-lutein cells appear to be under hormonal control.     

Endocrine milieu and developmental dynamics of ovarian cysts and persistent follicles in postpartum dairy cows

Ovarian follicular cysts and persistent follicles are follicular pathologies involved in reduced fertility of dairy cows. Two separate experiments were performed on high-yielding Holstein cows to characterize ovarian cyclicity and evaluate the developmental dynamics of follicle pathologies postpartum. In experiment 1, 58 cows were monitored by ultrasonography twice weekly from d 18±1 to 69±2 postpartum. First ovulation occurred 38±3, 27±2, 20±1, and 25±3 d postpartum in cows with 1 cycle (n=11), 2 cycles (n=21), 3 cycles (n=13), and 4 cycles (n=7), respectively. Follicular pathologies were developed in cows that were either acyclic (n=6) or had 1 or 2 cycles, but not in cows with more than 2 cycles. In experiment 2, 47 cows were monitored twice weekly from 10 d postpartum to second ovulation. Follicles ≥17 mm in diameter in 2 consecutive scans were aspirated, and concentrations of various hormones were measured. Cows were defined as cyclic (n=30; 64%) or with the potential to develop follicular pathology (n=17; 36%). Aspirated follicles (n=27) were classified into 3 main groups based on follicular growth rate, follicular diameter, and ovarian activity before and after follicular aspiration. Dominant follicles (n=4) were defined as large follicles (20 mm in diameter) with growth rate ≤1 mm/d and normal ovarian activity. Persistent follicles (n=6) had the same growth rate and diameter as the dominant follicles, but persisted at the same diameter for ≥10 d. Ovarian cysts (n=17) were defined as the largest follicular structures (19 to 32 mm in diameter), with abnormal growth rate (>1 mm/d) and abnormal ovarian activity. Single or turnover cysts did not differ in their growth parameters and were therefore combined and further classified according to follicular-fluid hormone concentrations. Estradiol-dominant cysts (n=7) were characterized by normal estradiol (284 to 659 ng/mL) and progesterone (20 to 113 ng/mL) concentrations, similar to those of the dominant follicle (554 to 993 ng/mL and 44 to 106 ng/mL, respectively). Progesterone-dominant cysts (n=5) were characterized by low estradiol (0.06 to 330 ng/mL) and high progesterone (586 to 3,288 ng/mL) concentrations. Low-steroidogenic active cysts (n=5) were characterized by low concentrations of both estradiol (23 to 61 ng/mL) and progesterone (17 to 205 ng/mL). Characterization of spontaneously forming cysts might enable definition of the formation of ovarian follicular pathologies in postpartum cows.     

Anogenital distance is associated with postpartum estrous activity,intensity of estrous expression,ovulation, and progesterone concentrations in lactating Holstein cows

The objectives of this retrospective observational study were to determine the associations of anogenital distance (AGD) with (a) postpartum estrous activity, (b) diameter of the preovulatory follicle, (c) intensity of estrous expression, (d) postestrus ovulation, (e) corpus luteum (CL) size, and (f) concentrations of progesterone at estrus and on d 7 after estrus. Lactating Holstein cows (n = 178; 55 primiparous, 123 multiparous) were enrolled into the study during the first postpartum week. All cows were continuously monitored by a pedometer-based automated activity monitoring (AAM) system for estrus. Postpartum estrous activity was assessed using the AAM estrus alerts, in which cows with at least one true estrus alert (i.e., a relative increase in steps from each cow's baseline detected by the AAM and the presence of at least one follicle >15 mm, a CL <20 mm, or no CL detected by ultrasound) by the first 50 d in milk (DIM) were considered to have commenced estrous activity. At the estrus alert >60 DIM, ovulation was determined by ultrasound at 24 h, 48 h, and 7 d after estrus, and blood samples were collected at estrus alert and on d 7 after estrus for progesterone analysis. The AGD was measured from the center of the anus to the base of the clitoris and classified as either short- or long-AGD using 2 cut-points of 148 mm (predictive of the probability of pregnancy to first insemination; short-AGD, n = 115; long-AGD, n = 63) and 142 mm (the median AGD; short-AGD, n = 90; long-AGD, n = 88). Regardless of the cut-point used, early postpartum estrous activity by 50 DIM (67 vs. 54%), duration of estrus (11.6 vs. 9.7 h), and preovulatory follicle diameter (20 vs. 19 mm) were greater in short-AGD than in long-AGD cows. Increased peak of activity at estrus in short-AGD cows (354 vs. 258% mean relative increase) was affected by an interaction between AGD and parity in which multiparous long-AGD cows had lesser relative increase in activity than primiparous cows (217 vs. 386%, respectively). Mean progesterone concentration at estrus was lesser in short-AGD (0.47 vs. 0.61 ng/mL) than in long-AGD cows. The ovulatory response at 24 h did not differ, but at 48 h (91 vs. 78%) and on d 7 after estrus (97 vs. 84%) it was greater in short-AGD cows. Although CL diameter on d 7 after estrus did not differ, short-AGD cows had greater progesterone concentration 7 d after estrus than long-AGD cows (4.1 vs. 3.2 ng/mL, respectively). In conclusion, greater proportions of short-AGD cows commenced estrous activity by 50 DIM, had larger preovulatory follicles, exhibited greater duration of estrus, had reduced progesterone concentration at estrus, had greater ovulation rates and progesterone concentration 7 d after estrus compared with long-AGD cows, with no difference in CL size between AGD groups. Because all the differences in physiological characteristics of short-AGD cows reported herein favor improved reproductive outcomes, we infer that these are factors contributing to improved fertility reported in short-AGD cows compared with long-AGD cows.     

Effects of whole cottonseed diet and recombinant bovine somatotropin on ovarian follicles in lactating dairy cows

The effects of whole cottonseed (WCS) in the diet and the administration of bovine somatotropin (bST) on ovarian follicular dynamics and plasma progesterone (P4) concentrations were examined in cows during a period of synchronized follicular growth. Lactating Holstein cows (n = 28) were randomly assigned to treatments in a 2 x 2 factorial arrangement. Diets consisted of WCS (15% of dry matter) or no WCS, and bST at a dose of 0 or 208 mg/14 d. Dietary treatments began within 24 h of calving and bST treatments began within 7 d postpartum. Cows received GnRH at 65 +/- 3 d postpartum (d 0), PGF2alpha, (d 7), a second dose of GnRH (d 9), and were inseminated 16 h later (d 10). Ovarian changes were monitored daily by ultrasonography from d 0 to 9. On d 9,93% of cows had a preovulatory follicle and 86% ovulated. For Class 2 (6 to 9 mm) follicles, a diet x bST interaction was detected, with bST stimulating Class 2 follicles in cows fed WCS, but not in cows on the control diet. Neither diet nor bST affected numbers of Class 1 (2 to 5 mm) or Class 3 (> or = 10 mm) follicles or sizes of the subordinate and dominant follicles. During the luteal phase of the cycle, lactating cows fed WCS tended to have elevated concentrations of plasma P4, whereas bST was without effect. Plasma concentrations of high-density lipoprotein cholesterol were increased in cows fed WCS. Number and diameter of corpora lutea did not differ among treatments.     

The FecB (Booroola) gene acts at the ovary: in vivo evidence

The aim of this study was to differentiate between pituitary and ovarian actions of the FecB gene by measuring the ovarian response to a standardized treatment with gonadotrophins designed to mimic the changes in FSH and LH that occur in the follicular phase of the ovarian cycle in ewes, with (Fec(B/-), n=6) and without (Fec(+/+), n=9) the gene, that were rendered hypogonadotrophic by pretreatment with a potent antagonist of GnRH. Ewes with ovarian autotransplants were used to facilitate the assessment of follicular function by the collection of ovarian venous blood and ultrasonography. The gonadotrophin regimen resulted in concentrations of FSH and LH that were similar to concentrations found in a normal cycle and did not differ between genotypes. Follicular development and ovulation occurred in all animals, and patterns of secretion of oestradiol, androstenedione and inhibin A were normal. Despite these endocrine similarities, the antral follicle population stimulated by FSH infusion retained the characteristic genotypic difference with the ovaries of Fec(+/+) animals containing a range of follicle sizes with decreasing proportions of small (<3.5 mm in diameter) and medium (3.5-4.5 mm in diameter) follicles as well as large follicles (> or =4.5 mm in diameter), whereas the ovaries of Fec(B/-) ewes contained no follicles of >4.5 mm in diameter. This genotypic difference was retained after ovulation with gene carriers having more preovulatory follicles/corpora lutea (3.8+/-0.3) of a smaller diameter (5.3+/-0.3 mm) than did non-gene carriers (1.7+/-0.3; 11.4+/-0.9 mm; P<0.05). As ewes carrying the FecB gene mutation were able to ovulate more follicles than non-gene carriers, despite identical concentrations and patterns of FSH and LH stimulation, the results of this study support the hypothesis that the FecB gene acts at the ovary to enhance ovarian sensitivity to gonadotrophic stimulation.     

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.     

Role of matrix metalloproteinase 2 in the ovulatory folliculo-luteal transition of ewes

Tissue dissolution and remodelling are associated with the processes of rupture of the ovulatory follicle and formation of the corpus luteum. Matrix metalloproteinase 2 (MMP-2) belongs to a family of endopeptidases that cleave extracellular proteins; its primary substrate is the lattice network of basement membranes that support epithelial cells and endothelium. The aim of this study was to ascertain a putative regulatory role of MMP-2 relevant to the folliculo-luteal transformation in ewes. Luteal regression and the preovulatory surge of gonadotrophins were synchronized by administration of PGF(2alpha) and GnRH on days 14.0 and 15.5 of the oestrous cycle, respectively. Dominant antral follicles present during pro-oestrus consistently ovulate approximately 24 h after GnRH administration. Normal IgG or a bioactivity-neutralizing MMP-2 monoclonal antibody was injected into the antral cavity of preovulatory follicles at 8 h after GnRH administration. Jugular blood samples were obtained for serum progesterone analysis and ovaries were removed for light microscopic morphometry on day 8. A definitive ovulation stigma was evident in control ewes. The antra of ruptured follicles had largely been supplanted with luteal tissue. In contrast, the ovarian surface contiguous with follicles injected with anti-MMP-2 was smooth and undisturbed, which is indicative of a failure of ovulation. Luteinized unruptured follicles were filled with (entrapped) fluid. Corpora lutea of control animals contained numerous connective tissue projections that provided a framework for cellular migration and angiogenesis. Luteal tissues that surrounded the cavity of antibody-treated follicles lacked trabeculae and were deficient in blood vessels. Systemic venous progesterone concentrations were lower in ewes with a luteinized unruptured follicle compared with those with a corpus luteum. It is proposed that MMP-2 is a mediator of ovulation and luteal development.     

Changes in the localization of MHC class II positive cells in hen ovarian follicles during the processes of follicular growth, postovulatory regression and atresia

The aim of this study was to determine the changes in the population of major histocompatibility complex class II positive (MHC-II(+)) cells in ovarian follicles during the processes of follicular growth, postovulatory regression and follicular atresia in hens. Cryostat sections of ovarian stroma containing cortical follicles, small white follicles, the largest (F(1)) and third largest (F(3)) preovulatory follicles, postovulatory and atretic follicles of laying hens were prepared. The sections were immunostained for MHC-II molecules using mouse anti-chicken MHC-II monoclonal antibody and positive cells were counted using a computer-assisted image analyser under a light microscope. MHC-II(+) cells were localized in the theca layer of normally growing follicles including cortical follicles, small white follicles and F(3) and F(1) preovulatory follicles, whereas they were found in both the theca and granulosa layers in postovulatory and atretic follicles. The frequency of MHC-II(+) cells in the theca layer was significantly increased during follicular growth from cortical follicles to F(3) preovulatory follicles. Although the population of MHC-II(+) cells did not differ between F(3) and F(1) preovulatory follicles, it increased significantly in postovulatory follicles (P < 0.01). The population of MHC-II(+) cells was significantly greater in the theca layer of atretic follicles than in non-atretic follicles (P < 0.01). These results indicate that the antigen-presenting function via MHC-II increases in association with follicular growth. A marked increase in MHC-II(+) cells indicates that these cells may be involved in regression of postovulatory and atretic follicular tissues.     

Heat stress diminishes gonadotropin receptor expression and enhances susceptibility to apoptosis of rat granulosa cells

Heat stress inhibits ovarian follicular development in mammalian species. We hypothesized that heat stress inhibits the function of follicular granulosa cells and suppresses follicular development. To test this, immature female rats were injected with pregnant mare serum gonadotropin (PMSG) at 48 h after the start of temperature treatment (control: 25 degrees C, 50% RH; heat stress: 35 degrees C, 70% Relative Humidity). The ovaries and granulosa cells of follicles at different developmental stages were analyzed for gonadotropin receptor levels and aromatase activity; estradiol levels were measured in follicular fluid. Before injection, heat stress diminished only the amount of FSH receptor on granulosa cells of antral follicles. During PMSG-stimulated follicular development, heat stress strongly inhibited gonadotropin receptor levels and aromatase activity in granulosa cells, and estradiol levels in the follicular fluid of early antral, antral and preovulatory follicles. To examine apoptosis and mRNA levels of bcl-2 and bax in granulosa cells, follicles harvested 48 h after PMSG injection were cultured in serum-free conditions. Heat-stressed granulosa cells showed a time-dependent increase in apoptosis. The bcl-2 mRNA levels were similar in control and heat-stressed granulosa cells; bax mRNA levels were increased in heat-stressed granulosa cells. According to these results, heat stress inhibits expression of gonadotropin receptors in granulosa cells and attenuates estrogenic activity of growing follicles, granulosa cells of heat-stressed follicles are susceptible to apoptosis, and the bcl2/bax system is not associated with heat-stress-induced apoptosis of granulosa cells. Our study suggests that decreased numbers and function of granulosa cells may cause ovarian dysfunction in domestic animals in summer.     

Localization and temporal regulation of tissue inhibitors of metalloproteinases 3 and 4 in bovine preovulatory follicles

The matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) are potential regulators of the focalized extracellular matrix degradation required for ovulation. The objectives of the present study were to determine localization and temporal regulation of TIMP-3 and TIMP-4 mRNA and protein in bovine preovulatory follicles. Ovaries containing preovulatory follicles were collected at 0, 12 and 20 h after GnRH injection for real-time PCR quantification of TIMP-3 and TIMP-4 mRNAs and immunohistochemical localization studies. Additional samples collected at 0, 6, 12, 18 and 24 h post GnRH injection were subjected to Western analysis to determine temporal changes in TIMP-3 and TIMP-4 proteins in the apex and base of preovulatory follicles. Results indicate the gonadotropin surge regulates TIMP-3 and TIMP-4 expression. TIMP-3 and TIMP-4 mRNAs increased within 12 h after GnRH injection. TIMP-3 protein was localized to granulosal and thecal layers of preovulatory follicles and adjacent ovarian stroma, whereas TIMP-4 immunoreactivity was localized to granulosal and thecal cells and ovarian blood vessels. Amounts of TIMP-3 and TIMP-4 proteins in the follicular apex peaked within 12 h post GnRH injection and subsequently declined by 24 h. However, amounts of TIMP-3 and TIMP-4 proteins in the base were not elevated after GnRH administration. Results demonstrate that mRNA and protein for both TIMP-3 and TIMP-4 are increased in bovine preovulatory follicles following the gonadotropin surge. Coordinate expression of TIMPs and MMPs may help regulate the extracellular matrix remodeling characteristic of the ovulatory process.     

Plasma FSH, inhibin A and inhibin isoforms containing pro- and -alphaC during winter anoestrus, spring transition and the breeding season in mares

Ten mares were studied from February (winter anoestrus) to their second ovulation in the breeding season to investigate the relationship between resumption of ovarian cyclicity in the spring and circulating concentrations of FSH, inhibin A and inhibin isoforms containing pro- and -alphaC immunoreactivity. An additional four mares were studied during one oestrous cycle. Growth and regression of ovarian follicles were monitored by transrectal ultrasonography. The frequency of blood sampling varied from three times a week to once a day, depending on the follicular activity present. Concentrations of FSH, oestradiol, inhibin A and pro- and -alphaC isoforms were low during deep winter anoestrus when minimal follicular activity was present in the ovaries. During spring transition, an increase in FSH concentration preceded the emergence of each follicular wave. Concentrations of inhibins were significantly higher (P < 0.05) during growth of anovulatory follicles in spring transition than during winter anoestrus. Plasma concentrations of oestradiol and inhibin A were significantly higher (P < 0.001, P < 0.05, respectively) during the growth of preovulatory follicles than during the growth of transitional anovulatory follicles, but concentrations of inhibin pro-alphaC isoforms did not differ between the two types of follicle. During the oestrous cycle, there was a significant inverse relationship (P < 0.001) between concentrations of FSH and the inhibins. Plasma inhibin pro-alphaC isoforms, but not inhibin A, reached a peak on the day of ovulation. The results strongly indicate that FSH regulates growth of spring anovulatory and preovulatory follicles. Inhibins are likely to contribute to negative feedback on the release of FSH from the pituitary gland both during the transitional period and the breeding season in mares.     

Contraception by induction of luteinized unruptured follicles with short-acting low molecular weight FSH receptor agonists in female animal models

During recent decades minor innovative drugs have been developed for the female contraceptive market and they all contain steroidal progestagens (and estrogens) that act centrally and have side effects that can be attributed to this central action. In this study, we present an innovative tissue-specific approach for female contraception by low molecular weight (LMW) FSH receptor (FSHR) agonists, which interact with the FSHR that is dominantly expressed in the granulosa cells. The oral administration of LMW FSHR agonists with a short circulation time, induced formation of luteinized unruptured follicles (LUFs) from the Graafian follicles, thereby preventing the release of the oocyte. The short-acting LMW FSHR compounds were fully agonistic to FSHR (EC(50)=4-5 nM). In an isolated mouse follicle culture, a short incubation period (2 h) resulted in inhibition of follicular rupture, where continuous incubation induced follicle growth. Pharmacokinetics after oral administration showed a surge-like exposure in rats and monkeys. Oral administration of short-acting LMW FSHR agonists inhibited ovulation at 10 mg/kg in rats and guinea pigs by generating LUFs without affecting cyclicity. Also, inhibition of follicular rupture was shown to be reversible within one cycle. Finally, LUFs were induced without affecting the hormonal cyclicity in cynomolgus monkeys, a mono-ovulatory species. In healthy women LUF formation occurs naturally, with a LUF acting as corpus luteum that produces enough progesterone to ensure normal menstrual cyclicity. Together with the presented data this indicates that the innovative approach with short-acting LMW FSHR agonists could lead to oral contraception for females at the ovarian level.     

Ovarian follicular activity in lactating Holstein cows supplemented with monensin

The objective of this study was to determine effects of monensin on ovarian follicular development and reproductive performance in postpartum dairy cows. Forty-eight multiparous Holstein cows were randomly assigned to receive either a control total mixed ration (n = 24) or the same diet plus 22 mg of monensin/kg (n = 24) from 21 d before anticipated calving until cows were either confirmed pregnant or were >180 d postpartum. Monensin had no effect on development of the first dominant follicle postpartum or the numbers of class 1 (3 to 5 mm), 2 (6 to 9 mm), or 3 (10 to 15 mm) follicles. Control cows had more class 4 (>15 mm) follicles at 10 to 13 d postpartum than cows in the monensin group. The first dominant follicle postpartum ovulated, regressed, or became cystic unrelated to differences between diets. However, the first ovulation postpartum occurred earlier in monensin-fed cows than in the control group (27.2 +/- 2.1 d vs. 32.4 +/- 1.5 d), with no dietary effects on the diameter of the ovulating follicle. Similarly, treatments did not differ in the proportion of cows with 2 or 3 waves of ovarian follicular development per cycle, nor in the number of follicles of all classes during the breeding period. Times of ovulation following treatment with prostaglandin F2alpha were not different between dietary groups. Pregnancy rates after timed artificial insemination were similar between diets. Supplementation with monensin resulted in a shorter postpartum interval to first ovulation but did not affect other reproductive measures in healthy, lactating dairy cows.     

Novel method for intrafollicular pressure measurements in the rat ovary: increased intrafollicular pressure after hCG stimulation

The ovulatory process in the rat comprises a period of about 12-15 h, from the time of the preovulatory LH surge to follicular rupture and extrusion of the oocyte. Follicular rupture is most likely caused, at least in part, by decreased tensile strength at the follicular apex due to degradation of collagen fibres of the extracellular matrix. It has been debated whether changes in intrafollicular pressure occur during the ovulatory process and whether such changes facilitate rupture of the follicle. In the present study, rats were primed with equine chorionic gonadotrophin (eCG, 10 iu) followed by hCG (10 iu) 48 h later. The intrafollicular pressure in the preovulatory follicle was recorded during 1 h at distinct time phases of the ovulatory process by use of an active servo-null pressure system based on the proportionality between electrical resistance and pressure within the tip of an inserted micropipette. The basal intrafollicular pressure was 16.6 +/- 1.0 mm Hg at the preovulatory phase (48 h after eCG) and increased gradually throughout the ovulatory process to 21.4 +/- 2.4 mm Hg at 4-7 h after hCG (mid-ovulatory phase) and 23.9 +/- 1.9 mm Hg at 8-12 h after hCG (late ovulatory phase; significantly higher (P < 0.01) than the preovulatory phase). Short-term peaks of increased pressure, possibly representing contractility, were not detected in follicles of the preovulatory phase, but were seen in most follicles of the mid- and late ovulatory phases. The mean amplitude of the short-term pressure increases was 12.3 +/- 3.2 mm Hg and the increases occurred at intervals of 24.7 +/- 3.6 s. These short-term increments in intrafollicular pressure were still present after hysterectomy had been performed. The wall tension index was calculated by measuring the follicular size and estimating the thickness of the follicle wall. The index increased from 93.9 +/- 13.3 at the preovulatory phase to 207.3 +/- 47.7 (mid-ovulatory phase) and to significantly higher values at the late ovulatory phase (320.9 +/- 33.5). In conclusion, this study shows that there is an increase in intrafollicular pressure in the ovulating follicle of the rat ovary during the late stages of the ovulatory process, and that short-term increases in intrafollicular pressure occur during the late phase of the ovulatory process. These changes in pressure may be essential for follicular rupture to proceed normally.