Oocyte maturation inhibitor, inhibin and steroid concentrations in porcine follicular fluid at various stages of the oestrous cycle

Oocyte maturation inhibitor (OMI), inhibin, progesterone and oestradiol 17 beta concentrations were measured in fluid collected from small (less than 3 mm), medium size (3-6 mm) and large (greater than 6 mm) porcine ovarian follicles, which were obtained on Days 5, 10, 15 and 18 of the oestrous cycle and at 24 h after the onset of oestrus. Concentrations of OMI decreased with increasing follicle diameter (P less than 0.05), independent of the stage of the oestrous cycle. Concentrations of inhibin showed a tendency to decrease with increasing follicle diameter on Days 10, 15 and 18, but not on Day 5 of the cycle. Concentrations of OMI and inhibin in the largest follicles were low before the onset of oestrus, and were essentially unaltered 24 h later. A positive correlation was found between OMI and inhibin concentrations, whereas the correlation between inhibin concentration and log (progesterone concentrations) was negative.     

In situ analysis of the changes in expression of ovarian inhibin subunit mRNAs during follicle recruitment after ovulation in pigs

In situ hybridization was used on frozen tissue sections with digoxigenin-labelled antisense riboprobes to inhibin/activin alpha and beta(A) subunits to determine whether inhibin/activin subunit mRNA expression was associated with development of growing, steroidogenically active follicles during follicle recruitment after ovulation. Cell proliferation-associated nuclear antigen Ki-67 protein and cytochrome P450 aromatase expression in granulosa cells were determined immunohistochemically and used as markers for granulosa cell proliferation and steroidogenesis, respectively, on days 3, 5 and 7 after the onset of oestrus. The amounts of inhibin/activin alpha and beta(A) subunit mRNA and P450 aromatase protein were greater (102, 93, and 238%, respectively; P < 0.05) in medium than in small non-atretic follicles and were positively correlated with Ki-67 and with each other. Inhibin/activin alpha and beta(A) mRNA, P450 aromatase, and Ki-67 in granulosa cells were reduced by 66-83% (P < 0.001) in atretic follicles compared with non-atretic follicles. In addition, inhibin/activin alpha and beta(A) mRNA and P450 aromatase in small (1-2 mm) non-atretic follicles decreased (P < 0.05) between day 3 and day 7 independently of morphological or biochemical signs of atresia. The pattern of inhibin/activin subunit mRNA expression supports the notion that activin and inhibin have roles in growth and steroidogenesis in follicle recruitment during the early luteal phase of the oestrous cycle.     

Cellular localization of inhibin mRNA in the bovine ovary by in-situ hybridization

Hybridization histochemistry has been used to detect the presence of mRNA for the alpha and beta A subunit of inhibin in tissue sections of the ovary of cows. 32P-labelled cDNAs, complementary to the bovine alpha or beta A subunit of inhibin or to a control segment of plasmid DNA (pBR 322), were used. The alpha subunit mRNA was located in the granulosa layer of antral follicles greater than 0.36 mm in diameter while the alpha and beta A subunit mRNA were both present in follicles of greater than 0.8 mm. In these latter follicles, the thecal layer hybridized with only the alpha subunit mRNA. No hybridization of the alpha or beta A subunit probe was found in the cells of the corpus luteum. Hybridization of both probes was abolished when the tissue sections were pretreated with ribonuclease (RNAse). The plasmid cDNA did not hybridize to any of the tissue sections. This study demonstrates that mRNA for the alpha inhibin subunit can be detected in granulosa and theca cells whereas the beta A inhibin subunit mRNA is restricted to the granulosa cells. These results provide evidence for an independent regulation of expression for the two subunits of inhibin.     

Changes in peripheral inhibin levels and follicular development following treatment of buffalo with PMSG and Neutra-PMSG for superovulation

Fourteen buffalo were synchronized by administration of a prostaglandin (PG) salt Lutalyse in a double injection schedule, with a single intramuscular (im) injection of 25 mg at Day -13, followed by 30 mg and 20 mg im 12 h apart on Day 0 of the experiment. The 30-mg PG injection was designated as 0 h of the experiment. Group I animals (n = 4) received saline and served as the controls, while animals in Groups II and III (n = 5 each) received PMSG (2500 IU im at -48 h. Group III animals were administered 5 ml Neutra-PMSG intravenously at 60 h. Blood samples were collected every 48 h from Day -12 to Day -4, every 24 h from Day -4 to Day 0, every 3 h from Day 1 to Day 4 and every 24 h from Day 5 to Day 10 of experiment for the measurement of peripheral plasma inhibin concentrations by RIA. The number of large follicles (> 10 mm diameter) in animals of Groups II and III was assessed by ultrasonography on Days -2, -1, 0, 1, 2, 5 and 7 of the experiment. Treatment with PMSG of Group II animals resulted in a significant increase (P < 0.05) in plasma inhibin concentrations over that of control animals of Group I at 24 to 99 h, with a peak inhibin concentration of 1.01 +/- 0.31 ng/ml at 48 h. Treatment with Neutra-PMSG in Group III animals caused a significant reduction (P < 0.05) in the peripheral inhibin concentrations at 84 to 120 h and in the number of large unovulated follicles at 168 h compared with that in Group II animals. Peripheral inhibin levels in Group III animals came down to those of Group I after 21 h of Neutra-PMSG treatment. These results suggest that treatment of buffalo with PMSG for superovulation causes a marked rise in peripheral inhibin concentrations. Administration of Neutra-PMSG after PG treatment reduces the peripheral inhibin concentrations and the number of large unovulated follicles.     

Changes in expression of inhibin subunits in the cyclic golden hamster (Mesocricetus auratus) and the regulation of inhibin alpha subunit production by luteinizing hormone

In the present study, changes in localization of each inhibin subunit in the ovary were investigated during the estrous cycle of the golden hamster. The effect of LH surge on changes in localization in inhibin alpha subunit in the ovary was also investigated. Inhibin alpha subunit was localized in granulosa cells of various stages of follicles throughout the estrous cycle. Inhibin alpha subunit was also present in numerous interstitial cells on days 1 and 2 (day 1 = day of ovulation), but the number of positive interstitial cells was fewer on days 3 and almost disappeared on day 4 of the estrous cycle. Newly formed luteal cells were also positive for inhibin alpha subunit on days 1 and 2. On the other hand, positive reactions for inhibin beta A and beta B subunits were only present in the granulosa cells of healthy antral follicles. However, a positive reaction for inhibin beta B subunit in peripheral mural granulosa cells disappeared on days 3 and 4 of the estrous cycle. Treatment with LHRH-AS at 1100 h on day 4 completely blocked the luteinizing hormone (LH) surge and ovulation, although relatively high concentrations of plasma follicle-stimulating hormone (FSH) were maintained throughout the experiment. There were few positive reactions for inhibin alpha subunit in theca and interstitial cells 24 hr after LHRH-AS injection. The effect of LHRH-AS treatment was blocked by a single injection of 10 IU human chorionic gonadotropin. These results suggest that the major source of dimeric inhibin in the cyclic hamster was granulosa cells of healthy antral follicles. Different distribution pattern of inhibin beta A from beta B subunits in large antral follicles on days 3 and 4 of the estrous cycle suggests different secretion patterns of inhibin A from B on these days. Furthermore, the LH surge may be an important factor to induce production of inhibin alpha subunit in interstitial cells of the cyclic hamster.     

The effect of stage of estrous cycle and follicular maturation on ovarian inhibin production in sheep

Twenty-four Scottish Blackface ewes (mean weight 50.0 +/- 0.1 kg with ovulation rate 1.3 +/- 0.1) were randomly divided into 4 groups of 6 animals. Under general anesthesia, following the collection of a timed sample of ovarian venous blood, the ovaries of these animals were collected either on Day 10 of the luteal phase or 12, 24, and 48 h after a luteolytic dose of a prostaglandin (PG) F2 alpha analogue (cloprostenol 100 micrograms i.m.) administered on Day 10. All follicles greater than 3 mm were dissected from the ovaries and incubated in Medium 199 (M199) at 37 degrees C for 2 h, following which the granulosa cells were harvested and incubated in triplicate for 24 h in M199 with or without ovine FSH or ovine LH. Plasma and culture media samples were assayed for inhibin, estradiol (E2), androstenedione (A4), and testosterone (T) by specific RIA. After correcting for hematocrit, ovarian secretion rates were calculated from the product of the plasma concentration and flow rate. The rate of ovarian inhibin secretion during the luteal phase was similar from ovaries categorized on the basis of presence of luteal tissue (1.0 +/- 0.3 and 0.9 +/- 0.5 ng/min for CL present and absent, respectively), confirming that the ovine CL does not secrete appreciable amounts of inhibin. Inhibin secretion was higher (p less than 0.05) at 12 h after PG-induced luteolysis but not at 24 or 48 h compared to values for luteal phase control ewes. Although ovaries containing large estrogenic follicles (greater than or equal to 4 mm in diameter and classified as estrogenic from in vitro criteria) secreted the most inhibin (55%; p less than 0.05), both ovaries containing large nonestrogenic follicles (33%) and small (11%; less than 4 mm in diameter) follicles secreted appreciable amounts of inhibin. This contrasted strongly with E2 where greater than 80% of the steroid was secreted by large estrogenic follicles. The rate of ovarian inhibin secretion was positively correlated (p less than 0.05) with the rate of E2, A4, and T secretion. Overall, there was no significant effect of stage of cycle on follicular inhibin content after 2 h incubation in vitro, release of inhibin by follicles incubated in vitro, or synthesis of inhibin by granulosa cells cultured in vitro. FSH and LH had no effect on the production of either inhibin or estradiol by cultured granulosa cells. Follicular diameter was positively correlated (p less than 0.001) with follicular inhibin and steroid release. Follicular inhibin content after 2 h incubation in vitro was more highly correlated with inhibin release by incubated follicles (r = 0.7; p less than 0.001) than with inhibin synthesis by granulosa cells in vitro (0.4; p less than 0.01).(ABSTRACT TRUNCATED AT 400 WORDS)     

Rapid changes in the expression of inhibin alpha-, beta A-, and beta B-subunits in ovarian cell types during the rat estrous cycle

Distributions of inhibin alpha-, beta A-, and beta B-subunits in different ovarian compartments were studied in cycling female rats by in situ hybridization with complementary RNA probes and using immunohistochemical localization with antibodies selective for each inhibin subunit. Consistent with earlier studies showing inhibin production by granulosa cells of maturing follicles, we also detected mRNAs for inhibin alpha-, beta A-, and beta B-subunits in granulosa cells of these follicles. However, based on immunohistochemistry and in situ hybridization, we found that inhibin alpha- is not only expressed in granulosa cells of mature follicles but in follicles at all stages of maturation, including primary to tertiary follicles. A number of primordial follicles also contained alpha mRNA and immunodetectable alpha-subunit. Interestingly, theca interna and interstitial gland cells contained inhibin alpha mRNA and alpha-subunit. Low levels of inhibin alpha immunoreactivity as well as specific hybridization to the complementary inhibin alpha mRNA probe were observed in newly formed luteal tissue. beta-Subunits, on the other hand, were detected exclusively in granulosa cells of healthy tertiary follicles. The changes in expression of inhibin alpha-, beta A-, and beta B-subunits were more pronounced during the follicular phase of the cycle: inhibin alpha reached its highest level in granulosa cells, theca interna, and interstitial gland cells a few hours after the LH/FSH surge, while at the same time the beta-subunits decreased dramatically in granulosa cells of mature follicles. Immediately before ovulation (estrus 0200 h), the alpha-subunit sharply declined in preovulatory follicles and was present mainly in granulosa cells from nonovulatory follicles at various stages of maturation. At that time, the beta A- and beta B-subunits could not be detected in preovulatory follicles but were localized mainly in small tertiary follicles (less than 300 microns). Unlike for the alpha- and beta B-subunits, beta A mRNA and immunoreactivity was present in large tertiary follicles (approximately 600 microns) immediately before ovulation. The present findings support the hypothesis that a decrease in inhibin production could be responsible for the secondary FSH surge observed early on estrus. This could be initiated by a change in the ratios of activin-inhibin production by decreasing first, the levels of beta-subunits, second, the levels of alpha-subunit, and third, by a resurgence of activin A produced mainly by granulosa cells from large tertiary follicles.(ABSTRACT TRUNCATED AT 400 WORDS)     

Response of estradiol and inhibin to experimentally reduced luteinizing hormone during follicle deviation in mares

The increase in LH concentrations at the time of the decrease in FSH concentrations during follicle deviation in mares was studied to determine the role of LH in the production of estradiol and immunoreactive inhibin (ir-inhibin). Ten days after ovulation, all follicles > or =6 mm were ablated, prostaglandin F(2 alpha) was given, and either 0 mg (control group, n = 15) or 100 mg of progesterone in safflower oil (treated group, n = 16) was given daily for 14 days, encompassing the day of diameter deviation. The follicular and hormonal data were normalized to the expected day of the beginning of diameter deviation when the largest follicle first reached > or =20 mm (Day 0). The experimentally induced decrease in LH concentrations during follicle deviation beginning on Day -4 delayed and stunted the increase in circulating concentrations of ir-inhibin and estradiol beginning on Days -3 and -1, respectively, but did not alter the predeviation FSH surge and the initiation of diameter deviation between the two largest follicles. Combined for both groups, the interval to the expected day of deviation was 16.6 days after ovulation when the largest follicle was a mean of 21.6 mm. After deviation, the largest follicle started to regress in the treated group beginning on Day 1 and was associated with decreased concentrations of ir-inhibin and estradiol, and increased concentrations of FSH. The negative influence of the dominant follicle on the postdeviation decrease in FSH observed in the control group was alleviated and concentrations resurged in the treated group. Apparently this is the first in vivo evidence that the increase in LH that precedes follicle deviation has a positive effect in supporting the production of inhibin during diameter deviation. It was concluded that the increase in LH concentrations before diameter deviation played a role in the production of estradiol and inhibin by the largest follicle during deviation.     

Regulation of follicle-stimulating hormone secretion by estradiol and dimeric inhibins in the infantile female rat.

Plasma and ovarian levels of the dimeric forms of inhibin and plasma estradiol-17beta were investigated and compared with changes in plasma gonadotropins from Postnatal Day (PND) 5 to PND 30 in the female rat. The inhibin subunit proteins were localized in follicular granulosa cells of the ovary. Plasma immunoreactive inhibin levels were low until PND 15 and increased thereafter. Plasma levels of inhibin B (alpha and beta(B) subunits) remained very low until PND 15 and then increased by approximately 24-fold. In contrast, plasma levels of inhibin A (alpha and beta(A) subunits) were relatively low and steady until PND 20, then increased by approximately 3-fold at PND 25. Changes in ovarian inhibin A and B levels closely resembled those in plasma levels. Plasma FSH levels were low at PND 10 but started to peak from PND 15 and remained high until PND 20, followed by a remarkable reduction at PNDs 25 and 30. This dramatic fall in FSH coincided with the rise of inhibin A. A significant inverse correlation was observed between plasma FSH and plasma inhibin A (r = -0.67, P < 0.0002), ovarian inhibin A (r = -0.48, P < 0.01), plasma inhibin B (r = -0.48, P < 0.05), and ovarian inhibin B (r = -0.54, P < 0.01). Plasma estradiol-17beta levels were elevated from PND 5 through PND 15, then fell sharply through PND 30. Plasma estradiol-17beta was significantly and positively (r = 0.75, P < 0.0002) correlated with plasma FSH. Plasma LH rose to higher levels at PND 15 and tended to be lower thereafter. The inhibin alpha, beta(A), and beta(B) subunits were localized to primary, secondary, and antral and large antral follicles, but the types of these immunopositive follicles varied with age. It appeared that, at PND 25 and afterward, all three subunits were mainly confined to large antral follicles in the ovary. We conclude that estradiol-17beta likely is the major candidate in stimulation of FSH secretion in the infantile female rat. We also conclude that inhibin regulation of pituitary FSH secretion through its negative feedback in the infantile female rat begins to operate after PND 20. We suggest that this negative feedback is achieved by increases in plasma levels of the two dimeric forms, and that inhibin A appears to be the major physiological regulator of FSH secretion at the initiation of this mechanism. We also conclude that large antral follicles in the ovary are the primary source of these bioactive inhibins that are secreted in large amounts into the circulation after PND 20.     

Localization of inhibin/activin subunit mRNAs within the primate ovary

In order to gain further understanding of the physiology of inhibin and activin in the primate, the expression of inhibin/activin subunit mRNAs in the monkey ovary was examined by in situ hybridization. Granulosa cells of small antral follicles were found to express mRNA for the beta B subunit, which decreased to undetectable levels in dominant follicles. In contrast, expression of alpha and beta A subunit mRNAs was detected in granulosa cells of dominant follicles and in corpora lutea, but not in small antral follicles. These results indicate that the expression of the beta A and beta B subunits is differentially regulated during the growth and development of ovarian follicles in the monkey.     

Ovarian dynamics and their associations with peripheral concentrations of gonadotropins,ovarian steroids,and inhibin during the estrous cycle in goats

Ovarian changes determined by daily transrectal ultrasound and its relationship with FSH, LH, estradiol-17beta, progesterone, and inhibin were investigated in six goats for three consecutive interovulatory intervals. Estrous cycles were synchronized using two injections of prostaglandin F2alpha analogue 11 days apart. All follicles 3 mm or greater in diameter and corpora lutea were measured daily. A follicular wave was defined as one or more follicles growing to 5 mm or greater in diameter. The day that the follicles reached 3 mm in diameter was defined as the day of wave emergence, and the first wave after ovulation was defined as wave 1. During the interovulatory interval (mean +/- SEM, 21.3 +/- 0.4 days; n = 18), follicular waves emerged at 0.3 +/- 0.5, 6.5 +/- 0.2, and 12.1 +/- 0.4 days for wave 1, wave 2, and wave 3, respectively, in goats with three waves of follicular development and at -0.6 +/- 0.3, 4.7 +/- 0.2, 9.4 +/- 0.5, and 13.4 +/- 0.5 days for wave 1, wave 2, wave 3, and wave 4, respectively, in goats with four waves of follicular development (Day 0 = the day of ovulation). The mean diameter of the largest follicle of the ovulatory wave was significantly larger than those of the largest follicles of the other waves. Corpora lutea could be identified ultrasonically at Day 3 postovulation and attained 12.1 +/- 0.3 mm in diameter on Day 8. Transient increases in plasma concentrations of FSH were detected around the day of follicular wave emergence. The level of FSH was negatively correlated with that of inhibin. These results demonstrated that follicular waves occurred in goats and that the predominant follicular wave pattern was four waves with ovulation from wave 4. These results also suggested that the emergence of follicular waves was closely associated with increased secretion of FSH.     

Uterine involution and ovarian changes during early post partum in autumn-lambing Corriedale ewes

The time of uterine involution and the changes in ovarian follicle populations were studied during early postpartum in multiparous, suckling Corriedale ewes lambing in the autumn. On Day 1 (n=5), Day 5 (n=4), Day 17 (n=4) and Day 30 (n=3) postpartum ewes underwent surgery to obtain ovaries and uteri. The weights of uteri and the lengths of the previous pregnant and nonpregnant horns were recorded as well as the presence of ovarian follicles larger than 1 mm in diameter. Uterine weight and length of uterine horns decreased (P2 to < 4 mm) follicles were also present. At days 17 and 30, aside from the small and medium follicles, all the ewes also had large (>/= 4 mm) follicles, and, at Day 30 2 ewes had large corpora lutea. We conclude that in autumn-lambing Corriedale ewes macroscopic uterine involution was complete around Day 17 post partum and that follicle development begins immediately after parturition, reaching preovulatory size before Day 17. In 2 of the 3 ewes studied until Day 30, ovulation (first progesterone increase) occurred after Day 17 (Days 18 and 25).     

Plasma concentrations of inhibin a and follicle-stimulating hormone differ between cows with two or three waves of ovarian follicular development in a single estrous cycle

Patterns of ovarian follicle development were monitored daily in Holstein-Friesian cows that had two (n = 4) or three (n = 4) waves of ovarian follicle development during a single estrous cycle. The plasma from daily blood samples was used in assays for inhibin A, FSH, progesterone, and estradiol-17beta. Mean cycle lengths for cows with two and three waves were 21.8 and 25.3 days, respectively (P < 0.02). Although the average number of follicles >3-mm diameter on each pair of ovaries was similar for two- and three-wave cows on Days 2, 3, and 4 (Day 0 = day of ovulation; 8.6 vs. 9.6 follicles), there were more follicles >6-mm diameter on the ovaries of cows with two waves on Days 3 and 4. This difference was associated with a shorter interval from wave emergence to peak concentrations of inhibin A during the first wave in two-wave cows (2.0 vs. 3.8 days; P = 0.03) and with higher peak concentrations (474 vs. 332 pg/ml; P = 0.03). Differences in peak FSH concentrations were not significant (1.7 vs. 1.3 ng/ml; P = 0.10) and were inversely related to inhibin A concentrations. The peak concentrations of inhibin A and FSH in the second nonovulatory wave in the three-wave cows were similar to the low concentrations measured in the first wave (292 vs. 332 pg/ml of inhibin A, 1.3 vs. 1.3 ng/ml of FSH; P > 0.20). Average peak concentrations of inhibin A and FSH were similar during the ovulatory wave for cows with either two or three waves in a cycle (432 vs. 464 pg/ml of inhibin A, 2.3 vs. 2.1 ng/ml of FSH; P > 0.3). The lower concentrations of FSH during the emergence of the first follicular wave in cows with three-wave cycles may have reduced the rate of development of some of the follicles and reduced the concentrations of inhibin A. This pattern of lower concentrations of FSH and inhibin A was repeated in the second nonovulatory wave but not in the ovulatory wave. Subtle differences in the concentrations of these two hormones may underlie the mechanism that influences the number of waves of ovarian follicle development that occur during the bovine estrous cycle.     

Changes in porcine oocyte germinal vesicle development as follicles approach preovulatory maturity

This study was conducted to determine the distribution of oocytes in meiotic arrest as a function of follicle maturation, atresia status, and follicular fluid steroid concentrations. Oocytes (n = 138) from > or = 3 mm follicles were recovered from gilts (n = 3/d) on Days 1, 3, 5, and 7 of the follicular phase initiated by withdrawal of altrenogest treatment. They were fixed in 4% paraformaldehyde, stained with Hoechst 33342, and examined by laser scanning confocal microscopy using combined bright field Nomarski optics and ultraviolet laser illumination. The number of oocytes in complete meiotic arrest increased (P < 0.05) as a function of the stage of maturation from 29% on Day 1 to 79 and 67% on Days 3 and 5, respectively. Oocytes showing complete germinal vesicle breakdown (GVBD) were found only on Day 7 (24 to 36 h after the preovulatory LH surge). The distribution of GV stages on Days 1 to 5 did not differ between atretic (n = 27) and nonatretic follicles (n = 81). In nonatretic follicles, GV stage was inversely related to the concentration of estradiol on Day 7 and to the concentrations of progesterone and androstenedione (P < 0.05) on Days 5 and 7 indicating that meiotically arrested oocytes were likely to be found in follicles with highest levels of steroidogenesis. In conclusion, a large proportion of oocytes present in 3 to 5 mm follicles had begun GVBD. The follicles in the ovulatory cohort may be recruited or selected from preexisting 3 to 5 mm follicles, or younger population with oocytes that are in complete meiotic arrest.     

Dynamics of messenger RNAs encoding inhibin/activin subunits and follistatin in the ovary during the rat estrous cycle

Quantitative changes in ovarian inhibin/activin subunit and follistatin mRNAs during the rat estrous cycle were examined by ribonuclease protection assay using digoxygenin-labeled RNA probes. Levels of ovarian inhibin alpha subunit mRNA remained low throughout estrus, metestrus, and diestrus; abruptly increased on the morning of proestrus; then rapidly decreased when the primary gonadotropin surge occurred. A similar changing pattern was observed in inhibin/activin beta(A) subunit mRNA. On the other hand, inhibin/activin beta(B) subunit mRNA showed a different changing pattern. Levels of beta(B) subunit mRNA remained constant during metestrus and diestrus, abruptly decreased on the afternoon of proestrus, then quickly recovered from the nadir by 1100 h on estrus. Throughout the rat estrous cycle, especially during the periovulatory period, alpha subunit mRNA levels were considerably higher than beta(A) and beta(B) subunit mRNA levels. In addition, changes in plasma concentrations of inhibin A and inhibin B were very similar to that in ovarian beta(A) and beta(B) subunit mRNA levels, respectively, with several-hour delays. These results suggest that levels of beta subunit mRNAs restrict secretion of dimeric inhibins. Levels of follistatin mRNA remained low from the midnight of metestrus to the midnight of diestrus, then increased until initiation of the primary gonadotropin surge. Thereafter, follistatin mRNA decreased, reached the nadir at 0200 h on estrus, then increased abruptly at 1100 h on estrus. Afterward, follistatin mRNA levels remained high until the morning of metestrus. The changing pattern of ovarian follistatin mRNA was similar to, and preceded, the changes in plasma concentrations of progesterone, suggesting that ovarian follistatin may modulate progesterone secretion during the rat estrous cycle.     

Localization and secretion of inhibins in the equine fetal ovaries

To clarify the source of inhibins in equine female fetuses, concentrations of immunoreactive (ir-) inhibin, inhibin pro-alphaC, and inhibin A in both fetal and maternal circulation and in fetal ovaries were measured. In addition, the localization of inhibin alpha and inhibin/activin beta(A), and beta(B) subunits and the expression of inhibin alpha(A) and inhibin/activin beta(A) subunit mRNA in fetal ovaries were investigated using immunohistochemistry and in situ hybridization. Concentrations of circulating ir-inhibin, inhibin pro-alphaC, and inhibin A were remarkably more elevated in the fetal than in the maternal circulation between Days 100 and 250 of gestation. Fetal ovaries contained large amounts of ir-inhibin, inhibin pro-alphaC, and inhibin A. In contrast, these inhibin forms were undetectable in both the maternal ovaries and placenta. The inhibin alpha and inhibin/activin beta(A) and beta(B) subunit proteins were localized to enlarged interstitial cells of the equine fetal ovary. Expression of inhibin alpha and inhibin/activin beta(A) subunit mRNAs were also observed in the interstitial cells. We conclude that the main source of large amounts of inhibins in fetal circulation is interstitial cells of fetal ovary and is not of maternal origin. Furthermore, these inhibins may play some important physiological roles in the development of gonads in the equine fetus.     

Inhibin production in vitro by granulosa cells from Booroola ewes which were either homozygous or non-carriers of a fecundity gene influencing their ovulation rate

The production of inhibin by granulosa cells was studied in vitro using cells from follicles of various sizes and health. Follicles were recovered on Days 10-13 of the oestrous cycle, from Booroola x Romney ewes which were homozygous (FF) carriers or non-carriers (++) of the fecundity (F) gene. Inhibin was measured using a bioassay based on the suppression of follicle-stimulating hormone (FSH) output by cultured pituitary cells from ovariectomized Romney ewes and, in some instances, for comparative purposes, by radioimmunoassay also. Geometric mean inhibin production by granulosa cells from nonatretic follicles increased with increasing follicle diameter, during the first 24 h of culture, for both genotypes. The geometric mean production of inhibin by cells from nonatretic 3-4.5 mm diameter FF follicles (the largest follicles found in FF ewes), was significantly higher (P less than 0.05) than that by cells from non-atretic 3-4.5 mm diameter ++ follicles, but similar to that of cells from non-atretic greater than or equal to 5 mm diameter ++ follicles. The production of oestradiol-17 beta by cells cultured in the presence of testosterone (1 microgram/ml) followed a pattern similar to cellular inhibin production. There was a positive linear correlation between inhibin and oestradiol-17 beta production during the first 24 h of culture, for both genotypes. In addition to acting as a substrate for oestradiol-17 beta synthesis, testosterone generally had a slight, stimulatory effect on inhibin production.(ABSTRACT TRUNCATED AT 250 WORDS)     

Use of high-resolution transrectal ultrasonography to assess changes in numbers of small ovarian antral follicles and their relationships to the emergence of follicular waves in cyclic ewes

Transrectal ovarian ultrasonographic studies have shown that, in cattle, follicular wave emergence is associated with a large increase in the number of small antral follicles (4-6mm in diameter); an analogous association has not been found for small follicles (2-3mm in diameter) in the ewe. In previous studies in ewes, accurate assessment of the number of follicles has been limited to follicles > or =2 or 3mm in size. Newer, high-resolution equipment allowed us to identify follicles > or =0.4mm and to quantify all antral follicles > or =1mm in diameter in seven cyclic Western White Face ewes. This allowed us to expand the small follicle pool examined, from 1 to 4 follicles/day (2-3.5mm in diameter) in earlier studies, to 8-18 follicles/day (1-3mm in diameter). Total number of small follicles (> or =1 and < or =3mm in diameter) increased between Days -1 and 0 (Day 0=day of ovulation), and declined between Days 1 and 3 (P<0.05). There were no significant changes in the number of small or medium (4mm in diameter) follicles around days of follicle wave emergence (+/-2 days). The 1-3 follicles in the 2-3mm size range, which constituted a follicle wave (i.e. grew to > or =5mm in size before regression or ovulation), were the only small follicles to emerge in an orderly succession during the estrous cycle, approximately every 3-5 days. Thus, unlike in cattle, there is no apparent increase in numbers of small follicles at follicle wave emergence in cyclic sheep, and little evidence for selection of recruited follicles and follicular dominance.