Natsuko Nagamine

Effect of passive immunization against inhibin on FSH secretion, folliculogenesis and ovulation rate during the follicular phase of the estrous cycle in mares.

Physiological roles of inhibin in mares were investigated by means of passive immunization using an antiserum to inhibin that had been raised in a castrated goat. Eight mares were given an intravenous injection of either 100 mL (n = 4) or 200 mL (n = 4) of inhibin antiserum 4 d after a single intramuscular injection of PGF2 alpha on Day 8 after ovulation, 4 control mares were treated with 100 mL castrated goat serum in the same manner. Jugular vein blood samples were collected after treatment with the serum until 192 h post treatment. Follicular growth and ovulations were monitored by ultrasound examination at 24-h intervals. The ability of the inhibin antiserum to neutralize the bioactivity of equine inhibin was examined in vitro using a rat pituitary cell culture system. Suppression of secretion of FSH from cultured rat pituitary cells by equine follicular fluid was reversed by the addition of increasing doses of the inhibin antiserum, thereby indicating its bioactivity. Plasma levels of FSH and estradiol-17 beta were higher in mares treated with the inhibin antiserum. The ovulation rate was significantly higher in mares treated with antiserum (100 mL = 3.75 +/- 0.63; 200 mL = 4.50 +/- 0.65) than in control mares (1.25 +/- 0.25). These results demonstrate that inhibin is important in regulating FSH secretion and folliculogenesis in mares. They also show that neutralization of the bioactivity of inhibin may become a new method for the control of folliculogenesis and ovulation rate in mares.

Plasma concentrations of ir-inhibin, inhibin A, inhibin pro-αC, FSH, and estradiol-17β during estrous cycle in mares and their relationship with follicular growth

The relationship among plasma levels of immunoreactive (ir)-inhibin, inhibin A, inhibin pro-αC, FSH, estradiol-17β and follicular growth were investigated during the normal estrous cycle in mares. Seven mares were used for two successive normal estrous cycles. Follicular diameters and ovulation were obtained by transrectal ultrasonography, and blood samples were collected daily by jugular venipuncture for hormonal assay. The results showed that inhibin A was inversely correlated (r=−0.59, p<0.0001) with FSH indicating its contribution to negative feedback control of FSH secretions from the pituitary gland. Estradiol-17β increased during the follicular phase reaching a peak (37.9±3.8 pg/mL) 2 d before ovulation. Estradiol-17β was positively correlated (r=0.78, p<0.0001) with inhibin A. The high levels of inhibin A and estradiol-17β were associated with the growth of the preovulatory dominant follicle and inversely correlated with FSH suggesting that both hormones are products of the large dominant follicles and were responsible for the decline in FSH secretion during the follicular phase of estrous cycle. In conclusion, an inverse relationship between inhibin A and FSH was clearly demonstrated indicating that inhibin A has a key role in the negative feedback control of FSH from the pituitary gland. In addition, inhibin A and estradiol-17β secretions were associated with the growth of the preovulatory dominant follicle and were positively correlated.

Mechanisms responsible for increase in circulating inhibin levels at the time of ovulation in mares

In female mammals, inhibin is secreted by the granulosa cells and selectively inhibits secretion of FSH. Although circulating immunoreactive (ir)-inhibin levels decrease after ovulation as a result of the disappearance of its main source, they abruptly increase at the time of ovulation in mares. To investigate the mechanisms responsible for this increase, 50 ml of equine follicular fluid (eFF) was administered into the abdominal cavity of mares during the luteal phase (eFF, n = 4). One hour after treatment, plasma levels of ir-inhibin and inhibin pro-alphaC (but not estradiol-17beta) were significantly higher in eFF-treated mares than in control mares (n = 4). The hormone profiles in eFF-treated mares were similar to those in mares with the spontaneous or hCG induced ovulations. The present study demonstrates that the release of follicular fluid into the abdominal cavity when the follicle ruptures is responsible for the ovulatory inhibin surge in the mare. These findings also suggest that circulating inhibin pro-alphaC may be useful for determining the time of ovulation in the mare.

A selective increase in circulating inhibin and inhibin pro-αC at the time of ovulation in the mare

The relationship between a selective increase in circulating immunoreactive (ir)-inhibin and the time of ovulation was investigated in mares. Concentrations of plasma ir-inhibin were measured every 4 h during the periovulatory period. Inhibin pro-αC, a precursor protein of the inhibin α-subunit, was also measured. The changes in ir-inhibin and inhibin pro-αC in circulation were parallel. Concentrations of both ir-inhibin and inhibin pro-αC in the plasma increased at the same time when ovulatory follicles ruptured, and the peak levels of circulating ir-inhibin and inhibin pro-αC were maintained for 4-8 h. There was no selective increase in plasma concentrations of estradiol-17β during the process of ovulation. These results suggest that the selective increase in ir-inhibin and inhibin pro-αC was caused by the absorption of follicular fluid after the rupture of ovulatory follicles. These results also suggest that the measuring of plasma concentrations of ir-inhibin or inhibin pro-αC in mares might be a useful method for detecting the time of ovulation.

Localization and Secretion of Inhibins in the Equine Fetal Ovaries

Abstract To clarify the source of inhibins in equine female fetuses, concentrations of immunoreactive (ir-) inhibin, inhibin pro-αC, and inhibin A in both fetal and maternal circulation and in fetal ovaries were measured. In addition, the localization of inhibin α and inhibin/activin βA, and βB subunits and the expression of inhibin αA and inhibin/activin βA subunit mRNA in fetal ovaries were investigated using immunohistochemistry and in situ hybridization. Concentrations of circulating ir-inhibin, inhibin pro-αC, 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-αC, and inhibin A. In contrast, these inhibin forms were undetectable in both the maternal ovaries and placenta. The inhibin α and inhibin/activin βA and βB subunit proteins were localized to enlarged interstitial cells of the equine fetal ovary. Expression of inhibin α and inhibin/activin βA 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.

Anti-inflammatory and Intestinal Barrier-protective Activities of Commensal Lactobacilli and Bifidobacteria in Thoroughbreds: Role of Probiotics in Diarrhea Prevention in Neonatal Thoroughbreds.

ABSTRACT We previously isolated the commensal bacteria lactobacilli and bifidobacteria from the Thoroughbred intestine and prepared the horse probiotics LacFiTM, consisting of Lactobacillus ruminis KK14, L. equi KK 15, L. reuteri KK18, L. johnsonii KK21, and Bifidobacterium boum HU. Here, we found that the five LacFiTM constituent strains remarkably suppressed pro-inflammatory interleukin-17 production in mouse splenocytes stimulated with interleukin-6 and transforming growth factor-β. The protective effects of the probiotic on impaired intestinal barrier function were evaluated in Caco-2 cells treated with tumor necrosis factor-α. Evaluation of transepithelial resistance showed that all the strains exhibited intestinal barrier protective activity, with significant suppression of barrier impairment by L. reuteri KK18. The LacFiTM constituent strains were detected in neonatal LacFiTM-administered Thoroughbred feces using polymerase chain reaction denaturing gradient gel electrophoresis and culture methods. These five strains were found to be the predominant lactobacilli and bifidobacteria in the intestinal microbiota of LacFiTM-administered Thoroughbreds. Administration of LacFiTM to neonatal Thoroughbreds decreased diarrhea incidence from 75.9% in the control group (n=29 neonatal Thoroughbreds) to 30.7% in the LacFiTM-administered group (n=101 neonatal Thoroughbreds) immediately after birth to 20 weeks after birth. LacFiTM treatment also prevented diarrhea especially at and around 4 weeks and from 10 to 16 weeks. The duration of diarrhea was also shorter in the probiotics-administered group (7.4 ± 0.8 days) than in the control group (14.0 ± 3.2 days). These results indicate that the LacFiTM probiotics regulates intestinal function and contributes to diarrhea prevention.