Yasuo Nambo

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.

Interleukin-1 receptor antagonist expression in the equine endometrium during the peri-implantation period.

To identify factors involved in the establishment of pregnancy in the mare, endometrium was collected from day 13 (day 0=day of ovulation) cyclic and day 13, 19, and 25 pregnant animals. From initial cDNA subtraction studies, interleukin-1 receptor antagonist (IL-1RN) mRNA was found as a candidate molecule expressed uniquely in the pregnant endometrium. Expression of IL-1RN mRNA was markedly increased in day 19 and 25 gravid endometrium. In situ hybridization analysis revealed that IL-1RN mRNA was localized to the glandular epithelium. Interleukin-1 receptor antagonist (IL-1RN) protein was found in the extracts of day 25 gravid endometrium and was immunochemically localized to the glandular epithelium/luminal cavity of the pregnant uterus. High concentrations of estradiol-17beta (E(2)) were detected in day 25 conceptuses. Concentrations of E(2) were higher in the gravid endometrial portion than in other endometrial regions. On the other hand, progesterone concentrations did not differ among endometrial samples analyzed. Furthermore, the expression of IL-1RN mRNA was up-regulated in endometrium culture samples treated with 10 ng/mL E(2) and 10 ng/mL progesterone. In the analysis of related gene expression, increased amounts of IL-1alpha and IL-6 mRNA were also found in the day 25 gravid endometrium; however, these expressions in endometrial culture samples were not up-regulated by the steroid treatment. These results indicate that expression of IL-1RN in the endometrium is likely regulated by E(2) and progesterone and suggest that IL-1RN regulates the degree of IL-1 signal transduction and thereby plays an important role in the establishment of equine pregnancy.

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.

The Application of Three-Dimensional Internal Structure Microscopy in the Observation of Mare Ovary

The ovary of the mare has a unique structure which differs totally from that of other mammals. However, because of its relatively large size, conventional histological techniques were unsuitable for the observation of the internal structure of the whole ovary. Three‐dimensional internal structure microscopy (3D‐ISM) consists of a cryotome–CCD camera–laser disc recorder–PC‐based control system coupled with a graphic workstation. The internal structure of the ovary is observed by processing over more than 1000 stored images of serially sliced surfaces of each frozen equine ovary. The 3D reconstruction was done using the full‐coloured, volume‐rendering method. The relationship between the localization of medulla, cortex and ovulation fossa was clarified. The ovulation fossa is localized in the centre of the ovary and is surrounded by a broad ovarian cortex. A trace of ovulation was observed only at the ovulation fossa. Medulla are localized in narrow peripheral areas. The phenomenon of the competition to occupy the cortical area ahead of the ovulation fossa by developing secondary follicles was visualized. Spatial localization of various sized follicles was identified from 3D‐reconstructed images. In this study, it has been clarified that application of this novel computerized technique can clarify the anatomical arrangements of the equine ovary and the complex mechanism of equine follicular development.

Pathology of lethal peripartum broad ligament haematoma in 31 Thoroughbred mares.

REASONS FOR PERFORMING STUDY Broad ligament haemorrhage in peripartum mares is a life-threatening disease and there are few reports on the aetiology and pathogenesis of broad ligament haematoma. OBJECTIVES To obtain information regarding the sites for the early diagnosis and pathogenesis of broad ligament haematoma of mares. METHODS Thirty-one mares that died of broad ligament haematoma peripartum were examined pathologically for bleeding sites. The arterial distribution of 5 young mares with several parities served as negative controls. RESULTS Age and/or multiparity were the predisposing factors for the disease. Arterial injuries were most commonly observed in the uterine artery (24 of 31 mares). Among these, the proximal uterine artery that lies within 15 cm of the bifurcation of the iliac artery was the most frequent site for rupture (18 mares). The lesions occurred preferentially at the bifurcations, lateral part of curvatures and abrupt flexures of the artery. The morphology of the injuries was classified into 3 types: ruptures with and without longitudinal fissures, and transections. Histologically, the arterial wall adjacent to the rupture showed atrophy of smooth muscle cells with fibrosis of the tunica media and disruption and/or calcification of the internal elastic lamina. CONCLUSIONS Arterial injuries that led to broad ligament haematoma in peripartum mares occurred most frequently in the proximal uterine artery, and atrophy of smooth muscle cells with fibrosis of the arterial wall was as one of the predisposing factors in aged and multiparous mares. POTENTIAL RELEVANCE Monitoring small aneurysms, mural tearing, medial fibrosis at the proximal uterine artery by transrectal echography could provide useful information for the early diagnosis and possible prevention of broad ligament haematoma of peripartum mares.

Antibody response in vaccinated pregnant mares to recent G3BP[12] and G14P[12] equine rotaviruses

BackgroundBoth the G3P[12] and the G14P[12] type of equine group A rotavirus (RVA) have recently become predominant in many countries, including Japan. G3 types are classified further into G3A and G3B. The G3A viruses have been circulating in Europe, Australia, and Argentina, and the G3B viruses have been circulating in Japan. However, only an inactivated vaccine containing a single G3BP[12] strain is commercially available in Japan. To assess the efficacy of the current vaccine against recently circulating equine RVA strains, we examined antibody responses in pregnant mares to recent G3BP[12] and G14P[12] strains by virus neutralization test.FindingsAfter vaccination in five pregnant mares, the geometric mean serum titers of virus-neutralizing antibody to recent G3BP[12] strains increased 5.3- to 7.0-fold and were similar to that against homologous vaccine strain. Moreover, antibody titers to recent G14P[12] strains were also increased 3.0- to 3.5-fold.ConclusionsThese results suggest that inoculation of mares with the current vaccine should provide foals with virus-neutralizing antibodies against not only the G3BP[12] but also the G14P[12] RVA strain via the colostrum.

Study of Early Pregnancy Factor (EPF) in Equine (Equus caballus)

PROBLEM: Early pregnancy factor (EPF) is an immunosuppressive protein detected in the early pregnancy serum. We have already reported that we developed the rosette inhibition test for mare EPF and detected EPF in thoroughbreds. The aim of this study was to determine whether or not our method could be used clinically.
 METHODS OF STUDY: The rosette inhibition test for equine EPF was carried out on serum from six nonpregnant and six pregnant Shetland ponies, a female and a male Chinese pony, and four nonpregnant and 13 pregnant thoroughbred mares. In the thoroughbreds, sera were collected during the pregnancy period. Furthermore, we measured progesterone and detected pregnant mare serum gonadotrophin (PMSG) in order to confirm pregnancy of the Chinese pony 3 and 6 months after mating.
 RESULTS: In the nonpregnant Shetland ponies, the rosette inhibition titre (RIT) was 6.0±1.0 and EPF was negative. In contrast, in the pregnant ponies, the RIT was 9.2±0.4 and EPF was positive. Based on these results, we diagnosed pregnancy of the Chinese pony. The RIT of the female Chinese pony (3 months after mating) was above 10 and EPF was positive. Furthermore, we detected PMSG and progesterone in the serum of this pony. EPF appeared in the maternal blood circulation at 24–72 hr after mating, it was detected until the second trimester, and after that it disappeared from the maternal serum.
 CONCLUSIONS: The ponys EPF was detected by using the same rosette inhibition test as in the thoroughbred and was present from 24 to 72 hr after mating until the second trimester. The results indicated that our method was useful for pregnancy diagnosis of Equine.