Immunoreactive Inhibin Concentrations Research Articles

Effects of an extended photoperiod on gonadal function and condition of hair coats in Thoroughbred colts and fillies

ABSTRACTThe effects of an extended photoperiod (EP) in Thoroughbreds colts and fillies from winter at one year old to spring at two years old on the gonadal functions, coat condition, and endocrine changes were investigated. Sixty-two Thoroughbreds (31 colts and 31 fillies) reared in the Hidaka Training and Research Center (Hidaka), Japan Racing Association were used. Thirty of them (15 colts and 15 fillies) were reared under EP conditions from December 20 to April 10, and the remaining 32 horses were reared under natural light alone as a control group. For EP, a 100-watt white bulb was set near the ceilings of stalls, and lighting conditions of 14.5-hr light and 9.5-hr dark periods were established. Blood was collected from the jugular vein once a month from October at one year old to February at two years old in both colts and fillies, and then twice a month in colts and weekly in fillies after March, and the coat condition was evaluated in January and April in 56 horses. To investigate endocrine changes, the plasma concentrations of prolactin, luteinizing hormone (LH), follicle-stimulating hormone (FSH), immunoreactive (ir-) inhibin, testosterone, estradiol-17β and progesterone were measured. No significant difference was noted in the coat condition between the two groups in January, but they changed from winter to summer coats (molting of winter coats) in April in the EP group compared with the control group. Regarding endocrine changes, the plasma concentrations of prolactin, FSH, ir-inhibin and testosterone were significantly higher in the EP colts than in the control group from January to April. The plasma concentrations of LH tended to rise in the EP colts from January to April compared with the control group. In the EP fillies, the plasma concentrations of prolactin, LH, ir-inhibin, estradiol-17β and progesterone were significantly higher during January and April, but a significantly high level of FSH was noted in the control than EP group in January. The ovulation day was advanced in the EP fillies compared with the control group. The present study clearly demonstrated that EP treatment during rearing advanced the molting of winter coats in both colts and fillies. These results suggested to be due to the action of prolactin being increased by EP treatment. In addition, EP treatment stimulated the hypothalamus-pituitary-gonadal axis even in yearlings, and advanced ovulation in fillies. Since EP treatment-induced changes in the yearlings were within the physiological range, and the method is safe and simple, EP treatment may be an effective technique in horse husbandry.

Role of the Double Luteinizing Hormone Peak, Luteinizing Follicles, and the Secretion of Inhibin for Dominant Follicle Selection in Asian Elephants (Elephas maximus)1

Elephants express two luteinizing hormone (LH) peaks timed 3 wk apart during the follicular phase. This is in marked contrast with the classic mammalian estrous cycle model with its single, ovulation-inducing LH peak. It is not clear why ovulation and a rise in progesterone only occur after the second LH peak in elephants. However, by combining ovarian ultrasound and hormone measurements in five Asian elephants (Elephas maximus), we have found a novel strategy for dominant follicle selection and luteal tissue accumulation. Two distinct waves of follicles develop during the follicular phase, each of which is terminated by an LH peak. At the first (anovulatory) LH surge, the largest follicles measure between 10 and 19.0 mm. At 7 ± 2.4 days before the second (ovulatory) LH surge, luteinization of these large follicles occurs. Simultaneously with luteinized follicle (LUF) formation, immunoreactive (ir) inhibin concentrations rise and stay elevated for 41.8 ± 5.8 days after ovulation and the subsequent rise in progesterone. We have found a significant relationship between LUF diameter and serum ir-inhibin level (r(2) = 0.82, P < 0.001). The results indicate that circulating ir-inhibin concentrations are derived from the luteinized granulosa cells of LUFs. Therefore, it appears that the development of LUFs is a precondition for inhibin secretion, which in turn impacts the selection of the ovulatory follicle. Only now, a single dominant follicle may deviate from the second follicular wave and ovulate after the second LH peak. Thus, elephants have evolved a different strategy for corpus luteum formation and selection of the ovulatory follicle as compared with other mammals.

Secretion of Inhibin during the Estrous Cycle in the Female Asian Elephant (Elephas maximus)

To define the source of circulating inhibin in female Asian elephants, the immunolocalizations of the inhibin α, β(A) and β(B) subunits, 3β-hydroxysteroid dehydrogenase (3β-HSD), aromatase cytochrome P450 (P450arom) and cytochrome 17α-hydroxylase P450 (P450 c17) were investigated. Concentrations of immunoreactive (ir-) inhibin, progesterone and follicle-stimulating hormone (FSH) during the estrous cycle were measured by radioimmunoassay. Inhibin immunoreactivity in follicular fluid and homogenate of corpora lutea was also measured. Immunolocalizations of inhibin subunits, 3β-HSD, P450arom and P450c17 were detected in the granulosa cells of antral follicles and luteal cells. The follicular fluid contained high levels of ir-inhibin and bioactive inhibin. The homogenate of corpora lutea also contained ir-inhibin. Serum ir-inhibin remained at low levels during the early non-luteal phase, began to increase from the late non-luteal phase and continued to increase during the early luteal phase. Serum ir-inhibin showed maximal levels in the middle of the luteal phase and gradually decreased to baseline three weeks prior to progesterone decline. The serum ir-inhibin levels were positively correlated with progesterone throughout the estrous cycle. On the other hand, ir-inhibin was negatively correlated with FSH during the late non-luteal and early luteal phases. These findings strongly suggest that the corpus luteum is one of the sources of inhibin as well as granulosa cells in the Asian elephant.

Effects of inhaled nanoparticle-rich diesel exhaust on regulation of testicular function in adult male rats

We investigated the effects of nanoparticle-rich diesel exhaust (NR-DE) on reproductive function. Eight-week-old male F344 rats were divided into 12 experimental groups and exposed to either whole NR-DE at low (15.37 μg/m3, 2.27 × 105 particles/cm3), middle (36.35 μg/m3, 5.11 × 105 particles/cm3), or high (168.84 μg/m3, 1.36 × 106 particles/cm3) concentrations or clean air for 4, 8, or 12 weeks (5 hours/day, 5 days/week). NR-DE exposure for 4 or 8 weeks did not affect body weight; however, body weight was significantly decreased in rats exposed to low- or high- concentration NR-DE for 12 weeks compared to the control group. Relative weights of testes, epididymides, seminal vesicles, and prostate had increased non-significantly in all NR-DE-exposed rats at 4, 8, and 12 weeks. Adrenal gland relative weights were significantly increased at 4 weeks in rats exposed to low-concentration NR-DE. Plasma luteinizing hormone and follicle stimulating hormone concentrations did not change significantly. Plasma testosterone concentrations were significantly increased after exposure to low- or middle-concentration NR-DE for 4 or 8 weeks compared to controls. Plasma immunoreactive (ir-) inhibin concentrations were significantly increased after exposure to high-concentration NR-DE for 4 weeks or middle- or high-concentration NR-DE for 12 weeks compared to controls. Testicular testosterone concentrations were significantly increased at 4, 8, and 12 weeks after exposure to low-concentration NR-DE compared to controls. In contrast, with exposure to low- or high-concentration NR-DE, testicular ir-inhibin concentrations were significantly greater than in controls, but only at 4 weeks. These results suggest that NR-DE inhalation disrupts the endocrine activity of the male reproductive system.

Follicle Deviation in Ovulatory Follicular Waves with One or Two Dominant Follicles in Mares

The follicle and hormone aspects of diameter deviation and development of one dominant (>/=28 mm) follicle (1DF) vs two dominant follicles (2DF) were studied in 32 ovulatory follicular waves in mares. Follicles were ranked each day as F1 (largest) to F3. The beginning of deviation was designated day 0 and preceded the first increase in the differences in diameter between F1 and F2 in the 1DF group and between a combination of F1 and F2 vs F3 in the 2DF group. One dominant follicle and 2DF developed in 21 (66%) and 11 (34%) waves, respectively. Double ovulations occurred in only one of the waves with 2DF. In 8/11 waves with 2DF, a second deviation occurred between F1 and F2 on 2.5 +/- 0.4 days after the first deviation. On day 0, 1DF and 2DF waves were similar in number of days after ovulation, number of follicles, difference in diameter between F1 and F2, and plasma concentrations of LH, estradiol and immunoreactive inhibin. The interval from maximum FSH concentration to day 0 was longer (p < 0.05) and FSH concentration was lower (p < 0.05) on days -1 to 4 in the 2DF group. The similarities on day 0 in the characteristics of 1DF and 2DF waves despite the differences in the declining portions of the FSH profile indicated that a specific day of the FSH decline or a specific concentration were not factors in initiating deviation. Unlike reported results in heifers, the results in mares did not indicate a hormonal basis for the development of 2DF or two deviations.

Immunodetection of NGF, trkA, p75 and Inhibin .ALPHA.-Subunit in Interstitial Cells of Golden Hamsters Treated with hCG

The aim of this study was to investigate the effect of luteinizing hormone (LH) surge on expression of nerve growth factor (NGF), trkA, p75 and inhibin alpha-subunit in ovarian interstitial cells of golden hamsters after human chorionic gonadotropin (hCG) treatment. NGF, two NGF receptors (trkA, p75) and inhibin alpha-subunit were immunolocalized by immunohistochemistry, and gonadotropins, steroid hormones and immunoreactive (ir-) inhibin concentrations were measured by radioimmunoassay. Stronger positive staining of NGF, trkA and p75 were found in interstitial cells at 6, 12 and 18 h after hCG injection in the treated group compared with the control groups. Inhibin alpha-subunit staining was found in interstitial cells at 12 and 18 h after hCG injection in the treatment group, but not in the control group. Plasma concentrations of progesterone increased significantly from 6 to 18 h after hCG treatment, whereas plasma concentrations of estradiol-17beta significantly decreased compared with the control group. An increased plasma concentration of FSH and decreased concentrations of ir-inhibin from 6 to 18 h after hCG treatment verified the negative relationship. There were no significant changes in the concentrations of LH in the hCG-treated group. In contrast, the typical preovulatory LH surge was found at 1700 h on day 4 (proestrus) in the control group. These results suggested that LH surge can induce expression of NGF, trkA, p75 and inhibin alpha-subunit in ovarian interstitial cells and that NGF, trkA, p75 and inhibin alpha-subunit may have a paracrine or autocrine role in modulation of ovarian interstitial cell function in golden hamsters.

Secretion of Inhibin in Female Japanese Quails (Coturnix japonica) from Hatch to Sexual Maturity

To clarify the cellular source and secretory pattern of inhibin in the Japanese quail during follicular development, the plasma concentrations of immunoreactive (ir) inhibin were measured from 1 to 7 weeks after hatching. Localization of the inhibin/activin alpha, beta A and beta B subunits was investigated by immunohistochemistry. To monitor development of the pituitary and ovarian functions, the plasma luteinizing hormone (LH) and progesterone concentrations were also measured. Ovarian weight increased gradually until 6 weeks of age and then abruptly increased at 7 weeks of age just at the onset of egg production. Plasma concentrations of LH increased significantly at 6 weeks of age. The plasma concentrations of ir-inhibin and progesterone and the pituitary contents of LH also increased significantly at 7 weeks of age. Immunohistochemically, the inhibin/activin alpha, beta A and beta B subunits were localized in the granulosa cells of all follicles during different stages of development from 1 to 7 weeks after hatching. The inhibin alpha, beta A and beta B subunits were also found in the interstitial cells but not theca cells of all follicles. These results demonstrated that the plasma concentrations of ir-inhibin of the female Japanese quails rose with ovarian development. The immunohistochemical results suggested that granulosa and interstitial cells are the major source of ovarian inhibins in female Japanese quails.

Changes in Serum Inhibin Levels and Immunolocalization of Inhibin/Activin Subunits During the Breeding Season in the Wild Male Japanese Black Bear (Ursus thibetanus japonicus)

The objective of this study was to investigate the changes in secretion of inhibin and cellular localization of inhibin alpha and inhibin/activin (beta(A) and beta(B)) subunits during the breeding season in the wild male Japanese black bear. Histological observations of testes were performed and seminiferous tubule diameters were measured. The sections of the testes were immunostained by the avidin- biotin-peroxidase complex method (ABC) using polyclonal antisera raised against porcine inhibin alpha, inhibin/ activin beta(A), and inhibin/activin beta(B) during the breeding season. Serum concentrations of immunoreactive (ir-)inhibin, testosterone, and luteinizing hormone (LH) were measured by radioimmunoassay. Higher values of seminiferous tubule diameters and all types of spermatogenic cells including mature-phase spermatozoa were found during the breeding season. There were seasonal changes in serum concentrations of ir-inhibin, testosterone, and LH. Ir-inhibin was positively correlated with testosterone, and LH. In addition, immunoreactivity of inhibin alpha, beta(A), and beta(B) subunits were also detected in Sertoli and Leydig cells during the breeding season. These results suggest that Japanese black bear testes may secrete bioactive inhibins during the breeding season and that the circulating inhibin may be a useful indicator of the testicular function in wild male Japanese black bears.

Reducing Estrogen Synthesis Does Not Affect Gonadotropin Secretion in the Developing Boar1

Boars have high concentrations of plasma and testicular estrogens, but how this hormone is involved in feedback regulation of the gonadotropins and local regulation of testicular hormone production is unclear. The present study examined the effects of reducing endogenous estrogens by aromatase inhibition on concentrations of plasma LH and FSH and on testicular and plasma concentrations of testosterone (T) and immunoreactive inhibin (INH). Thirty-six littermate pairs of boars were used. One boar from each pair was assigned to the control group (vehicle); the other boar to the treatment group (aromatase enzyme inhibitor, Letrozole, 0.1 mg/kg body weight [BW]). Weekly oral treatment started at 1 wk of age and continued until castration at 2, 3, 4, 5, 6, 7, or 8 mo. Plasma concentrations of gonadotropins, INH, T, estradiol (E2), and estrogen conjugates (ECs) were determined. Testicular tissue was collected at castration for determination of INH and T and for confirmation of reduced aromatase activity. The acute effects of aromatase inhibition on gonadotropins were monitored in two adult boars treated once with Letrozole (0.1 mg/kg BW). Treatment with the aromatase inhibitor reduced testicular aromatase activity by 90% and decreased E2 and ECs without changing acute, long-term, or postcastration LH and FSH. Plasma T, testicular T, and circulating INH concentrations did not change. Testicular INH was elevated in treated boars compared with controls. In conclusion, estrogen does not appear to play a regulatory role on gonadotropin secretion in the developing boar. This is in direct contrast to findings in males of several other species.

The Stimulatory Role of Estrogen on Sperm Motility in the Male Golden Hamster (Mesocricetus auratus)

To clarify the physiological roles of estrogens in the regulation of sperm motility in the golden hamster, two different approaches were used. In the first experiment, silastic tubes containing either low (low E2 group) or high (high E2 group) amount of estradiol-17beta were implanted (Exp 1). In the second experiment, male golden hamsters were actively immunized against estradiol-17beta (Exp 2). In Exp 1, all sperm motility parameters (including motility, straight velocity, curvilinear velocity, beat/cross frequency, and mean amplitude of lateral head displacement) were significantly increased except linear index in the high E2 group as compared with controls at 20 days after the treatment. In the high E2 group, plasma concentrations of luteinizing hormone (LH) significantly increased, whereas levels of circulating testosterone decreased significantly. Plasma concentrations of follicle-stimulating hormone (FSH) and immunoreactive inhibin were not affected by the treatment with estradiol-17beta. In the Exp 2, titer of circulating antibodies to estradiol-17beta consistently increased after the second immunization until the end of experiment (16 weeks). The sperm motility, straight velocity, and curvilinear velocity were significantly decreased after active immunization to estradiol-17beta. Concentrations of circulating LH and FSH were also decreased significantly by the treatment. In conclusion, the current observations indicate that estradiol-17beta affects sperm motility in adult male golden hamsters.

Secretion of Leptin Throughout Pregnancy and Early Postpartum Period in Japanese Monkeys: Placenta as Another Potential Source of Leptin

Leptin is one of the most important factors linking nutrition and reproduction. In the present study, plasma concentrations of leptin during pregnancy and early lactation in the Japanese monkey were determined. Plasma concentrations of gonadotropins, immunoreactive (ir-)inhibin, and steroid hormones were also measured. Plasma concentrations of leptin significantly increased during the second quarter of pregnancy and progressively elevated throughout pregnancy. During the fourth quarter of pregnancy, leptin levels reached up to 89 and 64 times of those during prepregancy and first quarter of pregnancy periods, respectively. After parturition, the circulating leptin level abruptly decreased. During the first 10 d of lactation, its average level decreased to the levels of the second quarter of pregnancy. Plasma irinhibin and estradiol-17beta were elevated throughout the pregnancy and decreased after parturition, and both of them were positively correlated with leptin levels during the whole pregnancy and early lactation. Plasma concentrations of progesterone significantly increased during the first quarter of pregnancy and kept at a higher level compared with prepregnancy and sharply decreased after parturition. Placental homogenates contain a large amount of leptin protein. These results suggest that placenta secretes a large amount of leptin and may be another source of leptin during pregnancy in Japanese monkeys. In addition, high correlations among leptin, irinhibin, and estradiol-17beta during these stages suggest that these hormones may have important regulating roles on leptin secretion during pregnancy in the Japanese monkey.

Secretion of Inhibin and Testicular Expression of Inhibin Subunits in Male Duck Embryos and Newly Hatched Ducks

Concentrations of immunoreactive (ir-) inhibin in circulation, amniotic fluid, and testes of embryos and newly hatched ducks were determined from d 21 of incubation to d 1 of age. Plasma concentrations of FSH and LH were also determined by chicken radioimmunoassay (RIA) systems. In addition, gene expression and cellular source of inhibin were investigated by in situ hybridization and immunohistochemistry. The results showed that plasma ir-inhibin gradually declined from d 21 to d 24, followed by an increase on d 25 and remained high until d 1 after hatching. FSH in plasma was high on d 21 followed by a sharp decline toward d 25 after which FSH levels stabilized. A reverse relationship was observed between inhibin and FSH during the late stage of incubation. Embryonic testes contained high ir-inhibin levels. Testicular ir-inhibin levels were relatively high at early time points with a peak on d 23, and significantly decreased from d 23 to d 24 and stabilized thereafter. Amniotic fluid concentrations of ir-inhibin were relatively low and remained constant between d 21 and d 25. In situ hybridization demonstrated that the expression of inhibin alpha- and betaA-subunit mRNA was coexisted in the cells in the seminiferous tubules of testes on d 25. The immunoreactivity of inhibin betaA- and betaB-subunits was colocalized in the cells in the seminiferous tubules of testes on d 25. The results of dimeric inhibins determined by the ELISA method showed that inhibin B can be measured in embryonic testicular homogenate and pooled embryonic plasma. Although inhibin A was detected in testicular homogenate, it was under the detection limit in pooled embryonic plasma. In conclusion, these results indicate that cells in the seminiferous tubules of embryonic testes in ducks may secrete dimeric (bioactive) inhibins to circulation and that the FSH-inhibin feedback loop may become operational during the late stage of the incubation.

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.

Relationship between Peripheral Plasma Inhibin and Progesterone Concentrations in Sahiwal Cattle (Bos Indicus) and Murrah Buffaloes (Bubalus bubalis)

The present study investigated peripheral plasma immunoreactive inhibin (ir-inhibin) concentrations in relation to the stage of oestrous cycle and progesterone concentrations in cycling Sahiwal cattle (Bos indicus) and Murrah buffaloes (Bubalus bubalis). Blood samples were collected once daily for thirty-two consecutive days from cattle and buffaloes (5 each) during winter months of January and February. Mean (±S.E.M.) plasma ir-inhibin concentrations ranged from 0.40±0.01 to 0.59±0.03 ng/ml in cattle and from 0.29±0.03 to 0.52±0.05 ng/ml in buffaloes. In cattle, ir-inhibin concentrations increased from 0.47±0.07 ng/ml on day -4 (day 0=day of oestrus) to reach a maximum value of 0.59±0.03 ng/ml on day -2. Thereafter, ir-inhibin concentrations showed a decline to reach a low of 0.40±0.01 ng/ml on day 11 of the oestrous cycle. In buffaloes, ir-inhibin concentrations increased from 0.38±0.04 ng/ml on day -4 to reach a maximum concentration of 0.52±0.05 ng/ml on day -2. Ir-inhibin concentrations then declined to reach a low of 0.29±0.03 ng/ml on day 9 of the cycle. In both cattle and buffaloes, ir-inhibin concentrations which were lowest (0.43±0.02 and 0.34±0.02 ng/ml, respectively) during the mid-luteal phase of the oestrous cycle increased (p<0.05) to 0.52±0.03 and 0.44±0.04 ng/ml, respectively, during the late luteal phase, and then further to the highest value of 0.53±0.02 and 0.49±0.04 ng/ml, respectively, during the perioestrus phase, following which these declined to 0.50±0.02 and 0.39±0.03 ng/ml, respectively, during the early luteal phase. The variations in peripheral plasma ir-inhibin profile in both the species appear to be related to the changes in characteristics of follicular populations during the oestrous cycle. Peripheral plasma ir-inhibin concentrations were negatively correlated with progesterone concentrations in cattle (r=-0.51, p<0.01) and buffaloes (r=-0.30, p<0.01) indicating that the corpus luteum is not a source of peripheral ir-inhibin in these species. (Asian-Aust. J. Anim. Sci. 2003. Vol 16, No. 1 : 6-10)

Effects of reduction of the number of primordial follicles on follicular development to achieve puberty in female rats

Effects of reduction of the number of primordial follicles on follicular development and concentrations of circulating hormones were examined in immature female rat offspring of dams given busulfan intraperitoneally on day 14 of gestation. The offspring of dams treated with 5 mg busulfan kg(-1) showed vaginal opening at an age comparable with the offspring of dams treated with 2.5 mg busulfan kg(-1) or with corn oil as a control, although they exhibited an irregular oestrous cycle until week 14 after birth. The serum concentrations of immunoreactive inhibin and FSH on day 26 after birth of the offspring treated with 5 mg busulfan kg(-1) were similar to those of age-matched controls. On day 15 after birth, however, the concentration of their immunoreactive inhibin was markedly lower than that of controls, whereas the concentration of their FSH was increased inversely. Comparison of the numbers of ovarian follicles in the controls and groups treated with 2.5 mg busulfan kg(-1) and 5 mg busulfan kg(-1) revealed that prenatal treatment with busulfan reduced the number of follicles in the primordial or primary phase and in the preantral phase on day 7 after birth. Although the increase of the ratio of the number of preantral follicles during days 7-13 after birth tended to vary with the prenatal dose of busulfan, the number of preantral follicles in the group treated with 5 mg busulfan kg(-1) was still smaller than in the controls. The concentration of serum immunoreactive inhibin of the offspring treated with busulfan was reduced on day 7 after birth without alteration of the concentration of gonadotrophin. On day 13 after birth, the concentration of serum immunoreactive inhibin was reduced only in the offspring treated with 5 mg busulfan kg(-1), and the concentration of serum FSH of the offspring was increased inversely as found on day 15 after birth. These results indicate that a reduction in the number of primordial follicles decreases the number of follicles that enter the growing phase, a major source of circulating inhibin in the neonatal and infantile ovary, and that consequently increased circulating FSH may accelerate follicular development to achieve puberty.

Effects of reduction of the number of primordial follicles on follicular development to achieve puberty in female rats.

Effects of reduction of the number of primordial follicles on follicular development and concentrations of circulating hormones were examined in immature female rat offspring of dams given busulfan intraperitoneally on day 14 of gestation. The offspring of dams treated with 5 mg busulfan kg(-1) showed vaginal opening at an age comparable with the offspring of dams treated with 2.5 mg busulfan kg(-1) or with corn oil as a control, although they exhibited an irregular oestrous cycle until week 14 after birth. The serum concentrations of immunoreactive inhibin and FSH on day 26 after birth of the offspring treated with 5 mg busulfan kg(-1) were similar to those of age-matched controls. On day 15 after birth, however, the concentration of their immunoreactive inhibin was markedly lower than that of controls, whereas the concentration of their FSH was increased inversely. Comparison of the numbers of ovarian follicles in the controls and groups treated with 2.5 mg busulfan kg(-1) and 5 mg busulfan kg(-1) revealed that prenatal treatment with busulfan reduced the number of follicles in the primordial or primary phase and in the preantral phase on day 7 after birth. Although the increase of the ratio of the number of preantral follicles during days 7-13 after birth tended to vary with the prenatal dose of busulfan, the number of preantral follicles in the group treated with 5 mg busulfan kg(-1) was still smaller than in the controls. The concentration of serum immunoreactive inhibin of the offspring treated with busulfan was reduced on day 7 after birth without alteration of the concentration of gonadotrophin. On day 13 after birth, the concentration of serum immunoreactive inhibin was reduced only in the offspring treated with 5 mg busulfan kg(-1), and the concentration of serum FSH of the offspring was increased inversely as found on day 15 after birth. These results indicate that a reduction in the number of primordial follicles decreases the number of follicles that enter the growing phase, a major source of circulating inhibin in the neonatal and infantile ovary, and that consequently increased circulating FSH may accelerate follicular development to achieve puberty.

Production of inhibin A not B in rams: changes in plasma inhibin A during testis growth, and expression of inhibin/activin subunit mRNA and protein in adult testis

Previous studies have shown that changes in the plasma concentrations of immunoreactive inhibin measured by radioimmunoassay occur in parallel with growth and regression of the testes during a reproductive cycle in adult Soay rams induced by exposure to an artificial lighting regimen of alternating 16 week periods of long days and short days. With the development of new two-site ELISAs for sheep inhibin A and inhibin B, we have re-examined the relationship between FSH and dimeric, biologically active inhibin in the reproductive cycle in adult Soay rams. No signal was generated by sheep testicular extract, ram or ewe plasma, or sheep ovarian follicular fluid in the inhibin B ELISA. In contrast, ram plasma contained significant activity in the inhibin A ELISA, which diluted in parallel to the inhibin A standard, and was abolished by preincubation of ram plasma with monoclonal antibodies specific for the betaA, but not the betaB, subunit. These results indicate that the ram is the first adult male mammalian species identified to date in which the testes produce and secrete dimeric inhibin A and not inhibin B. Northern blot analysis and immunocytochemistry confirmed the presence of alpha, betaA and betaB inhibin/activin subunit mRNA and protein in the testes of adult rams. Changes in plasma inhibin A concentrations occurred in parallel with the growth and regression of the testes during the long day: short day: long day lighting regimen in adult Soay rams, confirming our previous observations with immunoreactive inhibin. During the growth phase of the testes in the first 8 weeks of exposure to short days there was a positive correlation between plasma FSH and inhibin A concentrations, indicating that during this phase the secretion of inhibin A is stimulated by FSH and that inhibin A did not act as a negative feedback hormone on FSH secretion. From week 8.5 to week 16.0 of exposure to short days, there was a negative correlation between FSH and testosterone concentrations, but not inhibin, indicating that when inhibin concentrations are high, testosterone acts as the negative regulator of FSH secretion. Thus, in intact adult rams, when the testes are fully active it appears that inhibin A may sensitize the pituitary to the negative feedback effects of testosterone, at which time they act synergistically to maintain plasma concentrations of FSH.

Production of inhibin A not B in rams: changes in plasma inhibin A during testis growth, and expression of inhibin/activin subunit mRNA and protein in adult testis

Previous studies have shown that changes in the plasma concentrations of immunoreactive inhibin measured by radioimmunoassay occur in parallel with growth and regression of the testes during a reproductive cycle in adult Soay rams induced by exposure to an artificial lighting regimen of alternating 16 week periods of long days and short days. With the development of new two-site ELISAs for sheep inhibin A and inhibin B, we have re-examined the relationship between FSH and dimeric, biologically active inhibin in the reproductive cycle in adult Soay rams. No signal was generated by sheep testicular extract, ram or ewe plasma, or sheep ovarian follicular fluid in the inhibin B ELISA. In contrast, ram plasma contained significant activity in the inhibin A ELISA, which diluted in parallel to the inhibin A standard, and was abolished by preincubation of ram plasma with monoclonal antibodies specific for the betaA, but not the betaB, subunit. These results indicate that the ram is the first adult male mammalian species identified to date in which the testes produce and secrete dimeric inhibin A and not inhibin B. Northern blot analysis and immunocytochemistry confirmed the presence of alpha, betaA and betaB inhibin/activin subunit mRNA and protein in the testes of adult rams. Changes in plasma inhibin A concentrations occurred in parallel with the growth and regression of the testes during the long day: short day: long day lighting regimen in adult Soay rams, confirming our previous observations with immunoreactive inhibin. During the growth phase of the testes in the first 8 weeks of exposure to short days there was a positive correlation between plasma FSH and inhibin A concentrations, indicating that during this phase the secretion of inhibin A is stimulated by FSH and that inhibin A did not act as a negative feedback hormone on FSH secretion. From week 8.5 to week 16.0 of exposure to short days, there was a negative correlation between FSH and testosterone concentrations, but not inhibin, indicating that when inhibin concentrations are high, testosterone acts as the negative regulator of FSH secretion. Thus, in intact adult rams, when the testes are fully active it appears that inhibin A may sensitize the pituitary to the negative feedback effects of testosterone, at which time they act synergistically to maintain plasma concentrations of FSH.

Inhibin as a Possible Indicator of Follicular Development during Perinatal Period in Mares.

Plasma concentrations of immunoreactive (ir-) inhibin, FSH, LH, estradiol and progesterone were measured by RIA between -6 and 12 days after parturition in 6 mares. Follicular development was also monitored in the same animals after parturition by ultrasound scanner. Plasma concentrations of ir-inhibin were relatively low before parturition, then rose significantly after parturition, in accordance with the development of 10-30 mm follicles in diameter during the early period of postpartum estrus. In contrast, concentrations of both estradiol and progesterone remained high before parturition, then dropped significantly 1 day after parturition. The present study demonstrated that inhibin is more likely to be secreted from maternal ovaries into maternal circulation than the feto-placental unit during the prenatal period. This suggests that inhibin could be used as a potential indicator of follicular development around parturition in mares.

Changes in Inhibin Secretion during Development of the Female Duck Embryo

Concentrations of immunoreactive (ir-) inhibin in circulation, amniotic fluid and the ovary of embryos and newly-hatched ducks from Day 21 of incubation to one day of age were determined. The antiserum used was against bovine 31-kDa inhibin and was validated for RIA of inhibin using samples from female embryos. Plasma concentrations of FSH and LH were also determined by chicken RIA systems. Plasma concentrations of ir-inhibin were maintained at constant levels from day 21 to day 25, abruptly increased on day 26. After the increase, plasma concentrations of ir-inhibin quickly declined from day 27 to the day of hatch (day 28). Plasma concentrations of FSH were high on day 21, followed by an abrupt decline on day 22, gradually increased until day 24, and then decreased again on day 25. The maximal rise of plasma FSH was observed on day 27, and then rapidly decreased until the day after the hatch. No inverse relationship was observed between plasma concnetrations of ir-inhibin and FSH. Amniotic fluid concentrations of ir-inhibin were relatively low and remained constant between Day 21 and Day 25. Embryonic ovaries contained very low amounts of ir-inhibin. Ovarian inhibin levels were stabilized in early time points, temporally decreased on day 25, and abruptly increased on day 26. These results suggest that inhibin does not involved in the regulation of FSH secretion in the female duck embryo and the ovary of duck embryo is not the main source of circulating ir-inhibin.