母鸡抑制素的产生

母鸡抑制素主要是由排卵前卵泡颗粒细胞产生的,肾上腺是又一来源,ＬＨ在刺激体外颗料细胞产生抑制素方面的比ＦＳＨ有效。去除排卵前卵泡后,血浆免疫活性抑制素显著降低,而血浆ＦＳＨ急剧升高。卵泡颗粒层中,抑制素α亚基比β（Ａ）亚基表达充分。α亚基是由近似１．７ｋｂ的ｍＲＮＡ编码,主要的８．４ｋｂβ（Ａ）－ｍＲＮＡ带在排卵前卵泡的颗粒层中表达。     

Immunolocalization of inhibin/activin subunits in the shiba goat fetal, neonatal, and adult testes

The objective of this study was to investigate the cellular immunolocalization of inhibin alpha and inhibin/activin (betaA and betaB) subunits in the fetal, neonatal and adult testes of Shiba goats. The testes were obtained from a fetus at 90 days, a neonate at 15 days, and two adult Shiba goats (both of 3 years old). The sections of testes were immunostained by the avidin-biotin-peroxidase complex method (ABC) using polyclonal antisera raised against porcine inhibin alpha, inhibin/activin betaA, and inhibin/activin betaB. Inhibin alpha and inhibin/activin (betaA and betaB) subunits were expressed in Leydig cells, but not in the Sertoli cells of the fetus with a weak immunostaining. An increase in the number of positive cells and a more intense immunohistochemical signal for inhibin alpha and inhibin/activin (betaA and betaB) subunits were observed in the Leydig cells of neonatal testes. Moreover, inhibin alpha, betaA, and betaB subunits were expressed in the Sertoli cells and Leydig cells of adult testes, respectively. These results suggest that Shiba goats testes have the ability to synthesize inhibins in the fetus, neonate, and adult, and the cellular localization of inhibin/activin subunits showed age-related changes in fetal, neonatal, and adult testes of Shiba goats.     

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.     

Effects of vertically transferred 3,3',4,4',5-pentachlorobiphenyl on gene expression in the ovaries of immature Sprague-Dawley rats

We have previously shown that 3,3',4,4',5-pentachlorobiphenyl (PCB-126) vertically transferred from dams potentially exerts a direct effect on the ovaries of offspring and adversely affects female puberty. To investigate its toxicological targets in ovarian tissues, mRNAs encoding representative peptides that regulate follicular development in granulosa cells, theca cells, and oocytes were quantified using ovaries collected on postnatal days (PND) 5, 15, and 24 from the offspring of dams administered oral doses of 0, 1 or 3 microg/kg PCB-126 starting 2 weeks prior to mating and continuing until 20 days after delivery. Quantification using the real-time RT-PCR method revealed that PCB-126 lowered the amounts of mRNAs that encoded the inhibin alpha- and inhibin/activin beta A-subunits from PND 15 onwards; the amounts of mRNAs for inhibin/activin beta B-subunit, follicle-stimulating hormone (FSH) receptor, and aromatase on PND 15; and the amounts of luteinizing hormone receptor mRNA on PND 24 compared with those of the age-matched controls. In contrast, no differences were noted for mRNAs encoding c-kit, growth differentiation factor-9, bone morphogenetic protein-15, or kit ligand for any of the age groups examined. The serum FSH level on PND 24 was higher than that in the control. Since the earliest effects on the mRNAs in the rat ovaries were observed in those expressed in the granulosa cells of the growing follicles after the antral follicles had developed, molecules in granulosa cells but not in oocytes during the early stages of the antral follicles might be the primary targets of vertically transferred PCB-126.     

Histochemical localization of inhibin and activin alpha, beta A and beta B subunits in rat gonads.

The localization of alpha, beta A and beta B subunits of inhibin/activin polypeptides was studied in the ovary and testis of sexually mature, immature, and embryonic rats. Specific staining with these three subunits was also evident in the oocytes from embryonic to mature female rats. This result suggests that inhibin- and activin-like substances may be produced in the oocytes and these substances may play a role in the oocyte growth and differentiation. Judging from the intensity of immunoreaction in mature female rats, the three subunits should be produced more abundantly in luteal cells than in the granulosa cells. Immunoreactive alpha, beta A and beta B subunits were observed in the cummulus oophorous in the morning (11:00), but not in the evening (23:00) on proestrus. The results are in well agreement with the previous report that inhibin alpha and beta A subunit mRNA signals decline on proestrus evening. It is supposed that the cyclic change may be related with physiological phenomena prior to the ovulation, such as primary gonadotropin surges, loss of cummulus-oocyte gap junctions, or germinal vesicle breakdown. In both germ cells and Sertoli cells of the testis, alpha, beta A and beta B subunits were more abundant in the embryonic rat than in the mature rat. Although clear reactions with beta A and beta B subunits were detected in Leydig cells, alpha subunit was not detectable in the cells throughout the developmental stages examined.     

Localization of inhibin alpha-, betaA- and betaB-subunits and aromatase in ovarian follicles with granulosa theca cell tumor (GTCT) in 6 mares

To clarify the morphological and immunohistochemical characteristics in mares with granulosa theca cell tumor (GTCT), the localization of inhibin subunits (alpha, betaA, betaB) and aromatase in the granulosa cell layers and theca layers in the ovarian follicles were determined by immunohistochemical staining. The follicles were obtained from the ovaries of 6 mares with GTCT and 4 normal mares as controls. Immunohistochemically, inhibin alpha-subunit was localized in the granulosa cells of all follicles showing different sizes in all GTCT cases and betaA- subunit was localized in two GTCT cases in all sized follicles. But inhibin betaB- subunit and aromatase were not localized in GTCT cases. On the other hand, inhibin alpha-, betaA-, and betaB-subunits and aromatase were localized in the large and medium sized follicles, but inhibin betaA- and betaB-subunits and aromatase were not stained in the small sized follicles in normal cases. These findings suggest that some mares with GTCT can secrete dimeric inhibin (inhibin A), but all GTCT cases cannot secrete inhibin B. By the results of aromatase staining it is clear that testosterone is not converted into estradiol due to the lack of aromatase in the GTCT follicles.     

Secretion of inhibin in male Japanese quail (Coturnix japonica) from one week of age to sexual maturity

The objective of this study was to investigate the changes in secretion of inhibin and cellular localization of the inhibin alpha and inhibin/activin (beta(A) and beta(B)) subunits in male Japanese quail from 1 to 7 weeks after hatching. The post-hatch profile of plasma luteinizing hormone (LH), immunoreactive (ir) inhibin and testosterone were measured by radioimmunoassay. Testes were immunostained by the avidin-biotin-peroxidase complex method (ABC) using polyclonal antisera raised against inhibin alpha, inhibin/activin beta(A) and inhibin/activin beta(B) from one week of age to sexual maturity. Testicular weight increased gradually until 4 weeks and abruptly increased from 5 weeks of age onwards. The plasma concentrations of LH and ir-inhibin increased significantly at 5 weeks of age, and the plasma concentration of testosterone increased significantly at 6 weeks of age. Pituitary contents of LH showed a steady increase until 6 weeks of age and then abruptly increased at 7 weeks of age. Coincident to the increase in plasma testosterone, the testicular contents of testosterone significantly increased from 5 weeks through sexual maturity. Immunohistochemically, localization of the inhibin/activin alpha, beta(A) and beta(B) subunits was found in the Sertoli and Leydig cells at all ages of development from one week of age to sexual maturity. These results suggest that Sertoli and Leydig cells are the major source of inhibin secretion during development in male Japanese quail.     

Immunolocalization of inhibin/activin subunit proteins during the breeding season in testes and scented glands of muskrats (Ondatra zibethicus)

The objective of this study was to investigate the cellular immunolocalization of inhibin a and inhibin/activin (β(A) and β(B)) subunits in the muskrat testes and scented glands during the breeding season. Inhibin α and inhibin/activin (β(A) and β(B)) subunits were expressed in Sertoli cells and Leydig cells of testes and glandular cells of scented glands, respectively. Also, positive signals of inhibin α and inhibin/activin (β(A) and β(B)) subunits by Western blotting were both observed in testicular and scented glandular tissues. These results suggested that the testes and scented glands of the muskrats had the ability to synthesize inhibins and activins and that activins and inhibins might play an important role in testicular and scented glandular function in muskrats.     

Expression of Inhibin/Activin Subunits in the Equine Uteri during the Early Pregnancy

The establishment of equine pregnancy is a unique and long process during which a series of physical and possibly biochemical interactions are required between the conceptus and uterus. In this study, we investigated the expression pattern of inhibin/activin subunits in the uterus during early pregnancy. The uteri from four adult mares on cyclic day 13 or pregnancy day 25 were obtained. Immunohistochemical experiments suggested that inhibin/activin subunits were immunolocalized in the luminal and glandular epithelium on pregnancy day 25. In addition, the inhibin α and inhibin/activin β_B subunits were not detected, and inhibin/activin β_A subunit was detected, in the luminal and glandular epithelium on cyclic day 13. Real‐time polymerase chain reaction and Western blotting results for the inhibin/activin subunits suggested a significant increase in the expression of inhibin/activin subunit β_B and a significant decrease in the expression of inhibin/activin subunit β_A on pregnancy day 25 compared with those on cyclic day 13. Enzyme‐linked immunosorbent assays suggested a significant decrease in the concentration of activin A in endometrium extracts from cyclic day 13 to pregnancy day 25. These results suggest that inhibins or activins synthesized in the uterus, as endocrine factors and necessary nutriments, have different expression patterns and may play different, important roles during early embryonic development of the equine.     

Analysis of the function of activin betaC subunit using recombinant protein

Activins, TGF-beta superfamily members, have multiple functions in a variety of cells and tissues. Additional activin beta subunit genes, betaC and betaE, have been identified in humans and rodents. To explore the role of activin betaC subunit, we generated recombinant human activin C using Chinese hamster ovary cells. Recombinant activin C from the conditioned medium was purified by consecutive hydrophobic, size-exclusion, and high performance liquid chromatography. SDS-PAGE and Western blot analysis of the purified protein revealed that activin C formed disulfide bridges. However, activin C had no effect on the proliferation of cultured liver cells. Furthermore, there were no significant differences in erythroid differentiation and follicle stimulating hormone secretion in vitro. It was also shown that immunoreactive bands indicated the hetrodimer of activin betaC, and inhibin alpha subunits were detected in the conditioned medium from the activin C-producing cells, which were stably transfected with inhibin alpha subunit cDNA. This suggests that activin betaC subunit may have been present and that it may exert its effect as inhibin C.     

抑制素对动物生殖系统的影响及其在动物生产中的应用

抑制素是转化生长因子β超家族成员之一,是由α亚基和β亚基构成的异二聚体,主要由卵泡颗粒细胞和睾丸支持细胞分泌,在生殖系统的功能主要是通过调节FSH合成和分泌发挥的。作者就抑制素的分布与结构、在雌雄动物生殖系统的表达及功能、在动物生产中的应用进展进行综述。     

Involvement of activin and inhibin in the regulation of food and water intake in the rat

The expression of activin and inhibin has been demonstrated in the hypothalamus, but their physiological roles in the brain remain to be elucidated. In the present study, involvement of activin and inhibin in the regulation of food and water intake was examined. Male rats were deprived of food or water for 12 and 60 hr, and mRNA levels of activin/inhibin alpha, betaA and betaB subunits in the hypothalamus were estimated by RT-PCR. Gene expression of alpha subunit transiently decreased at 12 hr of food deprivation, while it did not change during water deprivation. Food and water deprivation for 60 hr increased mRNA levels of betaA and betaB subunits, respectively. These results indicated that gene expression of each subunit was independently regulated. Injection of activin A (0.5 and 4.0 microg) into the third ventricle decreased food intake. Water intake was suppressed by 4.0 microg, but not 0.5 microg, of activin A. Intracerebroventricular injection of inhibin A (0.5 and 4.0 microg) decreased water intake in a dose dependent manner without affecting food intake, suggesting that inhibin could act independently of activin. Taken together, it is suggested that activin and inhibin take part in the central regulation of nutrient and fluid balance, though further study is needed to determine precise molecular species involved.     

Follicular and hormonal dynamics during the estrous cycle in goats

Transrectal ultrasonography of ovaries was performed daily in 6 goats for 3 consecutive estrous cycles. Blood samples collected daily were measured for concentrations of FSH, inhibin A, and estradiol-17beta. Follicular and hormonal data were analyzed for associations between the follicular waves and hormonal concentrations. During the interovulatory intervals, follicular growth and regression occurred in a wave like pattern (2-5 waves), and the predominant patterns were three and four follicular waves. In addition, there was no significant difference among the diameters of dominant follicles during the growth phase of the follicular waves. The number of 3 mm follicles peaked on days 0, 7, and 11 in interovulatory intervals that had three follicular waves and on days -1, 5, 11, and 15 in those that had four follicular waves. Plasma concentrations of FSH increased around the day of follicular wave emergence and declined with the growth of follicles. Circulating FSH increased again concomitant with regression of dominant follicles in the anovulatory wave, whereas FSH levels remained low in the ovulatory wave. Inhibin A was negatively correlated with FSH, while it was positively correlated with estradiol-17beta, suggesting that inhibin A is a product of healthy growing follicles and that it contributes to the suppression of FSH secretion. In conclusion, the growth of ovarian follicles in goats exhibits a wave-like pattern, and follicular dominance is less apparent in goats. Moreover, inhibin A may be a key hormone for regulation of the follicular wave through suppression of FSH secretion in goats.     

Downregulation of the expression of inhibin α subunit and betaglycan in porcine cystic follicles

Inhibins, as members of the transforming growth factor beta (TGF-β) superfamily, downregulate the synthesis and secretion of follicle-stimulating hormone (FSH) in an endocrine manner. The role of inhibin/betaglycan in the ovary regulation recently gained attention. To date, no data exist on the function of inhibin α subunit and betaglycan in cystic follicles. In this study, the expressions of inhibin α subunit and betaglycan in cystic follicles were investigated using immunohistochemistry, real-time PCR and Western blot analysis. Both inhibin α subunit and betaglycan immunoreactivities were mainly localized in the granulosa cells of follicles. Expression of inhibin α subunit and betaglycan was inferior in cystic follicles compared with that in normal large follicles. However, the result of enzyme-linked immunosorbent assay showed no significant difference in the decreasing in concentration of inhibin α subunit in cystic follicular fluid compared with the control (P>0.05). In this study, we explored the effects of FSH on betaglycan expression in granulosa cells in vitro. As expected, a significant increase in the expressions of betaglycan mRNA and protein in granulosa cells was observed in response to exogenous FSH (30 ng/ml) (P<0.05) compared with the control. Consequently, this study provides evidence that the expressions of inhibin α subunit and betaglycan are inferior in cystic follicles, and this may be caused by the decrease in FSH in the presence of a cystic follicle.     

Immunohistochemical localization of inhibin subunits in the testis of the bull

The differential localization of the inhibin beta subunits betaA and betaB in the testis of adult bull was studied using specific monoclonal and polyclonal primary antibodies. Inhibin betaA- and betaB-subunits were localized only in the Sertoli cells. The inhibin betaA-subunit was observed in the cytoplasm while the betaB-subunit was localized in the nucleus. No specific findings depending on spermatogenic stages were observed among the seminiferous tubules. Moreover, the inhibin alpha-subunit was not detected in the testis of the bulls. In addition, no inhibin subunits were detected in the Leydig cells and spermatogenic cells. These findings indicate the presence of betaA- and betaB-subunits in the bull, which may suggest a possibility that activin is produced and/or stored in the Sertoli cells and regulates spermatogenesis in an autocrine/paracrine manner. Moreover, the inhibin betaB-subunit may be produced in the nucleus but the functional meaning of this is not yet clear.     

Cellular Localization of Inhibin alpha-subunit, PKB/Akt and FoxO3a proteins in the ovaries of minipigs

Experiments were conducted to examine the cellular localization of inhibin alpha-subunit, protein kinase B (PKB/Akt), and FoxO3a proteins in the ovaries of minipigs, Chinese Xiang pigs, by immunohistochemistry. The results indicated that inhibin alpha-subunits were localized in the granulosa cells of follicles at all stages but were not localized in corpora lutea. PKB was localized in the granulosa cells of primordial follicles and in the basal layers of the granulosa cells of preantral and antral follicles, but were not localized in atretic follicles and corpora lutea. FoxO3a was localized in the granulosa cells of follicles at all stages and was extensively localized in the cytoplasma of the luteinized granulosa cells of corpora lutea. Together, the stage- and cell-specific expression patterns of inhibin alpha-subunit, FoxO3a, and PKB suggest that these proteins might play potential roles in follicular development, atresia, and luteinization in the minipig.     

Seasonal changes in spermatogenesis and immunolocalization of inhibin/activin subunits in the wild male ground squirrel (Citellus dauricus Brandt)

The objective of this study was to investigate the seasonal changes in spermatogenesis and the immunolocalization of the inhibin alpha and inhibin/activin (betaA and betaB) subunits during the breeding and non-breeding seasons in the wild male ground squirrel. The testicular weight and size and seminiferous tubule diameter were measured, and histological observations of testes were performed. 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 betaA and inhibin/activin betaB during the breeding and non-breeding seasons. There were marked variations in testicular weight and size and seminiferous tubule diameter between the breeding and non-breeding seasons, and all types of spermatogenic cells, including spermatozoa, were found in the breeding season. In addition, immunoreactivity was also detected for the inhibin alpha, betaA and betaB subunits in Sertoli and Leydig cells during the breeding season, but immunostaining was only present for the inhibin alpha and inhibin/activin betaB subunits in Sertoli cells during the non-breeding season. These results suggest that seasonal changes in testicular weight and size and seminiferous tubule diameter of wild ground squirrels are correlated with changes in spermatogenesis, and the cellular localization of the inhibin/activin subunits showed season related changes in the breeding and non-breeding seasons.     

仙居鸡抑制素/活化素β_A亚基成熟区的cDNA克隆及序列分析

本研究根据发表的来航鸡抑制素 /活化素βA 亚基序列设计引物 ,运用RT PCR技术从仙居鸡卵泡的颗粒细胞总RNA中扩增出抑制素 /活化素βA 亚基成熟区序列 ,并进行了克隆和测序。结果显示 ,所测鸡成熟βA 亚基是由 116个氨基酸 (aa)残基组成的蛋白质 ,共有 9个半胱氨酸残基 ,与发表的鸡及哺乳类相应序列对比 ,其核苷酸序列的同源性分别为 99.2 %和 81%～ 85 .2 % ,其预测氨基酸序列的同源性分别为 10 0 %和 96 .6 %～ 97.4 % ,且所测鸡 βA 亚基成熟区半胱氨酸残基的数目和位置与发表的鸡和哺乳类相同 ,说明该亚基的序列及结构在不同物种间具高度保守性 ,揭示其可能具重要的生理功能     

Seasonal changes in immunolocalization of inhibin/activin subunits and testicular activity in wild male raccoon dogs (Nyctereutes procyonoides)

Thirty-four pairs of testes from wild adult raccoon dogs (Nyctereutes procyonoides) were obtained between September 2000 and May 2003. The cellular localization of the inhibin alpha and inhibin/activin (betaA and betaB) subunits in wild raccoon dog testes was investigated. The testicular weight and size and seminiferous tubule diameters were measured. There were marked seasonal variations in testicular weight and size and seminiferous tubule diameters, with values relatively low in September and high in March. Spermatogonia and primary spermatocytes were observed in September, and spermatogonia, spermatocytes, and round spermatids were present in January. All types of spermatogenic cells, including mature spermatozoa, were found in March, indicating that the breeding season is around March in Japan. Thereafter, spermatogonia and degenerating spermatocytes were observed in April. The sections of testes were immunostained by the avidin-biotin-peroxidase complex method (ABC) using polyclonal antisera raised against porcine inhibin alpha, inhibin/activin betaA and inhibin/activin betaB. The inhibin alpha and inhibin/activin (betaA and betaB) subunits were only expressed in Leydig cells in September. On the other hand, the inhibin alpha, betaA, and betaB subunits were observed in Leydig cells and Sertoli cells, but not in germ cells, in March. These results suggest that the testes of wild raccoon dogs have the ability to synthesize inhibins, and the cellular localization of inhibin/activin subunits showed season-related changes in the breeding and non-breeding seasons.     

Changes in the concentration of mRNAs for the inhibin subunits in ovarian follicles after administration of gonadotropins to progestin treated ewes.

RNA was extracted from single or small groups of ovine ovarian follicles after treatment of ewes with FSH and/or LH. The content of mRNA for the alpha-inhibin and beta A-inhibin subunits was analyzed by hybridization with specific cDNA probes. All ewes were treated with progestin vaginal pessaries to suppress spontaneous preovulatory follicle maturation and ewes were given three intramuscular injections of gonadotropins at 8-hr intervals starting 24 hr prior to collection of ovaries. In experiment I, both Schering-FSH and NIDDK-oFSH-17 (oFSH) significantly increased alpha- and beta A-inhibin mRNA per ewe in 2-5 mm follicles and tended to increase alpha- and beta A-inhibin mRNA in large (greater than 5 mm) follicles. In experiment II, oFSH and NIDDK-oLH-25 (oLH) were administered in a 2X2 factorial arrangement. Separate administration of oFSH or oLH increased (P less than .05) the alpha-inhibin mRNA concentration in large follicles. alpha-inhibin mRNA concentration in 4-5 mm follicles was also increased by oFSH but was decreased by oLH. Concomitant treatment with oFSH and oLH did not change alpha-inhibin mRNA concentrations from those measured in oFSH treated ewes. In experiment II, beta A mRNA concentrations followed a pattern similar to that of alpha A mRNA, but the differences were not statistically significant. We conclude that, in the ewe, exogenous FSH increases the concentration of inhibin mRNA in the whole follicle. The ability of exogenous oLH to alter expression of the inhibin subunit genes may depend upon the stage of follicle maturation.