Effects Of Inhibin On Goat's Fecundity

Inhibin is one kind of dimeric glycoprotein hormone that is secreted by sertoli-like cells of testis and granulosa cells of ovaries. Activin is also a kind of glycoprotein hormone that was first found in the gonad. Inhibin and activin have intimate closely function in female. Inhibin has the capacity to suppress secretion of FSH, while activin has stimulative function on the FSH secretion. Inhibin gene is composed of one a subunit and one of twoβsubunits (PA orβB), and generates dimeric inhibin A(a-βA) and inhibin B(a-βB) by disulfide bond, while activin is composed ofβA andβB and forms activin A(PA-βA), activin B(PB-βB) and activin AB(βA-βB) by disulfide bond.In order to try to reveal the genetic mechanism of inhibin regulating goat's fecundity, the effects of inhibin on goat fecundity in gene level and hormone level were taken up in goat breeds of different fecundity ability in this study.1. Plasma concentrations of INHB, ACTA and FSHINHB, ACTA and FSH in an estrous cycle of goat (including 5 Dazu black goats,5 Nanjiang brown goats and 4 Saanen dairy goats) were measured and analyzed by ELISA. The resluts were as followings:(1) An estrous cycle length was (20.1±0.5) days in Dazu black goat,(20.2=1.1) days in Nanjiang brown goat and (25.1±0.8) days in Saanen dairy goat.(2) In a whole estrous cycle, the time of peak points of FSH and lower points of INHB were coincident, the relationship of FSH and INHB were significantly negative in three goat breeds(P< 0.01). In a whole estrous cycle,The secretion of ACTA and FSH were also coincident, the correlation coefficient of FSH and ACTA was positive (P>0.05).(3) In the first peak point of FSH secretion in an estrous cycle, FSH level of Dazu black goat was significantly high than that of Nanjiang brown goat and Saanen dairy goat(P<0.05). While in the first lower point of INHB secretion, INHB level of Dazu black goat was significantly lower than that of Nanjiang brown goat and Saanen dairy goat(P<0.05).2. Cloning and sequences analysis of inhibin alpha, beta A and beta B subunit genes in goat Cloned cDNA sequences of inhibin alpha, beta A and beta B subunit genes in goats (including 3 Dazu black goats,3 Nanjiang brown goats and 3 Saanen dairy goats) by RT-PCR were submitted and received GenBank IDs (HQ699620 for INHa and HQ699621 for INHβA).The homologies of the sequences were higher compared with the sequences of other species in GenBank.(1) The length of cDNA sequences of inhibin alpha, beta A and beta B subunit genes were 1124 bp,1364 bp and 1499 bp, respectively. Their CDS regions were at 1083 bp,1278 bp and 1227 bp, encoded a polypeptide of 360,425 and 408 amino acids, included 18,21 and 20 amino acids signal peptides and 341,404 and 380 amino acids mature peptides, respectively. Compared with other species in GenBank, the homology in codogenic amino acids of CDS regions was more than 96%.(2) The relative molecular weight of the polypeptide of inhibin alpha, beta A and beta B subunit genes in goat were 94.655 KD,109.132 KD and 100.438 KD, respectively. The isoelectric points were 4.95,4.92 and 4.94, respectively; the minimum values of hydrophobicity were -0.467,-0.633 and -0.567, the maximum were 2.344,2.422 and 2.267, respectively. They had not protein transmembrane domains and had alpha helix (17.22%,21.65% and 23.04%), beta turn (5.28%, 3.53% and 4.41%), extended strand (18.89%,19.53% and 18.14%) and random coil (58.61%, 55.29% and 54.41%) in its secondary structure.116 amino acid residues of inhibin beta A and beta B subunit genes matched human inhibin beta A subunit gene in tertiary structure.(3) Compared with the sequence of exon 2 for inhibin alpha subunit gene in sheep, the cDNA sequence in goat had 19 mutation sites (including 17 transitionsal sites and 2 transversional sites) and 8 amino acids mutation sites. Compared with the partial sequence of exon 1 and 2 in Boer goat in GenBank, the cDNA sequence in goat had 3 mutation sites (including two transitionsal sites and one transversional site) and two amino acids mutation sites.One of the amino acids mutation sites was 338 C→G (i.e.32 C→G of exon 2) that resulted in amino acid change (L→V). the other was 345 A→G(i.e.39 A→G of exon 2) that resulted in one amino acid change (Q→R).(4) Compared with inhibin beta A subunit gene in sheep, the cDNA sequenceof goat had 10 mutation sites that were both transitionsal sites. Locus 198A→G of CDS region (i.e.211 A→G of cDNA sequence) resulted in amino acid change (P→K).(5) Compared with inhibin beta B subunit gene in sheep, the cDNA sequence in goat had 12 mutation sites (with 5 transitionsal sites,6 transversional sites and 1 insertion site) and 3 amino acid mutation sites. Loci 860 (A→G) and 1372 (G→T) respectively resulted in amino acid change (P→K) and (G→V); inserted G in locus 871 resulted in amino acid increased.3. Genetic polymorphism analysis for exons of inhibin alpha, beta A and beta B subunit genes in goatAfter sequencing and PCR-SSCP in larger population including 28 Dazu black goats and 27 Nanjiang brown goats, the results were as followings:(1) There were not polymorphic sites in exon 1 of inhibin alpha subunit gene in goat.32C→G and 39A→G in exon 2 of inhibin alpha subunit gene were found in all experimental goats. There were no polymorphic sites in partial sequence of exon of inhibin beta A subunit gene and two exons of inhibin beta B subunit gene in all goats.(2) There was a mutation site 57G→A (i.e.363G→A of cDNA sequence) in exon 2 of inhibin alpha subunit gene was found by sequencing in larger population.The mutation site resulted in one amino acid change (R→H).(3) Three genotypes (AA, AB and BB) were detected in two goat breeds after SSCP analysis. Genotype frequencies of AA, AB and BB were 0.071,0.429 and 0.500 in Dazu black goat, respectively, while three genotype frequencies were 0.391,0.435 and 0.174 in Nanjiang brown goat, respectively; Gene frequency of B allele (0.714) was higher than that of A (0.286) in Dazu black goat, while gene frequency of A allele (0.630) was higher than that of B (0.370) in Nanjiang brown goat. Theχ2 test showed that Dazu black goat and Nanjiang brown goat were in Hardy-Weinberg equilibrium(P>0.05).(4) For the first three litter size, the first two litter size, the first one litter size and average litter size in Dazu black goat, the least square means (LSM) of BB genotype was 0.595,1.01,0.703 and 0.769 more than those of LSM of AB(P<0.05),respectively. For the first three litter size, the first two litter size and average litter size in Nanjiang brown goat,LSM of BB genotype was 0.787,1.375 and 0.754 than LSM of AB (P<0.05).respectively and 0.849,1.486 and 1.131 than LSM of AA (P< 0.05).respectively. but there was not significantly difference between AA and AB (P>0.05). For the first one litter size, LSM of BB and AB were 1.01 and 0.983 more than AA (P<0.05),respectively, but there was not significantly difference between the frequencies of BB and AB(P>0.05).4. Quantitative expression analysis of inhibin alpha, beta A and beta B subunit genes in goatQuantitative expression of inhibin alpha, beta A and beta B subunit genes in goats (including 7 Dazu black goats,7 Nanjiang brown goats and 7 Saanen dairy goats) were analyzed by real-time fluorescent quantitation PCR. The results were as followings:(1) Expression of inhibin alpha, beta A and beta B subunit genes in pituitary gland, ovary, heart, liver, spleen, lung and kidney can be detected. The expression level of ovary was higher than that of pituitary gland(P<0.05). In ovaries and pituitary gland of three goat breeds, expression level of inhibin alpha subunit gene is the highest, inhibin beta A subunit gene is middle and inhibin beta A subunit gene is the lowest.(2) Expression level of inhibin subunit genes in every tissue of Dazu black goat was the highest in three goat breeds. Saanen dairy goat was in middle level and Nanjiang brown goat was in the lowest level(P<0.05).(3) The relationships between expression level of inhibin alpha, beta A and beta B subunit genes in ovary and the first one litter size of goat were in negative correlation (P<0.10).5. Conclusions(1) There was a negative correlation between FSH and INHB level and a positive correlation between FSH and ACTA level in an estrous cycle of the three goat breeds. The hormone variation tendency was basically coincided with the follicular development wave in goat.(2) Higher fecundity of goat may be correlation with higher FSH level, lower INHB level and lower expression of inhibin subunit genes in the last follicular development wave.(3) Exon 1 of inhibin alpha subunit gene, partial sequence of exon in inhibin beta A subunit and two exons of inhibin beta B subunit gene were not polymorphic sites in all goats and these sequences may faintly affect goat's fecundity.(4) The mutation site (57G→A) in exon 2 of inhibin alpha subunit gene in goat resulted in amino acid change (R→H), three genotypes (AA, AB and BB) were detected in the different individual of two goat breeds. B allele of exon 2 in inhibin alpha subunit gene may be a predominant gene that controls high fecundity of goat,and inhibin alpha subunit gene may be a candidate gene.(5) Expression levels of inhibin alpha, beta A and beta B subunit genes in ovaries were higher than that in pituitary gland. In goat's ovaries and pituitary gland,expression level of inhibin alpha subunit gene is the highest, inhibin beta A subunit gene is middle and inhibin beta A subunit gene is the lowest.