Chicken Follicle-stimulating Hormone Research Articles

Follicle-stimulating hormone increases the intramuscular fat content and expression of lipid biosynthesis genes in chicken breast muscle

Intramuscular fat (IMF) is a crucial factor in the quality of chicken meat. The genetic basis underlying it is complex. Follicle-stimulating hormone (FSH), well-known as an effector in reproductive tissues, was recently discovered to stimulate abdominal fat accumulation in chicken. The effect of FSH on IMF accumulation and the underlying molecular regulatory mechanisms controlling both IMF and abdominal fat deposition in vivo are largely unknown. In this study, two groups of chickens were treated with chicken FSH or a placebo. The lipid content of breast muscle, abdominal fat volume, and serum concentrations of FSH were examined. Related genes implicated in breast muscle and abdominal fat accumulation were also investigated. Compared to the control group, the triglyceride (TG) content of breast muscle and the percentage of abdominal fat in FSH-treated chickens were significantly increased by 64.9% and 56.5% (P<0.01), respectively. The FSH content in the serum of FSH-treated chickens was 2.1 times than that of control chickens (P<0.01). Results from quantitative real-time polymerase chain reaction (qRT-PCR) assays showed that relative expression levels of fatty acid synthase (FAS), lipoprotein lipase (LPL), diacylglycerol acyltransferase 2 (DGAT2), adipocyte fatty acid binding protein (A-FABP), and peroxisome proliferator-activated receptor γ (PPARγ) were significantly upregulated in breast muscle following FSH treatment (P<0.01). Treatment with FSH also significantly increased relative expression levels of FAS, LPL, DGAT2, A-FABP, and PPARγ in abdominal fat tissue (P<0.05). The results of principal component analysis (PCA) for gene expression (breast muscle and abdominal fat) showed that the control and FSH treatment groups were well separated, which indicated the reliability of the data. This study demonstrates that FSH plays an important role in IMF accumulation in female chickens, which likely involves the regulation of biosynthesis genes related to lipid metabolism.

Molecular characterization and identification of a novel polymorphism of 200 bp indel associated with age at first egg of the promoter region in chicken follicle-stimulating hormone receptor (FSHR) gene

Follicle-stimulating hormone receptor (FSHR) plays an important role in animal follicular development. Polymorphisms in FSHR promoter region likely impact transcription and follicle growth and maturation. In this study, a fragment of ~1.9 kb of cFSHR promoter for Zang, Xianju, Lohmann Brown, Jining Bairi and Wenchang breeds (line) was obtained. Totally 49 variations were revealed, of which 39 are single nucleotide substitutions, one is nucleotide substitution of (TTG) to (CAC) and nine are indels. Polymorphism at -874 site (a 200 bp indel mutation) was identified, and their effects on egg production traits as well as gene expression were analyzed. At this site, allele I(+) was dominant in Lohmann Brown and Xinyang Brown (a synthetic egg-laying line) lines, but very rare in three Chinese indigenous chicken breeds, namely Jining Bairi, Wenchang, Zang and one synthetic boiler line (Luqin). In Xinyang Brown population, the polymorphism was associated with age at first egg (AFE) (P < 0.05) and its effect on egg number at 37 weeks of age (E37) and egg number at 57 weeks of age (E57) was not significantly different (P > 0.05). The cFSHR mRNA level was not significantly different between three genotypes in small white and small yellow follicles of Xinyang Brown hens, however, allele I(+) tends to increase cFSHR transcription.

Characterization of the chicken follicle-stimulating hormone receptor (cFSH-R) complementary deoxyribonucleic acid, and expression of cFSH-R messenger ribonucleic acid in the ovary.

Studies were conducted to characterize the chicken (c) FSH receptor (R) cDNA, and to evaluate expression of cFSH-R mRNA in the hen ovary at known stages during follicle development. A total of 2.5 kb of nucleic acid sequence including the complete cFSH-R coding region was isolated by a combination of the reverse-transcription polymerase chain reaction and 5'- and 3'-rapid amplification of cDNA ends techniques. Overall, the nucleic acid sequence homology of the cFSH-R cDNA coding region is 71.8% and 72.2% compared to the rat and bovine FSH-R, respectively, while the deduced amino acid sequence identity for the receptor protein (693 amino acids) is 71.9% and 72.4%, respectively. By comparison, the cFSH-R nucleic acid and amino acid sequences are 60.1% and 49.4% identical to the respective cLH-R sequences. Northern blot analysis detected a single 4.3-kb cFSH-R mRNA transcript, which was selectively expressed in ovarian (granulosa, theca, and stromal) tissues, but not the oviduct, adrenal, liver, muscle, or brain. As the follicle developed from the prehierarchical (6- to 8-mm diameter) to the largest preovulatory (F1 follicle) stage, cFSH-R mRNA levels progressively declined within both the granulosa and theca layers (p < 0.05). Moreover, cFSH-R mRNA levels were lower in whole atretic than in morphologically normal 3- to 5-mm follicles (p = 0.0015). The pattern of cFSH-R mRNA expression within the granulosa layer during follicle development was notably different from that of the recently reported cLH-R, in that cLH-R mRNA levels increase to become readily detectable coincident with dramatically increased steroidogenic capacity during the last few days before ovulation of the follicle. On the other hand, highest levels of cFSH-R mRNA in 6- to 8-mm (prehierarchical) follicles were consistent with a role for the cFSH-R in maintaining the viability of prehierarchical follicles and in initiating granulosa cell differentiation at the time when follicles are selected into the preovulatory hierarchy.

Development of an homologous radioimmunoassay for chicken follicle-stimulating hormone and measurement of plasma fsh during the ovulatory cycle

1. 1. A highly specific and sensitive homologous radioimmunoassay was developed for measurement of chicken follicle stimulating hormone (cFSH). 2. 2. Mammalian gonadotropin releasing hormone (GnRH) significantly stimulated secretion of chicken luteinising hormone (cLH) but not cFSH when administered to 22 week non-laying hens. 3. 3. Chicken GnRH-I did not affect circulating cFSH concentrations but significantly stimulated cLH secretion when administered to 3 week cockerels. 4. 4. The plasma concentration of cFSH was low throughout the ovulatory cycle, but a significant decline in cFSH occurred prior to the pre-ovulatory LH surge and a significant increase occurred during the 3 hr prior to oviposition as LH levels decline.

Purification and characterization of chicken follicle-stimulating hormone

1. 1. Highly purified chicken follicle-stimulating hormone (cFSH) was isolated from chicken pituitaries by differential extraction, sequential chromatography on HPLC cation and anion exchange columns, and gel filtration chromatography. 2. 2. Purified cFSH (USDA-cFSH-K-1) had a potency of 77.44 units/mg in a chicken testes radioreceptor assay, and was biologically active in stimulating the secretion of progesterone by chicken granulosa cells. 3. 3. Purified cFSH contained negligible luteinizing hormone and thyroid stimulating hormone activity. 4. 4. The apparent molecular weight of cFSH was 38,000 Da and a single band on isoelectric focusing had a pI of 4.65.

Radioiodination of Chicken Luteinizing Hormone without Affecting Receptor Binding Potency1

By improving the currently used lactoperoxidase method, we were able to obtain radioiodinated chicken luteinizing hormone (LH) that shows high specific binding and low nonspecific binding to a crude plasma membrane fraction of testicular cells of the domestic fowl and the Japanese quail, and to the ovarian granulosa cells of the Japanese quail. The change we made from the original method consisted of 1) using chicken LH for radioiodination that was not only highly purified but also retained a high receptor binding potency; 2) controlling the level of incorporation of radioiodine into chicken LH molecules by employing a short reaction time and low temperature; and 3) fractionating radioiodinated chicken LH further by gel filtration using high-performance liquid chromatography. Specific radioactivity of the final 125I-labeled chicken LH preparation was 14 microCi/micrograms. When specific binding was 12-16%, nonspecific binding was as low as 2-4% in the gonadal receptors. 125I-Labeled chicken LH was displaced by chicken LH and ovine LH but not by chicken follicle-stimulating hormone. The equilibrium association constant of quail testicular receptor was 3.6 x 10(9) M-1. We concluded that chicken LH radioiodinated by the present method is useful for studies of avian LH receptors.

Direct demonstration of intrinsic follicle-stimulating hormone receptor-binding activity in acid-treated equine luteinizing hormone

After dissociating equine gonadotropins as a function of time at pH 3, we examined them by radioligand assay and sodium dodecyl sulfate polyacrylamide gel electrophoresis under nondissociating conditions (low, 0.1% SDS). Equine follicle-stimulating hormone (FSH) rapidly lost its receptor-binding activity, and low SDS-polyacrylamide gels demonstrated dissociation into subunits. Maximum dissociation occurred after 20–30 min of pH 3 incubation. Equine luteinizing hormone (LH), however, retained most biologic activity and was largely intact after 72 h of pH 3 incubation. Dose-response curves of acid-treated equine LH and FSH and intact equine LH and FSH were compared in five types of radioligand receptor assays. LH and FSH receptor-binding activities of equine LH were unaffected by pH 3. Equine LH showed 19- and 32-times more activity in the rat testis FSH assay than it did in chicken or horse FSH assays, respectively, directly demonstrating the intrinsic FSH receptor-binding activity of equine LH and the relative lack of specificity for these hormone preparations of the rat FSH receptor. Acid-treated equine FSH lost 95% of its biologic activity in FSH assays. In LH assays, the slight (0.2%) activity of equine FSH was relatively unaffected by acid treatment, suggesting that contamination by equine LH accounts for this activity.

Purification and properties of chicken growth hormone and the development of a homologous radioimmunoassay

Highly purified growth hormone (GH) has been isolated from pituitary glands of chicken ( Gallus domesticus), and a specific homologous radioimmunoassay (RIA) has also been developed. The purified chicken GH was active in the rat tibia bioassay and it gave a dose-dependent response which paralleled that of the bovine GH standard. High pressure liquid chromatography revealed that the purified chicken GH was homogenous. Chicken GH had an R f value of 0.2 in disc electrophoresis, and a MW of 26,000 from sodium dodecyl sulfate-gel electrophoresis. The isoelectric point was estimated to be 7.6 by gel isoelectric focusing. The amino acid composition of chicken GH was found to be similar to that of mammalian GH, and the NH 2-terminal amino acid was threonine. Partial sequencing (114 amino acids) of the chicken GH showed 79% homology with bovine GH. An antiserum was developed to the purified chicken GH in a rabbit, and it was used to develop a homologous RIA using 125I-labeled chicken GH as the ligand. The purified chicken GH was iodinated via the lactoperoxidase method to a specific activity of approximately 100 μCi/μg. Plasma from chickens, medium from incubation of pituitary glands, and homogenates of pituitary glands gave parallel dilution-response curves with the chicken GH standard. Mammalian GH, prolactin (PRL), follicle-stimulating hormone (FSH), and luteinizing hormone (LH) showed no cross-reaction with the 125I-labeled chicken GH. Purified turkey GH showed parallel dose response with the chicken GH, but purified turkey PRL did not cross-react. Chicken FSH and LH also showed no inhibition of binding. The minimum detectable concentration of the assay was 0.93 ng/tube, and the intraassay and interassay coefficients of variation were 9 and 16%, respectively. The specific binding of 125I-labeled chicken GH to a microsomal fraction isolated from chicken liver was identified, and the specific binding was generally low (1–4%). Turkey PRL, and chicken LH and FSH showed no inhibition of the 125I-labeled chicken GH hepatic binding and the ontogeny of the hepatic GH receptor binding sites in male and female chickens was examined.

Isolation and characterization of chicken follicle-stimulating hormone

Chicken FSH was purified from a glycoprotein fraction of broiler chicken adenohypophyses by a series of chromatographic separations on carboxymethyl (CM)-cellulose, calcium phosphate, diethylaminoethyl (DEAE)-cellulose, and Sephacryl S-200 superfine. The relative FSH potency of the final product was 74.6 × NIH-FSH-S12 in the radioreceptor assay using a homogenate of the testis from Japanese quail as the receptor preparation and rat FSH for the radioiodinated hormone preparation. The amino acid composition of the final product was similar to that of chicken FSH reported by P. M. M. Godden and C. G. Scanes (1975, Gen. Comp. Endocrinol. 27, 538–542) and turkey FSH reported by W. H. Burke, P. Licht, H. Papkoff, and A. Bona Gallo (1979, Gen. Comp. Endocrinol., 37, 508–520). Its molecular weight estimated by gel filtration with Sephacryl S-200 superfine was 37,000. Isoelectrofocusing of the FSH fraction obtained after the DEAE-cellulose chromatography showed that chicken FSH consisted of at least two components with different isoelectric points. In the component with p I 4.4 to 4.6 a high FSH potency was detected by both RRA and RIA. The second component with p I 4.1 to 4.2 had a high FSH potency in RIA but a slight FSH potency in RRA. Two more weakly immunoreactive components were found in more acidic ranges but they did not show any significant RRA potency.

Isolation and characterization of luteinizing hormone and follicle-stimulating hormone from pituitary glands of the turkey ( Meleagris gallopavo)

Follicle-stimulating hormone (FSH) and luteinizing hormone (LH) were purified from the pituitary glands of the turkey ( Meleagris gallopavo). These hormones were characterized biochemically and biologically and compared with chicken gonadotropins prepared in several independent laboratories. Amino acid and carbohydrate analyses demonstrated homology between turkey and other species of gonadotropin. The turkey LH purified here had significantly higher carbohydrate content than a previous preparation of turkey LH. Immunological studies further confirmed that the turkey FSH and LH were distinct from one another and that each was homologous to the respective gonadotropin from other vertebrates; the immunopotencies of the turkey hormones were similar to those from chickens. A variety of bioassays and radioreceptor assays (RRAs) confirmed the biological activity of the two turkey gonadotropins and revealed that the turkey LH was distinct from that of the chicken. As expected, the two types of turkey hormones were approximately equipotent in total gonadotropin bioassays (frog spermiation and 32P uptake by chick testes), and only the turkey LH was active in the rat Leydig cell assay and in RRA for LH in mammals. However, the turkey LH was also highly potent in several assays considered to be relatively FSH specific, including the Anolis lizard assay and several RRA systems using mammalian, turtle and avian gonadal receptors with 125I-labeled human FSH as tracer. Turkey and chicken FSH are similar in the RRAs, but the turkey LH was consistently more potent than either avian FSH in competing for FSH-binding sites. Chicken LH had relatively low activity by comparison. It is suggested that the evolution of the structure of active sites in turkey LH has involved convergence on those of the FSH molecule.

Purification of chicken pituitary follicle-stimulating hormone and luteinizing hormone.

SUMMARY Separation and partial purification of chicken pituitary follicle-stimulating hormone (FSH) and luteinizing hormone (LH) has been obtained by methods which were effective in the purification of the corresponding human and horse hormones. Increases in chicken LH activity were observed after chromatography on DEAE-cellulose and on Amberlite IRC-50 suggesting removal of an LH inhibitor. The biological potencies of chicken FSH and LH preparations when assayed in mammals were very much lower than those of the corresponding mammalian fractions on a weight basis. A weak immunological cross-reaction between chicken and human pituitary LH was used to estimate LH in chicken pituitary fractions and the results were compared with bioassays of the same fractions.