Buffalo Pituitaries Research Articles

Epitope-based in silico peptide design yields peptide-directed antibodies that recognize the buffalo luteinizing hormone

We present a novel peptide sequence identified through in silico epitope design and the later generation of peptide-directed antibodies recognizing the buffalo luteinizing hormone. Peptides and antibodies, specific to reproductive hormones, are valuable tools for developing point-of-care immunodiagnostic tools. The study predicted an epitope peptide in silico from buffalo luteinizing hormone and the generation of polyclonal antibodies against this peptide sequence. In this quest, we identified a novel epitope peptide sequence (luteinizing hormone peptide, LHP) through bioinformatics tools. The peptide was further synthesized and characterized. The polyclonal antibodies (anti-LHP) were raised against the peptide in the rabbit. Thereafter, we explored a strategy for detecting buffalo luteinizing hormone (LH) using the anti-peptide antibodies developed. The affinity of the peptide, bovine lutropin beta, and crude LH (prepared from buffalo pituitary) towards the raised antibodies was established by dot blot and ELISA. Specific recognition of the luteinizing hormone by the raised polyclonal antibodies highlights the ability of the identified peptide (LHP) and developed polyclonal antibodies (anti-LHP) as suitable diagnostic reagents for sensing the buffalo luteinizing hormone. Through this work, we analyzed and translated the “-omics” information in the LH gene sequence for the development of a novel peptide and antibodies as valuable immuno-reagents.

Indigenous Production of Bovine/Bubaline Reproductive Hormones

The research work of our laboratory on buffalo pituitary hormones is summarized here in the context of MOET programme of our country. All the anterior pituitary protein hormones of this species (i.e. Follicle stimulating hormone (FSH), Growth Hormone (GH) and others) have been purified from freshly frozen pituitaries. These hormones have been extensively characterized with regard to physico chemical, immunochemical and biological features. We have also produced buffalo Prolactin (PRL), Luteinizing Hormone (LH), FSH and Thyroid Stimulating Hormone (TSH) by recombinant DNA techniques.

Assessment of Pseudoaffinity Chromatography Using Textile Dyes for Isolation of Buffalo Pituitary Luteinizing Hormone

Extensive investigation has been carried out to elucidate the mechanisms involved in pseudoligand affinity chromatography using textile dyes, and, empirically, it has been attributed to the chemical and steric structures of dye and protein. Possibly, a variety of interactions especially ionic and/or hydrophobic influence with a varying share in the binding and differ from protein to protein and from dye to dye. In this study, we have attempted to understand the effect of various biophysical parameters like the nature of the eluant, pH, and ionic strength on the binding of crude luteinizing hormone (LH) with various triazine-based dyes and thus predict their nature. Based on the elution patterns, cibacron and reactive brown suggested a dual electrostatic and hydrophobic nature. Reactive blue and reactive yellow reflected a major electrostatic/ionic nature with yellow offering 50-fold purification in a single step, while reactive red and reactive green had a predominant hydrophobic nature. Appreciably, reactive red was binding LH very tightly unlike other dyes, and addition of the arginine in the elution buffer substantially weakened the protein-dye interactions. pH was observed to be a principal factor assisting the protein-dye binding as well as hydrophobicity of the dye and the proteins.

PURIFICATION AND BIOLOGICAL CHARACTERIZATION OF BACTERIALLY EXPRESSED RECOMBINANT BUFFALO PROLACTIN

Recombinant prolactin (PRL) from water buffalo (Bubalus bubalis) has been cloned and expressed in a prokaryotic expression system. The hormone was also successfully refolded into a biologically active form. Total RNA was purified from buffalo pituitaries and the buPRL cDNA was synthesized using primers designed on bovine PRL sequence. This prolactin cDNA was cloned in a pET 28a vector and expressed in Escherichia coli strain BL21(DE3)pLysS. Most of the expressed protein was present as insoluble inclusion bodies. The inclusion bodies were solubilized and buPRL was purified by Ni-NTA column. The purified protein was refolded by gradually decreasing the concentration of denaturant during dialysis. Total yield of the refolded and soluble prolactin was 22 mg/L from 100 mL bacterial culture in LB medium. The recombinant prolactin was as active as native prolactin in stimulating growth of Nb2 lymphoma cells.

Pseudo-affinity chromatographic approach to probe heterogeneity in buffalo pituitary luteinizing hormone: probable pseudolectin-like behavior of immobilized Cibacron Blue 3GA

The alpha (α) and beta (β) subunits of buffalo pituitary luteinizing hormone (LH) were chromatographed on Cibacron Blue 3GA agarose and their immunoreactivity was quantitated using anti-α and anti-β anti sera. Subsequent analyses showed α subunits were relatively more hydrophilic than β subunits. Further, the naturally occurring free α and β subunits were more hydrophobic than their native counterparts which were dissociated and isolated from heterodimeric LH. The lesser sugar content in freely occurring α and beta subunits may be attributed for increased hydrophobicity and consequent upon the existence of their uncombined free forms. In order to ascertain putative sugar–dye interaction, crude LH carrying free subunits, pure LH, and non-glycosylated recombinant β subunit of LH were loaded separately on Cibacron Blue. Methyl mannoside was able to elute 33% of the bound protein in case of crude and pure LH, whereas there was little (3%) elution in case of recombinant LH β subunit. This study suggests a compositional heterogeneity in free and native subunits of LH from the buffalo pituitary. In addition, our findings reveal the pseudolectin-like behavior of Cibacron Blue.

Purification of Monomeric Prolactin Charge Isoform from Buffalo Pituitaries

A standard protocol for isolation of buffalo prolactin (buPRL) was modified at the alcohol precipitation step. This modification could separate lower molecular weight prolactin from the higher molecular weight prolactin (PRL). Reloading the prolactin onto a Sephacryl S‐200 gel purified the buPRL monomer. The purity of buPRL monomer was confirmed by 15% SDS PAGE. The buPRL monomer was >90% pure. It was characterized by specific anti‐buPRL serum in ELISA and Western blot. A native PAGE of the PRL showed three charge isoforms. A protocol was standardized to separate prolactin monomeric least acidic isoforms using an anion exchanger.

A Reference Preparation of Buffalo Pituitary Follicle Stimulating Hormone using Lectin Affinity Chromatography

An improved and cost effective method to isolate FSH from buffalo pituitary glands is described here. The buFSH activity was monitored throughout by a highly sensitive heterologous radioimmunoassay (sensitivity 0.2 ng oFSH/mL) and the in vivo biological activity of the final preparation was also established. A biologically active buFSH‐enriched preparation with a moderate recovery (42%) was obtained. The yield of the final buFSH‐enriched preparation was 26.5 mg/kg of buffalo pituitary gland. In SDS‐PAGE, the purified buFSH resolved as a heterodimer of 30 kDa molecular size, with a 21 kDa presumptive α‐subunit. This preparation was also characterized in terms of biological and some of its physicochemical properties. A high‐titer antiserum to buFSH was also raised in rabbit using this preparation. The reagents generated, buFSH and buFSH‐specific polyclonal antisera, have possible diagnostic and therapeutic usage for improvement of reproductive health of water buffaloes.

Simultaneous Isolation of Prolactin and Growth Hormone from “Discarded Acid Pellet” Obtained from Buffalo Pituitaries

An acid pellet, obtained as a side fraction from a conventional gonadotropin purification pathway, has been found to contain the bulk of the pituitary lactogenic hormones (growth hormone or GH and prolactin or PRL). This discarded side fraction has been utilized to obtain buffalo lactogenic hormones (buGH and buPRL), simultaneously, and in bulk. The immunoreactivities of the purified semi‐pure buffalo GH and PRL (APECS and APP‐I, respectively) preparations were compared by direct binding ELISA with semi‐pure standard buGH and PRL (ECS and EP‐I, respectively) and were found to be as pure as standard semi‐pure buGH and buPRL. When checked by direct binding ELISA using buGH and buPRL antisera, it was observed that APECS and APP‐I were not cross‐immunoreactive. SDS‐PAGE and western blot analysis of APECS and APP‐I showed major bands located at the same positions as in the case of standard semi‐pure preparations (20 kDa for APECS and 23 kDa for APP‐I). The semi‐purified buGH and buPRL (APECS and APP‐I) were converted to a highly purified preparation by chromatographing them via Sephacryl S‐200 gel‐filtration chromatography.

Occurrence of Free Alpha Subunit of the Thyroid Stimulating Hormone Family in Pituitaries of Water Buffaloes ( Bubalus bubalis )

Presence of free alpha subunit has been noticed in a side fraction obtained during the processing of buffalo pituitary glands for thyrotropin and gonadotropins using standard isolation and fractionation procedures. In addition to the gel chromatographic data (Ve/Vo), when the material was analyzed with different antisera, it clearly reacted with alpha subunit directed antibodies. The NaCI precipitation method of Sairam for the separation of the free subunits from each other was applied to the material. The yield of the alpha subunit by the NaCI precipitation method with respect to the immunogenicity and the content was higher than what was obtained by the counter current distribution. The sugar content of the free alpha subunit was less (70 I¼g/mg) compared to the sugar content of the alpha subunit isolated by the dissociation from the intact hormone (204 I¼g/mg). Most of the free alpha subunit eluted as the unbound fraction from a Concanavalin A sepharose column thus supporting the idea that there was an alteration in carbohydrate structure in these naturally occurring free subunits.

Heterogeneity in buffalo pituitary prolactin.

The Ellis procedure of serial extraction of gonadotropins and growth hormone (GH) followed by alkaline ethanol extraction was adopted to process freshly frozen buffalo pituitaries. The procedure after slight modification was found very useful as more than 2 mg of GH free immunoreactive prolactin (PRL) could be isolated from each gram of wet pituitary tissue. Further, the biochemical purity and immunobiological potency of the extracted PRL, designated as P-I, was comparable with that of the highly purified samples of homologous and heterologous PRLs. No non-PRL protein was detectable in P-I. Micro-heterogeneity with regard to size, charge, co- and post-translational modifications was also investigated under different conditions of extraction and at different stages of purification. Immunological and biological potencies were compared in homologous competitive enzyme linked immunosorbent assay (ELISA) developed for buffalo PRL and in rat Nb2 lymphoma proliferation assay respectively. Structural heterogeneity was observed in all the preparations checked including fresh pituitary homogenate and highly purified hormone. Nevertheless a 25 K species corresponding to the hormone monomer was always the only paramount form comprising more than 90% of the total PRL protein in all the samples including P-I. Similar size forms were observed in all preparations and were found to be equivalents of monomers, dimers, covalent-and non-covalent multimers, disulphide bridged forms and cleaved fragments. Other sibling species identified were glycosylated PRL, charge isoforms and forms that perhaps differed in their extractability from the pituitary tissue. Strong apparent size heterogeneity was displayed by the monomeric buffalo PRL. In light of these observations and the information on the structural and functional significance and the consequences of polymeric forms, the use of a heterogeneous PRL (P-I) as a reference hormone is recommended for a valid assay.

Purification and characterisation of prolactin from sheep and buffalo pituitaries

A study of the problem of structural variants of proteins and their relative contribution to the expressed immunological and biological activity has been initiated using sheep and buffalo prolactins as models. The feasibility of obtaining immunologically and biologically active prolactin in high yields from the discarded ’acid pellet’ of sheep and buffalo pituitaries has been demonstrated. This permits use of the same batch of glands for purifying lutropin, follitropin and prolactin as side fractions. The major component in preparations of buffalo prolactin has a molecular size of 24 kDa. The preparations were active in a radioligand binding inhibition assay and in a rat liver based radioreceptor assay. Charge and size isomers of sheep prolactin and buffalo prolactin have been observed. The reference sheep prolactin did not, in preliminary work, give any indication of being glycosylated. However radioactive sulphate was found to be incorporated into prolactin-rich fractions of sheep and buffalo pituitariesin vitro. By physico-chemical and immunochemical criteria the [35S]-labelled material was similar to standard reference prolactin. The structural implications of sulphation have been probed.

Are sheep and buffalo prolactins sulfated?

Radioactive sulfate ( 35SO 4 2−) has been shown to be incorporated into immunoprecipitable prolactin-like material from incubated minces of sheep and buffalo pituitaries. The 35S-labelled prolactin could be purified by standard procedures. On SDS-PAGE, the 35S-labelled prolactin rich fraction gives two major Coomassie blue bands around 25KDa and these on Western blot analysis gave positive bands. Radioactive [ 14C]-mannose was also found incorporated into the prolactin like material. The nature of sulphate link to the peptide is not known. It could be sugar-SO 4 and/or Tyrosine-SO 4.

On the presence of sulphate in pituitary lutropin

The presence of sulphate in the carbohydrate of pituitary lutropin from different species has been investigated using a biosynthetic approach. Pituitaries from rats, rabbits, goats, and buffaloes were incubated in the presence of35SO4- and the35SO4- -labelled proteins in the tissue immunoprecipitated with a well characterized anti-sheep lutropin serum. The incorporation into immunoreactive lutropin was low in the case of rat, rabbit and goat pituitaries while, it was considerable in the case of buffalo pituitaries. Hence further characterization studies were carried out on35SO4- -labelled proteins of buffaloes. The physico-chemical, immunological and biological properties of radio-labelled buffalo pituitary material were shown to be similar to those of standard lutropin. Inin vitro conditions of incubations, most of the incorporation of35SO4- was observed into tissue lutropin while under similar conditions of incubation, [14C]-amino acids were found to get incorporated mostly into medium lutropin. The physiologically specific releasing hormone, lutropin-releasing hormone was found to stimulate the release of35SO4--labelled lutropin from the rabbit pituitaries into the medium. These results give indirect evidence that sulphate could be present in pituitary lutropin.

Incorporation of radioactive 35SO 2−4 into immunoreactive pituitary lutropin

The terminal hexosamines of bovine pituitary lutropin are thought to contain a sulfate moiety. In order to test this, a biosynthetic approach was adopted. When rat and buffalo (bovine) pituitaries were incubated with radioactive 35SO 2− 4 for 2 h in vitro it was observed that radioactivity gets incorporated into trichloroacetic acid-precipitable proteins. When the radioactive proteins were treated with an anti-sheep lutropin serum, radoactivity was found in the immunoprecipitate. The incorporation into rat lutropin like material was very marginal while it was very significant in the case of buffalo lutropin.

The Seminal Thinning Response of Carp (Cyprinus carpio) and Rainbow Trout (Salmo gairdnerii) after Injections of Pituitary Extracts

Twenty-four hours after an intraperitoneal injection of aqueous extracts of acetone-dried carp pituitary glands, the semen of previously reproductively quiescent carp increased similar to that commonly observed in fish immediately before or during spawning. Similar results were obtained with rainbow trout injected either with an extract of bigmouth buffalo (Ictiobus cyprinellus) pituitaries, or with chorionic gonadotropin. In this ripening process of out-of-season fishes, the rapid and extensive seminal thinning was studied by cell counts, seminal plasma percentage, relative water percentage, relative seminal plasma osmolarity, and seminal plasma sodium, potassium, magnesium, and calcium. The fluidity of the semen, reflected in a linear relationship between sperm cell count and relative water content, increased in direct proportion to the logarithm of the dose in the range 0.22 to 22.0 uAg/g. Although total osmotic pressure decreased with increasing fluidity, the ionic concentrations of sodium, potassium, magnesium, and calcium were unchanged, implicating depletion of osmotically active organic substances.