Effect Of Somatostatin On Hormone Secretion And Genes Expression Related To Growth And Fertility, Subsequent Embryo Development And Its Phylogenetic History In Mammals

Somatostatin(SST) is a regulatory peptide with two bioactive forms viz.SST-14 and SST-28.It acts on a wide array of tissue targets to modulate neurotransmission, inhibition of hormones secretion and regulation of cell growth and proliferation.Growth hormone(GH) secretion is controlled by SST and its patterns are also influenced by sex steroids,at least in part,through modulation of the secretion of hypothalamic SST and GH-releasing hormone.It is concluded that testosterone promotes and estradiol,at least at high doses,inhibits storage of GH in the anterior pituitary and these changes in GH production may be regulated,in part,by altered SST release from the median eminence into the hypophysial portal blood which provides an autocrine control of these hormones. Furthermore SST is an ancient gene family so,the objectives of the current study were to investigate the possible control of growth and fertility related genes viz.growth hormone, testosterone,estrogen and follicular stimulating hormone by SST in granulosa cells which in turn will explore the role of SST in oocyte development and maturation and trace back the phylogenetic origin of this novel gene and its receptors in mammals.A.Plasmid constructs,extraction,granulosa cells culture,transient transfection and GFP-expression studyObjectives:Objectives of these experiments were to construct vector containing fulllength normal mice somatostatin gene cloned from mice brain cDNA into the pEGFP-N1 vector,culture of E Coli and isolation of plasmid in bulk,separation of follicles from granulosa cells and finally culture for 24 h,transfection with the vector already constructed and study of GFP gene expression over next 96 h.Methods:Pooled brain first strand cDNA obtained from mice reverse transcriptase served as template for PCR amplification using Taq DNA polymerase and primer pairs provide by Sangon Pvt.Ltd. Shanghai.This PCR product was double digested with respective enzymes and cloned to the vector which was finally transfected and grown in E Coil to extract plasmid in bulk. Ovaries obtained from local abattoir were used to separate bovine granulosa cells(bGCs), which were cultured overnight to adhere in respective medium,and finally transfected with vector constructed already using Lipofectamin and GFP expression was studied over next 4 days.Results;somatostatin gene started expression after 24 h of transfection, gradually increased until 4^th day post transfection and then declined to minimum until day seventh.B.RNA extraction,reverse transcription and Real-Time PCR quantificationObjectives:The objectives of these experiments were to study somatostatin gene transfection effects on expression level of estrogen receptorβ,androgen receptor,growth hormone releasing hormone receptor and follicular stimulating hormone receptor transcript level.Methods:Total RNA was extracted from cells as per the manufacturer's protocol,cDNA synthesized from total RNA using gene-specific primers.Semiquantitative RT-PCR was used to get and estimate desired fragments of products.Realtime (RT)-PCR analysis was performed in individual sample with an ABI Prism 7900 Sequence Detection System(Perkin Elmer Applied Biosystems) using 6-carboxyfluorescein-and 6-carboxy-tetramethylrhodamine-labeled fluorogenic probes. The expression data were normalized againstβ-actin.Results:We found a significant (2.37X) increase in Erβexpression between experimental and controlled,with also a decrease in Ar,GHRHr and FSHr transcript level(P＜0.05).Conclusion:Somatostatin transfection affects growth and fertility related genes at transcriptional level.C.Testosterone,FSH,estradiol-17βand growth hormone concentrationsObjectives:The objectives of this experiment were to estimate the concentrations of testosterone,FSH,estradiol-17βand growth hormone in culture media.Method;Culture media testosterone,FSH,estradiol-17βand growth hormone concentrations were measured by kits supplied by Beijing Atom Hightech Co.Ltd.according to manufacturer's standards.Results:after 48 and 96 h of transfection,the culture media concentration of estradiol-17βwas increased significantly(P＜0.05) and testosterone, GH and FSH showed just opposite trend(P＜0.05) in experimental groups.Conclusion: Somatostatin transfection not only affects growth and fertility related genes at transcriptional level,but also affects hormonal profile of these genes.D.Oocyte MaturationObjectives:study somatostatin transfected granulos cells effect on maturation.Methods: The COCs were aspirated from ovaries using a syringe with a 20 gauge needle;COCs were washed and then transferred to 100μL drops of maturation medium containing bovine granulosa cells layers(transfected or control at the density of 5×10³ cells per drop) for 24 h at 38.5℃in atmospheric air with 5%CO₂.Results;Somatostatin transfected cells improved maturation rate significantly.E.In vitro fertilization and embryo cultureObjectives:To study effect of somatostatin transfected GCs on fertilization and embryo development.Methods:Following 24 h of maturation,oocytes transferred into 50 uL drops of fertilization medium(Be with 5 mg/mL BSA fraction V;10 mM caffeine and 10 mg/mL heparin) under mineral oil.One straw of semen was thawed at 38℃,the sperms were washed two times by centrifugation at 450×g for 10 min.Subsequently,the re-suspended sperms were added to each fertilization drop at concentration 1×10⁷ sperm/mL.Results:Somatostatin transfected cells had no effect on IVF & embryo development.D.Identification of putative somatostatin and somatostatin receptors within the draft genomes of D.rerio,Carassius auratus,X.tropicalis,G.gallus,Monodelphis domestica,Homo sapiens,Sus scrofa,Bos taurus,Mus musculus,Rattus norvegicus, Canis lupus familiaris,Ovis aries,Equus caballus,Pan troglodytes and Macaca mulattoObjective:The objective of this experiment was to identify all known and putative form of somatostatin and its receptors.Methods:All SST and SST receptors for G.gallus, Homo sapiens,Sus scrofa,Bos taurus,Mus musculus,Canis lupus familiaris,Ovis aries, Equus caballus,Pan troglodytes and Macaca mulatto and somatostatin sequences of D. rerio,X.tropicalis,Monodelphis domestica were identified from National Center for Biotechnology Information(www.ncbi.nlm.nih.gov) while somatostatin receptors for D. rerio,X.tropicalis and Monodelphis domestica from UCSC Genome Bioinformatics (http://genome.ucsc.edu).Results:DNA and Protein sequences for somatostain and its receptors were obtained.Conclusion:There were 5 forms of SST receptors with some having isoforms.E.Pairwise homology of putative somatostatin and somatostatin receptorsObjectives:The objective of this experiment was to determine the pairwise homology of somatostatin and its receptors.Methods:All somatostatin and somatostatin receptors were compared to each other somatostatin and somatostatin receptors including both known and putative from the examined species by Needlemen-Wunsch pairwise alignment using the needle program from the EMBOSS package with default parameters. Results:The percent identity of maximum scoring pair was listed.Conclusion:The percent identity was as high as 99%in some cases.F.Phylogenetie analysis of somatostatin and somatostatiu receptorsObjectives:The objectives of this experiment were to construct a phylogenetic tree and trace back phylogenetic history of SST and its receptors.Methods:All known and putative somatostatin and somatostatin receptors were aligned using ClustalX,and phylogenetic analysis was performed using both protein parsimony and nearest neighbor methods with a bootstrap score of 100 or 500 using the PHYLIP software package (Phylogeny Inference Package,version 3.5c;J.Felsenstein,University of Washington, Seattle,WA) and Mega 4.Results;History of somatostatin and its receptors can be traced back to 165 million years ago in mammals.Conclusion:Both protein parsimony and nearest neighbor methods yielded consisted trees.