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Somatostatin(SST)is a regulatory peptide with two bioactive forms viz.SST-14and SST-28.It acts Oa a wide array of tissue targets to modulate neuroWansmission,inhibition of hormones secretion and regulation of cell growth and proliferation.Growthhormone(GH) secretion is controlled by SST and its patterns are also influenced by sexsteroids，at least in part,through modulation of the secretion of hypothalamic SST andGH-releasing hormone.R is concluded that testosterone promotes and estradiol，at least athigh doses。
A.Plasmid constructs,extraction，granulosa cells culture，transient transfecfion and GFP-expreesion study
Objectives：Objectives of these experiments were to construct vector containing full-length normal mice somatostatin gene cloned from mice brain cDNA into the pEGFP-N1vector,culture of E Coli and isolation of plasmid in bulk,separation of follicles from granulosa cells and mlally culture for 24 h’transfection with the vector already conshucted and study of GFP gene expression over next 96 h.Methods：Pooled brainfirst strand cDNA obtained from mice reverse transcriptase served as template for PCR amplification using Taq DNA polymemse and primer pairs provide by Sangon Pvt.Lid.Shanghai.This PCR product Was double digested with respective enzymes and cloned tothe vector which was finaUy transfected and grown in E Coli 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 4th day post transfection and then declined to minimum until dayseventh.
B.RNA extraction，reveile transerlptiou and Real-Time PCR quantification
Objectives：The objectives of these experiments were to study somatostatin genetransfection effects on expression level of estrogen receptor β，androgenreceptor,growthhormone releasing hormone receptor and follicular stimulating hormone receptortranscript level.Methods:Total RNA Was extracted from cells as per the manufacturersprotocol,eDNA synthesized from total RNA using gene-specific primers.Semi-quantitative RT-PCR Was used to get and estimate desired fragments of products.Real-time(RT)-PCR analysis was performed in individual sample with an ABI Prism 7900Sequence Detection System(Perkin Elmer Applied Biosystems)using 6-carboxyfluorescein-and 6-carboxy-tetramethylrhodamine-labeled fluorogenic probes.The expression data were normalized against pactin.Result：We found a significant(2.37X)increase in Erp expression between experimental and controlled,with also a decrease in Ar,GHRHr and FSI-Ir transcript level(P<0.05).—ConCl-usion：Somatostatintransfection affects growth and fertility related genes at transcriptional level.
C.Testosterone，FSH,estradiol-17βand growth hormone concentrations
Objectives：The objectives of this experiment were to estimate the concentrations oftestosterone，FSH，estradiol-17β and growth hormone in culture media.Method：Culture media testosterone，FSH，estradiol-17β and 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-17β was 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 attransefiptional level，but also affects hormonal profile of these genes.