Date of this Version
Voss, A. M. 2013. Isolation of the Gonadotropin-Releasing Hormone II Receptor Within the Testis of the Pig. M.S. Thesis, University of Nebraska-Lincoln, Lincoln, NE.
The second isoform of gonadotropin-releasing hormone (GnRH II) has been linked to enhanced LH responsiveness of Leydig cells. Its receptor (GnRHR II) has been isolated in reproductive and non-reproductive tissues. Coding sequence for GnRHR II contains reading errors in many species, preventing production of a functional receptor. In contrast, the pig possesses coding sequence for a functional GnRHR II. Thus, the objectives of this study were to localize GnRHR II in developing swine testes, compare GnRHR II mRNA and protein levels within the maturing pig testis and develop a porcine model with reduced levels of endogenous GnRHR IIs to examine its biological function. Testicular tissue was collected at birth, pre-, and post-puberty. GnRHR II mRNA levels were higher in testes from post- compared to pre-pubertal boars (P < 0.05). GnRHR II protein was present in testes from all age groups, although, levels were reduced in testes from post- vs. pre-pubertal animals (P < 0.05). Immunofluorescence signal of GnRHR II was seen within the interstitial and tubular compartments of testes at birth and post-puberty, as well as within heads of spermatids in mature boars, suggesting multiple roles for GnRHR II in testicular function. Next, we wanted to develop an animal model with reduced levels of endogenous GnRHR IIs. Two target shRNA for GnRHR II were subcloned into a pLVX-shRNA2 vector, providing shRNA and fluorescent ZsGreen1 coexpression. Transduction of swine testis derived (ST) cells with shRNA1 and shRNA2 lentiviral particles reduced GnRHR II mRNA levels by 95 and 99%, respectively, compared to controls (P < 0.05). Next, zygotes were microinjected with shRNA2 lentiviral particles and transferred into a recipient, resulting in 5 live piglets. One female exhibited transgene expression with a single integration site on chromosome 14. The transgenic and 2 female littermates reached puberty at similar ages and had similar litter sizes at first parity. Five out of 15 piglets from the transgenic female were positive for green fluorescence, confirming germline transmission of the transgene.
Advisor: Brett R. White