Isolation of Gonadotropin-Releasing Hormone II Receptor Within the Testis of the Pig.

The second mammalian isoform of gonadotropin-releasing hormone (GnRH), GnRH-II, is ubiquitously expressed whereas the native isoform, GnRH-I, has been detected primarily within hypothalamic neurons and other reproductive tissues. Gonadotropin-releasing hormone-II has been linked to the interaction between energy balance and reproduction in females and has anti-proliferative effects on tumor-derived cell lines. In males, the release of testosterone from Leydig cells in response to LH challenges was significantly reduced in animals immunized against GnRH-II, likely through interactions with the GnRH-I receptor (GnRHR-I). The specific receptor for GnRH-II (GnRHR-II) is structurally similar to GnRHR-I, except GnRHR-II has an intracytoplasmic tail that is absent in GnRHR-I. Further, the receptor for GnRH-II has been isolated in both reproductive and non-reproductive tissues. Via confocal microscopy, our laboratory has localized GnRHR-IIs within the plasma membrane of a fetal swine testis-derived (ST) cell line using immunocytochemistry procedures with an antibody specific for GnRHR-II (Abnova, Taipei City, Taiwan), providing evidence for a functional GnRHR-II within the testis of the pig. Consistent with this, the objectives of this study were to determine GnRHR-II localization within the testis of swine and compare GnRHR-II mRNA and protein levels within the maturing pig testis. Toward this end, we collected testicular tissue from male pigs at 1, 56 and 592 d of age (n = 5 males/age group) for isolation of RNA and protein as well as tissue for immunohistochemistry procedures. Isolated tissue was fixed in 4% paraformaldehyde, embedded in paraffin, sectioned, placed on microscope slides and immunohistochemistry performed using the Vectastain ABC-AP Kit (Vector Laboratories, Burlingame, CA). Slides were blocked in normal serum for 20 min, incubated with an antibody specific for GnRHR-II (Santa Cruz Biotechnology, Santa Cruz, CA) for 1 h, and incubated for 30 min with secondary antibody. Alkaline phosphatase staining was detected within the interstitial compartment of testes from all three age groups, suggesting a possible role for GnRHR-II in Leydig cell function. Next, protein isolates were exposed to SDS-PAGE, transferred, and membranes incubated with Odyssey blocking buffer (LI-COR Biosciences, Lincoln, NE) overnight, a primary antibody for either GnRHR-II or actin (Santa Cruz) for 2.5 h (1:500) and a donkey-anti-goat secondary antibody (IR Dye 680LT; LI-COR) for 1 h (1:8000). Following standardization for actin protein levels, GnRHR-II protein was present in testes isolated from all males, regardless of age. Finally, isolated RNA was quantified, reverse transcribed to cDNA and quantitative PCR performed. Quantitative PCR results confirmed the Western blot analysis, indicating amplification of GnRHR-II within testicular tissue from all age groups. In addition, PCR data was analyzed using the GLM procedure of SAS and mRNA levels compared between age groups using Tukey's Studentized Range Test. Results revealed higher GnRHR-II mRNA levels in testes isolated from boars at 592 d of age (20.4-fold over 1 d age group) compared to testes from males at 56 (2.6-fold vs. 1 d) and 1 d of age (P < 0.05). In conclusion, this study represents the first report of GnRHR-II mRNA and protein within the interstitial compartment of developing porcine testes. The authors would like to thank the Nebraska Gene Center, Danbred North America for porcine tissue. (poster)