Location: Reproduction ResearchTitle: A putative role for GnRH-II and its receptor in spermatogenic function of boars Author
Submitted to: Society for the Study of Reproduction Annual Meeting
Publication Type: Abstract Only
Publication Acceptance Date: 3/20/2015
Publication Date: 6/2/2015
Citation: Desaulniers, A.T., Cederberg, R.A., Mills, G.A., Lents, C.A., White, B.R. 2015. A putative role for GnRH-II and its receptor in spermatogenic function of boars [abstract]. Society for the Study of Reproduction Annual Meeting. p. 210 (Abstract #530). Available: https://www.ssr.org/ Interpretive Summary:
Technical Abstract: Unlike the classical gonadotropin-releasing hormone (GnRH-I), the second mammalian isoform of GnRH (GnRH-II) is ubiquitously expressed with the most abundant transcript levels found in tissues outside of the hypothalamus. Moreover, GnRH-II is only an inefficient stimulator of gonadotropin release. Interestingly, the pig represents the only livestock species to maintain the full-length coding sequence needed to produce a functional receptor specific for GnRH-II (GnRHR-II). Together, GnRH-II and its receptor appear to modulate the interaction between feed intake and reproductive behavior in female marmosets and musk shrews. Despite its ubiquitous expression, other functions of the GnRHR-II remain elusive. We recently demonstrated that GnRH-II levels were higher in the porcine testis than in the anterior pituitary gland or hypothalamus, corresponding to greater GnRHR-II abundance in the testis versus the anterior pituitary gland. Previous data in our laboratory also suggest that testicular GnRHR-II is present on the plasma membrane of Leydig cells and has a role in localized steroidogenesis. Notably, we also detected GnRHR-II staining within the seminiferous tubules, suggesting that GnRHR-II may be involved in spermatogenesis and/or spermatogenic function. In the present study, our objective was to further localize GnRH-II and GnRHR-II in the reproductive tract of the boar. First, immunoblotting (n = 5 boars) revealed that GnRHR-II levels are 12-fold greater in the testis (P < 0.0001) compared to the epididymis, prostate, seminal vesicles or bulbourethral glands, suggesting an important role in testicular function. Next, we performed immunofluorescence on paraformaldehyde-fixed, paraffin-embedded boar testicular tissue (n = 6) using an antibody directed against GnRH-II and GnRHR-II. We identified GnRH-II immunostaining primarily within the tubular compartment, localizing to round germ cells. Moreover, we also observed intense GnRHR-II staining on elongating spermatids. In order to further explore the identification of the GnRHR-II on spermatids in fixed tissue, we collected Percoll-purified, ejaculated spermatozoa (n = 7) for use in immunoblotting and immunocytochemistry procedures. Indeed, a distinct band for GnRHR-II was detected by Western blot. Interestingly, the molecular weight between GnRHR-II detected in the testis (60 kDa) differed from purified sperm samples (54 kDa), indicating differential post-translational modification. We also performed immunocytochemistry on the purified spermatozoa samples and detected intense GnRHR-II signal on the connecting piece, suggesting a role in fertilization or acrosomal exocytosis. After discovering the GnRHR-II on mature spermatozoa, we next evaluated seminal plasma for the presence of GnRH-II via ELISA. Indeed, GnRH-II was detected in seminal plasma (n = 7; 122-332 pg/ml), which likely originates from the testis as GnRH-II levels were highest in testicular tissue homogenates (26.9 ± 2.0 ng/g tissue; P < 0.0001) compared to the epididymis (14.5 ± 2.5 ng/g tissue), prostate (3.4 ± 2.0 ng/g tissue), seminal vesicles (3.0 ± 2.0 ng/g tissue), or bulbourethral glands (1.4 ± 2.2 ng/g tissue). Together, these data suggest that GnRH-II and its receptor may have a role in spermatogenesis and/or spermatogenic function in boars. Partially supported by NIFA Hatch (NEB-26-199; BRW) and AFRI (2011-67015; CAL) funds.