212 GENE EXPRESSION PROFILE OF PROTAMINES AND TRANSITION NUCLEAR PROTEINS IN BOVINE TESTIS

Abstract

During spermiogenesis, haploid spermatids undergo complex morphological and physiological changes to differentiate into spermatozoa. These processes include chromatin remodelling mediated by the replacement of histones through transition nuclear proteins (Tnp) and protamines (PRM). These proteins have the function to compact and protect the chromatin, exerting great influence on human and mouse fertility. Several studies have demonstrated the positive relationship between unregulated production of protamines and infertility. In humans and mice, PRM1/PRM2 ratio is important to predict fertility. When the 1 : 1 ratio (ideal) is disrupted, in these species, the sperm DNA integrity is altered. Most of the infertility cases caused by protamine deficiency, in humans, are related to PRM2 (Aoki et al. 2005 Hum. Reprod. 20, 1298–1306). Expression levels of PRM2 have been correlated with the DNA damage in mice; however, its role on bull fertility is still unclear. The aim of this study was to determine the relative expression of PRM1, PRM2, PRM3, Tnp1, and Tnp2 in bovine testis. Evaluate the expression of these genes is of utmost importance to understand the role of each protamine during bull spermatogenesis. Testis from post-pubertal bulls (n = 10) were obtained from the slaughterhouse. The RNA extraction and cDNA synthesis were performed using commercial kits. The gene expression (P1, P2, P3, Tnp1, and Tnp2) was determined by real-time RT-PCR using bovine specific primers and β-actin as endogenous controls. A relative expression software tool (Pfaffl et al. 2002 Nucleic Acid Res. 30) was used to compare all samples of each group. The quantification of mRNA relative expression demonstrated a higher expression of PRM1, the relative expression of PRM2 was lower than the relative expression of PRM1 (5.008 ± 1.501 × 23.906 ± 6.174, respectively; P < 0.05). There was no difference between the relative expression of the mRNA for PRM2, Tnp1, and Tnp2 (5.008 ± 1.501, 5.023 ± 1.064, 4.266 ± 1.170, respectively; P > 0.05). The PRM3 mRNA had the lowest relative expression (2.003 ± 0.663). The PRM1/PRM2 ratio found in this study was 4.77 : 1.00. Differently from human and mice, the lower expression of PRM2 mRNA may be an evolutionary adaptation of the bull spermatogenesis, which makes the bovine sperm less susceptible to protamination changes that lead to infertility. More studies are being performed by our research group to evaluate the function of these proteins in bulls. It is fundamental to understand the biology of bovine spermiogenesis, providing knowledge to increase the fertility and be able to elucidate the evolutionary mechanisms that may have caused the possible loss of functionality of PRM2 in bulls.

This work was supported financially by FAPESP (09/17035-6 and 07/55294-8).