55 DIFFERENTIAL EXPRESSION OF AQUAPORINS AND SPERMADHESINS IN FROZEN–THAWED 'GOOD FREEZER' AND 'POOR FREEZER' BOAR SPERMATOZOA

Abstract

Cryopreservation is an excellent tool for sperm banking in livestock animals. Although its utilisation is widespread in species such as cattle, its application in pigs is limited due to various factors associated with either cryopreservation techniques or male-to-male variations. Indeed, the existence of so-called “good freezer” (or GF) and “poor freezer” (PF) males has been well established in swine and yet, the potential to screen for these characteristics is still being investigated. Few studies have reported the existence of molecular differences between both groups of boar spermatozoa, at the nucleic acid and protein levels. Here we hypothesise that cryotolerance in sperm may be associated with differential expression of proteins involved in membrane transport and or sperm capacitation. Thus, we conducted this study to compare the expression of spermadhesins, aquaporins (AQP), and glucose (GLUT) transporters in GF and PF boar frozen-thawed spermatozoa. Three ejaculates of 8 boars classified as GF (n = 4) or PF (n = 4), based on post-thaw sperm fertility records were used. Frozen-thawed spermatozoa were purified using a Percoll gradient, and washed 3 times with PBS/polyvinylpyrrolidone (PVP). Total sperm RNA extraction took place in 3 different sperm purifications of each sire and boar testis cells, and this was followed by cDNA synthesis. Primer sets designed for the detection of AQP isoforms 1 to 11, GLUT-3 and GLUT-5, and spermadhesins (AQN-1, AWN-1, SPM-I, PSP-1, and PSP-2) were used for real-time PCR (pool of ejaculates/boar), with GAPDH serving as a housekeeping gene, and testis cells cDNA as positive controls. Gene expression was analysed using the comparative quantitation (REST 2009 software; Qiagen Inc., Valencia, CA) via the Pfaffl method, and PCR amplicons were verified on gel electrophoreses. The expression of water (AQP-1, -5, and -11) and glycerol (AQP-3, -7, -9, and -10) transmembrane transporters, as well as GLUT-3 were detected in all samples. Expression levels of AQP-10 and AQP-11 were greater than other tested transporters (P < 0.05), and no differences in AQP expressions were found between both groups (GF v. PF; P > 0.05). The GLUT-3 transcript levels were lower in PF versus GF (0.705×; P < 0.05). Irrespective of groups, analysed spermadhesins were detected in spermatozoa, and AQN and AWN were the highest and lowest, respectively (P < 0.05). The PSP-1 mRNA level was reduced in PF versus GF (0.431×; P < 0.05), whereas other spermadhesins were comparable (P > 0.05). The findings indicate that frozen-thawed boar spermatozoa express aquaporins and glucose transporter transcripts that are essential for transmembrane fluxes occurring during cryopreservation. Additionally, the detection of spermadhesin mRNA in spermatozoa raises questions about their roles during early capacitation of frozen-thawed spermatozoa. Differential expression of these genes brings additional evidence that may contribute to a better description of GF from PF spermatozoa. Further investigations are still undergoing for a full characterisation of these genes in boar spermatozoa. Supported by the US Department of Agriculture-Agricultural Research Service (USDA-ARS) Grant #58-6402-3-0120 and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)-Brazil.