Influence of the temperature and the genotype of the HSP90AA1 gene over sperm chromatin stability in Manchega Rams

Abstract

The present study addresses the effect of heat stress on males' reproduction ability. For that, we have evaluated the sperm DNA fragmentation (DFI) by SCSA of ejaculates incubated at 37 °C during 0, 24 and 48 hours after its collection, as a way to mimic the temperature circumstances to which spermatozoa will be subject to in the ewe uterus. The effects of temperature and temperature-humidity index (THI) from day 60 prior collection to the date of semen collection on DFI were examined. To better understand the causes determining the sensitivity of spermatozoa to heat, this study was conducted in 60 males with alternative genotypes for the SNP G/C-660 of the HSP90AA1 promoter, which encode for the Hsp90α protein. The Hsp90α protein predominates in the brain and testis, and its role in spermatogenesis has been described in several species. Ridge regression analyses showed that days 29 to 35 and 7 to 14 before sperm collection (bsc) were the most critical regarding the effect of heat stress over DFI values. Mixed model analyses revealed that DFI increases over a threshold of 30 °C for maximum temperature and 22 for THI at days 29 to 35 and 7 to 14 bsc only in animals carrying the GG-660 genotype. The period 29-35 bsc coincide with the meiosis I process for which the effect of the Hsp90α has been described in mice. The period 7-14 bsc may correspond with later stages of the meiosis II and early stages of epididymal maturation in which the replacement of histones by protamines occurs. Because of GG-660 genotype has been associated to lower levels of HSP90AA1 expression, suboptimal amounts of HSP90AA1 mRNA in GG-660 animals under heat stress conditions make spermatozoa DNA more susceptible to be fragmented. Thus, selecting against the GG-660 genotype could decrease the DNA fragmentation and spermatozoa thermal susceptibility in the heat season, and its putative subsequent fertility gains.

Figure 1. Trends of daily average (Tave, °C) and maximun (Tmax, °C) temperatures, relative humidity (RH, %) and average (THI) and maximum (THImax) temperature humidity index along the year 2010 (Data from SIAR http://crea.uclm.es/siar/datmeteo/).

Dotted lines are days of semen collection.

Figure 2. Changes in xDFI, sdDFI and tDFI values with the incubation time (0 h, 24 h and 48 h) along the period of the year from which sperm samples were collected.

Figure 3. Ridge regression analyses relating DFI measures (xDFI, sdDFI and tDFI) from day 60 prior to semen collection to date of collection with weather measures (Tave  =  average daily temperature, Tmax = maximum daily temperature, and THI = temperature humidity index), for each HSP90AA1 genotype.

Fitted effects extending beyond dotted-lines (---) differ significantly (P<0.05) from zero. Four regions (gray regions) with a significant possible effect on sperm DFI levels were identified.

Figure 4. Regression coefficients from the mixed-effects model relating DFI values with summary measure of Tave, Tmax and THI for the days 7 to 14 before semen collection.

For each coefficient in the model, estimates (points) plus and minus 1 (bold line) and 2 (thin line) standard deviations are represented. ^*

Figure 5. Regression coefficients from the mixed-effects model relating DFI values with summary measure of Tave, Tmax and THI for the days 29 to 35 before semen collection.

For each coefficient in the model, estimates (points) plus and minus 1 (bold line) and 2 (thin line) standard deviations are represented. ^*