Transcripts of enriched germ cells responding to heat shock as potential markers for porcine semen quality

doi:10.1016/j.theriogenology.2007.11.020

Theriogenology

Volume 69, Issue 6, 1 April 2008, Pages 758-766

https://doi.org/10.1016/j.theriogenology.2007.11.020 Get rights and content

Abstract

A cDNA microarray-assisted experiment was conducted to survey genes that respond early to heat shock in enriched immature porcine germ cells; the 5′-UTR flanking the highest upregulated gene, heat shock 105/110 kDa protein 1 (Hsph1 or Hsp105), in response to heat shock was also investigated. We established a porcine testis cDNA microarray with 9944 transcripts from two libraries constructed from the testes of mature boars, with or without heat shock. After a mild heat shock treatment (39 °C for 1 h and recovered at 34 °C for 2 h), 380 transcripts demonstrated significant gene expression in enriched immature germ cells; 326 were upregulated and 54 were downregulated. Ten transcripts of interest exhibiting significance analysis of microarrays (SAM) scores higher than the median were subjected to quantitative real-time PCR; three (Hsp105, Hspa4l and Thap4) were upregulated >1.5-fold. The sequence of the 5′-UTR of Hsp105, the highest upregulated transcript, was cloned and analyzed. A single nucleotide polymorphism (SNP) was found at position −762 (C or T) upstream of the translational start site (ATG codon). Only two genotypes (CC or TC) were found in the mature boars that were studied (n = 31). A heterozygous genotype (TC) at this SNP site revealed an elevated percentage of morphologically normal sperm during hot and cold seasons; this SNP may be a useful marker for semen quality in boars. Furthermore, the cell-model established from enriched primitive germ cells has potential for the study of reproduction in mature animals.

Introduction

Elevated environmental temperatures can markedly reduce male fertility [1], [2]; high ambient temperatures and humidity have negative effects upon sperm morphology in boars [3], [4]. Many heat shock proteins (HSPs), e.g. Hsp70 [5], Hsp90 [6] and Hsp110 [7], exhibit protective roles in germ cells confronted with heat stress. In male germ cells, the expression of HSPs is activated by heat shock factor (HSF) in a lower temperature threshold [8]. This is especially prominent among germ cells, resulting in initiation of the protective process in advance. In our previous study on the Hsp70.2 promoter, we reported a significant correlation between single nucleotide polymorphisms (SNPs) and semen quality in boars [9]. However, the exact molecular mechanism associated with heat-tolerance in reproduction remains obscure.

To date, 1675 Quantitative Trait Loci (QTL), representing 281 different traits, have been discovered in pigs (PigQTLdb, http://www.animalgenome.org/QTLdb/pig.html) [10]. No QTL is related to male reproductive traits. Because the decline in fertility caused by heat shock impacts AI, it would be beneficial to clarify mechanisms behind heat-tolerance and discover essential biomarkers. The ratio of normal to abnormal sperm cells declines over the summer. Nevertheless, spermatogonia are more tolerant to heat than other types of germ cells involved in sperm development [1], [11]. Therefore, it would be of interest to identify these differences at a molecular level. Early responding genes in the advanced stages of spermatogenesis may play important roles in this protective machinery.

In order to investigate the transcripts that may contribute to the heat-tolerance of germ cells, a cDNA microarray-assisted approach was utilized, using an in vitro-enriched primitive germ cell model. The relative expressions of some highly regulated transcripts were confirmed by real-time PCR. The sequence of the 5′-untranslated region (5′-UTR) of the highest upregulated transcript was cloned and analyzed for single nucleotide polymorphisms (SNP). The genotypes in the reported polymorphism site of mature boars were determined and correlated to semen quality during both hot and cold seasons.

Section snippets

Animals

Testes from four purebred Duroc pigs (84 ± 15 d) were used for germ cell isolation and real-time PCR analysis. Three of the four animals (77 ± 11 d) were randomly selected for microarray analysis. Immature piglets, 2–3-mo-old, were selected to maximize the yield obtained during primitive germ cell isolation [12], [13]. To collect the testes, the animals were castrated following the guidelines published by the National Science Council, Republic of China. All assays, e.g. heat shock, microarray

Germ cell isolation

The cell morphology in fractions 4–10, equivalent to 2.4–3.0% BSA gradient, was similar to the gonocyte, as observed in testis sections of 2-mo-old swine (data not shown). After differential plating, most of the isolated cells showed typical characteristics of germ cells, i.e. spherical, with a conspicuous nucleus and a thin rim of cytoplasm [14]. The average yield of cell preparations from four individual animals was 1.0 ± 0.4 × 10⁶ cells/g. Vimentin is a cytoskeletal protein expressed in all

Discussion

In pigs, primitive gonocytes can be observed in the seminiferous tubules in testes from newborns to 2-mo-old piglets. At puberty (5–6 mo of age), mature sperm first appear in ejaculated semen [13]. Spermatogenesis, initiated in spermatogonia, is a complex process involving mitosis, meiosis and spermiogenesis. Environmental and/or genetic effects on germ cells will influence their development to normal sperm, leading to infertility.

The aims of this study were (1) to identify early responding

Acknowledgements

This research was funded by the Council of Agriculture of the Republic of China (grants nos. 91AS-1.1.3-AD-U1, 92AS-3.1.1-AD-U1, 93AS-3.1.1-AD-U1, 94AS-5.2.1-AD-U2, 95AS-6.2.1-AD-U1 (5), and 96AS-1.2.1-AD-U1).

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It has been extensively studied that exposure of the testes to temperatures at or above body temperature (40–43 °C) by experimental cryptorchidism model or local heating causes germ cell degeneration partly via apoptotic pathways in several mammalian species, including mice (Liang et al., 2013; Merico et al., 2008; Paul et al., 2009), rat (Sinha Hikim et al., 2003; Jia et al., 2009; Yamamoto et al., 2000), dogs (Henning et al., 2014), and monkeys (Zhang et al., 2003, 2005). Negative effects on spermatogenesis and sperm quality due to elevated environmental temperature have been reported in domestic animals such as sheep (Salces-Ortiz et al., 2015), goat (Xun et al., 2015), and pig (Gau et al., 2008). It has remained poorly understood, however, whether the degeneration of sperm under elevated ambient temperatures occurs via an apoptotic pathway or by other means.
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Transcripts of enriched germ cells responding to heat shock as potential markers for porcine semen quality

Abstract

Introduction

Section snippets

Animals

Germ cell isolation

Discussion

Acknowledgements

Anim Reprod Sci

Anim Reprod Sci

J Biol Chem

J Biol Chem

Anim Reprod Sci

Anim Reprod Sci

Methods

Biochim Biophys Acta

FEBS Lett

Cell

Cell

Trends Biochem Sci

Blood

Exp Hematol

Biochem Biophys Res Commun

Effects of hyperthermia on spermatogenesis, apoptosis, gene expression, and fertility in adult male mice

Biol Reprod

Scrotal heat stress effects on sperm viability, sperm DNA integrity, and the offspring sex ratio in mice

Mol Reprod Dev

Effect of temperature and humidity on sperm morphology in duroc boars under different housing systems in Thailand

J Vet Med Sci

Stage and lineage-regulated expression of two hsp90 transcripts during mouse germ cell differentiation and embryogenesis

Mol Reprod Dev

A QTL resource and comparison tool for pigs: PigQTLDB

Mamm Genome