Sus Scrofa

Animal Reproduction Science 119 (2010) 76–84

Contents lists available at ScienceDirect

Animal Reproduction Science journal homepage: www.elsevier.com/locate/anireprosci

Seasonal effect on sperm messenger RNA profile of domestic swine (Sus Scrofa) C.C. Yang a , Y.S. Lin a , C.C. Hsu a , M.H. Tsai b , S.C. Wu a,b , W.T.K. Cheng a,b,c,∗ a b c

Department of Animal Science and Technology, National Taiwan University, Taipei 106, Taiwan, ROC Institute of Biotechnology, National Taiwan University, Taipei 106, Taiwan, ROC Department of Animal Science and Biotechnology, Tunghai University, Taichung 407, Taiwan, ROC

a r t i c l e

i n f o

Article history: Received 19 August 2009 Received in revised form 24 November 2009 Accepted 3 December 2009 Available online 16 December 2009 Keywords: Swine Ejaculated sperm Seasonal effect mRNA

a b s t r a c t Seasonal infertility is a well-known problem in the modern swine (Sus scrofa) industry. The molecular mechanisms responsible for thermal effects on spermatogenesis are, however, just beginning to be elucidated. The existence of specific messenger RNA (mRNA) remnants contained within freshly ejaculated sperm has been identified in several species. Investigators have obtained differential RNA profiles of infertile men compared with fertile individuals; however, there are limited to the probes, which are mostly derived from nucleic acids of testicular tissues of either human or mice. The objective of this study was to investigate mRNA remnants from ejaculated sperm of the domestic swine and uncover important clues regarding the molecular regulation of spermatogenesis under environmental thermo-impacts. We utilized the remnant mRNA collected from swine ejaculated sperm as the target source to detect the global gene expression in summer and in winter by swine sperm-specific oligonucleotide microarray. Sixty-seven transcripts were differentially expressed with statistical differences between seasons of sperm samples collected, including forty-nine in winter (49/67) and eighteen in summer (18/67). There were only 33 of these transcripts that could be annotated to gene ontology hierarchy with the database of Homo sapiens and their functions mostly were involved in variety of metabolic processes. Moreover, these studies also confirmed that significant differences of gene expression profiles were found in swine sperm when comparisons were made between ejaculates collected during the winter and the summer season under the subtropical area such as Taiwan. Even though most of the genes found in our experiments are still poorly understood in terms of their true functions in spermatogenesis, bioinformatics analysis suggested that they are involved in a broad spectrum of biochemical processes including gamete generation. These concordant profiles should permit the development of a non-invasive testing protocol to assess the functional capacity of sperm as well as a new molecular selection scheme for fine breeding swine. © 2009 Elsevier B.V. All rights reserved.

1. Introduction

∗ Corresponding author at: Department of Animal Science and Biotechnology, Tunghai University, No. 181, Sec. 3, Taichung Port Road, Xitun District, Taichung 407, Taiwan, ROC. Tel.: +886 4 23590121x37110; fax: +886 4 23590385. E-mail address: [email protected] (W.T.K. Cheng). 0378-4320/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.anireprosci.2009.12.002

Seasonal infertility is a well-known problem in the modern swine industry (Love et al., 1993; Flowers, 1997). High ambient temperature may directly contribute to seasonal infertility by causing physiological stress and consequently decrease feed intake, which results in anestrus, extended weaning-to-estrus intervals, poor conception rates, higher

C.C. Yang et al. / Animal Reproduction Science 119 (2010) 76–84

embryo mortality rates and lower farrowing rates in sows (Dan and Summers, 1996; Peltoniemi et al., 1999; Suriyasomboon et al., 2006). These reproductive problems can also be present in boar studs, where results are lower in semen output and poor in ejaculated sperm quality during the summer and early fall (Kuo et al., 2000; Kunavongkrit et al., 2005; Suriyasomboon et al., 2005). Despite extensive histological and cytological studies of the testis under the influence of heat stress, the molecular mechanisms responsible for the thermal effects on spermatogenesis are, however, just beginning to be elucidated. It also should be noted that the testicular biopsy method used in previous studies is rather invasive and is not commonly used in routine practice. It is reported, nevertheless, that sperm RNA profiles coincide with those observed in the testes in fertile men (Ostermeier et al., 2002; Martins and Krawetz, 2005). In effect, they both echo spermatogenic gene expression. Therefore, mature sperm serve as a promising repository for information regarding both genetic and environmental influences. The observation that mammalian sperm retain RNAs has revolutionized the investigation of male infertility (Kramer and Krawetz, 1997; Miller et al., 1999; Yatsenko et al., 2006). Several scientists have obtained differential RNA profiles of infertile men as compared with fertile individuals (Wang et al., 2004; Ostermeier et al., 2005). In the previous studies, the facts of remnant mRNAs existed in swine freshly ejaculated sperm were successfully cloned, sequenced and identified. Here in the study, attempts were again made to utilize the remnant mRNAs collected from swine ejaculated sperm as the target source for further global detection of mRNAs expression profiles and seasonal variations (Summer vs Winter) were compared according results from high-throughput oligonucleotide microarray. It is anticipated that from these studies, important clues would be uncovered regarding the molecular regulation of spermatogenesis under environmental thermo-impacts and to provide potential tools for the improvement of the selection scheme based on molecular information of the superior swine. 2. Materials and methods 2.1. Semen collection and sperm processing All procedures of animal experiments in this study were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of National Taiwan University. Freshly ejaculated semen was collected from sexually mature healthy swine of the Duroc breed. All experimental swine were housed in a water pad barn with individual cages and were maintained under conventional conditions at the Changhua Animal Propagation Station, Livestock Research Institute (Council of Agriculture, Executive Yuan, Republic of China). This institute is located in the southern portion of Taiwan, which has typical subtropical weather (Fig. 1). The semen-collecting procedure was modified from the method described by Xu et al. (1996). Semen samples were collected weekly for eight continuous weeks staring from 4 weeks prior to the

77

Fig. 1. Monthly distribution of air temperature and precipitation plotted using a climograph for Changhua City (24◦ 01 N, 120◦ 30 E), Taiwan, Republic of China, from 2004 to 2007. The bar graph depicts the monthly accumulated precipitation; the line graph shows the monthly average temperature. Local weather records were collected from the Central Weather Bureau (Taiwan, Republic of China).

experimental periods, which were scheduled for March and September 2006 and 2007. Each ejaculate was quantified using a SpermaCue® spectrophotometer (Minitüb, Tiefenbach, Germany) for sperm concentration. Sperm motility and morphology were assessed microscopically at 37 ◦ C using phase contrast microscopy (Axioskop, Carl Zeiss, Oberkochen, Germany) with 100× and 400× magnification, respectively. The procedure was carried out by a routine operator to reduce subjective bias. Resulting data was denoted as mean and standard error of the mean (SEM) (Table 1). Student’s paired t-test algorithm was used to calculate the difference in same individual boar between the two seasons. Sperm morphology was also inspected with Diff-Quik (Sysmex, Kobe, Japan) stained smears. Sperm specimens were first fixed with Diff-Quik Fixative solution, then slides were immersed sequentially in Diff-Quik solutions I and II for 30 s each; rinsed in running water to remove excess dye. Slides were allowed to dehydrate in absolute alcohol and observed under inverted phase contrast microscopy (Leica DMIRB, Leica, Wetzlar, Germany) with 400× magnification. Samples were then washed and diluted following the steps described in Yang et al. (2009). Briefly, fresh semen was diluted with semen diluent (dglucose 60 g/L, sodium bicarbonate 1.2 g/L, EDTA 3.7 g/L, trisodium citrate 3.7 g/L, streptomycin 1 g/L, penicillin 0.5 g/L, pH 6.6–6.7) (Cheng, 1985) and incubated for 30 min at 37 ◦ C for non-motile sperm and other seminal debris including somatic cells to sink to the bottom of the tube so that they can be eliminated as much as possible at the next step. The upper half of the solution, the motile spermenriched fraction, was then diluted with an equal volume of phosphate buffered saline (PBS; Invitrogen, Grand Island, NY, USA) and centrifuged at 300 × g for 10 min at 18 ◦ C; the process was repeated and the upper half of the supernatant fraction was combined with an equal volume of PBS again but centrifuged at 1200 × g for 5 min at 18 ◦ C. After washing the pellet of sperm three times with PBS and centrifuging at 1200 × g for 5 min at 18 ◦ C, a small aliquot of the pelleted sperm was inspected by microscopy to ensure the reliability and reproducibility of this purification process, as well as to examine whether any adherent somatic cells or their debris remained with sperm. The sperm then were

78


Table 1 Semen characteristics of individual swine used in microarray and/or real-time polymerase chain reaction experimentsa . Sample

Winter (January–March) Ejaculate volume (mL)

For microarray Boar 1 273 Boar 2 191 Boar 3 152 Boar 4 155 Boar 5 146 Boar 6 133 For real-time PCR Boar 7 180 Boar 8 270 Boar 9 250 Boar 10 191 Boar 11 170 Boar 12 232

Summer (July–September)

Sperm concentration (×108 /mL)

Sperm motility (%)

Ejaculate volume (mL)

Sperm concentration (×108 /mL)

Sperm motility (%)

± ± ± ± ± ±

12 12 7 8 4 6

3.6 3.9 4.1 2.8 4.2 7.9

± ± ± ± ± ±

0.4 0.3 0.3 0.2 0.5 0.6

76 89 85 77 81 84

± ± ± ± ± ±

2 0 1 3 3 1

262 172 195 165 186 110

± ± ± ± ± ±

8 10 6 6 16 7

3.6 4.2 3.9 2.8 4.7 5.6

± ± ± ± ± ±

0.2 0.3 0.3 0.2 0.5 0.3

75 66 86 78 79 84

± ± ± ± ± ±

2 1 0 2 4 2

± ± ± ± ± ±

17 8 18 17 8 14

5.6 4.9 32 2.4 5.6 4.5

± ± ± ± ± ±

0.7 0.3 02 0.3 0.7 0.2

80 85 83 77 85 78

± ± ± ± ± ±

2 1 2 3 1 2

181 254 260 203 145 168

± ± ± ± ± ±

8 9 13 8 6 8

2.2 4.0 2.4 2.4 7.5 4.3

± ± ± ± ± ±

0.1 0.3 0.2 0.4 0.5 0.6

83 85 83 79 83 78

± ± ± ± ± ±

2 1 2 1 1 1

a All data were acquired from Changhua Propagation Station. Livestock Research Institute (Council of Agriculture, Executive Yuan, Republic of China) and expressed as the mean and standard error of the mean (SEM).

Table 2 The primer sequences used for real-time polymerase chain reactiona . Target

Accession number

Primer sequence

Location

Expected length (bp)

Beta-2-microglobulin (endogenous control)

DT331342 CX058018

Testis-specific serine kinase 6

CX062084

Testis-specific protein kinase 1

BX673497

96–112 142–164 600–620 647–664 221–239 277–293 27–44 99–118

69

Testis and spermatogenesis cell-related protein 1

5 -CGTCCCCCGAAGGTTCA 5 -GCAGTTCAGGTAATTTGGCTTTC 5 -TCCCAAACCTCCTCACCTTCT 5 -GCGCGAATTGCGATGAGT 5 -CTTGGGCGTCGTGCTCTAC 5 -GGGCAGGCCAGCGATAT 5 -TCCTGGCCATCCATTGCT 5 -CAGCTGCTCCAGGATCCATT

a

65 63 92

Primer sequences were designed by Primer Express” software, version 3.0 (Applied Biosystems, Foster City, CA, USA).

snap-frozen in liquid nitrogen and stored at −80 ◦ C until use. 2.2. Target RNA preparation for microarray All washed sperm samples obtained from the same individual swine (n = 6) in a single summer or winter 2006 were pooled together; therefore, twelve sperm samples were prepared in total. Total RNA was extracted from the washed pooled sperm using an RNeasy Lipid Tissue Mini Kit (QIAGEN, Hilden, Germany) following the manufacturer’s instructions. In order for differential expression for any other sample pair can then be calculated by assembling the ratios from their hybridizations with the reference. The antisense amplified RNA (aRNA) was amplified from 100 ng of the total RNA using a MessageAmpTM II aRNA Amplification Kit (Ambion, Austin, TX, USA), of which 1.5 ␮g of each test sample and reference target were labeled directly with Cy3- and Cy5-dye, respectively, using the non-enzymatic ULSTM Labeling Kit (Kreatech Biotechnology, Amsterdam, the Netherlands). In addition, the reference target was prepared from an equal molar mixture of aRNA from the total RNA pool of twelve sperm samples. Following combination of Cy3- and Cy5-labeled targets, the target mixtures were fragmented to 200 bases or shorter prior to hybridization using an RNA Fragmentation Reagents Kit (Ambion).

2.3. Analyses of gene expression profiles A sectored oligonucleotide array, including four microarrays containing 2240 probes each, was prepared (CombiMatrix, Mukilteo, WA, USA), and gene-specific probes, 35–40 bases in length, were selected and designed. There were 412 genes derived from cDNA sequences in our swine sperm cDNA library and an additional 296 genes were chosen from GenBank (NCBI, Bethesda, MD, USA) based upon their expression pattern related to spermatogenesis. The reference target was prepared from equal molar mixture of RNA from twelve sperm total RNA pool. Therefore, differential expression for any other sample pair can then be calculated by assembling the ratios from their hybridizations with the reference. All experimental procedures were conducted according to the manufacturer’s technical manual. Hybridized slides then were scanned at a 10 ␮m resolution with a GenePix 4000 scanner (Axon Instruments, Union City, CA, USA) at wavelengths of 532 and 635 nm for Cy3 and Cy5, respectively. The resulting 16 bit TIFF images were analyzed using GenePix Pro software (Axon Instruments). The data analysis method used was modified from Chuang et al. (2006). Briefly, the intensity ratios of the test sample over the reference for all of the targets were determined, and ratio normalization was performed in order to normalize the center of the ratio distribution to 1.0. Genes shown to


79

be significantly up- or down-regulated were identified by the significance analysis of microarrays (SAM) statistics program (Tusher et al., 2001), which utilizes a modified t-test and sample-label permutations to evaluate statistical significance. Hierarchical clustering was performed and displayed using Cluster and TreeView software (Eisen et al., 1998). These genes were analyzed for functional annotation using the gene ontology database with the Babelomics FatiGO web-based analysis tool (Al-Shahrour et al., 2007). 2.4. Reverse transcription real-time polymerase chain reaction analyses Validation of the gene expression profile of interest that was identified by microarray analysis was carried out using SYBR green chemistry with real-time detection instruments. Washed sperm samples from the same individual swine (n = 6) in the same season of summer or winter 2007 were pooled together in order to acquire a sufficient amount of total sperm RNA. Total RNA was extracted from the washed sperm pool using an RNeasy Lipid Tissue Mini Kit (QIAGEN) following the manufacturer’s protocol. Amplification reactions of genes of interest were performed using a set of Power SYBR® Green PCR Master Mix Kit (Applied Biosystems, Foster City, CA, USA) and a 7300 Real-Time PCR System (Applied Biosystems) according to the instruction manual but were scaled down to 25 ␮L per reaction. Each reaction, containing 20 ng of template, was performed in triplicates, and beta-2-microglobulin (B2M) mRNA was used as the internal control. The primer sequences used are described in Table 2. The thermal profile was as follows: 95 ◦ C for 10 min, 40 cycles of 95 ◦ C for 15 s and 60 ◦ C for 1 min, with a final dissociation stage, 95 ◦ C for 15 s, 60 ◦ C for 30 s and 95 ◦ C for 15 s, to complete the melting curve acquisition. Cyclic fluorescence signal measurements were taken at the end of the annealing phases. Relative differences for a gene in different seasons were determined using the comparative cycle threshold (CT ) method (Livak and Schmittgen, 2001). The resulting values were converted to fold-changes compared with the control by raising 2 to the CT power (2−CT ). 3. Results 3.1. Seasonal variation in gene expression Gene expression profiles for ejaculated sperm collected in different seasons were generated using CustomArrayTM 4X2K system microarrays (CombiMatrix). Each ejaculated sperm sample from six swine both in summer and winter were compared with the reference pooled samples to yield expression ratios, as described previously. All the data imported from GenePix (Axon Instruments) were analyzed and unreliable data (judged as absent or marginal) were filtered out before clustering. To identify significant changes in gene expression, we performed SAM using 1000 permutations and selected significant mRNAs at a false discovery rate (FDR) of 0%. Expression profiles of the 67 most significant transcripts among the 2240 probes identified by SAM and subjected to hierarchical clustering (Fig. 2A).

Fig. 2. Analysis of the change in seasonal transcription profiles of sperm mRNAs collected from freshly ejaculated swine sperm. (A) The differentially expressed transcripts (n = 67) identified from six swine in summer and winter were presented in a hierarchical clustering format, where rows indicate experimental swine (Boar 1 to Boar 6) and columns represent individual genes. The dendrogram on the top represents the swine hierarchy, while the one on the left shows the gene hierarchy. Color intensity is proportional to the magnitude of the deviation from the reference sample: red areas indicate increasing mRNA levels, green areas denote decreases in mRNA levels, and black areas show equal expression in both seasons. H: summer; C: winter. (B) A venn diagram is used to describe the proportion of specific or common transcripts between summer and winter. A total of 18 transcripts were found to have high-abundance signals in summer, while 49 transcripts showed high-abundance signals in winter. Of these transcripts, 641 transcripts did not reach statistical significance in both seasons through significance analysis of microarrays. (For interpretation of the references to color in this figure caption, the reader is referred to the web version of the article.)

A total of 18 transcripts had high-abundance signals in summer, while 49 transcripts showed high-abundance signals in winter (Fig. 2B). We found that only 33 of these transcripts could be annotated with known mammalian genes and could be mapped to gene ontology terms. Their major properties in the biological process at level 4 mostly

80


Table 3 The 33 sperm mRNAs remaining within sperm that could be annotated with known mammalian genes were further mapped to gene ontology terms and exhibited differential abundance in summer and winter. Original sequence for probe design Accession number

Predicted protein

Gene ontology classification on biological process at level 4

Unigene number

High-abundance of remnant spermatozoal mRNAs in summer AY609691 Ssc.54115 Activator of heat shock 90 kDa protein ATPase homolog 1 [Homo sapiens] CV869285 Ssc.58756 Testis-specific bromodomain protein [Bos taurus] BW980042 Ssc.20036 40S ribosomal protein S20 [Sus scrofa] NP 001020394

Ssc.33985

Speedy homolog 1 [Sus scrofa]

NP 001090946

Ssc.6705

60S ribosomal protein L10A [Sus scrofa]

BW979145

Ssc.22030

RANTES protein [Sus scrofa]

BX923878

Ssc.55408

Nucleolar protein family A, member 3 [Homo sapiens]

DY426860

Ssc.32915

Titin isoform N2-B [Mus musculus]

CX058501 BX914643

Ssc.47200 Ssc.16696

NP 999176 DN112619

Ssc.67 Ssc.7070

Lysozyme-like 4 [Bos taurus] Misshapen/NIK-related kinase isoform 1 [Homo sapiens] Sperm adhesion molecule 1 [Sus scrofa] Sestrin 1 [Homo sapiens]

DN112240 X92446 DN110557

Ssc.38317 Ssc.1717 Ssc.47022

Stannin [Rattus norvegicus] Heat shock 70 kDa protein 5 [Sus scrofa] Kinesin-like protein 2 [Bos taurus]

CJ015240

Ssc.54355

Ferritin, light polypeptide [Sus scrofa]

High-abundance of remnant spermatozoal mRNAs in winter CF179729 Ssc.18137 Mitogen-activated protein kinase kinase kinase kinase 1 isoform 1 [Homo sapiens] CV874937 Ssc.46959 DnaJ homolog, subfamily B, member 8 [Homo sapiens] AY610471 Ssc.9576 Calcium and integrin binding 1 [Mus musculus] DB816720 Ssc.32508 Death effector domain-containing DNA binding protein 2 [Homo sapiens] AK236260

Ssc.28477

AY609426

Ssc.772

AK233757

Ssc.53780

CX062084

Ssc.43158

NP 001008685

Ssc.42763

CX058018

Ssc.47204

CX058190

Ssc.47129

CX056776

Ssc.46925

NP 001090970

Ssc.54122

BX667222 NP 001026965

Ssc.22815 Ssc.20965

CX065760

Ssc.47028

CV877355

Ssc.46875

Testis-specific protein kinase 1 [Homo sapiens] Calcium regulated heat stable protein 1 [Rattus norvegicus] 60S ribosomal protein L8 [Rattus norvegicus] Testis-specific serine kinase 6 [Rattus norvegicus] Sperm mitochondria-associated cysteine-rich protein [Sus scrofa] Testis and spermatogenesis cell-related protein 1 [Mus musculus] G protein-coupled receptor 161 isoform 2 [Homo sapiens] CKLF-like MARVEL transmembrane domain-containing 2 [Bos taurus] Lectin, galactoside-binding, soluble, 3 [Sus scrofa] Lipocalin 8 [Bos taurus] PRA1 family protein 2 [Sus scrofa] Late cornified envelope-like proline-rich 1 [Bos taurus] Testis-specific histone 2a-like [Equus caballus]

Metabolic process Metabolic process; organelle organization and biogenesis Metabolic process; cellular biosynthetic process Metabolic process; cell cycle; response to DNA damage stimulus Metabolic process; cellular biosynthetic process Metabolic process; signal transduction: cell motility; cellular defense response Metabolic process; organelle organization and biogenesis; ribonucleoprotein complex biogenesis and assembly Metabolic process; cell differentiation; system development; cell cycle Cellular catabolic process Metabolic process; cell differentiation; system development Metabolic process; cell-cell recognition Regulation of cellular process; cell cycle; response to DNA damage stimulus Unclassification Cell differentiation; signal transduction Organelle organization and biogenesis; cell cycle; cellular localization Transport Metabolic process; cell differentiation; signal transduction; system development; Metabolic process; response to protein stimulus Metabolic process; cell differentiation; response to DNA damage stimulus Metabolic process; cell differentiation; signal transduction; organelle organization and biogenesis Metabolic process; gamete generation Metabolic process; signal transduction Metabolic process; cellular biosynthetic process Metabolic process; cell differentiation; organelle organization and biogenesis; gamete generation Cell motility; fertilization Cell differentiation; gamete generation Signal transduction Locomotory behavior System development Transport Transport; establishment of protein localization Cell differentiation; system development Metabolic process


Fig. 3. Confirmation of the microarray results by real-time polymerase chain reaction analysis of gene transcripts. Three genes, testis and spermatogenesis cell-related protein 1 (TSARG1), testis-specific serine kinase 6 (TSSK6) and testis-specific protein kinase 1 (TESK1), related to gamete generation were selected for verification of the microarray results. The fold-change was addressed as gene expression level in winter in proportion to that in summer, and then adjusted for the housekeeping gene beta-2-microglobulin for the same swine.

involved various metabolic processes, and are summarized in Table 3. 3.2. Validation of microarray data The stress imposed by elevated ambient temperatures may not be the same for all test subjects (Cameron and Blackshaw, 1980; Kunavongkrit et al., 2005). Hence, we used sperm RNA samples obtained from an additional six swine to objectively evaluate differential gene expression levels between summer and winter via reverse transcription real-time PCR. It is widely accepted that all cellular components must be properly packed within the sperm prior to spermiation; errors occurred in spermatogenesis being likely to influence fertility. Thus, focuses were made to the genes specifically related to spermatogenesis. Annotation of gene ontology revealed that candidate genes of testis-specific molecules such as testis and spermatogenesis cell-related protein 1 (TSARG1), testis-specific serine kinase 6 (TSSK6), and testis-specific protein kinase 1 (TESK1) apparently all play critical roles in male germ cells generation and/or maturation. It was clearly shown that transcriptional levels of TSARG1, TSSK6, and TESK1 genes were higher in winter than they were in summer (Fig. 3). This positive correlation demonstrated results similar to those observed in the microarray analysis. 4. Discussion 4.1. Utilization of sperm mRNA profile as a potential investigating tool In mammals, spermiogenesis is a highly complex process wherein the round spermatids undergo dramatic remodeling changes to form sperm (Wykes et al., 1997). All of the structures and regulation signals necessary to maintain male fertility must be correctly assembled within these streamlined cells before being released from the seminiferous epithelium and becoming transcriptionally dormant (Miller et al., 2005). As shown in the analyzing result of microarray, remnant mRNA within mature sperm that has almost lost all of its cytoplasm could still reflect the gene expression changes of spermatogenesis

81

under environmental thermo-impacts. This observation is in agreement with the results of a similar study exploring gene expression differences of fertile and infertile men using ejaculated sperm. It also has been reported that these transcripts represent remnants of stored mRNA from postmeiotically expressed genes (Ostermeier et al., 2002). There were no significant variation in sperm motility between the two seasons (p > 0.05), which possibly resulted from human manipulation in the semen collection and evaluation processes. Still, the sperm mRNA profile obtained by this non-invasive method is a valuable tool for studying the molecular mechanism of spermatogenesis under stresses. It has been reported that when motility is ≥60%; there is no relationship between the percentage of motile sperm and in vitro sperm penetration rate under conditions of artificial insemination (Flowers, 1997; Popwell and Flowers, 2004; Gadea, 2005). However, the apparent decline in gene expression related to heat stress may allow damage to accumulate, resulting in altered sperm functions (Hales et al., 2005; Yanagimachi, 2005), and may eventually lead to failure of conception. In order to accurately describe the variation of gene expression for resisting environmental stress during spermatogenesis, and to avoid achieving false positive results deriving from non-motile sperm that are unable to fertilize oocytes, the motile sperm-enriched fraction was used in our experiments. The risk of somatic cell contamination in sample preparations was also avoided in this study. 4.2. Effects of elevated environment temperature on reproduction efficiency The scrotal temperature is approximately 2–7 ◦ C lower than the core body temperature for maintaining normal spermatogenesis in mammals (Swiergiel and Ingram, 1987). High ambient temperature during summer has been described as having a negative impact on sperm quality (Suriyasomboon et al., 2005; Murase et al., 2007). Nevertheless, differentiation and maturation of premeiotic leptotene, zygotene and pachytene spermatocytes and early round spermatids are temperature-dependent (McNitt and First, 1970; Thonneau et al., 1998; Nakai et al., 2000; Kon and Endoh, 2001). It is known that swine has little capacity for sweating when the skin temperature is elevated (McNitt et al., 1972). Records of scrotal skin temperature for swine raised in subtropical areas have proven that it is indeed significantly higher in summer than in winter (Huang et al., 2002; Kunavongkrit et al., 2005). Therefore, the effect of temperature on the semen quality of swine has often been observed during or immediately after a hot summer (Kunavongkrit et al., 2005). In general, the first indication of abnormal sperm production is seen after a lag period of 2 weeks after the onset of high ambient temperature, and motile sperm do not usually return to a normal ratio until 5 weeks after the end of the period of high temperature (Wettemann et al., 1979; Flowers, 1997). Testicular germ cells are very sensitive to a wide spectrum of apoptotic stimuli, including exposure to mildly high ambient temperature (Blanco-Rodríguez and Martínez-García, 1998). Several lines of evidence indicate that high ambient temperature not only decreases the via-

82


bility and motility of sperm, but also has negative effects on the intrinsic functions and molecular properties of testicular germ cells, including impaired DNA, RNA and protein synthesis, abnormal chromatin packing and protein denaturation (Steinberger, 1991; Sailer et al., 1997), as well as reduced DNA integrity (Banks et al., 2005; Pérez-Crespo et al., 2008). Nevertheless, physiological spontaneous germ cell death is a continuous and frequent event that occurs in stages of mitotic and meiotic peaks in normal spermatogenesis to confine the germ cell population and remove abnormal sperm (Blanco-Rodríguez, 1998). It has been recognized that increased testicular germ cell apoptosis leads to abnormal spermatogenesis and sperm concentration reduction, pointing to a prominent role of programmed germ cell death in male infertility (Yin et al., 1997; Hikim et al., 2003). However, the mechanism of proapoptotic stimuli, which initiates testicular germ cell apoptosis, is not well understood. The mRNA of the TSARG1 gene, a DnaJ-like protein family member, existing in the swine sperm, and the mRNA levels were high in the sperm pools collected in winter. Yang et al. (2005) suggested that the expression of the TSARG1 gene may participate in the inhibition of testis spermatogenic cell apoptosis. Although the role of TSARG1 mRNAs within mature sperm in different season remains to be determined, our results support the hypothesis that the ambient temperature could influence sperm apoptosis. Previous studies regarding the sperm RNA profile provided valuable opportunity to identify molecular defects during spermatogenesis (Gilbert et al., 2007). According to microarray analyses, gene expression that showed that down-regulation occurred much more during summer than in winter. This observation is in agreement with a study of heat-induced stress results in the degradation of many mRNAs (Tramontano et al., 2000; Aguilar-Mahecha et al., 2001). 4.3. Serine/threonine protein kinase essential for male fertility Post-translational modifications of proteins through phosphorylation on serine/threonine or tyrosine residues of substrate proteins provide a fundamental regulatory mechanism in signal transduction during many biological processes, including spermatogenesis, oogenesis and fertilization (Wei et al., 2007). Among the protein kinases catalyzing phosphorylation, however, only a few are exclusively expressed in germ cells or in the testis (Toshima et al., 1995; Tseng et al., 1998; Visconti et al., 2001; Chen et al., 2004; Hao et al., 2004). 4.4. Testis-specific serine kinase 6 (TSSK6) Testis-specific serine/threonine kinase 6 (TSSK6), one of the TSSK family members, has the potential to phosphorylate basic proteins such as myelin basic protein (MBP), histone H1, H2A, H2AX, and H3 (Spiridonov et al., 2005). The highly specific gene expression of TSSK6 is confined to the late stages of spermiogenesis and the protein is detected both in the heads of elongated spermatids and in the equatorial segment of the ejaculated sperm of mice (Spiridonov et al., 2005; Xu et al., 2007). It has been reported

that the mRNA of the TSSK6 gene is a potential substrate bound by both germ cell-specific RNA binding proteins, deleted in azoospermia-like (DAZL) and Pumilio-2 (PUM2), which are essential for the germ cell development (Fox et al., 2005); this implies that the mRNA of the TSSK6 gene may play an important role, not only at the protein regulatory level, but also during the process of spermiogenesis. Furthermore, male infertility has been recognized as being associated with irregularity in the status of sperm chromatin condensation (Evenson et al., 1999; Irvine et al., 2000; Dadoune, 2003). Homozygous TSSK6 gene knockout mice (TSSK6–/– ) have been shown to exhibit male infertility associated with sperm number reduction and impaired chromatin condensation, and abnormal morphology and motility of sperm have been noted when heterozygous (TSSK6+/– ) mice are fertile but have reduced sperm numbers similar to their homozygous counter parts (Spiridonov et al., 2005). In addition, heat shock proteins, a family of highly conserved proteins, have been found to serve as an adaptive and protective mechanism response to physiological and/or environmental stresses (Rockett et al., 2001; Hayashida et al., 2006). Spiridonov et al. (2005) further demonstrated that interaction with heat shock proteins HSP90␤, HSC70 and HSP70 is required for TSSK6 activation and may play a critical role in targeting TSSK6 to specific subcellular sites. Although the exact molecular functions of the TSSK6 gene are still unknown, a series of studies has suggested that they are involved in post-meiotic germ cell differentiation and might hold an important position in sperm maturation, capacitation and fertilization. 4.5. Testis-specific kinase 1 (TESK1) The testis-specific kinase 1 (TESK1) protein is another member of the serine/threonine protein kinase family whose mRNA content was found to be high in ejaculated sperm during winter according to our results. TESK1 protein kinase with dual specificity is able to catalyze autophosphorylation and phosphorylation of exogenous substrates on both serine/threonine and tyrosine residues. Its expression pattern has been identified in many tissues and cell lines (Toshima et al., 1998, 1999). The true function of the TESK1 gene in testicular germ cells is not clear, but the predominant expression of TESK1 mRNA and protein at specific stages of testicular germ cells suggests that it is involved in the stages of meiotic cell division and early spermiogenesis in particular (Toshima et al., 1998). It is well-known that the first morphological indication of spermatogonial differentiation is incomplete cytokinesis, followed by the formation of a stable intercellular bridge between the daughter cells (de Rooij and Grootegoed, 1998; Cunto et al., 2002). Spermatocytes exhibit delayed centrosome migration and a defect in the contractile ring disassembly of two meiotic cells; as a consequence, actin cytoskeletal reorganization is aberrant (Toshima et al., 2001; Takahashi et al., 2003). Therefore, control of actin assembly and disassembly during cytokinesis is required for regular centrosome migration as well as normal contractile ring formation and dissolution. The molecular mechanisms of testis-specific cytokinesis are still largely obscure (Cunto et al., 2002). The main function


of TESK1 is its involvement in actin cytoskeleton reorganization through phosphorylates cofilin, which controls actin filament dynamics and reorganization by stimulating depolymerization and severance of actin filaments (McGough et al., 1997; Bamburg et al., 1999; Toshima et al., 2001). In a few words, TESK1 is important for regulating testicular germ cell differentiation during and after the meiotic phase of spermatogenesis. The preliminary results of an analysis of the stably retained mRNA remnants in swine ejaculated sperm were presented. These results may allow scientists to monitor genome-wide transcriptional profiles during germ cell development in responding to the environmental input. The different gene expression profiles of remnant mRNAs in summer and winter seasons could also provide insight into the molecular mechanisms of thermoregulation in spermatogenesis. Even though most of the genes found in our experiments are still poorly understood in terms of their true functions in spermatogenesis; bioinformatics analysis suggests that they are involved in a broad spectrum of biochemical processes including gamete generation. These concordant profiles should allow the development of a noninvasive testing protocol to assess the functional capacity of sperm as well as a new molecular selection scheme for fine breeding swine. Acknowledgements This research was conducted using funds provided by grant NSC95-2313-B-002-056 awarded by the National Science Council of Taiwan, ROC. We would like to express our sincere appreciation for this support. References Aguilar-Mahecha, A., Hales, B.F., Robaire, B., 2001. Expression of stress response genes in germ cells during spermatogenesis. Biol. Reprod. 65, 119–127. Al-Shahrour, F., Minguez, P., Tárraga, J., Medina, I., Alloza, E., Montaner, D., Dopazo, J., 2007. FatiGO +: a functional profiling tool for genomic data. Integration of functional annotation, regulatory motifs and interaction data with microarray experiments. Nucleic Acids Res. 35, W91–W96. Bamburg, J.R., McGough, A., Ono, S., 1999. Putting a new twist on actin: ADF/cofilins modulate actin dynamics. Trends Cell Biol. 9, 364–370. Banks, S., King, S.A., Irvine, D.S., Saunders, P.T., 2005. Impact of a mild scrotal heat stress on DNA integrity in murine spermatozoa. Reproduction 129, 505–514. Blanco-Rodríguez, J., 1998. A matter of death and life: the significance of germ cell death during spermatogenesis. Int. J. Androl. 21, 236–248. Blanco-Rodríguez, J., Martínez-García, C., 1998. Apoptosis pattern elicited by several apoptogenic agents on the seminiferous epithelium of the adult rat testis. J. Androl. 19, 487–497. Cameron, R.D., Blackshaw, A.W., 1980. The effect of elevated ambient temperature on spermatogenesis in the boar. J. Reprod. Fertil. 59, 173–179. Chen, K., Knorr, C., Moser, G., Gatphayak, K., Brenig, B., 2004. Molecular characterization of the porcine testis-specific phosphoglycerate kinase 2 (PGK2) gene and its association with male fertility. Mamm. Genome 15, 996–1006. Cheng, W.T.K., 1985. In vitro fertilization in farm oocytes. Ph.D. Thesis. Cambridge University, U.K. Chuang, E.Y., Chen, X., Tsai, M.H., Yan, H., Li, C.Y., Mitchell, J.B., Nagasawa, H., Wilson, P.F., Peng, Y., Fitzek, M.M., Bedford, J.S., Little, J.B., 2006. Abnormal gene expression profiles in unaffected parents of patients with hereditary-type retinoblastoma. Cancer Res. 66, 3428– 3433. Cunto, F.D., Imarisio, S., Camera, P., Boitani, C., Altruda, F., Silengo, L., 2002. Essential role of citron kinase in cytokinesis of spermatogenic precursors. J. Cell Sci. 115, 4819–4826.

83

Dan, T.T., Summers, P.M., 1996. Reproductive performance of sows in the tropics. Trop. Anim. Health Prod. 28, 247–256. Dadoune, J.P., 2003. Expression of mammalian spermatozoal nucleoproteins. Microsc. Res. Tech. 61, 56–75. de Rooij, D.G., Grootegoed, J.A., 1998. Spermatogonial stem cells. Curr. Opin. Cell Biol. 10, 694–701. Eisen, M.B., Spellman, P.T., Brown, P.O., Botstein, D., 1998. Cluster analysis and display of genome-wide expression patterns. Proc. Natl. Acad. Sci. U.S.A. 95, 14863–14868. Evenson, D.P., Jost, L.K., Marshall, D., Zinaman, M.J., Clegg, E., Purvis, K., de Angelis, P., Claussen OP, 1999. Utility of the sperm chromatin structure assay as a diagnostic and prognostic tool in the human fertility clinic. Hum. Reprod. 14, 1039–1049. Flowers, W.L., 1997. Management of boars for efficient semen production. J. Reprod. Fertil. Suppl. 52, 67–78. Fox, M., Urano, J., Reijo Pera, R.A., 2005. Identification and characterization of RNA sequences to which human PUMILIO-2 (PUM2) and deleted in Azoospermia-like (DAZL) bind. Genomics 85, 92–105. Gadea, J., 2005. Sperm factors related to in vitro and in vivo porcine fertility. Theriogenology 63, 431–444. Gilbert, I., Bissonnette, N., Boissonneault, G., Vallée, M., Robert, C., 2007. A molecular analysis of the population of mRNA in bovine spermatozoa. Reproduction 133, 1073–1086. Hales, B.F., Aguilar-Mahecha, A., Robaire, B., 2005. The stress response in gametes and embryos after paternal chemical exposures. Toxicol. Appl. Pharmacol. 207, 514–520. Hao, Z., Jha, K.N., Kim, Y.H., Vemuganti, S., Westbrook, V.A., Chertihin, O., Markgraf, K., Flickinger, C.J., Coppola, M., Herr, J.C., Visconti, P.E., 2004. Expression analysis of the human testis-specific serine/threonine kinase (TSSK) homologues. A TSSK member is present in the equatorial segment of human sperm. Mol. Hum. Reprod. 10, 433– 444. Hayashida, N., Inouye, S., Fujimoto, M., Tanaka, Y., Izu, H., Takaki, E., Ichikawa, H., Rho, J., Nakai, A., 2006. A novel HSF1-mediated death pathway that is suppressed by heat shock proteins. EMBO J. 25, 4773–4783. Hikim, A.P., Lue, Y., Yamamoto, C.M., Vera, Y., Rodriguez, S., Yen, P.H., Soeng, K., Wang, C., Swerdloff, R.S., 2003. Key apoptotic pathways for heat-induced programmed germ cell death in the testis. Endocrinology 144, 3167–3175. Huang, S.Y., Chen, M.Y., Lin, E.C., Tsou, H.L., Kuo, Y.H., Ju, C.C., Lee, W.C., 2002. Effects of single nucleotide polymorphisms in the 5 -flanking region of heat shock protein 70.2 gene on semen quality in boars. Anim. Reprod. Sci. 70, 99–109. Irvine, D.S., Twigg, J.P., Gordon, E.L., Fulton, N., Milne, P.A., Aitken, R.J., 2000. DNA integrity in human spermatozoa: relationships with semen quality. J. Androl. 21, 33–44. Kon, Y., Endoh, D., 2001. Heat-shock resistance in experimental cryptorchid testis of mice. Mol. Reprod. Dev. 58, 216–222. Kramer, J.A., Krawetz, S.A., 1997. RNA in spermatozoa: implications for the alternative haploid genome. Mol. Hum. Reprod 3, 473–478. Kunavongkrit, A., Suriyasomboon, A., Lundeheim, N., Heard, T.W., Einarsson, S., 2005. Management and sperm production of boars under differing environmental conditions. Theriogenology 63, 657– 667. Kuo, Y.H., Huang, S.Y., Lee, K.H., 2000. Effects of sperm number and semen type on sow reproductive performance in subtropical area, Asian Australas. J. Anim. Sci. 13, 6–9. Livak, K.J., Schmittgen, T.D., 2001. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method. Methods 25, 402–408. Love, R.J., Evans, G., Klupiec, C., 1993. Seasonal effects on fertility in gilts and sows. J. Reprod. Fertil. Suppl. 48, 191–206. Martins, R.P., Krawetz, S.A., 2005. RNA in human sperm. Asian J. Androl. 7, 115–120. McGough, A., Pope, B., Chiu, W., Weeds, A., 1997. Cofilin changes the twist of F-actin: implications for actin filament dynamics and cellular function. J. Cell Biol. 138, 771–781. McNitt, J.I., First, N.L., 1970. Effects of 72-hour heat stress on semen quality in boars. Int. J. Biometeorol. 14, 373–380. McNitt, J.I., Tanner, C.B., First, N.L., 1972. Thermoregulation in the scrotal system of the boar. II. Evaporative heat exchange. J. Anim. Sci. 34, 117–121. Miller, D., Briggs, D., Snowden, H., Hamlington, J., Rollinson, S., Lilford, R., Krawetz, S.A., 1999. A complex population of RNAs exists in human ejaculate spermatozoa: implications for understanding molecular aspects of spermiogenesis. Gene 237, 385–392. Miller, D., Ostermeier, G.C., Krawetz, S.A., 2005. The controversy, potential and roles of spermatozoal RNA. Trends Mol. Med. 11, 156–163.

84


Murase, T., Imaeda, N., Yamada, H., Miyazawa, K., 2007. Seasonal changes in semen characteristics, composition of seminal plasma and frequency of acrosome reaction induced by calcium and calcium ionophore A23187 in Large White boars. J. Reprod. Dev. 53, 853– 865. Nakai, A., Suzuki, M., Tanabe, M., 2000. Arrest of spermatogenesis in mice expressing an active heat shock transcription factor 1. EMBO J. 19, 1545–1554. Ostermeier, G.C., Dix, D.J., Miller, D., Khatri, P., Krawetz, S.A., 2002. Spermatozoal RNA profiles of normal fertile men. Lancet 360, 772–777. Ostermeier, G.C., Goodrich, R.J., Diamond, M.P., Dix, D.J., Krawetz, S.A., 2005. Toward using stable spermatozoal RNAs for prognostic assessment of male factor fertility. Fertil. Steril. 83, 1687–1694. Peltoniemi, O.A., Heinonen, M., Leppävuori, A., Love, R.J., 1999. Seasonal effects on reproduction in the domestic sow in Finland—a herd record study. Acta Vet. Scand. 40, 133–144. Pérez-Crespo, M., Pintado, B., Gutiérrez-Adán, A., 2008. Scrotal heat stress effects on sperm viability, sperm DNA integrity, and the offspring sex ratio in mice. Mol. Reprod. Dev. 75, 40–47. Popwell, J.M., Flowers, W.L., 2004. Variability in relationships between semen quality and estimates of in vivo and in vitro fertility in boars. Anim. Reprod. Sci. 81, 97–113. Rockett, J.C., Mapp, F.L., Garges, J.B., Luft, J.C., Mori, C., Dix, D.J., 2001. Effects of hyperthermia on spermatogenesis, apoptosis, gene expression, and fertility in adult male mice. Biol. Reprod. 65, 229–239. Sailer, B.L., Sarkar, L.J., Bjordahl, J.A., Jost, L.K., Evenson, D.P., 1997. Effects of heat stress on mouse testicular cells and sperm chromatin structure. J. Androl. 18, 294–301. Spiridonov, N.A., Wong, L., Zerfas, P.M., Starost, M.F., Pack, S.D., Paweletz, C.P., Johnson, G.R., 2005. Identification and characterization of SSTK, a serine/threonine protein kinase essential for male fertility. Mol. Cell. Biol. 25, 4250–4261. Steinberger, A., 1991. Effects of temperature on the biochemistry of the testis. Adv. Exp. Med. Biol. 286, 33–47. Suriyasomboon, A., Lundeheim, N., Kunavongkrit, A., Einarsson, S., 2005. Effect of temperature and humidity on sperm morphology in duroc boars under different housing systems in Thailand. J. Vet. Med. Sci. 67, 777–785. Suriyasomboon, A., Lundeheim, N., Kunavongkrit, A., Einarsson, S., 2006. Effect of temperature and humidity on reproductive performance of crossbred sows in Thailand. Theriogenology 65, 606–628. Swiergiel, A.H., Ingram, D.L., 1987. Effect of localized changes in scrotal and trunk skin temperature on the demand for radiant heat by pigs. Physiol. Behav. 40, 523–526. Takahashi, H., Funakoshi, H., Nakamura, T., 2003. LIM-kinase as a regulator of actin dynamics in spermatogenesis. Cytogenet. Genome Res. 103, 290–298. Thonneau, P., Bujan, L., Multigner, L., Mieusset, R., 1998. Occupational heat exposure and male fertility: a review. Hum. Reprod. 13, 2122– 2125. Toshima, J., Koji, T., Mizuno, K., 1998. Stage-specific expression of testisspecific protein kinase 1 (TESK1) in rat spermatogenic cells. Biochem. Biophys. Res. Commun. 249, 107–112. Toshima, J., Ohashi, K., Okano, I., Nunoue, K., Kishioka, M., Kuma, K., Miyata, T., Hirai, M., Baba, T., Mizuno, K., 1995. Identification and characterization of a novel protein kinase. TESK1, specifically expressed in testicular germ cells. J. Biol. Chem. 270, 31331–31337.

Toshima, J., Tanaka, T., Mizuno, K., 1999. Dual specificity protein kinase activity of testis-specific protein kinase 1 and its regulation by autophosphorylation of serine-215 within the activation loop. J. Biol. Chem. 274, 12171–12176. Toshima, J., Toshima, J.Y., Amano, T., Yang, N., Narumiya, S., Mizuno, K., 2001. Cofilin phosphorylation by protein kinase testicular protein kinase 1 and its role in integrin-mediated actin reorganization and focal adhesion formation. Mol. Biol. Cell 12, 1131–1145. Tramontano, F., Malanga, M., Farina, B., Jones, R., Quesada, P., 2000. Heat stress reduces poly(ADPR) polymerase expression in rat testis. Mol. Hum. Reprod. 6, 575–581. Tseng, T.C., Chen, S.H., Hsu, Y.P., Tang, T.K., 1998. Protein kinase profile of sperm and eggs: cloning and characterization of two novel testis-specific protein kinases (AIE1, AIE2) related to yeast and fly chromosome segregation regulators. DNA Cell Biol. 17, 823–833. Tusher, V.G., Tibshirani, R., Chu, G., 2001. Significance analysis of microarrays applied to the ionizing radiation response. Proc. Natl. Acad. Sci. U.S.A. 98, 5116–5121. Visconti, P.E., Hao, Z., Purdon, M.A., Stein, P., Balsara, B.R., Testa, J.R., Herr, J.C., Moss, S.B., Kopf, G.S., 2001. Cloning and chromosomal localization of a gene encoding a novel serine/threonine kinase belonging to the subfamily of testis-specific kinases. Genomics 77, 163–170. Wang, H., Zhou, Z., Xu, M., Li, J., Xiao, J., Xu, Z.Y., Sha, J., 2004. A spermatogenesis-related gene expression profile in human spermatozoa and its potential clinical applications. J. Mol. Med. 82, 317–324. Wei, Y., Fu, G., Hu, H., Lin, G., Yang, J., Guo, J., Zhu, Q., Yu, L., 2007. Isolation and characterization of mouse testis specific serine/threonine kinase 5 possessing four alternatively spliced variants. J. Biochem. Mol. Biol. 4, 749–756. Wettemann, R.P., Wells, M.E., Johnson, R.K., 1979. Reproductive characteristics of boars during and after exposure to increased ambient temperature. J. Anim. Sci. 49, 1501–1505. Wykes, S.M., Visscher, D.W., Krawetz, S.A., 1997. Haploid transcripts persist in mature human spermatozoa. Mol. Hum. Reprod. 3, 15–19. Xu, B., Hao, Z., Jha, K.N., Digilio, L., Urekar, C., Kim, Y.H., Pulido, S., Flickinger, C.J., Herr, J.C., 2007. Validation of a testis specific serine/threonine kinase [TSSK] family and the substrate of TSSK1 & 2, TSKS, as contraceptive targets. Soc. Reprod. Fertil. Suppl. 63, 87–101. Xu, X., Ding, J., Seth, P.C., Harbison, D.S., Foxcroft, G.R., 1996. In vitro fertilization of in vitro matured pig oocytes: effects of boar and ejaculate fraction. Theriogenology 45, 745–755. Yanagimachi, R., 2005. Male gamete contributions to the embryo. Ann. N.Y. Acad. Sci. 1061, 203–207. Yang, C.C., Lin, Y.S., Hsu, C.C., Wu, S.C., Lin, E.C., Cheng, W.T.K., 2009. Identification and sequencing of remnant messenger RNAs found in domestic swine (Sus scrofa) fresh ejaculated spermatozoa. Anim. Reprod. Sci. 113, 143–155. Yang, H.M., Liu, G., Nie, Z.Y., Nie, D.S., Deng, Y., Lu, G.X., 2005. Molecular cloning of a novel rat gene Tsarg1, a member of the DnaJ/HSP40 protein family. DNA Seq. 16, 166–172. Yatsenko, A.N., Roy, A., Chen, R., Ma, L., Murthy, L.J., Yan, W., Lamb, D.J., Matzuk, M.M., 2006. Non-invasive genetic diagnosis of male infertility using spermatozoal RNA: KLHL10 mutations in oligozoospermic patients impair homodimerization. Hum. Mol. Genet. 15, 3411–3419. Yin, Y., Hawkins, K.L., DeWolf, W.C., Morgentaler, A., 1997. Heat stress causes testicular germ cell apoptosis in adult mice. J. Androl. 18, 159–165.