RESEARCH ARTICLE Molecular Reproduction & Development (2014)

Identification and Characterization of Genes Differentially Expressed in X and Y Sperm Using Suppression Subtractive Hybridization and cDNA Microarray XIAOLI CHEN, YANG YUE, YANAN HE, HUABIN ZHU, HAISHENG HAO, XUEMING ZHAO, TONG QIN, AND DONG WANG* The Key Laboratory for Farm Animal Genetic Resources and Utilization of Ministry of Agriculture of China, Institute of Animal Science, Chinese Academy of Agriculture Sciences, Beijing, China

SUMMARY Differential expression of genes leads to variations in the phenotypes of X and Y sperm, although some differentially expressed gene products are shared through intercellular bridges. Genes differentially expressed in bovine X and Y sperm were identified by a combination of suppression subtractive hybridization (SSH), cDNA microarray, and sequence-homology analysis. Microarray data and Significance Analysis of Microarrays software were used to identify 31 differentially expressed genes, only four of which were previously identified. These genes are involved in fundamental life processes of mature sperm, and may be associated with the differences between X and Y sperm since 27 versus 4 were upregulated in X versus Y sperm, respectively. The levels of expression of seven genesincluding the known genes UTY, DPH3, CYTB, and ISCU, and the unknown genes X þ Y contig 41, X þ Y contig 18, and Y þ X contig 16were validated by quantitative real-time PCR, and some genes were clearly differentially expressed by X and Y sperm, despite the presence of intercellular bridges among spermatids. These results provide a theoretical basis for research on gene expression during sperm development, as well as on sex control at the level of sperm.



Corresponding author: The Key Laboratory for Farm Animal Genetic Resources and Utilization of the Ministry of Agriculture of China Institute of Animal Science Chinese Academy of Agriculture Sciences Beijing 100193, China. E-mail: [email protected]

Xiaoli Chen, Yang Yue and Yanan He contributed equally to this work.

Mol. Reprod. Dev. 2014. ß 2014 Wiley Periodicals, Inc. Received 15 April 2014; Accepted 29 July 2014

Abbreviations: Contig, continuous clone; EST, expressed sequence tag; qPCR, quantitative real-time PCR; SSH, suppression subtractive hybridization; Genes: ACTB, beta-actin; CYTB, cytochrome b; DPH3, dipthamide biosynthesis

ß 2014 WILEY PERIODICALS, INC.

Grant sponsor: National Science and Technology Support Program of China; Grant number: 2011BAD19B02; Grant sponsor: National ‘‘863’’ Hi-Tech Research and Development Program of China; Grant number: 2006AA10Z143; Grant sponsor: Agricultural Science and Technology Innovation Program (ASTIP) (cxgc-ias06); Grant sponsor: Beijing Innovation Team of Technology System in Dairy Industry Published online in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/mrd.22386

protein 3; GAPDH, glyceradehyde-3-phosphate dehydrogenase; ISCU, ironsulfur cluster assembly enzyme; UTY, histone methylation ubiquitously transcribed tetratricopeptide repeat Y

Molecular Reproduction & Development

INTRODUCTION In modern animal husbandry, the sex of offspring has significant economic benefits, making the ability to predetermine sex an important attribute of reproduction. Although flow cytometry can sort X and Y sperm up to 90% purity, the process is slow and costly when used for animal production. One simple and efficient alternative to sorting by DNA content is by immunological separation based on proteins differentially expressed by X and Y sperm. Separation based on the H-Y antigen, however, exhibited low specificity (Simpson et al., 1997; Sills et al., 1998; Hendriksen, 1999), while studies exploring proteins differentially expressed by X and Y sperm did not previously yield ideal results (Hendriksen et al., 1996; Hendriksen, 1999). A high-speed method of sorting sperm requires the systematic detection of genes expressed in a sex-specific manner. Differences between these two kinds of sperm have been explored extensively since X and Y sperm were first identified in 1923 (Painter, 1923; Cui and Matthews, 1993; Cui, 1997), although the discovery of intercellular bridges in groups of developing male germ cells during spermatogenesis (Burgos and Fawcett, 1955; Fawcett et al., 1959) has resulted in little progress identifying genes differentially expressed by X and Y sperm. Intercellular bridges facilitate the sharing of cytoplasmic components and allow the differentiation of germ cells to be directed by gene products from both parents (Erickson, 1973), thereby permitting all haploid spermatids from one spermatogonium to become functional equivalent gametes. This intercellular transport system is very important to haploid spermatids, ensuring the synchronized development of sperm for fertilization and protecting individual sperm from lethal consequences of mutations or the loss of a critical post-meiotically expressed gene (Caldwell and Handel, 1991). Yet, phase-contrast examination of intercellular bridges in rat sperm showed that, although a multitude of small granules moved continuously over these bridges, only 28% of the granules entering bridges were actually transported into other cells (Ventela et al., 2003). Confocal microscopy further showed that only about 50% of sperm were positively immunostained by antiserum against extra-embryonic tissue-spermatogenesis-homeobox gene 1 (ESX1), suggesting that ESX1 is a marker for X chromosome-bearing sperm (Yeh et al., 2005). Furthermore, X sperm do not share all their proteins with Y sperm (Chen et al., 2012; De Canio et al., 2014). Encouraged by this observed biological segregation, we constructed forward and reverse subtracted cDNA libraries by suppression subtractive hybridization (SSH) and then sequenced them to identify genes differentially expressed in X and Y sperm. The non-redundant clones were analyzed by microarray, and the results were validated by quantitative real-time PCR (qPCR). Our findings provide a theoretical and experimental basis of differential expression by X and Y sperm, which will be important for sex control and for protecting against sex-linked genetic diseases.

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RESULTS Quality Assessment of Total RNA Samples Lysis of 3  107 sperm with 1 mL TRIzol left small amounts of debris, but none were visible when b-mercaptoethanol was added to the lysis buffer (data not shown). To completely lyse sperm and obtain more RNA, total RNA was extracted using the hot-TRIzol method, which was modified by adding b-mercaptoethanol. The mean yield of total RNA extracted from 3  107 X and Y sperm was 650 ng per group. GAPDH and CD45 transcript were amplified from 30 ng of RNA from each preparation by endpoint reverse-transcriptase PCR to assess the quality of these total RNA preparations (Fig. 1). Amplification from each sperm preparation yielded a 249-bp GAPDH band specific to sperm, whereas none showed evidence of the 470-bp genomic GAPDH. The somatic-cell specific CD45 band was also not detected in any of the sperm RNA samples.

Construction of Subtractive cDNA Libraries To isolate genes differentially expressed in X and Y sperm, forward (XY) and reverse (YX) subtractive cDNA libraries were constructed. The same cDNAs expressed in X and Y sperm were effectively subtracted

Figure 1. A: Detection of contamination in XY sorted Bos taurus sperm total RNA samples, searching for genomic DNA. Positivecontrol testis tissue (T) was derived from a Beijing abattoir. PCR was performed with primers specific for the GAPDH and CD45 genes. The GAPDH primers yielded a 249-bp PCR product, consistent with the sequence of intact RNA, whereas the absence of a 470-bp band confirmed that the samples were not contaminated with genomic DNA. B: Quality assessment of total RNA for somatic-cell contamination. Although these CD45 primers amplified a 321-bp band in bovine testicular cDNA, these primers did not amplify anything from the sperm RNA samples, indicating that they were not contaminated with somatic cells, DNA markers of 600, 500, 400, 300, 200, and 100 bp (M) are indicated to the left. X1-X3 or Y1-Y3 represent three samples of X or Y sperm respectively. NC, negative control.

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during two rounds each of hybridization and amplification, whereas the differentially expressed genes were specifically amplified.

Expressed Sequenced Tag Sequencing and Contiguous Clone Assembly The insert in each clone was detected by PCR amplification and electrophoresis. Inserts longer than 100 bp were sequenced, yielding 1111 valid expressed sequence tags (ESTs), 566 for the forward and 545 for the reverse library. Pre-treatment yielded 565 and 541 high-quality ESTs, respectively, of average sizes (578 bp and 612 bp, respectively). Cluster and assembly analysis of these high-quality ESTs was performed using the fragment assembly program Phrap (http://www.phrap.org/phredphrap/phrap.html). The forward library yielded 89 unigenes with 84.25% sequence redundancy, including 28 singlets and 61 contigs, whereas the reverse library yielded 112 unigenes, with 79.29% sequence redundancy, including 52 singlets and 60 contiguous clones that overlap (contigs).

Verification of Differentially Expressed cDNAs Microarray analysis was performed to identify positive, differentially expressed cDNAs in the forward and reverse libraries. RNA samples of X and Y sperm were labeled in triplicate for microarray hybridization, including one fluorescence dye transfer. Each unigene was analyzed using Significance Analysis of Microarrays (SAM) software (Stanford University, Palo Alto, CA), and genes with |score (d)| 2 and false-discovery rate (FDR) 1  105), suggesting their novelty.

Mol. Reprod. Dev. (2014)

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qPCR Validation Of the differentially expressed genes (Table 1), the known genes histone methylation ubiquitously transcribed tetratricopeptide repeat Y (UTY), dipthamide biosynthesis protein 3 (DPH3), cytochrome b (CYTB), and iron-sulfur cluster assembly enzyme (ISCU) and the unknown genes X þ Y contig 41, X þ Y contig 18, and Y þ X contig 16were randomly selected for qPCR analysis. Five of these genes were up-regulated in X sperm and two were up-regulated in Y sperm, following normalization to betaactin (ACTB) levels (Fig. 2). The statistical significance of ISCU, CYTB, X þ Y contig 41, and X þ Y contig 18 transcription (Student’s t-test) and qPCR data are consistent with the results of SSH and microarray analysis.

DISCUSSION Total-RNA content is much lower in human sperm than in somatic cells, being 0.015 pg and 13 pg, respectively (Miller et al., 2005); total-RNA content of each bovine sperm is even lower, about 1.8  104 pg, versus 0.45 pg per spermatid; and both are much lower than the contents of somatic cells (Gilbert et al., 2007). Such low quantity of RNA per sperm has hampered research on gene expression in these gametes. Oxidation of sulfhydryl groups of protamine during sperm formation and maturation increases the number of disulfide bonds, making chromosomes more compact (Bedford and Calvin, 1974; Sylvester et al., 1984; Shalgi et al., 1989; Moore, 1998), further hampers RNA extraction and thus reduced yields. Thus, little is known about gene expression in sperm. Technically, we found that the addition of b-mercaptoethanol to the lysis solution, which effectively inhibits RNase activity (Meng and Feldman, 2010; Wang et al., 2010) and breaks disulfide bonds (Kawamura and Nagano, 1984; Kirley, 1990), resulted in more complete lysis of sperm and a higher yield of total RNA, producing about 600 ng of total RNAs from each sample of 3  107 sperm, or about 0.02 pg per sperm, which is consistent with 0.025 pg amount previously reported (Lalancette et al., 2008). Differences between X and Y sperm are due primarily to differences between their chromosomes and gene expression products. The finding that adjacent cells share gene products during spermatogenesis through intercellular bridges has dampened the enthusiasm to determine differences between X and Y sperm (Burgos and Fawcett, 1955; Fawcett et al., 1959). Yet, all expression products are probably not shared through these intercellular bridges; indeed, some differences have been reported (Ventela et al., 2003). Based on this report, we utilized SSH, microarray analysis, and sequence-homology analysis to assess differential gene expression in X and Y sperm. Subtractive cDNA libraries were generated, then ESTs were sequenced and sorted. To decrease screening and analysis, microarray analysis was performed to validate the SSH results. This method identified 31 genes differentially expressed by X and Y sperm, some of which may be involved in fundamental processes of

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TABLE 1. Differentially Expressed Unigenes Between X and Y Sperm of Bos Taurus Screened by Microarrays, and the Results of BLASTX and BLASTN Sequence Homology Analysis Unigene name

|Score (d)|a

Length

Accession No.

Description

E-valueb

n

7 E-13 4 E-17 4 E-24 2 E-41

11 2 3 4

1 E-83

3

3 E-111

1

2 E-70

1

2 E-168

7

7 E-15

6

9 E-18

1

0

6

5 E-29

6

2 E-16 2 E-05

1 5

4 E-26 8 E-92

5 3

2 E-168 3 E-16

3 7

8 E-32 1 E-05

1 1

c

Unigenes exhibiting high sequence homology with annotated proteins in the SwissProt database Y þ X contig45d 2.202487974 496 Q6B4Z3.1 Histone demethylase UTY [Pan troglodytes] 3.5646655 470 Q1LZC9.1 DPH3 homolog [Bos taurus] X þ Y contig 1d d 2.5503523 251 P00157.1 Cytochrome b [Bos taurus] X þ Y contig 13 2.3437931 475 Q9H1K1.2 Iron-sulfur cluster assembly enzyme ISCU, mitochondrial [Homo X þ Y contig 24d sapiens] Unigenes exhibiting high sequence homology with sequences in the NCBI nucleotide database (Bos taurus) 4.77180836 576 AC_000163.1 Bos taurus breed Hereford chromosome 6, Bos_taurus_UMD_3.1, Y þ X contig 16d whole genome shotgun sequence Y þ X4H1 3.35655878 310 AJ133269.1 Homo sapiens caveolin-1/-2 locus, Contig1, D7S522, genes CAV2 (exons 1, 2a, and 2b), CAV1 (exons 1 and 2) Y þ X2_E1 2.33019739 333 EU130451.1 Bos taurus prion protein (PRNP) gene, complete cds, alternatively spliced X þ Y contig 46 10.568588 1618 AC_000183.1 Bos taurus breed Hereford chromosome 26, Bos_taurus_UMD_3.1, whole genome shotgun sequence X þ Y contig 37 3.166568 575 AY062434.1 Homo sapiens elongation factor 1-alpha 1 (EEF1A1L14) mRNA, complete cds X þ Y1A12 2.106458 864 AC_000182.1 Bos taurus breed Hereford chromosome 25, Bos_taurus_UMD_3.1, whole genome shotgun sequence 3.8531496 466 AC109993.4 Homo sapiens chromosome X clone RP11-115M6 map q28, complete X þ Y contig 41d sequence X þ Y contig 43 3.8295829 573 AY062434.1 Homo sapiens elongation factor 1-alpha 1(EEF1A1L14) mRNA, complete cds X þ Y1F9 3.7812149 903 AB074968.1 Bos taurus mitochondrial DNA, complete genome, haplotype: JBC8 X þ Y contig 31 3.274684 501 AB098850.1 Bos taurus mitochondrial RNA, similar to 28S rRNA, clone: ORCS10668 X þ Y contig 35 2.8684649 795 AF277191.1 Homo sapiens PNAS-133 mRNA, partial sequence X þ Y contig 16 2.7647451 544 AC_000183.1 Bos taurus breed Hereford chromosome 26, Bos_taurus_UMD_3.1, whole genome shotgun sequence d 2.489133 345 FJ971088.1 Bos taurus isolate Mong01 mitochondrion, complete genome X þ Y contig 18 X þ Y contig 47 2.15915 1003 AB218812.1 Rattus norvegicus mRNA for hypothetical protein, complete cds, brain endothelial cell derived gene-2 X þ Y1A7 2.123656 705 HM045018.1 Bos taurus breed Heck cattle mitochondrion, complete genome X þ Y3G9 2.6695102 204 AC101745.9 Mus musculus chromosome 19, clone RP23-473I8, complete sequence Unigenes without homologous sequences in public databases X þ Y contig 48 3.0983816 378 X þ Y4G1 2.7715137 538 X þ Y5C11 2.7605442 485 X þ Y contig 57 2.6856292 837 e 2.5593111 596 X þ Y contig 29 X þ Y2D6 5.9064216 548 X þ Y1F2 5.8710012 524 X þ Y20 3.675124 273 X þ Y contig 3 3.2383862 334 X þ Y6D8 3.1690121 195 X þ Y2_E1 2.2084047 272

8 1 1 21 5 1 1 1 2 1 1

n, number of ESTs per unigene. The number following the contig is its probe number. X þ Y and Y þ X represent the forward library (XY) and reverse library (YX), respectively. A contig is a series of overlapping (‘‘contiguous’’) DNA sequences used to make a physical map that reconstructs the original DNA sequence. BLASTX (Nucleotide 6-frame translation-protein) and BLASTN (Nucleotide-nucleotide BLAST) both belong to the BLAST (Basic Local Alignment Search Tool) programs in NCBI (National Center for Biotechnology Information). a Selection criteria was |score (d)| 2 in the microarray. b Unigenes with E-value  1  105 were defined as exhibiting high sequence homology with nucleotide (nr/nt) databases. c The name, organism, accession number, and E-value given for the best match with SwissProt database using BLASTX. d Selected for quantitative PCR analysis. e Coding sequence.

mature sperm and may be associated with the differences between X and Y sperm. Despite their transcriptional arrest, developing spermatids and sperm rely on the translation of stored mRNAs to produce proteins necessary for development (Steger, 2001), for ATP production (Rodriguez-Martinez, 2001), and for sperm capacitation (Gur and Breitbart, 2006). Due to the absence of 80S ribosomal complexes from sperm, cytoplasmic mRNA translation cannot occur,

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although translation can occur in mitochondria (Miller et al., 1999; Miller and Ostermeier, 2006). Indeed, mRNAs are present in the mitochondria of mature mouse sperm, with cytoplasmic mRNAs translated on mitochondrial polysomes (Gur and Breitbart, 2006), and differentially expressed proteins can be detected after capacitation and cryopreservation (Chen et al., 2014). Similar to somatic cells, sperm mitochondria contain some key enzymes of the Krebs cycle and respiratory

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Figure 2. Relative expression levels of seven genes in X and Y Bos taurus sperm. RNA levels were assessed using qPCR, as described in Materials and Methods, and normalized to that of the ACTB (internal control).  P < 0.05,  P < 0.01,  P < 0.001 (Student’s t-test).

chain, which are important for ATP production (Schultz and Chan, 2001). Within our screen, we found X-enriched transcripts encoding the mitochondrial proteins CYTB and ISCU: CYTB encodes the cytochrome b protein, a subunit of the catalytic core of the cytochrome bc1 complex. This complex, which is found in the inner mitochondrial membranes of eukaryotic cells (Mitchell, 1976), is a central component of the mitochondrial respiratory chain, coupling electron transfer from ubihydroquinone to cytochrome c, and generating a proton gradient across the mitochondrial membrane (Schultz and Chan, 2001). ISCU, a nuclearencoded mitochondrial gene, encodes the iron-sulfur cluster scaffold protein, one of the core proteins involved in iron-sulfur assembly (Foster et al., 2000; Hoff et al., 2002). Iron-sulfur clusters are ubiquitous and functionally versatile prosthetic groups, are major components of the respiratory chain, define electron transport pathways in numerous membrane-bound and redox enzymes, and constitute the redox-active centers of ferredoxins (Johnson et al., 2005; Lill and Muhlenhoff, 2008). The up-regulation of CYTB and ISCU expression in X sperm suggests that these gametes may require more energy. This finding seems to be

Mol. Reprod. Dev. (2014)

consistent with size differences between X and Y sperm, namely that the heads, necks, and tails of X sperm are longer than that of Y sperm, and the heads of X sperm are larger in relative volume than Y sperm (Cui and Matthews, 1993; Cui, 1997). DPH3, another gene we found up-regulated in X sperm, was originally reported to encode the enzyme that catalyzes the first step in the biosynthesis of diphthamide (Wang et al., 2012). DPH3 is crucial for diphthamide modification of elongation factor 2 (eEF-2) in eukaryotes, with the latter being key to protein synthesis. dph3þ/ mice are phenotypically normal, whereas dph3-null mice showed a delay in embryonic development or embryonic lethality due to an absence of diphthamide-modified eEF-2 (Liu et al., 2006). The greater expression of DPH3 observed in X sperm by qPCR may reflect elevated protein synthesis in X compared to Y sperm. The gene encoding UTY, which contains a tetratricopeptide repeat (TPR) and a Jumonji-C (JmjC) domain, was upregulated in Y sperm. The structurally conserved TPR is thought to participate in protein-protein interactions (Blatch and Lassle, 1999) whereas JmjC-domain-containing

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proteins contain hydroxylase activity that can function as demethylases (Klose and Zhang, 2007). UTY is the apparent male counterpart of tetratricopeptide repeat X (UTX), and is encoded by the male Y chromosome. UTY and the ubiquitously transcribed UTX proteins are highly homologous, sharing the same domain structure as well as TPR protein interaction domains (Greenfield et al., 1998; Agger et al., 2007). The qPCR-detectable upregulation of UTY in Y sperm could have been predicted because the Y chromosome is present only in Y sperm; however, its function requires further study. The 11 novel sequences of differentially expressed genes were analyzed using Coding Potential Calculator software (Peking University, Beijing, China) (Kong et al., 2007), and 10 of these transcripts were classified as non-coding RNAs (ncRNAs). ncRNAs function as regulators of almost every aspect of biology, including epigenetic regulation in embryogenesis and disease (Eddy, 2001; Lee, 2009; Wilusz et al., 2009; Pauli et al., 2011). About 24,000 ncRNAsincluding microRNAs, small interfering RNAs, and piwi-interacting RNAshave been detected in individual sperm (Krawetz et al., 2011). A new 1.6kb ncRNA was found to be the precursor of a microRNA-like small RNA (mil-HongrES2), the overexpression of which suggested that it functions in sperm maturation in rat epididymis (Ni et al., 2011). The 10 ncRNAs that appear to play a very important role in differentiating between X and Y sperm require further study. The one remaining, differentially expressed gene, X þ Y contig 29, was classified as a coding sequence. Homologous sequences were not found in the NCBI database, however, so the function of X þ Y contig 29 requires further exploration. In conclusion, we observed four genes involved in fundamental processes in sperm that were differentially expressed in X and Y sperm, suggesting that they may be associated with physiological differences between these sperm. Additional, novel transcripts were also identified, most of which are ncRNAs, suggesting that these genes may play important roles during spermiogenesis and deserve further investigation.

MATERIALS AND METHODS Diethyl pyrocarbonate (DEPC) and TRIzol were purchased from Invitrogen (Carlsbad, CA). Sodium dodecyl sulfate (SDS), amplicillin, and b-mercaptoethanol were obtained from Sigma-Aldrich (St. Louis, MO). RNasefree DNase I was obtained from Takara Bio of Otsu, Japan. Super SMARTTM PCR cDNA Synthesis, Advantage1 2 PCR, and PCR-Select cDNA Subtraction Kits were all purchased from Clontech (Palo Alto, CA). pGEM-T Vector was obtained from Promega (Madison, WI). MessageAmpTM II aRNA Amplification Kit was obtained from Ambion (Austin, TX). Klenow enzyme, Nucleospin1 Extract II, and AffinityScriptTM QPCR cDNA Synthesis Kits were purchased from CapitalBio Corporation (Beijing, China), Macherey-Nagel (Düren, Germany), and Strategene (La Jolla, CA), respectively. The fluorescent dyes Cy5

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and Cy3 were purchased from GE Healthcare (Piscataway, NJ).

Collection and Sorting of Semen Samples In the present experiment, animal care and samples collection procedures were approved and conducted under established standard of the Institute of Animal Science, Chinese Academy of Agricultural Sciences, Beijing, China. Semen samples were obtained randomly from nine healthy, fertile Holstein bulls (23 years old) at the XY Breeding Livestock Co. (Tianjin, China). Ejaculate quality was assessed by microscopy, and semen with the desired quality (motility >80%, deformation ratio

Identification and characterization of genes differentially expressed in X and Y sperm using suppression subtractive hybridization and cDNA microarray.

Differential expression of genes leads to variations in the phenotypes of X and Y sperm, although some differentially expressed gene products are shar...
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