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Reproduction (2007) 133 1073-1086
DOI: 10.1530/REP-06-0292
Copyright © 2007 Society for Reproduction and Fertility
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RESEARCH

A molecular analysis of the population of mRNA in bovine spermatozoa

Isabelle Gilbert, Nathalie Bissonnette1, Guylain Boissonneault2, Maud Vallée and Claude Robert

Centre de Recherche en Biologie de la Reproduction, Département des Sciences Animales, Université Laval, Québec G1K 7P4, Canada, 1 Dairy and Swine Research and Development Centre, AAFC, PO Box 90, Lennoxville, Québec J1M 1Z3, Canada and 2 Département de Biochimie, Faculté de Médecine, Université de Sherbrooke, Sherbrooke, Québec J1H 5N4, Canada

Correspondence should be addressed to C Robert; Email: claude.robert{at}crbr.ulaval.ca


   Abstract
Top
 Abstract
Introduction
 Material and Methods
 Results
 Discussion
 Acknowledgements
 References
 
Spermiogenesis represents the transition from haploid spermatids to spermatozoa. This process entails an extreme condensation of the nucleus and a loss of nearly all cytoplasmic content. The presence of messenger RNAs in the spermatozoa has previously been shown. Generally, these transcripts are considered to be remnants of spermiogenesis. However, it has recently been proposed that there may exist a function for these sperm-associated RNAs. To address the possibility of a functional role for these transcripts, we sought to investigate and characterize the RNA pool found in bovine spermatozoa. The main goals of this study were to examine RNA integrity and survey the mRNA found in spermatids and spermatozoa. Assessment of mRNAs integrity was performed by three approaches: microelectrophoresis, comparative smearing after global amplification, and PCR amplification of target sequences located either in the 5' or the 3' ends, while mRNAs survey was performed by microarray hybridizations. RNA integrity studies in the spermatozoa showed a majority of low molecular size fragments indicating a natural segmentation of the mRNA population. The mRNA survey indicated that the sperm transcriptome harbors a complex mixture of messengers implicated in a wide array of cell functions and representing a large subset of transcripts found in spermatids. Subsequently, such sperm RNA profiling could allow the molecular diagnosis of male gamete quality.


   Introduction
Top
 Abstract
 Introduction
Material and Methods
 Results
 Discussion
 Acknowledgements
 References
 
During spermiogenesis, the differentiation of spermatids into spermatozoa involves a change in chromatin structure leading to a greater level of DNA compaction. This is accomplished through the replacement of most histones with protamines and a major loss of its cytoplasm. There is a high level of transcriptional activity in spermatocytes, prior to the first meiotic division, then a gradual decrease in the rate of transcription, before a short surge at the stage of the round spermatid (Dadoune et al. 2004). Transcriptional activity shuts down when spermiogenesis reaches the elongating spermatids stage, when a replacement of histone proteins by protamines occurs (Dadoune et al. 2004). This model is supported by a recent study of human sperm in which an in vitro assay using incorporation of radio-labeled UTPs showed no detectable signs of transcription in the spermatozoa (Grunewald et al. 2005). In addition, the translational activity in the spermatozoa is also compromised as it has been shown that the spermatozoa harbor no or few ribosomal RNAs (Ostermeier et al. 2002, Grunewald et al. 2005).

The presence of mRNA in spermatozoa is well established; yet little is known regarding its function and purpose (Dadoune et al. 2005). Given the inability of the spermatozoon to synthesize RNA, it is assumed that its RNA content originates from the trapped cytoplasmic content remaining after spermiogenesis, and consequently these RNAs may be not more than remnants. Recent interest in spermatic RNA has been motivated by the potential it may offer as a diagnostic tool for infertility. It has been proposed that, because of the delay between transcription and translation during spermatogenesis, the mRNA population present in spermatozoa should be representative of the past events of spermatogenesis (Ostermeier et al. 2002, Dadoune 2003, Steger 2003, Lambard et al. 2004).

In order to identify specific transcripts important for the production of functional spermatozoa, gene expression analyses have been conducted at specific stages of spermatogenesis as well as in different testicular somatic cells. In rodents, microarray analysis has revealed a large array of candidates that are differentially and specifically regulated during spermatogenesis (Wrobel & Primig 2005). This observation was confirmed by another large-scale study performed in the rat, showing different transcripts in spermatogonia, spermatocytes, and spermatids when compared with Sertoli cells and other testicular somatic cells (Schlecht et al. 2004). It has been reported that the transcriptional regulators, whose absence is often associated with infertility, are mostly transcribed in the meiotic or post-meiotic germinal cells but not in the somatic tissues (Eddy 2002).

Currently, the literature contains only a few reports regarding sperm-associated mRNAs. Some studies highlighted the subcellular localization of the RNAs in the male gamete. For example, the RNA of c-MYC has been localized in the midpiece and the tail (Kumar et al. 1993), while other reports showed the presence of RNA within the nuclear compartment of the spermatozoa (Pessot et al. 1989, Wykes et al. 1997). In addition, mRNAs for the transcription factors nuclear factor-kappa B (NF{kappa}B), homeobox 2A (HOX2A), interferon consensus sequence binding protein (ICSB), and the protein kinase c-jun n-terminal kinase 2 (JNK2), the growth factor heparin-binding EGF-like growth factor (HBEGF) as well as the retinoid X receptor b (RXRß) and epidermal growth factor receptor (ErbB3) receptors were all localized within the sperm head (Dadoune et al. 2005).

Recent studies reported that a quality assay for semen could be derived from RNA profiling. More specifically, the RNA stability of ejaculates of varying quality was examined by applying a freeze–thaw stress to the samples followed by a comparative analysis of RNA profiles using microarrays (Ostermeier et al. 2005b). Semen quality was found to be associated with transcript abundance as some transcripts were absent in the stress-sensitive spermatozoa. The potential involvement of spermatic transcripts for embryonic development was also proposed. Ostermeier et al.(2004) also detected two sperm-associated transcripts, protamine 2 and clusterin, in the fertilized egg therefore suggesting the contribution of spermatic transcripts to postfertilization development. Their finding has sparked considerable interest in the field since such a potential contribution of the male gamete to embryonic development challenges the well-accepted dogma, which restricted the male gamete to a DNA shuttling vector.

The objective of our present study was to characterize the bovine spermatic RNA pool first byassessing the integrity of the RNAs, then by a comparison of the transcripts found in the bovine spermatids and spermatozoa. This approach should provide important clues regarding whether the integrityand complexityof the mRNAs are compatible with a role in early embryonic development.


   Material and Methods
Top
 Abstract
 Introduction
 Material and Methods
Results
 Discussion
 Acknowledgements
 References
 
Spermatozoa preparation
To eliminate variations between individual, semen samples from ten different Holstein bulls were pooled. These samples were a courtesy of the Centre d’Insemi-nation Artificielle du Quebec (CIAQ, Sainte-Madeleine, QC, Canada). On an average, each ejaculate contained 7.04x109 spermatozoa. To eliminate damaged spermatozoa and contaminating somatic cells, the pooled semen samples were purified on a discontinuous gradient of Percoll 40:80 (Sigma-Aldrich). The Percoll solution (described by Parrish et al. 1988) was diluted in Tyrode’s sperm medium (Sp-TALP; 100 mM NaCl, 3.1 mM KCl, 25 mM NaHCO3, 0.3 mM NaH2PO4, 21.6 mM Na lactate, 2.0 mM CaCl2, 0.4 mM MgCl2, 10 mM Hepes,1 mM pyruvate, and 50 µg/ml gentamycin). The Percoll gradient purification was carried out as described by Parrish et al.(1988). Briefly, samples were deposited on the Percoll gradient and centrifuged at 700 g for 30 min. The pellets were washed and centrifuged twice at 250 g for 5 min in Sp-TALP solution. The pellets containing the motile spermatozoa were kept at –80 °C in RNAlater (Ambion, Austin, TX, USA) until RNA extraction.

Isolation of spermatids
Testes from ten bulls were collected at a local slaughter house and transported to the laboratory on ice immediately after excision. These tissues were used for spermatids isolation as well as for controls in several experiments. The purification of spermatids was carried out as described by Morin et al.(2005). Briefly, the haploid germ cells were isolated from a piece of fresh testis under the tunica albuginea. The tissue pieces were washed in D-PBS (137 mm NaCl, 2.7 mm KCl, 0.9 mm CaCl2, 0.5 mm MgCl2, 1.5 mm KH2PO4, 8.1 mm Na2HPO4, pH 7.4), digested with trypsin 0.1% (Sigma–Aldrich), treated with Turbo DNAse I (Ambion) then filtered on a nylon membrane with a mesh size of 70 µm. The cells were incubated at 37 °C, for half an hour in Hoeschst 33342 (Sigma-Aldrich). They were then sorted according to their ploidy using a fluorescence-activated cell sorter (Epics Elite ESP, Beckman Coulter, Miami, FL, USA), equipped with a laser Helium Cadmium (HeCd, Omnichrom Model 100 Chino, CA, USA) at a wavelength of 325 nm. The collected haploid cells were preserved in RNAlater (Ambion) and kept at –80 °C until RNA extraction.

RNA extraction
Heated TRIzol method
Extraction of total RNA from the spermatozoa, the spermatids and testis was carried out using TRIzol Reagent (Invitrogen). The protocol was followed according to the manufacturer’s recommendation with a minor modification: for extraction, the TRIzol reagent was heated at 65 °C and the samples were incubated for half an hour to completely dissociate the membranes (alternative extraction protocols were tested in Dr Bissonnette’s laboratory. In order to prevent any bias potentially arising from the RNA extraction procedure, the same method was used with the spermatid samples. The subsequent steps of the protocol were performed as recommended by the manufacturer. Total RNA sample pellets were dissolved in water and then washed on a RNA extraction column (RNeasy Mini Kit, Qiagen) upon which an RNAse-free, DNAse I treatment (Qiagen) was performed in order to eliminate contaminating genomic DNA from the samples. The integrity and the concentration of the total RNA samples were evaluated using a 2100-Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA) with the RNA PicoLab Chip (Agilent Technologies).

Guanidium thiocyanate–phenol–chloroform extraction
The sperm and testis sample extractions were carried out as described in Chomczynski & Sacchi (1987). Briefly, for the extraction step, total RNA was isolated from 300 mg of a pool of testicular tissues homogenized in 3 ml of solution D (4 M guanidium thiocyanate, 25 mM sodium citrate, pH 7, 0.5% sarcosyl, 0.1 M ß-mercaptoethanol; Sigma–Aldrich), while 500 µl of ejaculate was put in 5 ml of solution D. Then sequentially, 0.1 volume of 2 M sodium acetate, pH 4, 0.1 volume of phenol and 0.2 volume of chloroform–isoamyl alcohol mixture were added to the homogenates. Following centrifugation, the supernatants were transferred and a precipitation step was done by adding one volume of isopropanol and centrifugation. The pellets were dissolved in solution D and a second precipitation was performed by adding one volume of isopropanol and centrifugation. Finally, the RNA pellets were washed in 75% ethanol and resuspended in RNase-free water. In addition, a variation to the protocol was tested by replacing the 0.1 M ß-mercaptoethanol with 0.1 M dithiothreitol (DTT; Sigma–Aldrich). Total RNA samples were further purified by performing DNAse I treatment on RNeasy Mini Kit columns (Qiagen). The quantity and the quality of the total RNA extracts were measured using a 2100-Bioanalyzer (Agilent Technologies) with the RNA PicoLab Chip for the sperm samples and the RNA NanoLab chip for the testis samples (Agilent Technologies).

5 ' versus 3' target sequence comparisons
For each of the five candidates, two primer sets were designed. A first primer set was designed to overlap the start codon in 5' and the second primer set to overlap the stop codon in 3'. For both cell types, total RNA extracts (25 ng) from spermatids and spermatozoa samples were submitted to reverse transcription and global amplification using the BD SuperSMART PCR cDNA synthesis kit (BD Biosciences, Mississauga, ON, Canada) according to the manufacturer’s instructions. The PCR amplifications were performed using 1 µl of the reverse transcription reaction in a final volume of 25 µl containing 1.5 mM MgCl2, 0.4 mM of each dNTPs (Amersham), and 10 pM of each primers, in the provided reaction buffer at 1X concentration and 0.2 µl AmpliTaq Gold LD (Applied Biosystems, Foster City, CA, USA). The specific primers for the targeted gene transcripts were designed using the Primer3 Website (http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi) based on the appropriate sequence reported in Genbank. The primer sequences used are described in Table 1Go. The PCR conditions were 95 °C for 5 min, followed by 35 cycles of 30 s at 95 °C, 30 s at 57 °C, 45 s at 72 °C, followed by a final extension of 10 min at 72 °C. The PCR products were then resolved on a 2% agarose gel stained with ethidium bromide. Amplicons were detected under u.v. light using a Chemigenius Bioimaging.


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  		Table 1 Bovine primer sequences of the genes used.
 
Test to detect genomic DNA contamination of samples
Total RNA extracts (25 ng) from spermatids and spermatozoa samples were submitted to reverse transcription using the BD SuperSMART PCR cDNA synthesis kit (BD Biosciences) according to the manufacturer’s instructions. Genomic DNA contamination of the samples was tested by PCR under the conditions described above using a set of primers specific to protamine 1 (PRM1) that spans an intron. Leukocyte contamination was evaluated by PCR using two sets of primers respectively targeting the CD4 and CD45 antigens sequences. The positive controls for genomic DNA and leukocyte contamination were as follows: a purified bovine genomic DNA extract isolated from bovine hair follicles using an in-house phenol/chloroform-based protocol routinely used in Dr Bissonette’s laboratory for single nucleotide polymorphism (SNP) analysis; and total RNA extracted from a blood sample with TRIzol (Invitrogen) in accordance with standard procedures respectively. The PRM1, CD4, and CD45 primer sequence used are listed in Table 1Go.

Microarray probe preparation and hybridizations
Total RNA extracts (25 ng) from spermatids and spermatozoa samples were submitted to reverse transcription and global amplification using the BD SuperSMART PCR cDNA synthesis kit (BD Biosciences) according to the manufacturer’s instructions. The samples were confirmed free of genomic and white blood cell contaminations using the procedure described above. The amplified cDNA samples were labeled using an indirect procedure incorporating amino allyl-dUTPs (Ambion). The incorporation was done using the Klenow enzyme (New England Biolabs, Pickering, ON, Canada) in a DNA polymerization reaction primed with random decamers (Ambion). The incubation was carried out at 37 °C for 2 h. The fluorophores (Alexa Fluor 555 and 647, Invitrogen) were added chemically to the amino allyl groupings according to the vendor’s instructions. The spermatozoa and spermatids sample probes were hybridized for 16 h at 55 °C on the SS-Human19Kv7 slides containing 19 200 single-spotted human cDNA (University Health Network Microarray Center, Toronto, ON, Canada) in the SlideHyb1 buffer (Ambion). Hybridizations were performed in an ArrayBooster using the AC3C Advacard (The Gel Company, San Francisco, CA, USA). Following the overnight incubation, the slides were washed twice in a solution of low stringency (2X SSC–0.5% SDS) at 55 °C during 15 min followed by two washes in a high stringency solution (0.5X SSC–0.5% SDS) at 55 °C for 15 min and three final washes in a solution of 1X SSC. The experimental design included two biological replicates each with a dye-swap technical replicates (sub-replication) in order to account for any differences associated with the fluorophores for a total of four microarray hybridizations. The slides were scanned using a VersArray ChipReader System (Bio-Rad) and analyzed with the ArrayPro Analyzer software (Media Cybernetics, San Diego, CA, USA).

Microarrays analysis
Analysis of the microarray data sets was carried out to obtain a gene lists for each of the two cellular types used. The procedure was inspired by the article published by Vallée et al.(2005). The data were transformed into a base 2 log in order to obtain a curve of standard distribution. Uninformative data were removed from the analysis by determining a significant threshold of cutoff based on a degree of confidence associated with the variability associated with the negative controls. This cut-off threshold was calculated as follows: T=m+2x ST, where T is the calculated threshold for cut-off, m is the average of negative controls present on the slides and ST is the standard deviation of these negative controls. Moreover, all the data equal or lower to the cut-off threshold determined previously were not considered in the analysis. Finally, a gene is regarded as positive for the analysis and included in the gene list if the signal was higher than the background noise determined and present in both replicates of hybridizations and both of their sub-replications.

Validation of microarray results
For spermatozoa samples, the starting material was generated by SuperSMART as described above. For spermatid samples, total RNA (15–20 ng), treated with DNAse I as described above was reverse transcribed using the Sensiscript reverse transcriptase (Qiagen) according to the manufacturer’s recommendations in a volume of 20 µl. The reaction was primed using an oligo(dT) primer (1 µM; Ambion) and incubation was allowed to proceed for 1 h at 37 °C in a ThermoHybaid Hybrid Multiblock System (Bio-Rad). The PCR amplifications were performed and visualized on agarose gel as described above.


   Results
Top
 Abstract
 Introduction
 Material and Methods
 Results
Discussion
 Acknowledgements
 References
 
RNA extraction and integrity assessment
Because of the membrane sturdiness of the bovine spermatozoa, total RNA was extracted by incubating the spermatozoa samples in heated TRIzol reagent for half an hour. Extraction at room temperature resulted in poor RNA yields and was not harsh enough to completely destabilize the cellular membranes as debris were clearly visible at the bottom of the tube following centrifugation (data not shown). To avoid creating a bias, the spermatid samples were extracted with the same method. The impact of this protocol modification to the usual protocol was assessed in order to determine if it affected the integrity of the isolated mRNAs. In order to do so, the integrity of the spermatozoa and spermatid RNA extracted by the heated TRIzol was evaluated by microelectrophoregram using the 2100-bioanalyzer (Fig. 1A and BGo). The RNA isolated from the spermatid extract shows a normal profile, e.g. the presence of two peaks corresponding to the 18S and 28S rRNA and a lower baseline shift indicative of the mRNA smearing profile. By contrast, the spermatic RNA sample clearly shows an absence of rRNA and the overall RNA profiling displays a majority of short-length fragments. This uncommon profile was further investigated in order to make sure whether the preponderance of short size RNA is a typical spermatic RNA characteristic and not an artifact of the RNA extraction method.


Figure 1
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  		Figure 1 Total RNA microelectrophoretic profiles of spermatids (A) and spermatozoa (B) samples. Total RNA was extracted with the heated TRIzol procedure. M, marker; FU, fluorescence; S, seconds.

 
To determine if the observed spermatic RNA profiles were resulting from the extraction procedures or truly representative of the spermatic RNA population, a comparative electrophoresis profiling analysis was performed with sperm and testis total RNA samples extracted under diverse conditions. Electrophoregrams are shown in Fig. 2Go, which displays the conventional or heated TRIzol methods (Fig. 2Go panels A–C) and the single-step RNA isolation method described by Chomczynski & Sacchi (1987) (Fig. 2Go panels D and E). Additionally, the Chomczynski & Sacchi (1987) protocol was modified by substituting the ß-mercaptoethanol with dithiothreitol for a greater reducing potential to enhance dissolution of the sperm structure (Fig. 2Go panels F and G). All of the RNA isolation methods show the same short size RNA profile for the spermatozoa and a normal profile for the testis. Comparison between heated and room temperature TRIzol treatment can be seen in Fig. 2B and CGo. The electrophoregrams indicate that the peak corresponding to the 28S rRNA is clearly lower in the heated TRIzol samples (Fig. 2Go panel B, white arrow) comparatively to the room temperature TRIzol treatment (Fig. 2Go panel C). This translates into a higher abundance of midsize fragments in the range of the 18S rRNA (1.8 kb) and smaller than the 28S rRNA (4.5 kb; rRNA product sizes based on reported sequence Genbank DQ222453).


Figure 2
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  		Figure 2 Comparison of total RNA micro-electrophoresis profiles between the spermatozoa and testis samples extracted using diverse methods. Upper panels: total RNA extracted from sperm (A) and testis (B and C) obtained either by heating the TRIzol (A and B) or using the conventional room temperature TRIzol extraction procedure (C). Middle panels: total RNA from sperm (D) and testis (E) extracted with the guanidium–phenol–chloroform method supplemented with ß-mercaptoethanol. Lower panels: total RNA from sperm (F) and testis (G) isolated with guanidium–phenol–chloroform method supplemented with DTT. The white arrow indicates the reduction of the 28S rRNA caused by the heated TRIzol treatment. M, marker; FU, fluorescence; S, seconds.

 
Based on the starting cell concentration used for the RNA extraction and the RNA content measured using the electrophoregram data, each bovine spermatozoon contains ~1.8x10–4 pg total RNA, whereas the quantity of total RNA was estimated at 0.45 pg per spermatid.

Global amplification of mRNA
Considering the small amount of RNA contained in spermatic samples, a global amplification step was necessary to obtain a sufficient amount of cDNA for the microarray hybridization. To avoid any bias that might be introduced by this amplification step, the spermatic and spermatid samples were amplified in parallel using the same procedure. The global amplification step was carried out using the SuperSMART (Clontech) kit. This PCR-based approach follows a typical sigmoid curve associated with an exponential amplification that reaches a plateau phase. It is necessary to prevent over cycling, which could result in a serious bias in the proportionality of the transcripts, as well as under cycling, which could result in an insufficient amount of material for the microarray probe. Therefore, the manufacturer recommends to perform an optimization reaction in parallel and taken to completion (with aliquots taken periodically), while the experiment reaction is held at 15 cycles. These aliquots are then separated on gel to visualize the amplification profiles. This step is used to determine the optimal number of cycles suitable for the experiment holding reaction. Figure 3Go shows the distribution profiles of cDNA populations resulting from aliquots taken at various cycles of the amplification reaction. It is worth noting that the smears generated by the spermatic sample are lower than the one produced by the spermatid sample. This also confirms the preponderance of RNA species of smaller size present in the spermatozoa when compared with those from the spermatid population.


Figure 3
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  		Figure 3 Electrophoresis on a 1.2% agarose gel stained with ethidium bromide of the aliquots of cDNA taken at various cycles during the Super-Smart preamplification step. Each lane is numbered according to the number of PCR cycles carried out. M:1 KB plus DNA marker.

 
PCR amplification of target sequences located either in the 5' or the 3' ends
The short size spermatic transcriptome was examined more thoroughly using two sets of primers designed either to cover the initiation codon (5' end) or near the stop codon (3' end; Fig. 4AGo). The presence of amplicons was compared between the two cell types (Fig. 4BGo). In all cases, the 3' target sites resulted in the amplification of the respective sequences. However, for the 5' end targets, all amplicons were detected in the spermatids, whereas only four out of five amplicons were detected in the spermatozoa. Since the reverse transcription reaction is primed using an oligo(dT), the 3' end targets are most likely to be amplified if the transcript population is fragmented. Figure 4BGo shows that the transcripts for sperm associated antigen 4 (SPAG4), P80, clusterin (CLU), and A kinase anchor protein 4 (AKAP4) are truncated, while bearing at least a short poly (A) tail enabling the annealing of the oligo(dT) primer. The fact that the 5' end target sites were mostly not amplified is indicative that the messenger population in the spermatozoa is segmented contrarily to the one found in the spermatids. However, the sperm transcriptome contains some full-length mRNAs as demonstrated by the 5' and 3' amplicons for actin beta (ACTB; Fig. 4BGo).


Figure 4
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  		Figure 4 Comparison between the spermatids and the spermatozoa for the presence of PCR amplicons targeting the 5' or 3' ends. (A) Schematic of the localization of the primers sets for each gene candidates. The 5' end primer set overlaps the start codon (ATG), while 3' end primer set is localized near or overlapping the stop codon. (B) Electrophoresis of the PCR amplification products using both primer sets (5' or 3' ) for both cell types. N, negative control; M, 1 kb plus DNA marker.

 
Verification of sample purity
The purity of the samples was checked by RT-PCR. The PRM1 gene was targeted to detect the presence of genomic DNA (gDNA) contamination, since the intron-spanning primers were used (Fig. 5Go). Hence, the presence of gDNA in the sample produces an amplicon of 315 bp (Fig. 5Go, Lane 1). No genomic DNA contamination was detectable in the samples used to prepare the microarray probes. An assessment of contamination by cells of hematopoietic origin was carried out by RT-PCR using the CD4 and the CD45 markers (data not shown). The presence of contamination by hematopoietic cells remained undetected in all of the samples used to prepare the microarray probes.


Figure 5
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  		Figure 5 The intron-spanning protamine 1 (PRM1) primer set was used to detect the presence of genomic DNA contamination. Lane 1: positive control, a spermatozoa RNA sample was spiked with genomic DNA to produce the amplicon of 315 bp corresponding to genomic contamination and an amplicon of 234 bp corresponding to PRM1 cDNA. Lane 2: without contamination.

 
Microarray hybridizations
Due to the lack of well-developed bovine microarray commercially available, a microarray containing 19 200 human cDNAs was used to survey the transcripts present in the spermatids and those from spermatozoa. In our experience and others, it is known that cross-species hybridization is a powerful and robust method that allows the identification of transcripts (Adjaye et al. 2004, Shah et al. 2004, Vallée et al. 2005, 2006). Moreover, comparative analyses of expressed sequence tags (ESTs) between human and bovine show an average identity level higher than 85% (unpublished observations). Using the stringent cut-off limit described in the Material and Methods section and the requirement that a target must be present in all four replicates in order to be considered, the array analysis revealed 2583 and 1117 positive sequences for the spermatids and the spermatozoa samples respectively (see supplementary data for transcripts identity which can be viewed online at www.reproduction-online.org/supplemental/). Of these positive signals, 996 were found in both cell types (Fig. 6Go). To evaluate the diversity of function associated with the transcripts, the candidates were grouped according to their known cellular functions (Fig. 7Go). For each cell type, a list of 45 gene transcripts that produced the strongest signal is reported in Tables 2Go and 3Go.


Figure 6
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  		Figure 6 Venn diagram of the proportion of specific or common transcripts between spermatids and spermatozoa. A total of 2583 sequences were found positive in the spermatid, while spermatozoa contain 1117 positive signals. Of these positive signals, 978 were found in both cell types. None of the 121 candidates considered to be present in the spermatozoa were absent in any of the four hybridizations performed with the spermatids.

 

Figure 7
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  		Figure 7 Illustration of the various cellular functions associated with the positive candidates detected by microarray. The upper diagram represents the spermatids, while the lower diagram represents the spermatozoa. The results are expressed as a percentage.

 

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  		Table 2 List of the 45 microarray probes producing the strongest signals for spermatids cDNA targets.
 

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  		Table 3 List of the 45 microarray probes producing the strongest signals for the spermatozoa cDNA targets.
 
Validation of the microarray hybridization results
For microarray validation, specific candidate genes were chosen according to their known implication in spermatogenesis or their contribution to early embryonic development. The microarray analysis indicated that male-enhanced antigen (MEA), sperm protein SSP411 (SSP411), SPAG4, kelch domain containing 3 (KLHDC3), testis enhanced gene transcript (TEGT), nuclease sensitive element binding protein 1 (NSEP1), peptidylprolyl isomerase H (PPIH), and flotillin 1 (FLOT1) amplicons were present in both cell types, whereas 2A histone family member Z (H2AFZ), spermatid perinuclear RNA binding protein (STRBP), and eukaryotic translation initiation factor 2B subunit 2 beta (EIF2B2) sequences were exclusive to spermatids (Fig. 8Go). During the validation process, all candidate genes were undetectable in the unamplified spermatozoa samples. However, cell type specificity for the spermatozoa became clear only when using a preamplified sample indicating the low level of abundance of the targeted transcripts. In contrast, cell-type specificity for the candidate genes could be confirmed in the spermatids without prior amplification. For all the selected candidates, RT-PCR results confirmed the microarray data. FLOT1 amplicon was selected as a spermatozoa-specific transcript, since it did not meet the selection requirement in the spermatid samples as it was only positive in one of the biological microarray replicates (positive for both sub-replications). The RT-PCR validation detected the presence of FLOT1 messenger in both cell types.


Figure 8
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  		Figure 8 Validation of the microarray results for selected candidates by RT-PCR. For all candidates, tissue specificities found by microarray were confirmed by RT-PCR. It is important to mention that none of the candidates could be detected by RT-PCR in the unamplified spermatozoa samples. Tissue specificity for spermatozoa samples was confirmed in SuperSMART preamplified samples, whereas confirmation of specificity for spermatids was conducted in non-amplified samples. aCommon transcripts found in spermatids and spermatozoa. bSpermatid-specific transcripts. cSpermatozoa-specific transcripts (but positive in half of the spermatid replicates. SPZ, spermatozoa (pre-amplified); SPD, spermatid; N, negative control; M, 1 kb plus DNA marker.

 

   Discussion
Top
 Abstract
 Introduction
 Material and Methods
 Results
 Discussion
Acknowledgements
 References
 
The possibility that the role of the male gamete goes beyond the well-established DNA shuttling function has gained recent interest (Ostermeier et al. 2002, 2004, 2005a). Although the presence of RNAs trapped within the spermatozoa is well documented (Kumar et al. 1993, Ostermeier et al. 2002, Dadoune et al. 2005, Grunewald et al. 2005), both the identity and the role of these transcripts remain largely uncharacterized. The goal of the present study was to characterize and survey the RNA population found in the bovine spermatozoa. Many precautions were taken to obtain cellular samples free of hematopoietic cells as well as RNA samples free of genomic DNA. This ensured that the following data would be representative of the cell type under study. The use of the discontinuous Percoll gradient to purify the semen samples eliminated the damaged spermatozoa, ensuring that the downstream characterization is specific to intact cells. In addition, pooled samples were used to avoid conclusion based on individual specificities. The spermatid samples were isolated by staining the DNA with Hoechst in order to sort the cells according to their ploidy. It is noteworthy that this approach does not discriminate between the round, the elongating, and the elongated spermatids. Thus, for this study, the spermatids samples include all the three types of spermatids.

Even though the general structure of the spermatozoa is similar between species, very harsh methods must be used to destabilize the membrane structure of bovine spermatozoa. This is not the case for all species, for instance, the porcine spermatozoa are completely destabilized in extraction buffer at room temperature (data not shown). Ostermeier et al. (2005a, 2005b) prepared their human spermatic RNA samples by heating the lysis buffer provided by Qiagen. This approach was clearly ineffective for bovine spermatozoa because substantial cellular debris is deposited at the bottom of the tube following lysis, which resulted in poor RNA yields (data not shown). Despite the harsh conditions in which the bovine spermatozoa had to be lysed, the impact of this treatment showed no sign of mRNA damage but resulted in the partial degradation of the 28S rRNA. Such observation has been reported for bacterial RNA extraction where heated lysis buffer is used to eliminate the rRNAs without damaging the mRNAs (Sung et al. 2003). Interestingly, the two major rRNAs were absent from the spermatic RNA samples as shown by the electrophoregram profile. This observation is in agreement with previous reports (Ostermeier et al. 2002, Grunewald et al. 2005). Indeed, it is generally accepted that mature spermatozoa are not translation-ally active so that rRNAs essential for ribosome assembly may not be available. Despite such assumption, the presence of 80S cytoplasmic ribosomes containing the 18S and the 28S rRNAs has been detected in bovine spermatozoa (Gur & Breitbart 2006). In this report, the presence of 18S rRNA was indeed detected by RT-PCR and a translational activity was attributed to the mitochondrial ribosomes while those of the cytoplasm were inactive. The presence of 18S rRNA only detectable by RT-PCR is therefore in agreement with previous reports showing that the rRNAs are present at an extremely low level in the spermatozoa.

Comparative size profiling of the transcript population performed by the microelectrophoresis approach and further confirmed by the preamplification step revealed that when compared with the spermatid and testis samples, the spermatic RNA population is predominantly composed of small-size RNAs (<1 kb). This observation is divergent from the smear length previously reported in the human where it spanned over the 28S ribosomal RNA size (Ostermeier et al. 2002, Grunewald et al. 2005). These discrepancies could be species specific as it is known that the human spermatic population is much more heterogeneous than the bovine, which has been submitted to intensive breeding programs. Indeed, human semen is composed of sperm with varying degrees of structural and functional differentiation and normality (Buffone et al. 2004), which is not the case for bull semen samples.

The presence of mainly small size RNA is not a confirmation of the complete absence of full-length transcripts. This question was addressed using the 5' versus the 3' ends PCR amplifications. This test clearly shows that the spermatid RNA contains full-length messenger RNA, whereas for four out of five candidates of the spermatic transcripts were totally truncated. The use of an oligo(dT) to prime the reverse transcription reaction is not informative on whether it is degraded or rather cut-off from the 3'end. In eukaryotes, there are two general mRNA decay pathways that are both initiated with the removal of the poly(A) tail (Meyer et al. 2004). Following deadenylation, the mRNA is either degraded from the 5' end, which involves the removal of the cap structure and the activation of a 5'–3' exonuclease or simply from the 3' end through the activity of a 3'–5' exonuclease, which ends by the hydrolysis of the cap structure (Meyer et al. 2004). In the case of the spermatic transcriptome, the integrity assessment described herein supports the presence of short 3' ends bearing at least a short poly(A) tail.

The majority of functional mRNAs must have a poly(A) tail to be translated. It is known that during spermatogenesis, mRNAs found at the elongated spermatid steps undergo deadenylation (Kleene 1993, 1996, Schmidt et al. 1999). It would therefore be logical to find deadenylated mRNAs in the spermatic transcriptome. This processing of the poly(A) tail is typical of a cellular state of translational silencing and generally incomplete, leaving behind a short stretch of poly(A) residues. This is also the case in the oocyte, where the maternal RNAs are stored under a deadenylated form that will be read-enylated upon recruitment for translation (Bachvarova et al. 1985, Huarte et al. 1992). Interestingly, microRNAs are known translational inhibitors, but have also recently been associated with directing the rapid deadenylation of mRNAs (Wu et al. 2006) reminiscent of the situation that prevails in the mature male gamete.

Therefore, these observations strongly suggest that the spermatic RNA population is composed mainly of naturally truncated mRNAs, which are at least partly adenylated. This is not typical of RNA turnover or decay since the general pathways involve exonucleases rather than endonucleases (Meyer et al. 2004). Such internal cleavage could be the signature of an unidentified nonspecific endonuclease or perhaps the RNA interference pathway, which is also triggered by microRNAs called small interfering RNAs (siRNA; Preall & Sontheimer 2005).

Aside from the transcriptional activity found in the mitochondria, the spermatozoon is transcriptionally inactive (Alcivar et al. 1989, Grunewald et al. 2005). Spermatic RNAs must therefore originate from the previous stages of spermatogenesis. Therefore, a comparative survey between spermatids and spermatozoa was undertaken to evaluate the level of similarity between the two populations of mRNAs. The objective of this analysis was clearly not of quantitative nature considering that equal amounts of starting RNA correspond to very different cell numbers between both cell types. The data generated by the microarrays was analyzed by a statistical method inspired by Vallée et al.(2005). To be considered significant, the signal for a candidate had to be above a threshold value determined according to the fluorescence output of the negative controls printed on the microarray. Furthermore, to be included in the cell-type specific transcript list, the gene candidates had to be considered present based on the above threshold value in all four of the microarray hybridizations. The microarray slide used contains 19 200 human ESTs of which 2583 and 1117 positive sequences met the requirements to be qualified as present in the spermatids and the spermatozoa respectively. This detection rate is lower than the one reported earlier by about a twofold difference (Ostermeier et al. 2002, Zhao et al. 2006). This lower detection rate is attributed to the high stringency of the analysis rather than lack of transcript diversity in these cell types or the cross-species hybridization.

The microarray hybridizations were performed as cross-species between the bovine probes targeting human cDNAs. Previous reports of cross-species micro-array hybridizations revealed that they are informative and precise because of their high overall correlation with homologous hybridizations (Adjaye et al. 2004, Shah et al. 2004, Vallée et al. 2005, 2006). In the study of Adjaye et al.(2004), only 5.7% of the candidates were shown to yield less reproducible hybridization results across the four replicates when compared with 4.0% for the human homologous hybridization.

The comparison of the transcript inventories between the two cell types revealed that most of the transcripts present in the spermatozoa are present in the spermatids. As Fig. 6Go shows, a total of 121 transcripts were classified as specific to the spermatozoa relative to the spermatid. This is attributable to the high stringency of our analysis, which is expected to produce very few false-positives, however, this level of confidence is counterbalanced by the loss of many true-positives. Indeed, for every one of the 121 transcripts classified as spermatozoa specific, at least one of spermatid hybridization generated a positive signal. When the raw data is analyzed individually, 98 out of 121 (81%) transcripts were present in three of the four spermatid hybridizations. Furthermore, 18 (15%) of the remaining spermatic-specific transcripts showed signals in two of the spermatid replicates and all of the five (4%) remaining candidates were detected in one of the spermatid hybridization. Interestingly, the identities of these last five transcripts, which seem to be more prevalent in the spermatozoa relatively to the spermatid, remain unknown.

Confirmation of the tissue specificity by RT-PCR validated the results of the microarray analysis. The fact that none of the transcripts could be confirmed in the unamplified spermatic cDNA samples is indicative of a very low abundance of the targeted sequence in the spermatic RNA population. By contrast, the tissue specificity of all the spermatid candidates was directly validated in unamplified samples. However, the RT-PCR validation approach only allows to detect the presence of a targeted section of the mRNA thus, the amplicon represents a fragment of the mRNA sequence.

Recently, it was proposed that spermatic RNA could be an important factor determining the success of early embryonic development (Ostermeier et al. 2004). The survey of the bovine spermatic population reveals transcripts involved in a wide array of cellular functions. The absence of genes associated with cell cycle regulation in the spermatozoon is in agreement with the fact that the mature gamete does not require the cell cycle machinery. Similar reports in the human used different platforms to survey the spermatic transcrip-tome, either cDNA microarrays (Ostermeier et al. 2002) or serial analysis of gene expression (Zhao et al. 2006). They both also found a list of candidates involved in a very wide spectrum of cellular functions. Considering the limited capacity of the spermatozoon to store RNA relatively to the female gamete, a specific role of the spermatic RNA for early embryonic development could imply the targeting of a particular pathway by the accumulation of specific transcripts during the later stages of spermatogenesis. Otherwise, it is expected that an extremely low content of a heterogeneous mixture of transcripts would simply be diluted relatively to the large pools of stored maternal RNA also containing a similar diversity of transcripts (Robert et al. 2000). All the reported survey including the present report does not highlight such specificity in the spermatic RNA population.

The characterization of spermatic RNA indicates a clear fragmentation of the mRNA population that is a subset of the transcriptome found in previous stages of the spermatogenesis. The nature of the spermatic mRNA population does not seem to target a specific metabolic pathway, but is rather a global representation of a wide array of basic cellular functions. This is by definition an overlap to the maternally stored RNAs pools that are known to support early development at least during the embryonic transcriptional silence period, which lasts until the 8 cell stage in the bovine (Barnes & First 1991, Memili et al. 1998). These facts in addition to the lack of RNA integrity do not lead to any clear evidence towards the potential to translate these as an essential paternal contribution to early embryonic development. An interesting recent publication has provided evidence of novel epigenetic influences of spermatic micro-RNAs originating from the fragmentation of cellular mRNA (Rassoulzadegan et al. 2006). Therefore, instead of the traditional translational fate of the mRNA, the contribution of the paternal RNA could rather be associated with epigenetic events. These novel roles of the paternal RNAs still remain to be validated. Finally, as the spermatic RNA profiles are associated with the previous steps of spermatogenesis, infertile phenotypes arising from an abnormal spermatogenesis could potentially be detected in the spermatic RNA population. The development of such a tool in species of economical interest such as the bovine could be used for the identification of fertility markers for animal breeding purposes.


   Acknowledgements
Top
 Abstract
 Introduction
 Material and Methods
 Results
 Discussion
 Acknowledgements
References
 
Our thanks to Dr Pierre Leclerc for giving the spermatid isolation protocol and Dr Maurice Dufour for his help in sorting the spermatid cells. The authors also thank Steve Perrault for critical reading of the manuscript. This project was supported by a grant of the Fond Québecois de la Recherche sur la Nature et les Technologies (#107924) within the new investigator program. Some complementary funding was provided by DairyGen and NSERC to test the RNA extraction protocol. Isabelle Gilbert’s wages were paid by an NSERC grant (#155182-05) supplemented by funds from Agriculture and Agri-Food Canada. The authors declare that there is no conflict of interest that would prejudice the impartiality of this scientific work.


   Footnotes
 
Received 23 October 2006
First decision 16 November 2006
Revised manuscript received 23 January 2007
Accepted 14 February 2007


   References
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 Abstract
 Introduction
 Material and Methods
 Results
 Discussion
 Acknowledgements
 References
 

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