Molecular Human Reproduction, Vol. 7, No. 12, 1115-1122,
December 2001
© 2001 European Society of Human Reproduction and Embryology
Testis and spermatogenesis |
Meiotic human sperm cells express a leucine-rich homologue of Caenorhabditis elegans early embryogenesis gene, Zyg-11
Unité INSERM 99, Hôpital Henri Mondor, 94010 Créteil, France
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and methodsResultsDiscussionAcknowledgementsReferences |
|---|
We cloned a human protein (Hzyg) homologue to Caenorhabditis elegans Zyg-11, an essential protein for cell division at the initial developmental stages of this species, and to a Drosophila melanogaster gene product (Mei-1) which is likely to be involved in meiosis. Hzyg mRNA encodes a protein of 766 amino acids (88 kDa), 14% of which are leucine residues, with some being arranged in four leucine rich repeat motives usually involved in protein–protein interactions. Hzyg is encoded by a single gene, located on chromosome 9q32-q34.1, and transcribed as two mRNA: a 5 kb transcript strongly expressed in testis and skeletal muscle and barely detectable in other human tissues, and an abundant 3.1 kb mRNA detected only in the testis. By using in-situ hybridization and immunohistochemistry, we clearly established the presence of Hzyg expression in pachytene spermatocytes (stage V) and spermatids (stage I and/or II) around the time of meiosis. The cell specific expression of Hzyg transcripts in testis, and the conservation of this gene among distant species, suggest that this protein may have an important role during meiosis.
/meiosis/spermatogenesis/Zyg-11
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionAcknowledgementsReferences |
|---|
We have produced a large series of human testis expressed sequence tags (ESTs) (Pawlak et al., 1995
). During the course of their characterization, the clone A11107 (EST Hzyg) exhibited a significant homology to the Caenorhabditis elegans Zyg-11 gene, a gene which contributes to the cytoplasmic organization and spindle orientation at the one-cell stage in this worm (Kemphues et al., 1986). Actually, temperature-sensitive Zyg-11-defective mutants of Caenorhabditis elegans embryos exhibit development retardation, arrest of meiosis at metaphase II, a delay in the formation of the pronuclei and incorrect placement of the first cleavage furrow. All of these effects can be rescued by injection of Zyg-11 cDNA (Carter et al., 1990).
EST Hzyg also displayed a significant homology to a Drosophila melanogaster gene located in region 62A1–62A2 of the chromosome 3L. Mutations in this region disrupt male meiotic division I (Ivy, 1981) and are supposed to target a yet unidentified gene named Mei-1 [this gene is not related to the gene mei-1 from Caenorhabditis elegans (Clak-Maguire and Mains, 1994)]. Analysis of this genomic region revealed several genes and the gene homologous to EST Hzyg seems the sole likely candidate for Mei-1.
Based on the similarities of EST Hzyg with these Caenorhabditis elegans and Drosophila melanogaster genes, both of which are potentially important for meiosis, we investigated the expression of Hzyg mRNA and the associated protein in human testis. Hzyg was found to be expressed specifically at meiotic stages of spermatogenesis. The conservation of this gene among distant species suggests that this protein has a putative important role during human male meiosis.
|
Materials and methods |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionAcknowledgementsReferences |
|---|
Tissues and preparation of DNA and RNA
Testicular samples and genomic DNA and mRNA were obtained and prepared as previously described (Pawlak et al., 1995
; Levy et al., 1996).
cDNA cloning and sequencing
The initial Hzyg cDNA (1 kb-clone A11107) is part of a series of human testis cDNA clones already described (Pawlak et al., 1995). The 5' end region of the open reading frame was obtained by extending this 1 kb by two successive primer extensions. First, a 28 bp oligonucleotide (nucleotides 341–369 of clone A11107) was hybridized to 1 µg of human testis mRNA and elongated by using the Marathon cDNA amplification kit (Clontech, Palo Alto, USA). The elongated products were ligated at their 5' end with an adaptator (Clontech) and PCR-amplified using two oligonucleotides complementary to the adaptator and to the position 314–341 of clone A11107 respectively. A fragment of 1.2 kb was obtained. A second primer extension was done using the same protocol, and two oligonucleotides located on the 1.2 kb fragment at nucleotide positions 400–423 and 384–406 were used for reverse transcription and amplification steps respectively. This second primer extension generated a fragment of 900 bp. These two overlapping fragments were cloned into the pSPORT2 plasmid. The A11107, 1.2 kb and 0.9 kb cDNA clones were sequenced using the Erase-a-base system (Promega, Madison, USA) as previously reported (Giuili et al., 1992). The fragments were sequenced at least five times on each strand as previously described (Pawlak et al., 1995).
The nucleotide and protein sequences were analysed on the server Infobiogen (www.infobiogen.fr) using several programs including Protparam (Expasy), SAPS (ISREC), Blocks, Psort and NetOglyc WWW server.
Southern and Northern blot analysis
Southern blotting of human genomic DNA was performed as previously reported (Levy et al., 1996), using 20 µg per lane of human genomic DNA digested with the restriction enzymes HindIII, PstI, PvuII, EcoRI or KpnI (New England Biolabs, Hertfordshire, UK). Northern blots of human tissue RNAs were obtained from Clontech. The insert of clone A11107 was [32P] labelled by random priming using the Megaprime protocol (Promega). The probe was hybridized to the Southern and Northern blots according to the Express Hyb protocol (Clontech). The Southern blot was washed at 68°C in 0.1xSSC (1xSSC: 150 mmol/l NaCl, 15 mmol/l sodium citrate pH 7.0), 0.1% SDS and exposed overnight on a ß-max X-ray film (Amersham France, Les Ulis, France) with one intensifying screen. The Northern blots were washed at 65°C in 0.05xSSC, 0.1% sodium dodecyl sulphate (SDS) and treated as for the Southern blots apart from 48 h instead of overnight exposure.
In-situ hybridization
An EcoRI-HindIII fragment of 555 bp (nucleotides 228–783 of clone A11107) was cloned into the corresponding restriction sites of pSPORT1 (Life technologies, Gibco BRL, Cergy Pontoise, France). The resulting plasmid, linearized using either the BamHI or RsrII restriction enzymes was used as a template for sense and antisense single strand RNA probes, using Sp6 or T7 RNA polymerase respectively. The labelling reactions were done as described previously (Matsuoka et al., 1992), using [33P] labelled uridine triphosphate (UTP) instead of [33S] UTP.
Testis tissue samples from a normal adult male were frozen in OCT-compound and sectioned at 10 µm. The sections were fixed and hybridized as previously reported (Matsuoka et al., 1992), except that the probe (6x105 cpm) was added to the tissue sections in 30 µl. The sections were rinsed twice in 4xSSC and 2xSSC for 5 min and in 1xSSC at room temperature for 10 min. The non-specific signal was removed by treatment with a solution containing 20 µg/ml RNAse A, 20 IU/ml RNAse T1, 10 mmol/l Tris pH 8, 0.5 mol/l NaCl and 1 mmol/l EDTA for 45 min at 37°C. The sections were then washed at 42°C in 1xSSC for 30 min, 0.1xSSC for 15 min, at 60°C in 0.05xSSC three times for 30 min and finally at room temperature in 0.05xSSC for 10 min. The sections were dehydrated in ethanol in the presence of 0.3 mol/l ammonium acetate, air dried and exposed to ß-max film (Amersham France) for 4 days. The sections were then coated with LM-1 emulsion (Amersham France) exposed for 3 weeks, developed in Kodak reactives and counterstained with O-toluidine. Controls were carried out on adjacent sections hybridized to the sense probe.
Antibodies
A 13 amino acid peptide (CSNFKEENMDTSR), corresponding to the carboxy terminal part of Hzyg, was coupled to keyhole limpet haemocyanin (KLH) as an immunogen (Synt:em, Nimes, France). Hzyg antibodies against this antigen were raised in rabbits and purified on an NHS-activated column (Amersham France) coupled to the specific peptide following manufacturer's instructions.
Western blot analysis
Human testis and kidney tissues were homogenized in 25 mmol/l Tris-HCl buffer pH 6.8 containing 10% glycerol, 2% SDS, 0.7 mol/l ß-mercaptoethanol, and 125 µg/ml bromophenol blue. After centrifugation, the supernatants were boiled for 5 min. Proteins were separated on 10% SDS-polyacrylamide gel electrophoresis (PAGE), blotted onto a polyvinylidene difluoride (transfer membrane; Millipore, St Quentin Yvelines, France), and probed with the anti-Hzyg serum. Treatment of the membrane was carried out according to the enhanced chemifluorescence protocol (ECF; Amersham France). Sheep anti-rabbit IgG coupled to horseradish peroxidase (1/10000) was used as secondary antibody. The membrane was incubated for 7min in ECF solution and fluorescent signals were visualized using a phosphofluoroimager STORM 840 (Molecular Dynamics, Sunnyvale, USA).
Immunohistochemistry
Normal human testis paraffin sections (6 µm), picked up on polylysine slides, were deparaffinized and heated in a microwave oven for 3x5 min for antigen retrieval. Slides were incubated in 1x PBS containing 10% sheep serum and 0.3% Triton X100 for 30 min, and then incubated with affinity-purified anti-Hzyg antibodies at room temperature for 45 min. The sections were then washed and overlaid with CY3-conjugated anti-rabbit IgG (Amersham France) at room temperature for 30 min. DNA was stained using 4,6-diamidino-2-phenylindole (DAPI). Sections were analysed by using haematoxylin/eosin staining for stage determination.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionAcknowledgementsReferences |
|---|
Cloning of Hzyg
In a precedent study, we identified a large series of human testis EST (Pawlak et al., 1995
). One of them, when translated into an amino-acid sequence, exhibited a significant homology (blast score = 124; P: 4 e–27) with amino acids 552–619 of the Caenorhabditis elegans Zyg-11 protein. The corresponding cDNA clone (clone A11107, 1 kb), elongated by two successive 5' primer extensions, gave two overlapping fragments of 1.2 kb and 900 bp respectively. From the sequences of the initial clone and these two fragments, we generated a 2548 bp unique nucleotide sequence (Accession number: X99802).
The ATG, at nucleotide 39, in a correct context for the initiation of translation (Kozak, 1991), opens a reading frame (2298 bp) which would encode a protein (Hzyg) of 766 amino acids (Figure 1). This sequence, at its 5' end, perfectly matches with two human ESTs (Genbank Acc. No. AL036600 and AI903478), which extend 160 nucleotides upstream of Hzyg cDNA, and contain several stop codons in frame with the ATG initiation codon at nucleotide 39. Four ESTs (Genbank Acc. No. AA025815, AA687640, AA431332 and AW204619) extended the 3' Hzyg nucleotide sequence (212 bp) for 46 nucleotides. Thus, the 3' UTR mRNA is 258 nucleotides long and contains an AACAAA sequence as a non-canonical polyadenylation signal located 10 bp upstream of the polyA tail. Such an unusual polyadenylation signal has already been observed in several human genes including hRPS27 (Tsui et al., 1996), ARF3 (Tsai et al., 1991) and factor XI (Fujikawa et al., 1986) as well as in the Drosophila melanogaster tropomyosin gene (Boardman et al., 1985) and in the chicken type II procollagen gene (Sandell et al., 1984).
|
The Hzyg open reading frame encodes a 766 amino acid sequence, giving a total molecular weight of 88 kDa. The sequence of this protein, analysed using Psort and Protparam programs, revealed that Hzyg is likely to be cytoplasmic and relatively unstable. It contains 103 acidic residues (Asp + Glu) and 83 basic residues (Arg + Lys) not arranged in clusters. There are three potential N-glycosylation sites (Asn 294, 464, 553) and one potential O-glycosylation site (Ser 308). Hzyg is very rich in leucine with 108 residues (14.1% of the total residues). At positions 105, 130, 297 and 354 (Figure 1
), several of these Leu are part of consensus motives for leucine-rich repeats (LRR) (Kobe and Deisenhofer, 1994, 1995). LRRs contain 20–29 amino acids; the 10 amino terminal ones fit the consensus motif LXXLXLXX(N/T/C), in which X is any amino acid, while the carboxy terminal region of the LRR is variable in length and contains several aliphatic amino acids such as A, V, L, I, F, Y or M.
The amino acid sequence of Hzyg exhibited 24% identity and 45% similarity with Caenorhabditis elegans Zyg-11 (Figure 2), and also matched with several unknown proteins in this species, indicating that Zyg-11 belongs to a Caenorhabditis elegans multigene family. In the Drosophila melanogaster genome, Hzyg matched with only a single gene product, probably Mei-1 (Genbank Acc. No. AAF47500) (Figure 2). The gene, located on chromosome 3L, codes for a putative yet uncharacterized protein, which exhibited 38% identical residues and 54% similar residues with Hzyg. Zyg-11 and the Drosophila melanogaster protein exhibited 2 and 3 LRRs respectively.
|
Southern blot analysis
A Southern blot of human genomic DNA, digested using KpnI, EcoRI, PvuII, PstI or HindIII restriction enzymes and hybridized to Hzyg cDNA, revealed 1–4 bands per lane (Figure 3
). Such a simple pattern argues for the presence of a single Hzyg gene in the human genome.
|
Chromosomal localization
Four sequence tag sites (STS) corresponding to the Hzyg nucleotide sequences (Genbank Acc. No. A005A01, stSG4875, stSG4266 and A007G01) have been mapped on chromosome 9 between D9S1821 and D9S159 (137.6–142.7 cM) (Unigene database—Accession number: Hs.29285). In addition, the comparison of the Hzyg nucleotide sequence with the human genome sequence (Genbank Acc. No. AL359678) confirmed the location of the Hzyg gene on chromosome 9. Thus, it clearly appears that Hzyg is encoded by a single gene located on the long arm of chromosome 9 in q32-q34.1.
Hzyg mRNA expression
Northern blot analysis of Hzyg mRNA expression in several tissues revealed, under high stringency conditions, a strong signal at 3.1 kb, detected only in human testis. An abundant 5 kb transcript was also detected in testis and skeletal muscle and to a lower level in prostate, ovary, small intestine, heart, brain and pancreas (Figure 4).
|
The cellular localization of Hzyg mRNA expression was determined by in-situ hybridization on human testis sections. Dark field examination at low magnification revealed a distribution of the grains inside the tubules (Figure 5A and B
). At higher magnification, the signal was mainly located on cells with large nuclei corresponding to pachytene spermatocytes (Figure 5E and F) as well as on smaller cells located in the centre of the tubule and identified as spermatids (Figure 5G and H). Under identical conditions the sense probe did not give any signal (Figure 5C and D). A very low signal was observed in the interstitial parenchyma. A careful examination of the grain distribution clearly showed that the signal was concentrated in the cytoplasm of round cells at the central and adluminal compartments of seminiferous tubules, suggesting that the spermatocytes and spermatids were the main source of Hzyg transcripts. The signal was low near the boundaries of tubules, thus excluding a major contribution of Sertoli cells and spermatogonia, which are located along the basement membrane. Therefore, in the human testis, Hzyg appears to be expressed mainly in germ cells that undergo the meiotic division.
|
Hzyg protein expression
Antibodies raised against the carboxy-terminal part of Hzyg revealed a single band at the expected size (88 kDa), on human testis extracts (Figure 6
). By using these antibodies on human testis sections, we observed a very specific expression of Hzyg in the cytoplasm of late pachytene spermatocytes (mainly stage V) and in the cytoplasm of round spermatids (stage I and/or II) (Figure 7).
|
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionAcknowledgementsReferences |
|---|
We have cloned from a human testis library a new human protein (Hzyg), which is expressed at meiotic stages of human spermatogenesis.
In the testis, Hzyg mRNA is transcribed from a single gene as two major transcripts (5 and 3.1 kb), whereas a single 5 kb band is detected in skeletal muscle and to a lower extent in prostate, ovary, small intestine, heart, brain and pancreas. Such a specific expression pattern of a single gene in testis as compared with somatic tissues is often observed in germinal cells. For example, this is the case for the farnesyl pyrophosphate synthetase transcript, which displays a longer 5'UTR in testis as compared with the transcript expressed in somatic tissues (Teruya et al., 1990), and for c-abl mRNA (Meijer et al., 1987) and ß1-galactosyl transferase mRNA (Shaper, 1990) which are polyadenylated at a different site in testis as compared with other tissues. The structure of the two Hzyg transcripts detected in testis is not yet known, however they may differ in their 3'UTR sequences as indicated from the ESTs present in databases. Nevertheless, if both Hzyg transcripts are functional, the detection of a single band on Western blot of testis extracts suggests that either they code for the same protein or that post-transcriptional events modify the epitope reacting with the antibody.
Hzyg contains 766 amino acids (88 kDa) and the main feature of its structure is the presence of leucine residues at a level of 14%. Several of the leucine residues are gathered in four consensus motives for LRRs (Kobe and Deisenhofer, 1994, 1995). LRR motives are generally involved in protein–protein interactions and in 50% of cases in signal transduction pathways (Kobe and Deisenhofer, 1994, 1995). This structural feature will aid in future investigations in order to decipher the role of Hzyg in human testis.
The most interesting aspect is a possible function of Hzyg in meiosis. We observed specific expression of Hzyg in the cytoplasm of late pachytene spermatocytes (mainly stage V), as well as in the cytoplasm of round spermatids (stage I and/or II), two stages which correspond to meiotic division. The observation of Hzyg transcripts in somatic tissues is not contradictory to a specific role of Hzyg during meiosis. For example, proteins such as SPO11, which initiates homologous recombination during meiosis (Romanienko and Camerini-Otero, 1999), are also expressed in somatic tissues. In addition, Hzyg exhibits significant homology with the Caenorhabditis elegans Zyg-11 protein, which has a determinant role during meiosis, as shown by the meiosis arrest at metaphase II and an incorrect placement of the first cleavage furrow in Zyg-11-deficient worms. Finally, Hzyg is the orthologue of a Drosophila melanogaster gene located in region 62A1–62A2 of the chromosome 3L likely to correspond to the gene Mei-1, and mutations at this locus promote a non-disjunction of the chromosome at the first meiotic division. With these mutations, the second phase of meiosis appears normal except for the non-haploid complements resulting from metaphase I chromosome misbehaviour (Ivy, 1981).
While Hzyg may be involved in meiosis, this protein is apparently not indispensable for somatic mitosis since it was not detected in all human somatic tissues undergoing cell division, and we have not detected this protein in rapidly dividing tissues such as human testis tumours (data not shown).
In summary, Hzyg is likely to play a role during male germ cell division. Already two animal models with knock out genes related to Hzyg are available. In fact, major alterations of meiosis have been reported in Drosophila melanogaster and Caenorhabditis elegans deficient in Hzyg homologous genes. Further investigations are needed to decipher the role of Hzyg in human germ cells.
|
Acknowledgements |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionAcknowledgementsReferences |
|---|
We thank Dr Yannick Laperche and Dr Jacques Hanoune for critical reading of the manuscript. We thank Dr Mohamed Benahmed for providing us with normal human testis mRNA. This work was funded by the Groupement de Recherche sur l'étude des Génomes and by the Association pour la Recherche contre le Cancer. Chloé Féral is a recipient of a fellowship from the Ministère de la Recherche et de l'Enseignement.
|
Notes |
|---|
1 To whom correspondence should be addressed. E-mail: guellaen{at}im3.inserm.fr
|
References |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionAcknowledgementsReferences |
|---|
Boardman, M., Basi, G.S. and Storti, R.V. (1985) Multiple polyadenylation sites in a Drosophila melanogaster tropomyosin gene are used to generate functional mRNAs. Nucleic Acids Res., 13, 1763–1776.
Carter, P.W., Roos, J.M. and Kemphues, K.J. (1990) Molecular analysis of zyg-11, a maternal-effect gene required for early embryogenesis of Caenorhabditis elegans. Mol. Gen. Genet., 221, 72–80.[Web of Science][Medline]
Clak-Maguire, S. and Mains, P.E. (1994) Mei-1, a gene required for meiotic spindle formation in Caenorhabditis elegans, is a member of a family of ATP-ases. Genetics, 136, 533–546.[Abstract]
Fujikawa, K., Chung, D.W., Hendrickson, L.E. et al. (1986) Amino acid sequence of human factor XI, a blood coagulation factor with four tandem repeats that are highly homologous with plasma prekallikrein. Biochemistry, 25, 2417–2424.[Medline]
Giuili, G., Scholl, U., Bulle, F. et al. (1992) Molecular cloning of the cDNAs coding for the two subunits of soluble guanylyl cyclase from human brain. FEBS Lett., 304, 83–88.[Web of Science][Medline]
Ivy, J. (1981) Mutations that disrupt meiosis in males of Drosophila melanogaster. (Thesis) University of California, San Diego, California, USA.
Kemphues, K.J., Wolf, N., Wood, W.B. et al. (1986) Two loci required for cytoplasmic organization in early embryos of Caenorhabditis elegans. Dev. Biol., 113, 449–460.[Web of Science][Medline]
Kobe, B. and Deisenhofer, J. (1994) The leucine-rich repeat: a versatile binding motif. Trends Biochem. Sci., 19, 415–421.[Web of Science][Medline]
Kobe, B. and Deisenhofer, J. (1995) Proteins with leucine-rich repeats. Curr. Opin. Struct. Biol., 5, 409–416.[Web of Science][Medline]
Kozak, M. (1991) An analysis of vertebrate mRNA sequences: intimations of translational control. J. Cell Biol., 115, 887–903.
Levy, I., Wu, Y.Q., Roeckel, N. et al. (1996) Human testis specifically expresses a homologue of the rodent T lymphocytes RT6 mRNA. FEBS Lett., 382, 276–280.[Web of Science][Medline]
Matsuoka, I., Giuili, G., Poyard, M. et al. (1992) Localization of adenylyl and guanylyl cyclase in rat brain by in situ hybridization: comparison with calmodulin mRNA distribution. J. Neurosci., 12, 3350–3360.[Abstract]
Meijer, D., Hermans, A., von Lindern, M. et al. (1987) Molecular characterization of the testis specific c-abl mRNA in mouse. EMBO. J., 6, 4041–4048.[Web of Science][Medline]
Pawlak, A., Toussaint, C., Levy, I. et al. (1995) Characterization of a large population of mRNAs from human testis. Genomics, 26, 151–158.[Web of Science][Medline]
Romanienko, P.J. and Camerini-Otero, R.D. (1999) Cloning, characterization, and localization of mouse and human SPO11. Genomics, 61, 156–169.[Web of Science][Medline]
Sandell, L.J., Prentice, H.L., Kravis, D. et al. (1984) Structure and sequence of the chicken type II procollagen gene. Characterization of the region encoding the carboxyl-terminal telopeptide and propeptide. J. Biol. Chem., 259, 7826–7834.
Shaper, N.L., Wright, W.W. and Shaper, J.H (1990) Murine beta 1, 4-galactosyltransferase: both the amounts and structure of the mRNA are regulated during spermatogenesis. Proc. Natl Acad. Sci. USA, 87, 791–795.
Teruya, J.H., Kutsunai, S.Y., Spear, D.H. et al. (1990) Testis-specific transcription initiation sites of rat farnesyl pyrophosphate synthetase mRNA. Mol. Cell. Biol., 10, 2315–2326.
Tsai, S.C., Haun, R.S., Tsuchiya, M. et al. (1991) Isolation and characterization of the human gene for ADP-ribosylation factor 3, a 20-kDa guanine nucleotide-binding protein activator of cholera toxin. J. Biol. Chem., 266, 23053–23059.
Tsui, S.K., Lee, S.M., Fung, K.P. et al. (1996) Primary structures and sequence analysis of human ribosomal proteins L39 and S27. Biochem. Mol. Biol. Int., 40, 611–616.[Web of Science][Medline]
Submitted on May 30, 2001; accepted on September 5, 2001.
CiteULike 
Connotea 
Del.icio.us What's this?
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||