1 Service de Biologie de la Reproduction 2 Service de Gynécologie Obstétrique - SIHCUS-CMCO, CHU de Strasbourg, 19, rue Louis Pasteur, BP120, 67303 Schiltigheim cedex, 3 Institut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS/INSERM/ULP, BP163 1, rue Laurent Fries, F-67400 Illkirch Cedex, CU de Strasbourg, 4 CECOS-Mulhouse, Hopital Emile Muller, 20, rue Laennec 68000 Mulhouse, 5 Institut de Médecine de la Reproduction, Marseilles and 6 IRH/Laboratoire Marcel Merieux, Bron, France
7 To whom correspondence should be addressed. Email: viville{at}igbmc.u-strasbg.fr
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Abstract |
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Key words: casein kinase/globozoospermia/male infertility/round-headed sperm
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Introduction |
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Intracytoplasmic sperm injection (ICSI) is currently offered as a treatment for severe male infertility. ICSI circumvents natural barriers and raises questions concerning the risk of injecting an abnormal spermatozoon. Even though the chances of conceiving a chromosomally normal baby appear reasonable, a male child still risks the transmission of his father's phenotype. In such cases, ICSI may introduce a new concept in genetics: the heredity of infertility (Bourne et al., 1995).
Human globozoospermia was first described by Holstein et al. (1973). It is a rare type of teratozoospermia, characterized by round spermatic heads, the absence of acrosome vesicles and a disorganization of the midpiece and tail. Different types of globozoospermia have been described (Singh, 1992
) with 100% affected sperm or only partial teratozoospermia and complete lack of acrosome and spherical nucleus or with some remains of acrosome vesicles detached from the nucleus (Aitken et al., 1990
). Nuclear damage was also described with abnormal chromatin packaging (Larson et al., 2001
) and mitochondria abnormalities (Battaglia et al., 1997
). Globozoospermia, found in <0.1% of infertile male patients, leads to the inability of the sperm to fertilize an oocyte. ICSI makes oocyte fertilization possible but with a poor fertilization rate and only a few babies born (Liu et al., 1995
). This poor fertilization rate can probably be explained by an abnormal oocyte activation (Battaglia et al., 1997
; Nardo et al., 2002
).
The existence of family cases suggests a genetic origin of this pathology but the mode of inheritance remains unknown, and so far no responsible genes have been identified (Kullander and Rausing, 1975; Florke-Gerloff et al., 1984
; Syms et al., 1984
; Viville et al., 2000
). Recently, a mouse lacking the casein kinase II
' catalytic subunit (encoded by the Csnk2a2 gene) was described. This mutant mouse presents a single phenotype reminiscent of that seen in human globozoospermia syndrome. In epididymal sections, 100% of surviving sperm cells appear abnormal with round heads, an absence of acrosomes and occasionally kinked or bent flagella (Xu et al., 1999
).
Casein kinase II (CKII) is a ubiquitously expressed, pleiotropic and constitutively active serine/threonine kinase. CKII has been highly conserved during evolution and is present in all eukaryotes. A great number of studies deal with substrates of CKII, but the fact that >160 different substrates exist is more confusing than elucidatory (Pinna and Meggio, 1997). Even if an intervention of CKII in the cellular cycle is proved (Faust and Montenarh, 2000
; Lebrin et al., 2001
), cellular functions still remain unclear. In humans and mice, there are two catalytic subunits, a and a', able to join indifferently with two regulatory subunits b to form various heterotetramers
/
;
'
'/
;
'
/
. Csnk2a2 mRNA is preferentially expressed in the more differentiated germ cells and
' subunit protein is present in adult testis and sperm (Xu et al., 1999
).
Interestingly, the yeast Schizosaccharomyces pombe orthologue (orb5) of Csnk2a2 exhibits, when mutated, a spherical phenotype. This suggests that this gene is implicated in the re-establishment of the polarized growth of yeasts after cellular division (Snell and Nurse, 1994).
Based on the phenotypes observed in mice and yeast, the high level of evolutionary conservation, their profile of expression and the association of CKII' with the cytoskeleton, we tested Csnk2a2 and Csnk2b as candidate genes in human globozoospermia patients. In order to identify a mutation in one of these genes, we compared the entire Csnk2a2 and Csnk2b coding region and introns/exons boundaries of patients with globozoospermia to fertile control individuals.
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Materials and methods |
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The control group was made up of 10 fertile men, each having at least one child, who volunteered for the study. No morphology assessment was carried out for this group.
Mapping and expression profile of Csnk2a2 and Csnk2b genes
Mouse and human cDNA sequences were obtained by searching through databases (http://www.ensembl.org/). Alignment between human and mouse nucleic and amino acid sequences (http://www.ebi.ac.uk/clustalw/) made it possible to evaluate the percentage of homology and similarity of the two genes. Human expression of Csnk2a2 and Csnk2b was established in silico through the database (http://genome-www5.stanford.edu/cgi-bin/SMD/source/expressionSearch?).
PCR reactions, DNA purification and sequencing
Genomic DNA from each patient was extracted from whole blood using the standard saline protocol (Rousseau et al., 1992). PCR primer sequences and amplification conditions are given in Table I. PCR reactions were carried out in the T-Gradient PCR system (Biometra) in a volume of 50 µl and 35 cycles. DNA amplicons were purified and both strands of each DNA fragment were sequenced with primers used for the PCR amplification. Sequence products were analysed on an ABI3100 automated sequencer (Applied Biosystems, UK) and sequences analysed using Seqscape software (Applied Biosystems). Every time a sequence difference was identified, a new PCR was done and re-sequenced.
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Results |
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Case 2 is a 32 year old patient with no medical history and no assisted procreation attempts. No family information was obtained.
Case 3 is a 33 year old patient without medical history and no assisted procreation attempts. Case 3 has four sisters and one brother 22 years old without a child.
Case 4 is a 42 year old patient who suffered from a left varicocele and was operated on at age 25 years. He never had any assisted reproduction attempts. Case 4 has two brothers of reproductive age, one with five children and one presenting, according to the patient, the same phenotype. We did not have the possibility to control this hypothesis. Case 4's parents are first cousins.
Case 5 is a 43 year old patient with no medical history. The couple had one ICSI attempt resulting in no fertilization and one sperm donor intrauterine insemination resulting in the birth of a child. Case 5 has one single brother without a child and one sister with two children.
Case 6 is a 40 year old patient with no notable medical history. The couple had one ICSI attempt and five IVF attempts with donor sperm without a pregnancy. Case 6's parents are first cousins. No more family information was obtained.
All patients presented a normal hormonal (LH, FSH, testosterone) profile and karyotype.
Characterization of the Csnk2a2 and Csnk2b genes and profile of expression
Mouse and human cDNA sequences were obtained by searching through databases (http://www.ensembl.org/). Comparison of Csnk2a2 or Csnk2b human, mouse, Zebrafish, drosophila and yeast cDNA and amino acid sequences was performed using the Clustalw alignment program. Human Csnk2a2 cDNA sequence is 91.8% homologous to mouse, 80.2% to Zebra fish, 82.4% to drosophila and 68% to yeast. Human CKII' protein sequence is 63.0% homologous to mouse protein, 83.0% to Zebra fish, 81.7% to drosophila and 62.3% to yeast. Human Csnk2b cDNA sequence is 93.1% homologous to mouse, 85.3% to Zebra fish, 76.6% to drosophila and 36.0% to yeast. Human CKII
protein sequence is 100% homologous to mouse protein, 99.0% to Zebrafish, 82.0% to drosophila and 52.0% to yeast.
In silico studies showed an over-expression of Csnk2a2 in the testis (three times in human and two times in mice). Csnk2b is ubiquitously expressed.
Human and mouse Csnk2a2 genes consist of 12 exons ranging from 51 to 444 bp located on chromosome 16q13.3-p13.2 and 8C5 respectively. Human and mouse Csnk2b genes consist of seven exons ranging from 80 to 243 bp located on chromosome 6p21.3 and 17B1 respectively.
PCR amplification and sequence analysis of the Csnk2a2 and Csnk2b genes
PCR amplification of all the exons of both genes was carried out for all six patients and 10 men in the control group. Figure 1 shows the gel electrophoresis of the 12 exons of the Csnk2a2 gene and the seven exons of the Csnk2b gene. No mutation was characterized either on Cskn2a2 or Cskn2b among the six patients studied. This analysis included the CGG triplet sequence of the CsnK2a2 gene promoter and no differences were found. In the control group, four and three already-known polymorphisms were identified for Csnk2a2 and Csnk2b respectively. These polymorphisms were already identified and have no consequences on the protein function.
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Discussion |
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For this purpose, we recruited six globozoospermic patients and 10 men for the control group. PCR amplification and sequencing of the exons and exon/intron boundaries of both genes from all patients and controls did not reveal any mutations. The search for a mutation by PCR has its limits and cannot categorically exclude a loss of function of CK2' and/or CK2
. Unfortunately, we were unable to obtain a sufficient number of sperm cells from the ejaculate to perform kinase activity tests or western blotting, which would have revealed a loss of kinase activity or a different pattern of post-translational modification respectively. The promoters of Csnk2a2 and Csnk2b genes are not known in spite of bio-informatics studies (Xu et al., 1998
). Therefore, they have not been fully analysed. However, we have sequenced 150 bases upstream from exon1 of Csnk2a2 in order to identify the number of repetitions of the CGG triplet included in the promoter region. Indeed, according to a recent study (Mangel et al., 1998
) this number of CGG repetitions might vary, as is the case for fragile X syndrome, and alter the level of expression of Csnk2a2. We could not find any differences between patients and controls. This result does not exclude a loss of expression due to promoter alterations.
Despite the absence of mutation, this work has prompted some information on globozoospermia. Two of our patients mentioned the existence of consanguinity in their family and the presence of at least one brother claiming to suffer from the same phenotype. This argues that the syndrome could be autosomal recessive. Unfortunately, we do not have access to these brothers and we could not control the claimed phenotype. Furthermore, we observed tiny differences among our patients in the expression of the phenotype (see Table II). Such a finding suggests that there could be different forms of globozoospermia, which could nvolve different genes. This is supported by several reports where globozoospermia is associated with extra morphological criteria, namely an abnormal chromatin condensation and/or mitochondrial sheath (Singh, 1992; Larson et al., 2001
; Vicari et al., 2002
). Furthermore, the recent description of two mouse lines mutated for Herb or Gopc genes reinforces this hypothesis. Indeed, these two lines present a unique phenotype highly reminiscent of globozoospermia (Kang-Decker et al., 2001
; Yao et al., 2002
).
The clinical interest of our research remains essential. Our work attempts to answer a crucial question that every patient asks himself, namely the aetiology of his illness and the risk of transmitting it to his future son. Moreover, the discovery of a mutation would open the way to the physiopathological understanding of globozoospermia, which might in the more distant future lead to the development of a treatment. Conscious of the limited number of patients studied so far, we are currently pursuing this work by recruituing more patients and by performing a similar study with Herb and Gopc genes as candidates. We hope to obtain family cases, in order to be able to perform positional cloning.
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Acknowledgements |
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Notes |
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* The results of these studies were presented in part at the 20th Annual Meeting of the ESHRE, Berlin, Germany, 2004.
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References |
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Submitted on December 15, 2004; resubmitted on January 14, 2005; accepted on January 19, 2005.