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Department of Obstetrics & Gynecology,3 Virginia Commonwealth University, Richmond, Virginia 23298
Pulmonary and Critical Care Medicine,4 University of North Carolina, Chapel Hill, North Carolina 27599
Department of Obstetrics & Gynecology,5 and Pulmonary and Critical Care,6 University of Arkansas for Medical Sciences, Little Rock, Arkansas 72205
Laboratorio de Andrología,7 Unidad de Reproducción Humana, Clínica Tambre, 28002 Madrid, Spain
INSERM U. 428, Faculte de Medecine,8 Universite Paris-Descartes, 75006 Paris, France
INSERM U. 654, Hôpital Henri-Mondor,9 94010 Créteil cedex, France
Center for Research on Reproduction and Women's Health,10 University of Pennsylvania, Philadelphia, Pennsylvania 19104
ABSTRACT
The SPAG16 gene encodes two major transcripts, one for the 71-kDa SPAG16L, which is the orthologue of the Chlamydomonas rheinhardtii central apparatus protein PF20, and a smaller transcript, which codes for the 35-kDa SPAG16S nuclear protein that represents the C-terminus (exons 11–16) of SPAG16L. We have previously reported that a targeted mutation in exon 11 of the Spag16 gene impairs spermatogenesis and prevents transmission of the mutant allele in chimeric mice. In the present report, we describe a heterozygous mutation in exon 13 of the SPAG16 gene, which causes a frame shift and premature stop codon, affording the opportunity to compare mutations with similar impacts on SPAG16L and SPAG16S for male reproductive function in mice and men. We studied two male heterozygotes for the SPAG16 mutation, both of which were fertile. Freezing-boiling of isolated sperm from both affected males resulted in the loss of the SPAG16L protein, SPAG6, another central apparatus protein that interacts with SPAG16L, and the 28-kDa fragment of SPAG17, which associates with SPAG6. These proteins were also lost after freezing-boiling cycles of sperm extracts from mice that were heterozygous for an inactivating mutation (exons 2 and 3) in Spag16. Our findings suggest that a heterozygous mutation that affects both SPAG16L and SPAG16S does not cause male infertility in man, but is associated with reduced stability of the interacting proteins of the central apparatus in response to a thermal challenge, a phenotype shared by the sperm of mice heterozygous for a mutation that affects SPAG16L.
axoneme, central apparatus, mutation, sperm motility, SPAG16,, Spag16L
Cilia and flagella are evolutionarily conserved eukaryotic organelles that extend from the cell body. They comprise a microtubular backbone, the axoneme, which is surrounded by a plasma membrane and organized by the basal body, a cylindrical structure that consists of nine microtubule triplets and that is located underneath the cell membrane [1–3]. Depending on their function, cilia can be divided into motile and primary (nonmotile) cilia. Motile cilia are characterized by a typical "9+2" architecture, with nine outer microtubule doublets and a central pair of microtubules. These 9+2 structures are present in sperm and ciliated cells of the lung, brain, oviduct, and other tissues [4]. Attached to the outer nine microtubules are the outer and inner dynein arms. The outer arms generate power and adjust beat frequency [5, 6], while the inner arms generate the axonemal waveform [7, 8]. The radial spokes and central pair structures are key regulators of dynein activity [9]. More than 200 different polypeptides have been identified in the axoneme [10–12], and cDNAs encoding some of them have been cloned from Chlamydomonas rheinhardtii, trypanosomes, and mammals [13–18]. Mutations in some of these genes result in paralyzed flagella and cilia.
Polypeptide mapping of the Chlamydomonas central pair suggests the presence of at least 23 different proteins in addition to tubulin [19], including PF6 (SPAG17), PF16 (SPAG6), and PF20 (SPAG16) [17, 18, 20–25]. SPAG6, SPAG16, and SPAG17 form part of an interactome, with SPAG6 binding to SPAG16 and to SPAG17. SPAG16L has contiguous tryptophan and aspartic acid (WD) repeats in the C-terminus, which mediate interactions with SPAG6. These proteins are essential for flagellar motility [26–28]. Targeted mutations of the Spag16 and Spag6 genes in mice cause male infertility due to severe defects in sperm motility.
The mouse Spag16 gene encodes a 2.5-kb transcript from exons 1 to 17 and a 1.4-kb transcript from mouse exons 11–17, and these transcripts are expressed in different patterns during spermatogenesis, yielding proteins of 71 kDa (SPAG16L) and 35 kDa (SPAG16S), respectively. The SPAG16L protein is expressed meiotically (in diploid cells) and is incorporated into the central apparatus of the sperm tail. The SPAG16S protein, which is derived from a testis-specific transcript in the mouse, accumulates in both meiotic and postmeiotic (haploid) male germ cells, is abundant in the nucleus, and appears to function as a transcription factor. Our previous studies have revealed that these two mouse SPAG16 proteins perform different functions; SPAG16L is essential for sperm flagellar motility, whereas SPAG16S appears to be required for postmeiotic germ cell viability [23, 27, 28]. Analysis of a targeted mutation in exon 11 of the Spag16 gene, which would affect the expression of both SPAG16L and SPAG16S, has suggested that spermatogenesis would be markedly impaired by a heterozygous mutation that affects the expression of both proteins [27]. A targeted mutation that deletes exons 2 and 3 of the Spag16 gene, which affects only SPAG16L expression, results in male infertility and severe sperm motility defects in nullizygous males, but causes only subtle motility defects in heterozygous males, which are fertile [28].
In the course of investigating a female subject with symptoms of primary ciliary dyskinesia, we identified the first known mutation in the SPAG16 gene. This discovery allowed us to compare the phenotypes of mice and humans carrying mutations that have similar impacts on the expression of SPAG16L and SPAG16S. We now report studies on two related male subjects, who carry one mutant allele (heterozygotes) involving two different single base insertions in cis in exon 13 of the SPAG16 gene (GenBank accession no. NM_024532). This mutant allele results in a frame shift and premature stop codon, which interrupts the WD repeats that interact with SPAG6. Although both males are fertile, this heterozygous mutation is associated with biochemical instability of central apparatus components in response to freezing-boiling, similar to that observed for the sperm of mice heterozygous for an inactivating mutation that affects the expression of SPAG16L.
The present study was approved by the institutional review board of the Ethics Committees of the University of Pennsylvania, Virginia Commonwealth University, and University of North Carolina. Procedures involving animals were conducted with the approval of the Institutional Animal Care and Use Committee of the Virginia Commonwealth University in accordance with the Guide for Care and Use of Laboratory Animals (protocol no. 06013417). Human subject research was approved by the Human Research Committee of the University of North Carolina (protocol no. FWA 4801). All human sperm were obtained after receiving informed consent from all of the subjects involved in the study.
The two Caucasian adult male subjects in the present study were identified as a result of extensive genetic studies of a child with primary ciliary dyskinesia (PCD) or Kartagener Syndrome (MIM 244400 and 242650), and members of her extended family (see Pedigree in Supplemental Fig. 1, available online at www.biolreprod.org). The diagnosis of PCD (subject #367 in Pedigree, Supplemental Fig. 1) was based on situs inversus, sino-pulmonary disease, low nasal nitric oxide (1.9 nl/min; normal: 
376 ± 124 nl/min) [29], and abnormal ciliary ultrastructure. In particular, the ciliary electron micrographs (EMs) showed an abnormal ultrastructure, which consisted of axonemal disorganization with absence of radial spokes, probably associated with inner dynein arm defects. In addition, the ciliary motility of a sample of nasal ciliated epithelium was severely defective, with decreased ciliary beat frequency, stiffness of the waveform, and lack of co-ordination of beat among the cilia. This patient was one of the 78 PCD patients described in a previous report [29]. Owing to the abnormal ciliary EMs, efforts were focused on identifying mutations in central complex genes, i.e., SPAG16 and SPAG6. All 16 exons and adjacent intronic regions of SPAG16 were sequenced in the propositus (subject #367; see Pedigree), and two individual single base-pair insertions were identified in cis in exon 13, termed c.[1464–1465insC; 1469–1470insA]. This mutation leads to a frame shift (over 140 bp) and a premature stop codon (p.F489LfsX46). Thus, this mutation causes changes in the structure (amino acid sequence) and the loss of three of the five WD repeat domains (see Fig. 1A). No other significant coding sequence changes were noted in SPAG16, and there was no evidence of splicing variants/abnormalities of SPAG16, using RT-PCR amplification of RNA from a lymphoblastoid cell line that contained the exon 13 mutation. Finally, all 11 exons of SPAG6 were sequenced to look for a digenic mode of inheritance (coupled to SPAG16) for PCD. However, no mutations were found in the coding region or adjacent intronic sequences of SPAG6. We assume that the PCD in this child is not the result of mutations in SPAG16 and/or SPAG6, since two mutant alleles could not be identified. Thus, it seems that another gene is implicated in causing disease in this child with PCD.
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Since the father of the PCD child (subject 1, and #369 in pedigree) had a history of a prior infertility evaluation and oligospermia at 26 years of age, additional studies were undertaken. The father was 33 years of age at the time of the present study. He had no clinical evidence of sinus-pulmonary disease or situs inversus. He had fathered three children, and two other pregnancies spontaneously terminated at 6.5 wk and 17 wk, respectively. Analysis of the kinship indicated that the SPAG16 mutation was transmitted to subject 1 from his father. For the purpose of the present study, two semen samples were obtained from subject 1 at a 3-mo interval and analyzed separately. Subject 2, who is a first cousin once removed of subject 1 (see Pedigree in Supplemental Fig. 1), also carried the exon 13 mutation in SPAG16. He was studied at 42 years of age, and had fathered one child and had no significant medical history. This subject provided one semen sample for the purpose of the present study. Both study subjects are Caucasian of Irish/German ancestry.
5'-Rapid Amplification of cDNA Ends (5'-RACE)
5'-RACE was carried out to identify the human SPAG16S that corresponds to the mouse orthologue, using the human testis Marathon cDNA amplification kit (Clontech, Palo Alto, CA) according to the manufacturer's instructions. Briefly, a primer was designed from exon 12 of full-length SPAG16 (5'-CACTTGAAGTAGCCAATTTGTCGCC-3') and used together with the Marathon cDNA adaptor primer to generate the 5'-RACE products, which were then cloned into the pCR2.1-TOPO TA vector and subjected to DNA sequence analysis.
Semen Sample Collection, Analysis, and Selection Criteria
Semen samples were collected from the two study subjects and normal males, who were sperm donors of proven fertility between 23 and 40 years of age, and whose general health was confirmed by history and physical examination. An abstinence period of at least 48 h was requested prior to semen collection. Samples were collected by masturbation in sterile propylene cups and allowed to liquefy for at least 30 min at room temperature.
Semen analyses were performed according to the World Health Organization (WHO) criteria. The concentration of spermatozoa, presence of white and red blood cells, percentage of total motile sperm, and normal morphology were evaluated in each sample according to previously described methods [30, 31]. Briefly, sperm samples were diluted to 2 x 106 sperm/ml and motility was analyzed in a 100-µm chamber (Cell Vision–USA, Hopedale, MA) using the IVOS Sperm Analyzer (Hamilton-Thorne Research, Beverly, MA). The parameters that were used for the analysis set-up are described in the technical guide for the IVOS system, version 12.2 (Human set-up 1). For each sperm sample, a total of ten fields were analyzed. Morphology was evaluated following the WHO recommendations with a modification of the Papanicolaou method, and the percentages of sperm cells with apparent defects were estimated.
Sperm proteins were extracted as previously described [32]. Briefly, sperm were washed three times in PBS. Prior to the final centrifugation, the volume, sperm concentration, and total sperm numbers were determined for each sample. After the final wash, sperm pellets were dissolved in SDS sample buffer (50 mM Tris-HCl [pH 7.5], 150 mM NaCl, 1% SDS, 40 mM dithiothreitol, and protease inhibitor) and boiled for 5 min. The samples were centrifuged at 10 000 rpm for 3 min and the supernatants were saved. Protein concentration was determined using the bicinchoninic acid assay (BCA; Pierce).
Freezing-Boiling of Sperm Extracts
Sperm proteins were extracted as above and frozen at –80°C for 2 days. The samples were thawed and heated at 100°C for 5 min, then sonicated with a 2-mm probe for three 10-sec bursts at setting 7 of the Heat Systems-Ultrasonics Model W-220F Cell Disruptor. After this procedure, the samples were refrozen at –80°C. This cycle was repeated four times.
Male Mice Heterozygous for an Inactivating Mutation of the Spag16 Gene
Mice heterogeneous for a mutation in the Spag16 gene, which interferes with the expression of SPAG16L, but not SPAG16S, were generated as previously described [28], and epidydimal sperm were collected and prepared as previously reported.
Mutation Identification in Sperm
To confirm the mutation in sperm, PCR was performed to amplify the full length of exon 13 from sperm using primers that flank exon 13: forward primer within intron 12, 5'-GCATTGTAT AGACAGCATAGA-3' and reverse primer within intron 13, 5'-GACCGAGAGTAA TCAGATTTA-3'. The PCR products were subjected to direct sequencing analysis or cloned into the pCR2.1 TA vector, and four separate clones were selected for sequencing.
Equal amounts of sperm protein (40 µg/lane) were prepared and subjected to immunoblot analysis using antibodies against AKAP4, SPAG6, SPAG16, and SPAG17, as previously described [23]. The genomic structure of SPAG16 is shown in Figure 1A, as well as the exons that code for the epitopes used to develop the anti-N-terminus and anti-C-terminus antibodies [27].
For ultrastructural analysis, sperm were fixed with 2% glutaraldehyde, exposed to 1% osmium tetroxide, and transferred onto an embedding material. The sections were stained and examined by transmission electron microscopy (TEM).
Identification of Transcript for the Human Orthologue of SPAG16S
To determine the human orthologue of SPAG16S, which to date has been identified only in the mouse, 5'-RACE was performed using a primer from exon 12 of full-length SPAG16 (SPAG16L). Two major PCR products were amplified, the longer of which was identified as being from SPAG16L; the 5'-UTR was identified and the sequence was submitted to GenBank (accession no. EF530039). The smaller product was identified as being from SPAG16S. Similar to the murine Spag16S transcript, human SPAG16S contains an untranslated exon ahead of the first coding exon (exon 11 of SPAG16L). This noncoding exon is 148 kb upstream of exon 11 and 234 kb downstream of exon 10. This sequence and the 5'-UTR of human SPAG16S have been deposited in GenBank (accession no. EF591776).
Two Major SPAG16 Proteins Are Present in Human Sperm
Protein was isolated from sperm collected from normal donors, and Western blot analysis was performed with antibodies that recognize the N-terminus and C-terminus of SPAG16L (Fig. 1B). The anti-N-terminus antibody reacted with a 71-kDa protein (Fig. 1Ba), and the anti-C-terminus antibody recognized proteins of 71 kDa and 35 kDa (Fig. 1Bb). In addition, both antibodies reacted with proteins of between 50 kDa and 71 kDa in normal human sperm (arrowheads in Fig. 1, Ba and Bb). The latter proteins, which may represent the translated products of alternatively spliced transcripts previously reported by Pennarun et al. [25] or they may be unrelated cross-reacting proteins that contain WD repeats.
Confirmation of Insertion Mutations in Spag16 Gene in Sperm
PCR was performed on sperm from the two patients, to confirm the presence of the exon 13 mutation. A single band was amplified using the primer set described in the Methods section. The PCR products were either sequenced directly with the forward PCR primer or cloned into the pCR2.1 TA vector. Plasmid DNAs isolated from four different clones were sequenced with the M13 reverse primer.
Direct sequencing of the PCR products demonstrated that a cytosine (C) and an alanine (A) were inserted at different sites into one of the alleles, giving rise to a frame-shift mutation in the downstream region c.[1464–1465insC; 1469–1470insA] (p.F489LfsX46). Sequencing of the four clones from each subject revealed two wild-type and two insertion mutations, consistent with the heterozygous state (Fig. 2, Aa and Ab). The insertion mutations resulted in a premature stop codon in the translated sequence, which would yield a truncated protein of 534 amino acids from the mutant SPAG16L allele, thereby lacking C-terminal sequences in both SPAG16L and SPAG16S (Fig. 2B). This is most certainly a private mutation, since our previous analysis of the SPAG16 gene in subjects with PCD [25], and subsequent screening of over 100 unrelated individuals with PCD did not reveal a similar mutation (unpublished data). The two insertions are probably not the result of independent events, but are more likely the consequence of replacement of the downstream sequence, which is very similar to the region that bears the two insertions, during recombination.
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The sperm count and motility were significantly below the normal levels in one subject with the heterozygous mutation when tested on two separate occasions at a 3-mo interval, while the other subject had a sperm count and motility within the normal range (Table 1).
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Sperm were collected for TEM analysis. In the oligospermic male (subject #1), approximately 35% of the sperm lacked the central pair, and 20% lacked one to four outer doublet microtubules in two independent analyses. These findings were consistent across two independent analyses on two different semen samples. The other male (subject #2) had normal axonemal architecture at the ultrastructural level (Fig. 3 and Table 2).
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SPAG16 Protein Expression and Evidence for Instability of SPAG16L, SPAG6, and the 28-kDa Fragment of SPAG17 in Mutant Sperm
The anti-N-terminus antibody reacted with the 71-kDa SPAG16L protein, but also recognized other proteins with molecular masses between 50 kDa and 67 kDa (Figs. 1A and 4Aa) in normal donor sperm, whereas the antibody recognized only the 71-kDa SPAG16L protein in sperm extracts from the two study subjects (Fig. 4Ab). The anti-C-terminus antibody recognized the 71-kDa and 35-kDa SPAG16L and SPAG16S proteins in the patient sperm, although additional proteins were recognized in the sperm extract from healthy donors (Fig. 4Ac). This suggests that splice variants exist and may include alternative splicing of exon 12, which is consistent with our previous analysis of the SPAG16 gene [25].
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In an unanticipated discovery, we found that when we froze and boiled the sperm protein extracts four times, both SPAG16L and its interacting partner SPAG6, as well as the 28-kDa fragment of SPAG17, which interacts with SPAG6, were lost from mutant sperm, while SPAG16S and the major sperm fibrous sheath protein, AKAP4, as well as the 72-kDa fragment of the SPAG17 protein, which does not interact with SPAG6, were retained (Fig. 4B). The retention of SPAG16S and loss of SPAG16L in mutant sperm is consistent with the different subcellular locations of these proteins in the mouse. In addition, the proteins with molecular masses of 50 kDa to 67 kDa, which were detected in fresh extracts of normal sperm, also disappeared from the normal sperm extracts after freezing-boiling (Fig. 4B).
Instabilities of SPAG16L, SPAG6, and the 28-kDa Fragment of SPAG17 in Sperm of Mice Heterozygous for the Spag16L Mutation
SPAG16L mutant mice were previously generated in our laboratory [28]. The nullizygous male mice are infertile owing to a severe motility defect. On the other hand, the heterozygous mutants are fertile. Epididymal sperm isolated from wild-type and heterozygous mutant mice were collected for Western blot analysis. There was an approximately 50% reduction of SPAG16L protein in the heterozygous mutant sperm compared to the wild-type sperm, even though the same amount of SPAG6 protein was present (Fig. 5A). When the samples were frozen and boiled four or more times, SPAG16L, SPAG6, and the 28-kDa fragment of SPAG17 were markedly reduced in the extracts of sperm from heterozygous mutant mice, whereas these three proteins were present in the sperm from wild-type mice, even though the amount of protein was lower than in the fresh samples (Fig. 5A). As for the human samples, the AKAP4 and 72-kDa SPAG17 proteins were not affected by freezing-boiling.
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The SPAG16 gene encodes the orthologue of C. reinhardtii PF20. It has been shown that PF20 is required for the structural integrity of the axoneme and flagellar motility in C. reinhardtii and Trypanosoma brucei [24, 33]. The human SPAG16 gene, similar to its murine counterpart, encodes two major transcripts of 2.5 kb and 1.4 kb, the former being the mRNA for SPAG16L and the latter encoding the 35-kDa SPAG16S [23]. Both the 71-kDa SPAG16L and 35-kDa SPAG16S proteins are present in human sperm. The SPAG16S transcript was not discovered in our initial report on the isoforms of human SPAG16 because the primer used was located upstream of the transcriptional start site of SPAG16S [25]. We conclude that SPAG16S exists in humans based on the results of 5'-RACE and Western blotting with an antibody to the C-terminus of SPAG16L. The anti-N-terminus and anti-C-terminus antibodies recognized proteins of 50 kDa to 67 kDa in human sperm but not in mouse sperm (Fig. 1). At least four SPAG16 splice variants have been reported [25]. The immunoreactive 50-kDa to 67-kDa proteins most likely represent proteins that are translated from these alternatively spliced transcripts. Although it appears that the splice variants differ in abundance among individuals, all carry the full-length 71-kDa protein (Figs. 1 and 4A).
The human mutation in exon 13 of the SPAG16 gene described in the present study should affect the expression of both SPAG16L and SPAG16S. The findings that one subject with this heterozygous mutation had normal semen parameters and that both were fertile indicate that haploinsufficiency of SPAG16L/SPAG16S does not impair male fertility in humans. This is in contrast to mice, in which a mutation introduced in exon 11 of the Spag16 gene affected the expression of both SPAG16L and SPAG16S, whereas haploinsufficiency resulted in markedly impaired spermatogenesis. Subject 1 was oligospermic and had significant structural abnormalities in sperm tail architecture, features that mirror to some extent those seen in the heterozygous exon 11 Spag16 mutant. However, there are multiple causes of teratooligospermia, and it is not possible to determine whether the abnormal semen parameters of subject 1 are the direct result of the heterozygous mutation in the SPAG16 gene or other factors. Since subjects 1 and 2 were fertile, as were other male carriers in the pedigree, and subject 2 had normal semen parameters, we conclude that the heterozygous state for mutations that disrupt the expression of both SPAG16L and SPAG16S do not impair male fertility.
The two insertion mutations in exon 13 result in a frame shift and premature stop codon, which would theoretically result in a truncated SPAG16L protein. We were unable to obtain evidence of truncated proteins from Western blots of sperm extracts from the study subjects who carried the SPAG16 mutation, although the 50-kDa to 67-kDa immunoreactive proteins were missing. We suggest that the corresponding RNAs are unstable and/or that these truncated proteins are unstable, if they are generated from the mutant transcripts.
The 71-kDa SPAG16L protein was present in freshly prepared sperm extracts from both study subjects. However, when the samples were subjected to four rounds of freezing/boiling and Western blotting was repeated, the 71-kDa protein had disappeared from the sperm of affected subjects along with another central apparatus protein, SPAG6, with which it interacts. The 28-kDa SPAG17, which associates with SPAG6, was also missing. We presume that these proteins are degraded during the process. AKAP4, a protein that is localized to the fibrous sheath of the sperm, the 72-kDa SPAG17, and the nuclear protein SPAG16S were not affected. These findings suggest that normal levels of SPAG16L are required to maintain the integrity of central apparatus proteins in response to freezing/boiling. These observations are generally consistent with our studies in the mouse, which have revealed an essential role for SPAG16L in sperm motility and the interaction of SPAG16L with SPAG6, which in turn interacts with SPAG17. However, the defects in human sperm associated with the heterozygous mutation do not impair fertility, so the instability of central apparatus proteins observed is of biochemical but not physiologic significance. Unfortunately, we were not able to determine if the heterozygous SPAG16 mutation is associated with subtle sperm motility defects (reduced VCL), as we have previously shown for mice heterozygous for mutations that affect the expression of SPAG16L [28], due to the limited availability of study subjects and semen samples from the two males that we studied.
To explore further this notion, the relative abundances of SPAG16L, SPAG6, and the 28-kDa SPAG17 fragment were investigated in sperm from heterozygous Spag16L mutant mice. When Western blotting was performed with fresh samples, the SPAG16L level in heterozygous sperm was half that in the sperm of wild-type littermates, although there were no differences in SPAG6 and SPAG17 protein expression, which is consistent with our previous observations [28, 34]. However, when the samples were frozen/boiled four times and Western blotting was repeated, as seen for the human samples, SPAG16L, SPAG6, and the 28-kDa fragment of the SPAG17 protein were markedly reduced in the heterozygous mutant sperm but not in the sperm of wild-type mice. Again, AKAP4 and the 72-kDa fragment of SPAG17 were not affected. Thus, at least in terms of responses to thermal challenge, the phenotypes of heterozygous mutations that affect SPAG16L expression are similar in mice and humans.
It should be noted that instability of central apparatus components is characteristic of mutations that affect the PF20 and PF16 (orthologue of SPAG6) genes in Chlamydomonas. The central apparatus is missing in the pf20 Chlamydomonas mutant [24], and the C1 microtubule disappears from isolated axonemes from the pf16 mutant, suggesting instability in response to the physical process of the isolation procedure. The reductions in SPAG16L, SPAG6, and 28kDa SPAG17 levels in frozen/boiled sperm from humans and mice with heterozygous mutations that affect SPAG16L expression presumably reflect susceptibilities to proteolysis as a consequence of fragility of the central apparatus in the face of a physical (thermal) challenge.
In summary, we have discovered the first mutation in the SPAG16 gene, which appears to affect the stability of interacting proteins in the sperm axoneme. However, this biochemical phenotype, which is expressed in response to a thermal challenge, is evidently not physiologically significant as it does not cause male infertility.
ACKNOWLEDGMENTS
TEM was performed in the Imaging Core of the University of Pennsylvania's Diabetes Center (DK19525). We thank Susan Minnix, RN for co-ordinating multiple aspects of the studies of the propositus and that family.
FOOTNOTES
1Supported by National Institutes of Health Grants HD37416, HD06724, and U54 RR019480. 
Correspondence: 2Zhibing Zhang, Department of Obstetrics & Gynecology, Virginia Commonwealth University, 1101 East Marshall Street, Room 11-028A, Richmond, VA 23298. FAX: 804 828 0573; e-mail: zzhang4{at}vcu.edu
Received: 1 June 2007.
First decision: 26 June 2007.
Accepted: 30 July 2007.
REFERENCES
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