| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Research Article |
Center for Research on Reproduction and Women's Health,3
Department of Ophthalmology,4 University of Pennsylvania Medical Center, Philadelphia, Pennsylvania, 19104
Department of Histology and Embryology,5 University of the Republic, CP 11800 Montevideo, Uruguay
Department of Obstetrics and Gynecology,6 Virginia Commonwealth University, Richmond, Virginia, 23298
ABSTRACT
The axonemes of cilia and flagella contain a "9+2" structure of microtubules and associated proteins. Proteins associated with the central doublet pair have been identified in Chlamydomonas that result in motility defects when mutated. The murine orthologue of the Chlamydomonas PF20 gene, sperm-associated antigen 16 (Spag16), encodes two proteins of Mr
71 x 103 (SPAG16L) and Mr 35 x 103 (SPAG16S). In sperm, SPAG16L is found in the central apparatus of the axoneme. To determine the function of SPAG16L, gene targeting was used to generate mice lacking this protein but still expressing SPAG16S. Mutant animals were viable and showed no evidence of hydrocephalus, lateralization defects, sinusitis, bronchial infection, or cystic kidneys—symptoms typically associated with ciliary defects. However, males were infertile with a lower than normal sperm count. The sperm had marked motility defects, even though ultrastructural abnormalities of the axoneme were not evident. In addition, the testes of some nullizygous animals showed a spermatogenetic defect, which consisted of degenerated germ cells in the seminiferous tubules. We conclude that SPAG16L is essential for sperm flagellar function. The sperm defect is consistent with the motility phenotype of the Pf20 mutants of Chlamydomonas, but morphologically different in that the mutant algal axoneme lacks the central apparatus.
gamete biology, sperm, sperm motility and transport, testis
INTRODUCTION
The "9+2" microtubular structure of the axoneme is conserved in most cilia and flagella. The primary role of the nine outer doublet microtubules has been well elucidated, i.e., the associated dynein motors power the microtubule sliding that ultimately results in flagellar bending [1–3]. However, the roles of the radial spokes (RS) and the central pair of microtubules (CP) are not understood well. Much of the current knowledge of CP/RS function is derived from genetic, structural, and functional investigations in Chlamydomonas. These studies suggest that the CP/RS serve as mechanochemical sensors to control motility in 9+2 cilia and flagella [4, 5].
A number of central pair proteins have been identified in Chlamydomonas. PF6 localizes to the 1a projection on the C1 microtubule. Flagella lacking this protein display a twitching motion [6]. PF16 and PF20 are respectively localized to the C1 microtubule and the intermicrotubule bridge connecting C1 and C2. Mutations in these proteins result in paralyzed flagella with alterations in the organization of the central pair [7, 8]. Other proteins, including PF15, PP1c, KLP1, KRP, AKAP240, and calmodulin, also localize to the CP and its associated structures and have roles in regulating flagella motility [9–14].
Because relatively little is known about the central apparatus in mammalian sperm flagella, we cloned the cDNAs encoding mouse and human SPAG6 and SPAG16 [15–19], orthologues of Chlamydomonas PF16 and PF20, respectively, and investigated the functions of these proteins by gene targeting. Within 8 wk of birth, approximately 50% of the SPAG6-deficient mice die from hydrocephalus. Males surviving to maturity are infertile, because their sperm have motility defects and are morphologically abnormal, frequently missing their heads and displaying disorganized axonemal structure [20].
The mouse Spag16 gene uses alternative promoters to transcribe two major mRNAs (Spag16_pr1, 2.5 kb, and Spag16_pr2, 1.4 kb), which are differentially expressed during spermatogenesis and encode proteins of Mr 71 x 103 (SPAG16L) and Mr 35 x 103 (SPAG16S), respectively. Both proteins contain contiguous WD repeats in their carboxyl termini, indicative of protein-protein interactions. The meiotically-expressed SPAG16L is found in the cytoplasm of germ cells and then becomes part of the central apparatus of the sperm axoneme. In contrast, the SPAG16S protein is expressed postmeiotically and accumulates in nuclei of spermatids [18, 21]. We previously targeted both the SPAG16L and SPAG16S proteins in embryonic stem (ES) cells by disrupting domains within the Spag16 gene that encode the WD repeats. Highly chimeric mice carrying the mutant Spag16 allele have impaired spermatogenesis with a significant loss of germ cells at the round spermatid stage. In addition, a striking disorganization of the sperm axonemal structure is found. The mutated Spag16 allele is never transmitted, indicating that Spag16 haploinsufficiency caused the spermatogenetic defects. These findings reveal essential roles for the Spag16 gene during mouse spermatogenesis: germ cell viability and the integrity of the axoneme [21].
The different patterns of expression and localization of SPAG16L and SPAG16S, coupled with the spermatogenic phenotype of the Spag16+/– mouse, suggest that these protein variants may have distinct roles during male germ cell development. To study the functions of each protein, we embarked on studies to target the two proteins separately. In the present work, the SPAG16L protein was specifically eliminated by disrupting exons 2 and 3 of Spag16_pr1. We found that SPAG16L-deficient male mice were infertile; in particular, their sperm counts were lower than normal, and the sperm had marked motility defects. However, unlike the phenotype of SPAG6-null animals, flagella from SPAG16L-null mouse sperm had no major morphological or ultrastructural defects. Sperm from male mice heterozygous for the targeted mutation of Spag16_pr1 showed modest but statistically significant motility defects upon computer-assisted motion analysis. The observations suggest that a deficiency in SPAG16L impairs the function of the sperm tail axoneme without causing gross structural changes.
MATERIALS AND METHODS
Targeted Mutation of the Spag16 Gene Region Encoding the SPAG16L Protein
A targeting vector containing a neomycin selection cassette was generated to replace exons 2 and 3 of Spag16_pr1. The vector was linearized with Cla I and electroporated into TL-1 129/Sv ES cells. ES cell clones that survived G418 selection were evaluated for homologous recombination by Southern blot analysis. Three correctly targeted ES cell clones were used to generate chimeric mice with the assistance of the University of Pennsylvania Medical School Transgenic Mouse Core. These mice were then crossed with C57BL/6J females to obtain heterozygous mutants, and the mouse lines were maintained in on-campus animal facilities. Mice used in these studies were the offspring of crosses between the F1 and/or F2 generations (129/Sv/C57BL/6J genetic background). Mice were genotyped by PCR. Two sets of primers were used in the PCR experiments. One set of primers corresponded to the neomycin selection cassette (antisense: 5'-GAACTTCGGAATAGGAACTTC-3') and Spag16 gene (sense: 5'-CCATGGTATGGAGATTAGAGGACAACCTC-3'). The other set of primers corresponded to the deleted region of Spag16_pr1 (sense: 5'-GGTCACCATAACGGAAACATC-3'; antisense: 5'-CAGGAGCATGTTTTGCAGAGC-3'). The mutated gene is officially designated as the Spag16tm2Jfs allele. For simplicity and to designate the specific elimination of the expression of SPAG16L while maintaining expression of SPAG16S, this allele will be referred to as Spag16_pr1– throughout this report.
Assessment of Fertility and Fecundity
To assess fertility and fecundity, littermate males (5–7 mo old) of different genotypes (Spag16_pr1+/+, Spag16_pr1+/–, and Spag16_pr1–/–) were each placed in cages with two mature wild-type females for at least 2 mo. Littermate females of different genotypes were each caged with a wild-type fertile male for a similar period of time. The number of mice achieving a pregnancy and the number of offspring from each mating set or pregnancy were recorded.
Northern and Southern Blot Analysis
Northern blots containing total testicular RNA (30 µg/lane) were probed with a full-length Spag16_pr1 cDNA. Blots were stripped and reprobed for mouse Akap4 [22], Spag6, and Pf6. For Southern blotting, ES cell DNA (15 µg/lane) was digested with Kpn I and separated on a 0.8% agarose gel. The DNA was transferred to nylon membranes and probed with a Spag16-specific probe downstream of the homologous recombination region.
Immunoblot Analysis and Generation of an Anti-SPAG6 Antibody
Equal amounts of testicular or sperm protein (40 µg/lane) were prepared and subjected to immunoblot analysis using antibodies against SPAG6, PF6, AKAP4, and the N- and C-termini of SPAG16 [15–19, 22]. A cDNA encoding the C-terminus (amino acids 250–507) of mouse SPAG6 was cloned in frame into the pET28a vector. The fusion protein was purified and rabbit polyclonal antibodies were generated as reported previously [18].
Motility Assays
Sperm were collected after swimming out from the caudae epididymides into modified Whitten's medium (15 mM Hepes-sodium salt, 1.2 mM MgCl2, 100 mM NaCl, 4.7 mM KCl, 1 mM pyruvic acid, and 4.8 mM lactic acid) for 10 min at 37°C. The solution had a final pH of 7.35. Sperm were counted and diluted to 2 x 106 sperm/ml, and motility was analyzed in a 100-µm chamber (Cell Vision-USA) using the IVOS Sperm Analyzer (Hamilton-Thorne Research). The parameters that were used for the analysis setup are described in the technical guide for the IVOS system, version 12.2 (mouse setup 1). Sperm populations were analyzed as soon as possible after their release from the epididymides. For each sperm sample, a total of 10 fields were analyzed.
Histology, Immunochemistry Microscopy, and Transmission Electron Microscopy
Cauda epididymal sperm, testis, tracheal tissue, kidney, and ependymal tissue were prepared for light and transmission electron microscopy using standard methods, as previously described. Immunochemistry on testes and sperm were performed as previously described [21]. The same mice that were used to assess fertility were used for histological analysis of the testis.
Measurement of Serum Hormone Levels
Testosterone was quantified using a radioimmunoassay kit (TKTT1; Diagnostic Products Corp). FSH and LH were measured by the University of Virginia Center for Research in Reproduction Ligand Assay and Analysis Core (U54-HD28934).
Ethics
Procedures involving animals were conducted under the approval of the Institutional Animal Care and Use Committee of the University of Pennsylvania in accordance with the Guide for Care and Use of Laboratory Animals (Protocol #4365–01).
RESULTS
Targeted Disruption of Spag16_pr1 Eliminates SPAG16L in Germ Cells and Sperm
We disrupted the Spag16_pr1 gene in murine ES cells by replacing the second and third exons with the Neo-resistance gene (Fig. 1A). This manipulation prevented expression of the Mr 71 x 103 SPAG16L protein, but allowed expression of the Mr 35 x 103 SPAG16S protein. To generate chimeric mice, ES cells carrying the targeted allele of the Spag16 gene (Spag16_pr1–; Fig. 1B) were injected into blastocysts which were transferred into pseudopregnant mice. Mutant mice were produced from the chimeric offspring. Disruption of the Spag16_pr1 genomic region was confirmed by PCR analysis (Fig. 1C) and Southern blotting (data not shown). The proportion of wild-type (37 of 139; 27%), heterozygous (72 of 139; 51%), and nullizygous (30 of 139; 22%) offspring from mating of heterozygous males and females was not significantly different from the expected Mendelian pattern of inheritance (Hardy-Weinberg equilibrium test, Pearson chi square [1] = 0.207, Pr = 0.649). Spag16_pr1–/– mice were viable and showed no gross abnormalities. The mutant mice did not display abnormalities in non-reproductive tract tissues (e.g., polycystic kidneys) nor were there laterality defects (situs inversus)—abnormalities associated with defects in ciliogenesis and immotile cilia syndrome [23, 24]. Hydrocephalus was not found in the analysis of brains from 10 mutant mice.
|
The absences of the 2.5 kb Spag16_pr1 mRNA and its corresponding Mr 71 x 103 SPAG16L protein in the testes of Spag16_pr1–/– mice were confirmed by both Northern and immunoblot analyses (Fig. 2, A and B). Although a truncated 0.5 kb mRNA was found in the testes of heterozygous and null animals, a truncated protein corresponding to SPAG16L was not detectable. As might be expected, both the 1.4 kb Spag16_pr2 mRNA and the encoded Mr 35 x 103 SPAG16S protein were present in normal amounts in testicular cells. The mRNA and protein levels of other central pair proteins, e.g., PF6 and SPAG6, did not change in testes and sperm of null animals when compared to their wild-type littermates (Fig. 2, A and B). In addition, the testes of Spag16_pr1–/– mice contained Akap4 mRNA and its encoded protein, AKAP4, the major fibrous sheath component of the sperm flagellum (Fig. 2, A and B).
|
An antibody that specifically recognizes SPAG16L, but not SPAG16S, showed that the protein is expressed in the cytoplasm of germ cells and along the entire length of the flagella of epididymal sperm in wild-type animals (Fig. 3, A and B). Similar but less robust staining patterns were seen in the testis and sperm of heterozygous animals. No detectable staining was observed in germ cells and sperm of null mice.
|
SPAG16L is Required for Male but Not Female Fertility
Spag16_pr1–/– males that were mated with wild-type females produced no pregnancies after more than 2 mo of continuous cohabitation even though vaginal plugs were observed in the females (Table 1). The Spag16_pr1+/– littermates were all fertile, producing as many offspring per pregnancy as wild-type littermates. All 6 Spag16_pr1–/– females achieved a pregnancy during the observation period, and the time to establish a pregnancy was the same as that of wild-type and heterozygous littermates.
|
Spermatogenic Defects Are Present in Some Spag16_pr1–/– Mice
Testicular histology of Spag16_pr1+/+ and Spag16_pr1+/– mice showed that a normal structure of the seminiferous tubules was present (Fig. 4, A–D). Although the testes from three Spag16_pr1–/– mice also were normal (Fig. 4, E and F), three other mice of the same genotype from two different ES clones showed abnormalities in spermatogenesis at the round spermatid stage; degenerated germ cells were seen (Fig. 4, G and H). Two of these animals from the same ES clone also had smaller testes (about two thirds and one half normal size). Although a general trend toward a smaller size was observed, the weights of testes and seminal vesicles of Spag16_pr1–/– males were not statistically different from those of their wild-type littermates. Serum testosterone, FSH, and LH levels were also examined in several animals of each genotype but were not found to vary in a meaningful way (Table 2).
|
|
SPAG16L-Deficient Sperm Have Motility Defects
Sperm were present in the testes and efferent ducts of null animals; however, the numbers of sperm recovered from the caudae epididymides were significantly lower when compared to wild-type males (Table 3). In addition, computer-assisted sperm analysis demonstrated that sperm motility differed between wild-type and mutant animals. Whereas wild-type sperm displayed vigorous flagellar activity and progressive long track forward movement, only a small percentage of the Spag16_pr1–/– epididymal sperm were motile and showed progressive forward motility (Table 3, Figs. 5 and 6, and Supplemental Movie available online at http://www.biolreprod.org). Generally, flagellar movement was limited to a slow, erratic waveform with a very low amplitude. In addition, the mean curvilinear velocity (VCL) of motile sperm, an estimate of instantaneous sperm swimming speed, was significantly less in Spag16_pr1–/– mice. Interestingly, sperm from Spag16_pr1+/– mice had an intermediate VCL value, suggesting that, although these animals were fertile, the inactivation of one Spag16_pr1 allele impairs flagellar activity because of a reduction in SPAG16L protein (Figs. 2B and 3B). There were no differences in linearity of motile sperm, an estimate of the straightness of the sperm track, between wild-type and mutant sperm (Table 3).
|
|
|
Despite these motility defects, light microscopic examination revealed that more than 99% of the epididymal sperm from Spag16_pr1–/– animals were morphologically normal. Heads were not detached from the tails, something that was common in sperm from SPAG6-deficient mice, and no obvious disorganization of the axoneme was observed. Further, transmission electron microscopic analysis of testicular and epididymal sperm revealed that Spag16_pr1–/– testes and sperm had a normal axonemal structure (Fig. 7 and Table 4). In particular, the central pair of microtubules was present.
|
|
DISCUSSION
Both the mouse Spag16 and human SPAG16 genes encode two proteins of Mr 71 x 103 (SPAG16L) and Mr 35 x 103 (SPAG16S), which have different locations in male germ cells and sperm. SPAG16L is found initially in the cytoplasm of germ cells and then in the central apparatus of the sperm axoneme, whereas SPAG16S is present in nuclei of germ cells. Previously, a targeted deletion that eliminated both proteins resulted in impaired spermatogenesis and a disorganized sperm axoneme in chimeric mice [21]. The inability of the chimeric mice to transmit the mutant Spag16 allele, presumably because of haploinsufficiency, suggested that the gene has an important role during spermatogenesis. However, this approach did not allow the individual functions of SPAG16L and SPAG16S to be ascertained.
To study the function of SPAG16L, we deleted exons that were not present in the SPAG16S protein by gene targeting. Unlike the targeted deletion of both SPAG16L and SPAG16S, chimeric mice were able to transmit the mutant Spag16_pr1– allele. The resulting null mice were viable even though they did not express SPAG16L. These animals showed no evidence of hydrocephalus, lateralization defects, sinusitis, bronchial infection or cystic kidneys—conditions typically associated with ciliary defects. However, males were infertile; although some mice had a mild spermatogenetic defect, others did not. In general, these animals had significantly lower sperm counts compared to their wild-type littermates. Most striking, sperm from Spag16_pr1–/– animals had marked motility defects, even though the axonemal structure was normal at the ultrastructural level. The absence of a severe spermatogenic defect in this SPAG16L mouse model indicates that it is SPAG16S that causes the serious deficiency in spermatogenesis seen in males in which both SPAG16L and SPAG16S were eliminated. SPAG16S is related to a Drosophila testis-specific transcription factor, Cannonball [25], raising the possibility that it has an essential role in mouse spermatogenesis. In contrast, SPAG16L has a different function from SPAG16S, in that it appears to play an important role in sperm motility.
Although both the murine SPAG16L and the Chlamydomonas PF20 proteins are critical for flagellar motility, distinctly different phenotypes are observed when the two proteins were mutated/eliminated. When Chlamydomonas PF20 is mutated, flagella become paralyzed and the axoneme lacks its entire central apparatus [8]. In contrast, sperm from Spag16_pr1–/– mice showed a very shallow beat with little bend formation, suggesting a defect in the attachment of the spoke heads to the central pair apparatus or a deformation of this structure [26]. In addition, these sperm had no obvious structural defects in the axoneme, i.e., the central pair was present. The differences between these two phenotypes suggest a number of possibilities. The locations of the proteins in the central pair may differ. Although PF20 is localized to the bridge connecting C1 and C2 in the central apparatus in Chlamydomonas, it is not known whether SPAG16L which by immunoelectron microscopy is localized on or around the central apparatus, shares an identical position in the bridge connecting the two central microtubules. In addition, the presence of WD repeats suggests that SPAG16L interacts with other central pair proteins. Such a protein complex may be more stable in the absence of SPAG16 in the axoneme of the mouse compared to Chlamydomonas because of differences in the composition of the interacting proteins.
The phenotype of the SPAG16L-null mouse also is different from mice in which another central pair protein, SPAG6, has been targeted. SPAG6-null animals have a more severe phenotype as it affects the axonemal structure in both cilia and flagella of males and females. These mice have a lower body weight, and 50% die from hydrocephalus within 8 wk of age, presumably reflecting abnormalities in the function of cilia of ependymal cells. Unlike the SPAG6 mutant mice, body weight was not affected in the SPAG16L-null mice, and none of the mutant mice developed hydrocephalus. This suggests that SPAG6 has a more important role in maintaining the flagellar structure and function in mammals. Our previous studies indicate that SPAG6 may be the nexus linking SPAG16 and PF6, because in vitro studies showed that SPAG6 recruits both SPAG16L and PF6 to microtubules [17]. Thus, the sperm motility defects in the SPAG16L-deficient mice may reflect a disruption in the functional interactome of central apparatus proteins.
The presence of a mild spermatogenetic defect in SPAG16L-null mice is intriguing. Some, but not all, of these animals had smaller than normal testes and impaired spermatogenesis at the round spermatid stage. In contrast, when both SPAG16L and SPAG16S were eliminated, a severe spermatogenetic defect was evident. Although this suggests that SPAG16S is primarily responsible for the spermatogenetic defect, it is possible that SPAG16L contributes to the phenotype. In this regard, we have found that SPAG16L interacts with the chromosome condensing-like protein (CCLP) in a yeast 2-hybrid assay (unpublished data). Although relatively little is known about CCLP, its message is highly expressed in testis between 20 and 30 days after birth, when the germ cells are undergoing dramatic change, including nucleus condensation [27]. Thus, it is possible that SPAG16L interacts with CCLP to affect events in nuclear remodeling that occur during spermatogenesis.
The male infertility phenotype caused by a marked reduction in sperm motility and the absence of gross abnormalities in tissues containing ciliated cells indicates that SPAG16L could be a good molecular target for male contraception. Hormonal suppression of spermatogenesis by administration of exogenous hormones to interfere with the hypothalamic-pituitary-testicular axis has been the primary approach for male contraception, but concerns about side effects from these endocrine methods are substantial [28]. Nonhormonal approaches that affect only male germ cell function represent a more innovative strategy for the development of novel contraceptives for the male. There are increasing numbers of meiotically and postmeiotically expressed germ-cell-specific gene products involved in different aspects of spermatogenesis and sperm function that theoretically could serve as targets for intervention [29]. Our findings highlight the possibility of a nonhormonal approach for male contraception by the targeting of SPAG16L function.
ACKNOWLEDGMENTS
We thank Jean Richa and Peifu He (Transgenic Core, University of Pennsylvania Medical Center, Philadelphia) for help with injection of the ES cells. Transmission electron microscopy was performed in the Imaging Core of the University of Pennsylvania's Diabetes Center (DK19525). Follicle-stimulating hormone and luteinizing hormone levels were measured at the University of Virginia Center for Research in Reproduction Ligand Assay and Analysis Core (U54-HD28934).
FOOTNOTES
1 Supported by National Institutes of Health grants HD37416, HD06724, and TW06223–01, and the Foundation Fighting Blindness (FFB). 
2 Correspondence: Jerome F. Strauss, III, School of Medicine, Virginia Commonwealth University, Sanger Hall, 1101 East Marshall Street, Room 1–071, P.O. Box 980565, Richmond, VA 23298. FAX: 804 828 7628; e-mail:jfstrauss{at}vcu.edu
Received: 15 November 2005.
First decision: 1 December 2005.
Accepted: 22 December 2005.
REFERENCES
This article has been cited by other articles:
|
|
 |
|
  Z. Zhang, X. Shen, B. H Jones, B. Xu, J. C Herr, and J. F Strauss III Phosphorylation of Mouse Sperm Axoneme Central Apparatus Protein SPAG16L by a Testis-Specific Kinase, TSSK2 Biol Reprod, July 1, 2008; 79(1): 75 - 83. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. Lee, D. R. Campagna, J. L. Pinkus, H. Mulhern, T. A. Wyatt, J. H. Sisson, J. A. Pavlik, G. S. Pinkus, and M. D. Fleming Primary Ciliary Dyskinesia in Mice Lacking the Novel Ciliary Protein Pcdp1 Mol. Cell. Biol., February 1, 2008; 28(3): 949 - 957. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Z. Zhang, M. A Zariwala, M. M Mahadevan, P. Caballero-Campo, X. Shen, E. Escudier, B. Duriez, A.-M. Bridoux, M. Leigh, G. L Gerton, et al. A Heterozygous Mutation Disrupting the SPAG16 Gene Results in Biochemical Instability of Central Apparatus Components of the Human Sperm Axoneme Biol Reprod, November 1, 2007; 77(5): 864 - 871. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
