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BOR - Papers in Press, published online ahead of print December 26, 2003.
Biol Reprod 2003, 10.1095/biolreprod.103.024802
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BIOLOGY OF REPRODUCTION 70, 1213–1221 (2004)
DOI: 10.1095/biolreprod.103.024802
© 2004 by the Society for the Study of Reproduction, Inc.

Gamete Biology

The Novel Dominant Mutation Dspd Leads to a Severe Spermiogenesis Defect in Mice1

Masayuki Kai4,5, Masahito Irie4, Tomohisa Okutsu3,4, Kimiko Inoue5,6, Narumi Ogonuki5,6, Hiromi Miki6, Minesuke Yokoyama5,7, Rika Migishima7, Kaori Muguruma8, Hisako Fujimura9, Takashi Kohda4,5, Atsuo Ogura5,6, Tomoko Kaneko-Ishino5,10, and Fumitoshi Ishino2,5,11

Division for Gene Research,4 Center for Biological Resources and Informatics, Tokyo Institute of Technology, Midori-ku, Yokohama 226-8501, Japan CREST,5 Japan Science and Technology Agency (JST), Kawaguchi, Saitama 332-0011, Japan BioResource Center,6 RIKEN, Tsukuba, Ibaraki 305-0074, Japan Mitsubishi Kagaku Institute of Life Sciences,7, Tokyo 194-8511, Japan Central Institute for Experimental Animals,8 Miyamae-ku, Kawasaki 216-0001, Japan Exploratory Toxicology and DMPK Research Laboratories,9 Tanabe Seiyaku Co., Ltd., Chuo-ku, Osaka 541-8505, Japan School of Health Sciences,10 Tokai University, Bohseidai, Isehara, Kanagawa 259-11, Japan Medical Research Institute,11 Tokyo Medical and Dental University, Chiyoda-ku, Tokyo 101-0062, Japan


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Spermiogenesis is a complex process that is regulated by a plethora of genes and interactions between germ and somatic cells. Here we report a novel mutant mouse strain that carries a transgene insertional/translocational mutation and exhibits dominant male sterility. We named the mutation dominant spermiogenesis defect (Dspd). In the testes of Dspd mutant mice, spermatids detached from the seminiferous epithelium at different steps of the differentiation process before the completion of spermiogenesis. Microinsemination using spermatids collected from the mutant testes resulted in the birth of normal offspring. These observations indicate that the major cause of Dspd infertility is (are) a defect(s) in the Sertoli cell-spermatid interaction or communication in the seminiferous tubules. Fluorescent in situ hybridization (FISH) analysis revealed a translocation between chromosomes 7F and 14C at the transgene insertion site. The deletion of a genomic region of chromosome 7F greater than 1 megabase and containing at least six genes (Cttn, Fadd, Fgf3, Fgf4, Fgf15, and Ccnd1) was associated with the translocation. Cttn encodes the actin-binding protein cortactin. Immunohistochemical analysis revealed localization of cortactin beside elongated spermatids in wild-type testes; abnormality of cortactin localization was found in mutant testes. These data suggest an important role of cortactin in Sertoli cell-spermatid interactions and in the Dspd phenotype.

sertoli cells, spermatid, spermatogenesis


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Human infertility is observed in 10–15% of couples and the contributing factors are attributed to males and females equally. Spermatogenic arrest is the major cause of male infertility [1, 2]. The most common cause of spermatogenic arrest identified genetically is a microdeletion of the Y chromosome; however, this deletion accounts for less than 5% of male infertility [3]. There are likely many other autosomal genes and loci that affect spermatogenesis, but only a limited number have been identified.

The random integration of foreign DNA during the production of transgenic mice often leads to genomic rearrangements. Nakanishi et al. reported that chromosomal translocation was observed in about 5% of their EGFP transgenic lines [4]. Translocation is often accompanied by a genomic deletion that can vary enormously in size. It has not been easy to determine the size of deletions, but recently improved mouse genome databases that contain information on single nucleotide polymorphisms (SNPs) provide platforms for the quick size determination of even massive deletions.

A number of mutant mice showing abnormalities in spermatogenesis, including some insertional mutants [5–7], have provided important models for male infertility [3]. However, much remains to be understood. One of the obstacles to the analysis of spermatogenesis is that the spermatogenic process has not been reproduced in vitro [8]. The interaction between somatic Sertoli cells and germ cells is an important factor in spermatogenesis, making it difficult to reconstitute spermatogenesis in vitro. Sertoli cells are multifunctional nurse cells that provide nutritional and physical support for spermatogenic cells throughout sperm development. The Sertoli cell-spermatid interaction in particular is thought essential for the formation of a normal sperm head and for the release of sperm at the correct time. Filamentous (F-) actin plays an important role in Sertoli cell-spermatid interactions. The ectoplasmic specialization (ES) [9], a specialized junctional structure between Sertoli cells and elongated spermatids after step 8, contains an actin layer [10, 11]. The ES is damaged by treatment with the actin-disrupting drug cytochalasin D [12]. Mice treated with bisphenol-A or ß-estradiol 3-benzoate exhibit an abnormality of the ES. They also show abnormal spermatid morphology and the detachment of spermatids from the seminiferous epithelium, suggesting that an abnormal ES leads to spermatid abnormalities [13].

In this report, we identified a novel insertional/translocational mutation, which we named dominant spermiogenesis defect (Dspd), that results in a severe spermiogenesis defect manifested as head dysmorphology in elongated spermatids and a significant decrease in the number of elongated spermatids. Immature spermatids found in the mutant epididymides were released from the seminiferous epithelium inappropriately, suggesting inefficient Sertoli cell-spermatid interactions. The mutation loci of Dspd were mapped to two chromosomes (chromosomes 7F [chr.7F] and 14C [chr.14C]) by analyzing the translocation, which produced a genomic deletion of greater than 1 megabase (Mb) in chr.7F. Based on its location within the deleted region of chr.7F and the putative function of its gene product, Cttn (cortactin) is the most likely candidate gene responsible for the Dspd phenotype. Analysis of Dspd may provide a novel approach to understanding the molecular mechanisms and roles of Sertoli cell-spermatid interactions in spermiogenesis.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
All procedures described within were reviewed and approved by Tokyo Institute of Technology Institutional Animal Care and Use Committee and were performed in accordance with the Guiding Principles for the Care and Use of Laboratory Animals.

Transgenic Mice

The full-length human PEG8 cDNA (GenBank Accession Number AB030733) [14] was cloned downstream from the cytomegalovirus immediate early (CMV-IE) enhancer and the chicken ß-actin promoter (Fig. 1A). The DNA was injected into the male pronuclei of (C57BL/6 x C3H) F1 eggs using standard techniques. The transgenic mice were selected after screening the tail DNA by Southern-blot analysis using 32P-labeled human PEG8 cDNA as a probe.



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  		FIG. 1. The PEG8 transgene and mutant pedigree. A) The PEG8 transgene is composed of the CMV-IE enhancer, chicken ß-actin promoter, human PEG8 cDNA, and rabbit ß-globin polyA signal. The length of the construct is 4.5 kilobases. B) Pedigree for the 1L line. The squares represent male mice, and circles represent female mice. The half-colored symbols represent mice that inherited the transgene. Asterisks indicate individuals analyzed by FISH. C) Transgene expression analysis by RT-PCR using testes of 1L line (heterozygous for the Dspd mutation; Dspd/wt) and other PEG8 transgenic lines (11L, 16L, and 19L). In 16L, the level of PEG8 expression was relatively low. The expression in 1L (Dspd/wt) and 19L was similar. D) Western blot analysis of testis whole-cell extract with anti-PEG8 antibody. No detectable PEG8 protein was produced in 1L (Dspd/wt) or 19L. Recombinant His-tagged PEG8 protein (His-PEG8) was used as a positive control

Mating of Mice

The founder female (1L) was mated to a male C57BL/6 mouse, and two male offspring having the transgene were obtained. The two F1 males, at 3–6 mo of age, were mated to C57BL/6 females. They made 11 vaginal plugs; however, no offspring were obtained after natural matings.

Microinsemination

Spermatogenic cells were collected from recipient Dspd/wt male mice and frozen, as described previously [15, 16]. After thawing, elongated and round spermatids were directly injected into the ooplasm of wild-type mature oocytes using a Piezo-driven micromanipulator. Mature oocytes were collected from B6D2F1 females, which had been superovulated by injection with 5 IU of eCG followed 48 h later by 5 IU of hCG. Fertilized oocytes were cultured for 48 h, and four- to eight-cell embryos were transferred into the oviducts of pseudopregnant ICR females on the day following sterile mating with vasectomized ICR males.

Reverse Transcription-Polymerase Chain Reaction

Total RNA was prepared from the testes of Dspd/wt mice and another PEG8 transgenic mouse using ISOGEN (Nippon Gene, Toyama, Japan); cDNA was synthesized from 1 µg of total RNA using SuperscriptII reverse transcriptase (Life Technologies, Grand Island, NY) and an oligo(dT) primer. The polymerase chain reaction (PCR) was performed with EX Taq DNA polymerase (Takara, Kyoto, Japan). The primers for PEG8 amplification were PEG8-F: 5'-TGGACACACAGCTCTGCTTG-3' and PEG8-R: 5'-CCTGGGAATGCTCATTCATG-3'.

Western Blot Analysis

Recombinant His-tagged PEG8 (His-PEG8) protein was produced in Escherichia coli and purified with Probond Nickel-Chelating Resin (Invitrogen, San Diego, CA). Rabbits were immunized with the purified His-PEG8 protein. The polyclonal antiserum was collected and used for Western blot analyses. Whole cell extract (WCE) was prepared from adult testes. WCE samples containing 10 µg of total proteins were electrophoresed on 10% SDS-polyacrylamide gels and electroblotted to Hybond-P membranes (Amersham, Tokyo, Japan). His-PEG8 was used as a positive control. The enhanced chemiluminescence Western blotting detection system (Amersham) was used to visualize the results.

Histological Examination

For light microscopic examination, testes and epididymides were fixed in Bouin solution (Sigma, St. Louis, MO) and embedded in paraffin using standard techniques. Sections (3–4 µm) were cut, deparaffinized in xylene, rehydrated with a series of ethanol solutions, and stained with hematoxylin-eosin. Fifteen Dspd/wt mice were examined by light microscopy, and they showed the same phenotype. The numbers of spermatids per tubule at stages I–VI (steps 13–15) and at stages VII–VIII (step 16) were counted under light microscopy. Ten tubules of each stage (stages I–VI and stages VII–VIII) per testis were examined. Three mice for each genotype (Dspd/wt and wt/wt) were examined. Statistical analysis was performed by the unpaired t-test.

For electron microscopic examination, small blocks of testes were immersed in 2.5% glutaraldehyde, 2% paraformaldehyde in 0.1 M phosphate buffer (pH 7.3), and postfixed with 1% osmium tetraoxide. After dehydration, specimens were embedded in Epon/Araldite. Ultrathin sections were prepared, stained with uranyl acetate and lead citrate, and examined with an electron microscope (JEM-1210; JEOL, Tokyo, Japan). Four Dspd/wt mice were examined by electron microscopy, and they showed the same phenotype.

Fluorescent In Situ Hybridization (FISH)

Fluorescent in situ hybridization (FISH) was performed using a standard method, using the entire transgene as a probe. One F1 male and one F3 female (marked with asterisks [*] in Fig. 1B) having the transgene were examined. They had the same karyotypes, except for the sex chromosomes.

Genomic Library Screening

Genomic DNA was prepared from a Dspd/wt mouse, partially digested with the restriction enzyme Sau3AI, and ligated into the cosmid vector Supercos I (Stratagene, La Jolla, CA), which had been digested previously with XbaI and BamHI. After it was packaged (Gigapack III XL; Stratagene), the phage was used to transfect E. coli XL1-blue. The cosmid library was screened using PEG8 cDNA as a probe.

Genomic Deletion Analysis Using Single Nucleotide Polymorphisms

(DBA2 x C57BL/6) F1 mutant mice were produced by the microinsemination of eggs from superovulated DBA2 females with spermatogenic cells from a male Dspd/wt (C57BL/6 background) mouse. Genomic DNA from three transgene-positive F1 mice and three transgene-negative F1 mice (wild-type controls) was prepared. DNA fragments containing polymorphisms between DBA2 and C57BL/6 were amplified by PCR using the primers listed below, which are based on sequences in the Mouse RefSNP database (Celera Discovery System, Celera Genomics, http://www.celera.com). The PCR products were analyzed by direct sequencing. All three transgene-positive F1 mice gave the same results.

Primer sets for chromosome 7 mCV24660825: 5'-CATGGGACTGTGATGGTAAC-3' and 5'-GCATGTGTATCTGCTCAAGG-3; mCV24854750: 5'-ACACGCAGATGCTCTGTAG-3' and 5'-ATGTATCTAACAGTGAAGGCC-3'; mCV23705239: 5'-GAAGCATGCAAACACAGC-3' and 5'-GTAAAGTCTGTGGTGCAGTG-3'; mCV22810840: 5'-GCTGCTTTGACTTCTGCTC-3' and 5'-AACTCTCCCAGGTTTGTT-3'; mCV24856115: 5'-CCATGTATCAAAGCCAGG-3' and 5'-GATCTGTCATTGAGTGTACCC-3'; mCV24317904: 5'-TCGGAGCAGTAACTCTGTG-3' and 5'-GGAGTACAGGTACTGCAGG-3'; mCV22532403: 5'-GCAGGAGGTCACACTAAGC-3' and 5'-AGTAAATGCAGAAATAGCTGG-3'; and mCV22989836: 5'-CCAAGGTTTCCAACAAAAG-3' and 5'-ACCACCACCTATGTGATCAG-3'.

Primer sets for chromosome 14 mCV24975960: 5'-CAACTGTGTCACAGTATGG-3' and 5'-GGTTTCTGGTGATTGTGG-3'; mCV22764767: 5'-CTCCTGCTGTGATTGATCTC-3' and 5'-TGCAGAGGATGAAAGATTGG-3'; mCV24986264: 5'-TGTGCACACTGTAACAAACC-3' and 5'-GCACTGTTTTGCATTGTTCC-3'; mCV24616564: 5'-CTGCAGCCATAAAACTTCC-3' and 5'-CAAGGGTCTTTATCACCAC-3'; and mCV23859822: 5'-TTCAGGTTGCTTGTAAGCC-3' and 5'-TAAGCAGGAGTGATTGCTG-3'.

Immunohistochemistry

Testes removed from adult mice were fixed overnight in 20% (v/v) formalin containing 5% sucrose. They were embedded in paraffin using standard techniques and sections (3–4 µm) were prepared. Sections were deparaffinized in xylene, rehydrated through a series of ethanol solutions, boiled in 1 mM EDTA to activate antigens, treated with 1% H2O2 to block endoperoxidases, and incubated in 5% (v/v) normal goat serum in PBS. Sections were reacted with anti-cortactin rabbit polyclonal antibody (#3502; Cell Signaling Technology, Beverly, MA) at dilution 1:50, visualized with anti-rabbit Envision Plus (DAKO, Glostrup, Denmark), and counterstained with methylgreen.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Four lines of transgenic mice (1L, 11L, 16L, and 19L) carrying the human PEG8 transgene [14] (Fig. 1A) were produced. Male infertility was observed only in 1L progenies; two F1 males inherited the transgene (Tg) from a founder female mouse and were infertile (Fig. 1B). No phenotypic abnormalities were observed in the other transgenic lines expressing the PEG8 message (Fig. 1C; 11L and 19L), and no detectable PEG8 protein was produced in the testes of 1L or 19L mice (Fig. 1D). Therefore, we concluded that the infertility observed in the 1L line was not caused by PEG8 expression, but by an insertional mutation of the transgene. The two affected F1 males had few mature spermatozoa in the cauda epididymidis, and no offspring were obtained after natural mating although 11 vaginal plugs (total number from the two F1 males) were recorded. We could not perform in vitro fertilization due to the inability of retrieving spermatozoa from the epididymidis of F1 heterozygous males. However, when round and elongated spermatids collected from the mutant testes were injected into oocytes directly (see Microinsemination in Materials and Methods), normal offspring were obtained (Fig. 1B), indicating that the spermatids had normal haploid genomes. Intracytoplasmic sperm injection enabled us to maintain this mutant strain and to carry out histological and genetic analyses. All the heterozygous males examined were infertile, but wild-type littermates and heterozygous females were fertile and phenotypically normal. However, the litter sizes of heterozygous females in mating with wild-type males were relatively low (average = 3.0, SD = 1.0, n = 12), whereas the ratios Tg(+):(-) and male:female followed the Mendelian law (Tg[+]:[-] = 20:16, male:female = 20:16, 36 offspring). In this study, we have not tried to make homozygous mice because of the infertility of heterozygous males; therefore, phenotype of homozygotes have been unknown; however, they were speculated to be embryonic lethal (see Discussion).

Histological analyses were performed to precisely determine the phenotype. Examinations of the testes from heterozygous mutant mice revealed a severe spermiogenesis defect (Fig. 2, A and C). In the mutant testes, round spermatids seemed to be normal, but the number of elongated spermatids was significantly decreased (steps 13–15, P = 4.3 x10-10; step 16, P = 6.5 x10-15). In Dspd/wt mice, the average number of steps 13–15 spermatids remaining in the epithelium per tubule (stages I–VI) was about 30% of the wild type, and the arrangement of elongated spermatids was disrupted. Some of the elongated spermatids had misshapen heads (inset of Fig. 2C). There were only a few mature (step 16) spermatids at the luminal edge of the seminiferous epithelium. The average number of step 16 spermatids per tubule (stages VII–VIII) was 7.7% of the wild type. In addition, multinucleated spermatids were often found (data not shown). Leydig cells were morphologically normal. In the cauda epididymidis of wild-type males, the lumen was filled with mature spermatozoa (Fig. 2, F and H). By contrast, the number of spermatozoa found in the mutant epididymides was reduced, and almost all spermatozoa had variously misshapen heads (Fig. 2, E and G, arrows). Furthermore, immature spermatids and their debris were seen detached inappropriately from the seminiferous epithelium in the mutant epididymides (Figs. 2G and 3). The detachment of immature spermatids from the seminiferous epithelium is clearly observed in the electron micrographs (Fig. 3). Immature spermatids were found detached from the seminiferous epithelium in the lumens of Dspd/wt seminiferous tubules. Elongated spermatids in the lumen had abnormal head shapes, and some of them were often enclosed by a nucleus-free, membrane-bound cytoplasm that was probably Sertoli-cell in origin. Round spermatids were also found in the lumen. The decrease in the number of spermatids suggests that many spermatids are detached from the seminiferous epithelium during maturation. Spermatids detached from the seminiferous epithelium at different steps of the differentiation process before the completion of spermiogenesis. There seemed to be no particular step at which detachment of spermatids occurs. Based on these observations, the abnormalities associated with this mutation can be summarized as 1) head dysmorphology of elongated spermatids and 2) detachment of immature spermatids from seminiferous epithelium. Therefore, we named the mutation dominant spermiogenesis defect (Dspd) for its genetic and phenotypic features.



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  		FIG. 2. Light microscopy of the histology of adult testes and epididymides. Seminiferous tubules from a heterozygous Dspd mouse (Dspd/wt) (A, bar = 200 µm; C, bar = 100 µm; inset of C, bar = 5 µm) and a wild-type littermate (wt/wt) (B, bar = 200 µm; D, bar = 100 µm) are shown. In Dspd/wt seminiferous tubules, Sertoli cells and germ cells from spermatogonia to round spermatids appeared normal; however, there were only a few mature spermatids. Furthermore, misshapen elongated spermatids were frequently found in mutant seminiferous tubules (inset of C). The cauda epididymidis from Dspd/wt (E, bar = 200 µm; G, bar = 100 µm) and wt/wt (F, bar = 200 µm; H, bar = 100 µm) are shown. The wt/wt epididymal tubule was filled with spermatozoa. Much fewer spermatozoa were observed in the mutant epididymal tubule; almost all the spermatozoa had morphologically abnormal sperm heads (G, arrows and inset, bar = 5 µm)



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  		FIG. 3. Electron micrograph of Dspd/wt seminiferous tubule. Lu, lumen; Epi, epithelium of the seminiferous tubule. Immature spermatids are shown detaching from the seminiferous epithelium. Some elongated spermatids (Sd) were accompanied by cytoplasm that was probably Sertoli-cell in origin, and they had abnormal head morphology. Wandering round spermatids (Rsd) were often found in the lumen. Bar = 5 µm

The chromosomal localization of the transgene insertion site was determined by FISH using the transgene as a probe. In the mutant mice, a translocation was observed between chr.7F and chr.14C that resulted in a fusion chromosome longer than chr.1 and a short chromosome containing a proximal fragment of chr.14. A signal from the transgene was detected at the junction of the long fused chromosome (Fig. 4A). The same results were obtained with two 1L mice (one F1 male and one F3 female, marked by asterisks [*] in Fig. 1B). The low litter size of the mutant females (Fig. 1B) suggests that only offspring inheriting the two mutant chromosomes as a pair can survive.



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  		FIG. 4. Genomic analyses of Dspd mice. A) FISH analysis revealed that translocation had occurred between chr.7F and chr.14C and that the transgene had been inserted into the junction of the two chromosomes. B) Deletion analysis using SNP markers. Red arrows point to the transgene-genome junctions cloned from the genomic library. Genes mapped near the junctions are shown. The accession numbers of the SNPs are based on the Celera Mouse RefSNP database. + indicates the allele was present and - indicates the allele was absent. These data show a deletion of greater than 1 Mb on chr.7 of Dspd mice. C) Summary of the genomic structure of Dspd mutation loci. Chr.7 was joined to the distal fragment of chr.14 by the inserted transgene, resulting in a long fusion chromosome (violet line). The proximal fragment of chr.14 was likely joined to the telomeric region of chr.7 (blue line). The blue broken line represents the genomic region that could not be analyzed (see Results). A region greater than 1 Mb containing at least six genes (Cttn, Fadd, Fgf3, 4, 15, and Ccnd1) was found to have been deleted

Next, a genomic library of a Dspd/wt mouse was screened using PEG8 cDNA as a probe. Two clones containing transgene (Tg)-genome junctions were obtained; Southern-blot analysis confirmed that they were adjacent to the transgene (data not shown). The DNA sequences of the two junction sites were mapped on chr.7 and chr.14 using mouse genome sequence databases (Fig. 4B, red arrows) in conjunction with the FISH results.

To determine whether there were any deletions of the genome at the mutation loci, we carried out SNP analysis between two laboratory mouse strains, C57BL/6 (B6) and DBA2 (D2). Mapping information for the mouse SNPs was provided by Celera Mouse RefSNP database. The (D2 x B6) F1 mice with the Dspd mutation were produced by injecting spermatids of the Dspd/wt mice with a B6 background into D2 oocytes. DNA fragments containing SNPs were amplified by PCR and checked by direct sequencing of the PCR products. The results are shown in Figure 4B. We detected both D2 and B6 alleles at all markers analyzed on chr.14. The physical distance from the Tg-genome junction to the nearest marker, mCV24616564, is about 1 Mb. Therefore, these data showed that no deletion greater than 1 Mb occurred at the junction of chr.14. We confirmed that the nearest gene, Sftpd, was intact on both alleles. However, the 1-Mb region between mCV24616564 and the Tg-genome junction could not be analyzed because the region consists of highly repetitive sequences and the genomic assembly has not yet been completed. It is unlikely that any functional genes exist in this region.

Conversely, at the junction site of chr.7, only the D2 allele was detected as a marker on the distal side of the Tg-genome junction, while the other side of the junction was intact, as expected. All the markers from mCV24854750 to mCV22532403 had been deleted on the B6 (mutant) allele. The mCV22532403 marker is about 1.2 Mb away from the Tg-genome junction. The B6 allele was present at the marker closest to the telomere, mCV22989836. These data indicate that a region greater than 1 Mb between the Tg-genome junction and mCV22532403 was deleted in the mutant chromosome. No genes have been mapped between mCV22532403 and mCV22989836 in the telomeric region of chr.7. The Dspd mutation loci are summarized in Figure 4C. Chr.7 was joined to the distal fragment of chr.14 by the inserted transgene, resulting in a long fusion chromosome (Fig. 4C, violet line). The proximal fragment of chr.14 was likely joined to the telomeric region of chr.7 (Fig. 4C, blue line). A region greater than 1 Mb was deleted from chr.7.

At least six genes are reported to be within the deleted region: Cttn, Fadd, Fgf3, Fgf4, Fgf15, and Ccnd1. To verify the possibility that the translocation, genomic deletion, or transgene insertion might activate genes that are not normally expressed in testicular tissue, we analyzed the expression of genes in regions flanking the mutation loci (Dhcr7, Sftpd, Rbmxrt, and Ear1) by reverse transcription-polymerase chain reaction (RT-PCR). In the Dspd/wt testes, these genes were expressed at nearly the same levels as in wild-type testes (data not shown).

As shown in Figure 4C, the gene that encodes the actin-binding protein cortactin, Cttn, was haploid deleted in Dspd/wt mice. The importance of cortactin in the Sertoli cell-spermatid interaction and spermiation, i.e., the release of sperm from Sertoli cells, was suggested by a previous study of rat testis [17]. Therefore, we carried out immunohistochemical analyses of cortactin in mouse seminiferous tubules. Relatively weak signals were observed at basal areas and around elongated spermatids (steps 9–15) (Fig. 5, B and C, arrows).



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  		FIG. 5. Immunohistochemistry of cortactin in seminiferous tubules. (A, bar = 100 µm) Dense staining in wild-type (wt/wt) stages VII–VIII tubules demonstrates that cortactin is heavily concentrated beside step 16 spermatids. (Inset of A, bar = 5 µm) The dense stains shown at higher magnification. (B, bar = 100 µm) Weak staining was observed around the heads of elongated spermatids at early stages (stages III–IV, step 15). (C, bar = 5 µm; Lu, lumen; Ba, basal area) Cortactin localized around elongated spermatids (arrows in C). (D, bar = 100 µm) No dense staining was observed in Dspd/wt testis. A few condensed sperm heads were occasionally found (arrows in D) in the Dspd/wt seminiferous tubule (stages VII–VIII); however, they were not accompanied by dense staining

Importantly, dense staining was observed, especially in stage VII–VIII tubules of wild-type mice (wt/wt), which represented cortactin concentrated beside the heads of step 16 spermatids (Fig. 5A). These dense stains more likely belong to Sertoli cells than to spermatids, as referred to in a previous report [17]. Conversely, in Dspd/wt seminiferous tubules, localization of cortactin was disrupted. A weak diffuse signal was observed in Sertoli cells. No dense staining beside step 16 spermatids was found (Fig. 5D). There were no free dense stains (i.e., stains not associated with mature spermatids) at the seminiferous epithelium and no wandering stains in lumens.

A few sperm heads were occasionally found at the luminal edge of the seminiferous epithelium; however, they were not accompanied by the dense staining (Fig. 5D, arrows).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Transgene insertions are often accompanied by genomic deletions of various sizes. It is not a simple matter to determine the size of a deletion, especially when it is coupled with a translocation and the inability to produce homozygotes due to infertility, as with Dspd. In this case, SNPs can be used to determine the mutant loci effectively. Using SNPs, several alleles at the translocation sites of chromosomes can be promptly checked by direct sequencing. Even if a part of the genomic assembly is imperfect, we believe that a method using SNPs is an efficient way of analyzing a mutation with a massive rearrangement of the genome.

The mutation loci of Dspd were mapped to chr.7F and chr.14C. On chr.7F, a region greater than 1 Mb was deleted. By contrast, no deletions this large were found on chr.14, and no genes on chr.14 seemed to be affected by the mutation. Therefore, the deleted region of chr.7F is likely responsible for the Dspd phenotype. There are six well-characterized genes and at least five predicted genes (confirmed by ESTs) within the deleted region of chr.7F. The well-characterized genes are, from the proximal end, Cttn, Fadd, Fgf3, Fgf4, Fgf15, and Ccnd1. Targeted mutations of all these genes except Cttn have been reported [18–23]; none of the knockout mice showed any type of spermatogenic abnormality. Therefore, these five genes are unlikely the cause of the phenotype observed here, at least not independently. However, the possibility that combinations of hemizygosity might lead to unexpected effects on Sertoli or spermatogenic cells cannot be ruled out. Embryonic lethality was reported in Fadd-null mutant mice [18, 19], implying that homozygotes of Dspd (which we have not tried to make) would die in utero, if produced. The functions of the five predicted genes in this region (LOC233977, AU040576, 2210010N10Rik, BC025890, and BC019711) are unknown, making these genes possible candidates responsible for the observed phenotype.

Cttn is the most likely gene responsible for the Dspd phenotype because its gene product is localized where Sertoli cell-spermatid interactions occur and it has a putative function in spermiogenesis. Cttn encodes the 80–85-kDa actin-binding protein cortactin, which has HS1 repeats and an SH3 domain. At least three isoforms spliced with different numbers of HS1 repeats have been reported. Cortactin was initially identified as a pp60Src substrate [24] and a target of the Src family of protein kinases [25]. A number of cell and biochemical studies have shown that cortactin interacts with the Arp2/3 complex and N-WASP in the formation of actin filament networks [26, 27]. Therefore, cortactin connects a protein phosphorylation signal with the organization of the actin cytoskeleton. However, the functions of cortactin in vivo are not yet clear. Dspd is the first reported mutation affecting Cttn in mice.

The Sertoli cell-spermatid ES is a specialized actin-related junctional structure in Sertoli cells, which consists of actin bundles between the Sertoli plasma membrane and endoplasmic reticulum [10–12]. The functions of the ES in Sertoli cell-spermatid interactions are not clear. However, the ES is considered essential for normal morphology of elongated spermatids and for maintaining the Sertoli cell-spermatid connection until spermiation. The localization of cortactin in mouse testis resembles that of the actin-binding protein espin, a major component of the ES [28]. Cortactin is reported to interact with ß1-integrin, which is also included in the ES [17]. Furthermore, cortactin is a target of Fyn, a member of the Src family of tyrosine kinases [25, 29]. Fyn is concentrated in the ES in mouse testis, and fyn-/- mutant mice show an abnormality of the ES at 3–4 weeks of age [30]. These lines of evidence suggest that cortactin is included in the ES as a regulator of this actin-related junctional structure. The reduced cortactin concentration might lead to abnormalities of the ES.

Chapin et al. reported that cortactin and other junctional/cytoskeletal proteins localize around mature spermatids in rat testis and suggested that they may form some Sertoli cell-spermatid junctional structure (maybe the ES), and may function to adhere mature spermatids to Sertoli cells until spermiation [17].

The diffuse stain of cortactin in Sertoli cells and the absence of the stage-specific dense staining in the lumen and epithelium of Dspd/wt seminiferous tubules indicates an abnormality of the cortactin localization. We think it is likely that the haplo insufficiency of cortactin might lead to a disrupted localization of cortactin and an inefficient Sertoli cell-spermatid junctional structure and to the subsequent spermiogenesis defect.

It is interesting that Dspd is genetically dominant because insertional mutations generally cause a simple loss of function and do not become apparent until bred to homozygosity. However, the dosage effect of some genes encoding cytoskeletal proteins or structural components can lead to phenotypical expression. For example, the haplo insufficiency of desmoplakin, a constitutive component of desmosomal plaques, causes a striate subtype of palmoplantar keratoderma [31]. Cttn may be in this category.

Other possibilities might explain the genetic dominance of Dspd. Perhaps the deletion of part of the genome or the insertion of the transgene truncated a gene product, resulting in a dominant-negative protein that interfered with the function of its normal counterpart. To date, however, no genes or ESTs have been found at the Tg-genome junctions or at the edge of the deletion in Dspd. The presence of a transgene (containing a strong enhancer) might have activated genes that are not normally expressed in testicular tissue, except the intact genes neighboring the mutation loci (Dhcr7, Sftpd, Rbmxrt, and Ear2) were expressed in both wt/wt and Dspd/wt testes at almost the same levels (data not shown). A third possibility, genomic imprinting, can be excluded because both paternal and maternal transmission of Dspd appeared with the same phenotype, although the mutation locus at distal chr.7 was close to the imprinted gene cluster [32].

Follicle stimulating hormone and testosterone are major regulators of spermatogenesis. Abnormal levels of these hormones can lead to spermatogenic defects similar to Dspd mice [33, 34]. It is possible that secretions of these hormones might have been affected in Dspd mice. However, in this study, we have not examined these hormone levels. We suppose that there was little, if any, hormonal disorders in Dspd mutant mice for the following reasons: 1) as far as we know, the deleted genes are not involved in the regulation of these hormone levels; 2) if regulation of the hormone levels were genetically disrupted, it would be likely to affect the pubertal development of gonads; however, reproductive organs of both male and female Dspd mice had a normal appearance.

In this study, we isolated a mouse strain with a novel mutation that we named Dspd. We mapped the mutation loci to chr.7F and chr.14C and found a genomic deletion at chr.7F. We detected the absence of cortactin localization beside mature spermatids in the mutant testis and suggested that the haplo insufficiency of cortactin reduced the efficiency of the Sertoli cell-spermatid junctional structure, which resulted in the spermiogenesis defect. This is the first report of a mutation affecting cortactin in mice and the first proposal of the importance of cortactin haplo insufficiency to male infertility. Further study of Dspd will shed light on the molecular mechanisms of the last stage of spermatogenesis and will promote an understanding of the genetic causes of male infertility.


   FOOTNOTES
 
1 This work was supported by grants to F.I. from CREST, the research program of the Japan Science and Technology Agency (JST), the Uehara Memorial Life Science Foundation, and the Ministry of Health, Labour for Child Health and Development (14-C). Back

2 Correspondence: Fumitoshi Ishino, Medical Research Institute, Tokyo Medical and Dental University, 2-3-10 Kandasurugadai, Chiyoda-ku, Tokyo 101-0062, Japan. FAX: +81 3 5280 8073; fishino.epgn{at}mri.tmd.ac.jp Back

3 Current address: Ajinomoto Co., Inc., 1-1 Suzuki-cho, Kawasaki-ku, Kawasaki, Kanagawa 210-8681, Japan Back

Received: 30 October 2003.

First decision: 26 November 2003.

Accepted: 10 December 2003.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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