Male infertility, impaired spermatogenesis, and azoospermia in mice deficient for the pseudophosphatase Sbf1

Pseudophosphatases display extensive sequence similarities to phosphatases but harbor amino acid alterations in their active-site consensus motifs that render them catalytically inactive. A potential role in substrate trapping or docking has been proposed, but the specific requirements for pseudophosphatases during development and differentiation are unknown. We demonstrate here that Sbf1, a pseudophosphatase of the myotubularin family, is expressed at high levels in seminiferous tubules of the testis, specifically in Sertoli’s cells, spermatogonia, and pachytene spermatocytes, but not in postmeiotic round spermatids. Mice that are nullizygous for Sbf1 exhibit male infertility characterized by azoospermia. The onset of the spermatogenic defect occurs in the first wave of spermatogenesis at 17 days after birth during the synchronized progression of pachytene spermatocytes to haploid spermatids. Vacuolation of the Sertoli’s cells is the earliest observed phenotype and is followed by reduced formation of spermatids and eventual depletion of the germ cell compartment in older mice. The nullizygous phenotype in conjunction with high-level expression of Sbf1 in premeiotic germ cells and Sertoli’s cells is consistent with a crucial role for Sbf1 in transition from diploid to haploid spermatocytes. These studies demonstrate an essential role for a pseudophosphatase and implicate signaling pathways regulated by myotubularin family proteins in spermatogenesis and germ cell differentiation.

Protein tyrosine and dual-specificity phosphatases participate in diverse physiological processes. In conjunction with kinases, they regulate the phosphorylation states of proteins and lipids in various subcellular compartments, thereby impinging on many aspects of cellular function (1). Recent studies have identified an additional component of this catalytic hierarchy, the so-called pseudophosphatases (2, 3), which display extensive sequence similarities to phosphatases, but harbor inactivating mutations in their active-site consensus motifs that render them catalytically inactive. Several proteins with pseudophosphatase features have been identified (2), most of which are conserved throughout metazoan evolution. Several are similar to myotubularin, a phosphatase that was originally discovered by virtue of its mutation in a subset of patients with X-linked myotubular myopathy (4, 5). Myotubularin belongs to a growing subgroup of phosphatases that dephosphorylate lipid substrates (6) and is one of several lipid phosphatases whose mutations are associated with heritable disorders of aberrant growth or differentiation (6–8).

Sbf1 (SET-binding factor 1 or MTMr5) is the most extensively characterized of the myotubularin-related pseudophosphatases. It is a 220-kDa cytoplasmic protein that was initially identified based on its in vitro interaction with MLL, a proto-oncogenic protein involved by chromosomal translocations in mixed lineage leukemias (5, 9). Sbf1 contains several domains (e.g., pleckstrin and Rab3 GEF homology motifs) that are conserved in signaling proteins, and in vitro studies suggest a role for Sbf1 in cellular growth control (5, 9–11). The ability of Sbf1 to modulate cellular growth, however, is abrogated by restoring catalytic activity to its phosphatase homology domain. Based on these and other observations, it has been hypothesized that pseudophosphatases may contribute to cellular homeostasis by opposing the actions of phosphatases (3), perhaps as naturally occurring substrate-trapping mutants analogous to experimentally inactivated phosphatases, which bind phosphorylated substrates and prevent their dephosphorylation (12). Alternatively, the inactivated catalytic pockets of pseudophosphatases may serve as docking motifs for phosphorylated substrates analogous to the roles of SH2 and PTB motifs (2). Regardless of their biochemical mechanisms of action, little is known about the developmental roles of pseudophosphatases. Here, we demonstrate that the presence of Sbf1 is required for male reproductive function. Our studies further reveal that Sbf1 is specifically required for spermatogenesis through contributions to male germ cell differentiation and survival.

Differential expression of Sbf1 in the seminiferous tubules. Northern blot analysis of RNA isolated from adult mouse tissues demonstrated expression of Sbf1 at high levels in the testis and at lower levels in the brain and colon. Analysis of human tissues similarly revealed highest levels of Sbf1 expression in the testis (Figure 1a). Western blot analysis of proteins isolated from testes at postnatal days 20–90 showed that expression of Sbf1 was relatively constant throughout progression of spermatogenesis, which is highly synchronized during this time period (Figure 1b). Notably, Sbf1 was also detected in prepubertal testis (day 10), suggesting that it was expressed by either spermatogonia, preleptotene spermatocytes, or Sertoli’s cells. These observations raised the possibility of a role for Sbf1 in germ cell function during spermatogenesis, the ordered process of germ cell mitosis, meiosis, and differentiation into sperm (14).

Figure 1

Tissue-specific expression of Sbf1. (a) Northern blot analysis of total RNA prepared from human tissues or adult mice. The hybridization probe consisted of an Sbf1 cDNA (upper panel). Abundance of 28S rRNA is shown for the mouse blot for comparison. (b) Western blot analysis of protein extracts from testes at different stages of spermatogenic development. Migration of Sbf1 (upper panel) and β-actin (loading control, lower panel) is indicated. Faint bands in some lanes at 150 kDa represent apparent Sbf1 degradation products. (c) Anti-Sbf1 Western blot analysis of protein extracts from wild-type, Sbf1^–/–, and W^v/W^v adult mice.

To investigate the role of Sbf1 in spermatogenesis, its expression was evaluated by in situ analyses of testes from adult and juvenile mice. In situ hybridization demonstrated that Sbf1 mRNA was most prominently expressed within seminiferous tubules of adult testis at the basal regions that contain spermatogonia, spermatocytes, and Sertoli’s cells (Figure 2a). In agreement with the in situ hybridization data, immunohistochemical analysis of Sbf1 protein distribution in testes at different days of development revealed that it was prominently expressed in Sertoli’s cells, spermatogonia, and pachytene spermatocytes, but was notably absent in postmeiotic round spermatids (Figure 2, c and e). Its predominant cytoplasmic localization was consistent with more extensive studies of its subcellular distribution (5). Western blot and immunohistochemical analysis of testes from W^v/W^v mice, which lack germ cells but contain Sertoli’s cells, confirmed that the latter express cytoplasmic Sbf1 (Figure 1c and Figure 2g). The observed expression profiles of Sbf1 in the seminiferous tubules of pubertal mice suggested a function in the early stages of germ cell differentiation.

Figure 2

Sbf1 expression in testis. (a and b) In situ hybridization of wild-type adult mouse testis demonstrates specific expression of Sbf1 mRNA transcripts in the basilar portions of the seminiferous tubules. Sections were hybridized with antisense (AS) and sense (S) Sbf1-specific riboprobes as indicated (×200). (c and d) Immunohistochemical analysis demonstrates Sbf1 protein expression in cells at the periphery of seminiferous tubules in wild-type (+/+) but not Sbf1^–/– (–/–) mice at 4 weeks of age (×400). Scale bars, 25 μm. (e and f) Sbf1 protein is detected by immunohistochemistry in cells of the immature testis (day 16) in wild-type (+/+) but not Sbf1^–/– (–/–) mice (×200). (g and h) Immunohistochemical detection of Sbf1 protein in seminiferous tubules of adult W^v/W^v mice shows Sbf1 expression in Sertoli’s cells. Control (h) represents secondary Ab alone.

Male Sbf1^–/– mice are infertile with azoospermia. To establish the role of Sbf1 in normal mouse development and physiology, a loss-of-function mutation was introduced into the gene by homologous recombination. The targeting vector was engineered to inactivate Sbf1 by replacing exons 21–28 with a Pgk-Neo cassette (Figure 3a) to prematurely terminate the recombined allele. The expected wild-type and mutated Sbf1 alleles were observed by Southern blot analyses of DNA extracted from targeted ES cell lines (not shown) and mouse tissues (Figure 3b). Western blot analysis showed that Sbf1^–/– mice expressed neither full-length nor truncated portions of Sbf1 (Figure 3c). Immunohistochemical analysis of seminiferous tubules of Sbf1^–/– juvenile and prepubertal mice confirmed the absence of Sbf1 in spermatogonia, pachytene spermatocytes, and Sertoli’s cells (Figure 2, d and f).

Figure 3

Targeted disruption of the Sbf1 gene. (a) Schematic depiction of the Sbf1 protein, gene, targeting construct, and recombined allele. Conserved motifs present in Sbf1 include an N-terminal Rab GEF homology domain, internal MTM homology motifs, and a C-terminal PH domain. Following homologous recombination, a Pgk-Neo cassette replaces Sbf1 exons 21–28, which encode amino acids 653–1120 of the Sbf1 protein as indicated. Locations are shown for external probes A and B used for genotype analyses. Restriction enzyme sites: E, EcoRI; X, XhoI; S, SacI; H, HindIII; B, BamH1. (b) Southern blot analysis of genomic DNA isolated from wild-type (+/+), Sbf1^+/– (+/–), and Sbf1^–/– (–/–) mice. Probes are indicated below the respective panels. (c) Western blot analysis of brain extracts of wild-type (+/+), Sbf1^+/– (+/–), or Sbf1^–/– (–/–) mice. Primary Ab’s (indicated below panels) consisted of mAb’s specific for the N-terminal (mAb 8) or C-terminal (mAb 68) portions of Sbf1. Faint bands in some lanes at 150 kDa represent apparent Sbf1 degradation products.

Intercrossing of F1 Sbf1^+/– mice showed that Sbf1^–/– progeny were born at the expected Mendelian ratios, were viable, and reached adulthood. Nullizygous males from backcrossed generations, however, exhibited reduced postnatal viability (wild-type, 17; heterozygote, 44; homozygous null, 7; 0 males, at 3 weeks). Sbf1^–/– males were infertile and incapable of siring offspring, in contrast to the normal fertility and fecundity of Sbf1^+/– males and Sbf1^–/– females (Table 1). Evaluation of the mating behavior of Sbf1^–/– males showed that they copulated with females at a rate comparable to wild-type males (data not shown), suggesting that behavioral factors were not the cause of infertility. The external genitalia and testicular descent of Sbf1^–/– males appeared normal, but testis sizes and weights were markedly reduced compared with either heterozygous or wild-type males (Table 2, Figure 4, a and b). Furthermore, sperm counts and epididymal histology revealed the lack of sperm within the epididymi of Sbf1^–/– mice (Table 2, Figure 4, c and d).

Figure 4

Failure of spermatogenesis in Sbf1^–/– mice. Comparative anatomy and histology are shown for wild-type (+/+) and Sbf1^–/– (–/–) testes. (a and b) Male reproductive organs are shown for mice at 20 weeks of age. Sbf1^–/– testis, but not epididymis, is considerably smaller compared with wild-type (×2). (c and d) Sections of epididymis from 20-week-old mice were stained with hematoxylin and eosin (×200). Note absence of spermatozoa in Sbf1^–/– epididymis. (e–n) Sections of wild-type and Sbf1^–/– testes at different stages of spermatogenic development (specific time points indicated in the respective panels) were stained with hematoxylin and eosin (×200). Testes from Sbf1^–/– mice show extensive vacuolar degeneration that is apparent at day 17 and is followed by complete depletion of germ cells by 52 weeks.

Table 1

Infertility phenotype of male Sbf1 mutant mice

Table 2

Sterility of Sbf1^–/– male mice

The pituitary hormones FSH and LH (through testosterone secretion) regulate spermatogenesis and prevent the germ cell compartment from undergoing apoptosis. These hormones are first expressed during puberty in the male mouse and are required throughout adulthood to maintain spermatogenesis. Serum levels of FSH, LH, and testosterone at 12 weeks of age were not significantly different in Sbf1^–/– compared with wild-type littermates (Table 3), indicating that the testicular defects were not caused by reduced levels of these hormones.

Table 3

Serum hormone levels of wild-type and Sbf1^–/– mice at 12 weeks of age

The seminiferous tubules of Sbf1^–/– mice were evaluated for perturbations in spermatogenesis. Histological examination of adult testes revealed extensive vacuolation of the tubules accompanied by a decrease in the number of round spermatids, very few elongating spermatids, and no mature sperm in Sbf1^–/– mice (Figure 4, k and l). Germ cell differentiation was disorganized, lacking the characteristic basal-to-luminal maturation observed in normal seminiferous tubules. By 52 weeks of age, few germ cells remained within Sbf1^–/– tubules, although Sertoli’s cells persisted (Figure 4, m and n) and marked interstitial hyperplasia of Leydig’s cells was observed. These data indicated that the primary phenotype of adult Sbf1^–/– mice was a progressive spermatogenic failure.

Sbf1^–/– mice display defects in the first wave of spermatogenesis. The severe pathological findings in testes of adult Sbf1^–/– mice prompted examination at earlier times of postnatal development. Spermatogenesis begins neonatally in the mouse, and the first wave of germ cell differentiation is synchronized such that all tubules are at the same stage of spermatogenesis (15). In sexually immature mice at 10 days of age, a time at which germ cell meiosis has begun, seminiferous tubules of Sbf1^–/– mice were comparable in both size and morphology to their wild-type littermates (Figure 4, e and f). Examination of testes at days 14 and 16 revealed no histopathology (data not shown). Histologic aberrations were first detected at day 17 in Sbf1^–/– testes (Figure 4, g and h) and consisted of vacuoles that appeared to be associated with Sertoli’s cells as seen by light microscopic analysis (Figure 5a). Electron microscopy revealed that the vacuoles were completely within the cytoplasm of Sertoli’s cells in multiple electron microscope montages examined (Figure 5b).

Figure 5

Vacuolization of Sertoli’s cells at 17 days of age. (a) Section of Sbf1^–/– seminiferous tubules demonstrating a Sertoli’s cell with intracytoplasmic vacuole (*) (×400). (b and c) Electron microscopic analysis of a representative Sertoli’s cell and associated vacuole at low (b) (×1,500) and high (c) (×4,000) magnification. Sertoli’s cell (S), spermatogonial cell (Sg), spermatocyte (Sc), and vacuole (V) are indicated.

By 20 days of age, when germ cells in testes of wild-type mice had completed the second meiotic division and progressed to the haploid round spermatid stage, the seminiferous tubules of Sbf1^–/– mice showed reduced numbers of round spermatids and increased vacuolization (Figure 4, i and j). Tubular pathology progressively worsened in subsequent days with further decrease in spermatids at all stages of differentiation and the appearance of apoptotic bodies. These observations localized the onset of the spermatogenetic defect to the stage when pachytene spermatocytes progress to round spermatids.

To more clearly delineate when the Sbf1 nullizygous defect occurs, we examined molecular markers expressed at specific stages of germ cell development. These included genes expressed in leptotene to zygotene spermatocytes (DMC1), leptotene to pachytene spermatocytes (Hsp25), and spermatids (calspermin) (16–18). Northern blot analysis of DMC1 and Hsp25 transcripts through the first wave of spermatogenesis showed that they were expressed at comparable levels in wild-type and Sbf1^–/– testes. Conversely, calspermin RNA expression was reduced significantly in testes of Sbf1^–/– mice (Figure 6). These data are consistent with the histological findings that spermatid production is significantly reduced and indicate that spermatocyte viability in Sbf1^–/– mice is preserved at least into early adulthood.

Figure 6

Expression analysis of different germ cell markers in Sbf1^–/– mice. Northern blot of total RNA prepared from mouse testis at different time points of spermatogenic development. Autoradiogram shows transcript levels in Sbf1^–/– versus wild-type mice probed with ³²P-labeled DMC1, Hsp25, and calspermin cDNAs. The same blot was hybridized with a β-actin probe as a control.

The possible role of apoptosis in depletion of the germ cell compartment in Sbf1^–/– male mice was examined by conducting TUNEL assays on testes at different stages of development. Although significant vacuolation (the earliest morphological abnormality) was first observed in Sbf1^–/– testes at day 17 (Figure 4, g and h), the number of TUNEL-labeled apoptotic nuclei per tubule remained comparable between Sbf1^–/– and wild-type mice at this time point. At 4–8 weeks of age, however, there was a dramatic increase in the number of apoptotic nuclei in the seminiferous tubules of Sbf1^–/– mice. At later time points, the apoptotic index decreased in the Sbf1^–/– tubules as fewer germ cells remained (Figure 7). Taken together, these observations suggested that in the absence of Sbf1 there was a failure in the differentiation of the prehaploid stages of spermatogenesis with consequent induction of programmed cell death in the germ cell compartment. The presence of vacuolization in the Sertoli’s cells prior to the onset of apoptosis suggests that Sertoli’s cell dysfunction may be the primary stimulus for the aberrant spermatogenic progression and germ cell apoptosis that ensues.

Figure 7

Germ cell death in juvenile and adult mouse testis. (a) Sections of testis from 25-day-old mice were labeled by TUNEL assay and counterstained with methyl green (×200). Scale bars, 25 μm. (b) Quantitation of apoptotic cells per seminiferous tubule based on TUNEL labeling of testis at different stages in mouse testicular development (three mice per time point). *P < 0.05 compared with wild-type.

Specific roles for myotubularin family phosphatases in organogenesis and differentiation were suggested previously by their tissue-restricted expression profiles (4, 15) and organ-specific phenotypes in heritable human diseases (4, 7), but the roles for myotubularin-related pseudophosphatases were undefined. Using a loss-of-function mouse model, our current studies demonstrate an essential role for the myotubularin-related pseudophosphatase Sbf1 in spermatogenesis. Hormonal and copulatory studies showed no changes in Sbf1^–/– mice, indicating a primary spermatogenetic defect. The onset of the spermatogenetic defect occurs in the first wave of spermatogenesis at 17 days after birth during the synchronized progression of pachytene spermatocytes to haploid spermatids. Early disruption of spermatogenesis was evidenced by Sertoli’s cell vacuolization and defective germ cell differentiation and tubular disorganization. This was followed by extensive apoptosis and progressive depletion of germ cells, which were completely absent in the seminiferous tubules at 1 year of age. Late onset depletion of spermatogonia and spermatocytes suggests that these features are secondary effects of the disordered spermatogenetic environment.

The temporal onset of the spermatogenic defect in Sbf1^–/– mice corresponds to the initiation of the meiotic division of pachytene spermatocytes to haploid spermatids at 17–20 days after birth (19). The nullizygous phenotype in conjunction with high-level expression of Sbf1 in premeiotic germ cells and Sertoli’s cells is indicative of either a direct or indirect role for Sbf1 in the transition from spermatogonia to haploid spermatocytes. Our findings that vacuolization of Sertoli’s cells precedes germ cell apoptosis suggest that dysfunction of Sertoli’s cells may be the primary defect in Sbf1^–/– mice. Sertoli’s cells serve a particularly important role during the meiotic stages of the first spermatogenetic cycle. During this time they mediate formation of a tubular lumen, undergo extensive cytoplasmic differentiation, and stimulate synthetic activity of secretory proteins that direct germ cell differentiation (20). Vacuolization is a common feature of Sertoli’s cell dysfunction and injury (21, 22) and is thought to result from swelling of membrane-bound organelles such as the endoplasmic reticulum (23). Since Sbf1 is expressed in Sertoli’s cells and a subset of germ cells, it is not clear whether the defect in germ cell differentiation in Sbf1^–/– mice is cell autonomous or solely the consequence of Sertoli’s cell dysfunction. To definitively determine this will require germ cell transplantation experiments (24). Nevertheless, our data demonstrate that Sbf1 is necessary for early events of spermatogenesis that are initiated in the pubertal mouse.

Several proteins with pseudophosphatase features have been identified. The most extensively characterized is STYX, which has been shown recently to be essential for spermatid development (25). Recent observations that myotubularin is also expressed in both Sertoli’s and germ cells (26) raise the possibility that phosphatases and pseudophosphatases of this family may function on convergent pathways in spermatogenesis. Several lines of evidence strongly suggest that these proteins are likely to impact specific aspects of lipid-mediated signaling. Sbf1 contains a pleckstrin homology (PH) domain that is responsive to phosphatidylinositol 3-kinase in yeast (27) and thus likely to bind phosphatidylinositol lipids. Sbf1 also contains a motif of unknown function shared with Rab3 GEFs (7) involved in vesicular transport or secretory pathways controlled by the Rab family of GTPases (28). Myotubularin itself dephosphorylates phosphatidylinositol 3-phosphate [PI(3)P] (6, 29) and the presence of PI(3)P-binding FYVE domains in other members of this family (15) implies that myotubularin phosphatases regulate signaling by lipid second messengers. Germline mutations of myotubularin are associated with X-linked myotubular myopathy (XLMTM), a congenital disorder characterized by impaired terminal differentiation of myoblasts (4). Several XLMTM mutations consist of single amino acid substitutions in the phosphatase catalytic pocket of myotubularin and abrogate its ability to dephosphorylate PI(3)P (6), implying that phosphatase activity is critical for its function. Furthermore, a phosphatase-defective myotubularin mutant causes an accumulation of PI(3)P in mammalian cells when hyperexpressed (6). Similar inactivating mutations in the myotubularin-related phosphatase MTMr2 are associated with Charcot-Marie Tooth syndrome (7). Since Sbf1 shares extensive sequence similarity with the myotubularin catalytic pocket, these data raise the possibility that myotubularin-related pseudophosphatases may also regulate, or be regulated by, cellular levels of PI(3)P.

Although Sbf1 or other myotubularin-related proteins have not yet been linked to specific signaling pathways, several pathways are known to affect the premeiotic and meiotic progression of pachytene spermatocytes. For instance, the MAP kinase ERK1 is specifically activated during the G2/M transition in spermatocytes (30). Furthermore, mice harboring mutations in the c-kit tyrosine kinase receptor display defective spermatogenesis characterized by a block at the premeiotic stage (31). The c-kit mutation in these mice disrupts PI 3′-kinase binding and abrogates phosphatidylinositol signaling via Akt (32). Structural features suggest that Sbf1 may function in response to phosphatidylinositol-mediated signaling, however the implicated lipid substrate and Sbf1^–/– testicular phenotype are different from those associated with c-kit. In vitro studies suggest a possible role for Sbf1 in modulating the properties of SET domain proteins such as MLL and Suv39h1 (9, 11). In support of this, a purified protein complex containing Drosophila trithorax (homologue of MLL) also contained dSbf1 and dCBP (33). Interestingly, mice that are compound null for Suv39h1 and the related Suv39h2 display chromosomal mis-segregation during meiosis and apoptosis of pachytene spermatocytes (34). Since Sbf1 interacts with and modulates the transcriptional properties of Suv39h1 in vitro (11), and the timing of the testicular defect in Sbf1^–/– mice also occurs in meiosis, an etiologic relationship of Sbf1 deficiency with Suv39h protein malfunction is possible. However, our own data using spermatocyte gene markers show that pachytene spermatocytes are not initially lost in Sbf1^–/– mice, suggesting that the Sbf1 and Suv39h testicular defects may result from unrelated mechanisms. Nevertheless, this issue warrants further investigation.

Approximately 40% of cases of human male infertility are idiopathic (35). Gene-knockout studies in mice have identified autosomal genes involved in spermatogenesis, but only three candidate genes (AZFa–AZFc) have been reported in humans. Mutations in the AZF genes are the etiological factors in 10–15% of cases of idiopathic azoospermia and severe oligozoospermia (36, 37). Since these genes account for a small proportion of inherited spermatogenic defects, our studies raise the possibility that Sbf1 deficiency may contribute to human male sterility. Therefore, further studies would appear to be warranted to search for hereditary mutations of Sbf1 in appropriate families.

This work was supported by a grant from the NIH (CA-55029). R. Firestein was supported by a training grant from the National Institute of General Medical Sciences (5T32GM07365). We thank Bich-Tien Rouse for Ab preparation, Eva Pfendt for immunohistochemistry, and Caroline Tudor for photographic assistance.

1. Denu, JM, Stuckey, JA, Saper, MA, Dixon, JE. Form and function in protein dephosphorylation. Cell 1996. 87:361-364.
   View this article via: PubMed CrossRef Google Scholar
2. Wishart, MJ, Dixon, JE. Gathering STYX: phosphatase-like form predicts functions for unique protein-interaction domains. Trends Biochem Sci 1998. 23:301-306.
   View this article via: PubMed CrossRef Google Scholar
3. Hunter, T. Anti-phosphatases take the stage. Nat Genet 1998. 18:303-305.
   View this article via: PubMed CrossRef Google Scholar
4. Laporte, J, et al. A gene mutated in X-linked myotubular myopathy defines a new putative tyrosine phosphatase family conserved in yeast. Nat Genet 1996. 13:175-182.
   View this article via: PubMed CrossRef Google Scholar
5. Firestein, R, Cleary, ML. Pseudo-phosphatase Sbf1 contains an N-terminal GEF homology domain that modulates its growth regulatory properties. J Cell Sci 2001. 114:2921-2927.
   View this article via: PubMed Google Scholar
6. Taylor, G, Maehama, T, Dixon, JE. Myotubularin, a protein tyrosine phosphatase mutated in myotubular myopathy, dephosphorylates the lipid second messenger, phosphatidylinositol 3-phosphate. Proc Natl Acad Sci USA 2000. 97:8910-8915.
   View this article via: PubMed CrossRef Google Scholar
7. Bolino, A, et al. Charcot-Marie-Tooth type 4B is caused by mutations in the gene encoding myotubularin-related protein-2. Nat Genet 2000. 25:17-19.
   View this article via: PubMed CrossRef Google Scholar
8. Ali, IU, Schriml, LM, Dean, M. Mutational spectra of PTEN/MMAC1 gene: a tumor suppressor with lipid phosphatase activity. J Natl Cancer Inst 1999. 91:1922-1932.
   View this article via: PubMed CrossRef Google Scholar
9. Cui, X, et al. Association of SET domain and myotubularin-related proteins modulates growth control. Nat Genet 1998. 18:331-337.
   View this article via: PubMed CrossRef Google Scholar
10. De Vivo, I, Cui, X, Domen, J, Cleary, ML. Growth stimulation of primary B cell precursors by the anti-phosphatase Sbf1. Proc Natl Acad Sci USA 1998. 95:9471-9476.
    View this article via: PubMed CrossRef Google Scholar
11. Firestein, R, Cui, X, Huie, P, Cleary, ML. Set domain-dependent regulation of transcriptional silencing and growth control by SUV39H1, a mammalian ortholog of Drosophila Su(var)3-9. Mol Cell Biol 2000. 20:4900-4909.
    View this article via: PubMed CrossRef Google Scholar
12. Flint, AJ, Tiganis, T, Barford, D, Tonks, NK. Development of “substrate-trapping” mutants to identify physiological substrates of protein tyrosine phosphatases. Proc Natl Acad Sci USA 1997. 94:1680-1685.
    View this article via: PubMed CrossRef Google Scholar
13. Salanova, M, et al. Type 4 cyclic adenosine monophosphate-specific phosphodiesterases are expressed in discrete subcellular compartments during rat spermiogenesis. Endocrinology 1999. 140:2297-2306.
    View this article via: PubMed CrossRef Google Scholar
14. Print, CG, et al. Apoptosis regulator bcl-w is essential for spermatogenesis but appears otherwise redundant. Proc Natl Acad Sci USA 1998. 95:12424-12431.
    View this article via: PubMed CrossRef Google Scholar
15. Laporte, J, et al. Characterization of the myotubularin dual specificity phosphatase gene family from yeast to human. Hum Mol Genet 1998. 7:1703-1712.
    View this article via: PubMed CrossRef Google Scholar
16. Yoshida, K, et al. The mouse RecA-like gene Dmc1 is required for homologous chromosome synapsis during meiosis. Mol Cell 1998. 1:707-718.
    View this article via: PubMed CrossRef Google Scholar
17. Wakayama, T, Iseki, S. Specific expression of the mRNA for 25 kDa heat-shock protein in the spermatocytes of mouse seminiferous tubules. Anat Embryol (Berl) 1999. 199:419-425.
    View this article via: PubMed CrossRef Google Scholar
18. Wu, JY, Means, AR. Ca(2+)/calmodulin-dependent protein kinase IV is expressed in spermatids and targeted to chromatin and the nuclear matrix. J Biol Chem 2000. 275:7994-7999.
    View this article via: PubMed CrossRef Google Scholar
19. Bellve, AR, et al. Spermatogenic cells of the prepuberal mouse. Isolation and morphological characterization. J Cell Biol 1977. 74:68-85.
    View this article via: PubMed CrossRef Google Scholar
20. Gondos, B., and Berndston, W.E. 1993. Sertoli cell toxicants. In The Sertoli cell. L.D. Russell and M.D. Griswold, editors. Cache River Press. Clearwater, Florida, USA. 552–575.
21. Chapin, RE, Morgan, KT, Bus, JS. The morphogenesis of testicular degeneration induced in rats by orally administered 2,5-hexanedione. Exp Mol Pathol 1983. 38:149-169.
    View this article via: PubMed CrossRef Google Scholar
22. Boekelheide, K. 1993. Sertoli cell toxicants. In The Sertoli cell. L.D. Russell and M.D. Griswold, editors. Cache River Press. Clearwater, Florida, USA. 552–575.
23. Creasy, DM, Foster, JR, Foster, PM. The morphological development of di-N-pentyl phthalate induced testicular atrophy in the rat. J Pathol 1983. 139:309-321.
    View this article via: PubMed CrossRef Google Scholar
24. Ogawa, T. Spermatogonial transplantation: the principle and possible applications. J Mol Med 2001. 79:368-374.
    View this article via: PubMed CrossRef Google Scholar
25. Wishart, MJ, Dixon, JE. The archetype STYX/dead-phosphatase complexes with a spermatid mRNA-binding protein and is essential for normal sperm production. Proc Natl Acad Sci USA 2002. 99:2112-2117.
    View this article via: PubMed CrossRef Google Scholar
26. Li, JC, et al. Rat testicular myotubularin, a protein tyrosine phosphatase expressed by Sertoli and germ cells, is a potential marker for studying cell-cell interactions in the rat testis. J Cell Physiol 2000. 185:366-385.
    View this article via: PubMed CrossRef Google Scholar
27. Isakoff, SJ, et al. Identification and analysis of PH domain-containing targets of phosphatidylinositol 3-kinase using a novel in vivo assay in yeast. EMBO J 1998. 17:5374-5387.
    View this article via: PubMed CrossRef Google Scholar
28. Martinez, O, Goud, B. Rab proteins. Biochim Biophys Acta 1998. 1404:101-112.
    View this article via: PubMed Google Scholar
29. Blondeau, F, et al. Myotubularin, a phosphatase deficient in myotubular myopathy, acts on PI 3-kinase and phosphatidylinositol 3 phosphate pathway. Hum Mol Genet 2000. 9:2223-2229.
    View this article via: PubMed Google Scholar
30. Sette, C, et al. Activation of the mitogen-activated protein kinase ERK1 during meiotic progression of mouse pachytene spermatocytes. J Biol Chem 1999. 274:33571-33579.
    View this article via: PubMed CrossRef Google Scholar
31. Kissel, H, et al. Point mutation in kit receptor tyrosine kinase reveals essential roles for kit signaling in spermatogenesis and oogenesis without affecting other kit responses. EMBO J 2000. 19:1312-1326.
    View this article via: PubMed CrossRef Google Scholar
32. Blume-Jensen, P, et al. Kit/stem cell factor receptor-induced activation of phosphatidylinositol 3′-kinase is essential for male fertility. Nat Genet 2000. 24:157-162.
    View this article via: PubMed CrossRef Google Scholar
33. Petruk, S, et al. Trithorax and dCBP acting in a complex to maintain expression of a homeotic gene. Science 2001. 294:1331-1334.
    View this article via: PubMed CrossRef Google Scholar
34. Peters, AH, et al. Loss of the Suv39h histone methyltransferases impairs mammalian heterochromatin and genome stability. Cell 2001. 107:323-337.
    View this article via: PubMed CrossRef Google Scholar
35. Kretser, DM. Male infertility. Lancet 1997. 349:787-790.
    View this article via: PubMed CrossRef Google Scholar
36. Ferlin, A, Moro, E, Garolla, A, Foresta, C. Human male infertility and Y chromosome deletions: role of the AZF-candidate genes DAZ, RBM and DFFRY. Hum Reprod 1999. 14:1710-1716.
    View this article via: PubMed CrossRef Google Scholar
37. Hargreave, TB. Genetics and male infertility. Curr Opin Obstet Gynecol 2000. 12:207-219.
    View this article via: PubMed CrossRef Google Scholar