Stephanie Schubert

Hspa4l-Deficient Mice Display Increased Incidence of Male Infertility and Hydronephrosis Development

ABSTRACT The Hspa4l gene, also known as Apg1 or Osp94, belongs to the HSP110 heat shock gene family, which includes three genes encoding highly conserved proteins. This study shows that Hspa4l is expressed ubiquitously and predominantly in the testis. The protein is highly expressed in spermatogenic cells, from late pachytene spermatocytes to postmeiotic spermatids. In the kidney, the protein is restricted to cortical segments of distal tubules. To study the physiological role of this gene in vivo, we generated mice deficient in Hspa4l by gene targeting. Hspa4l-deficient mice were born at expected ratios and appeared healthy. However, approximately 42% of Hspa4l−/− male mice suffered from fertility defects. Whereas the seminiferous tubules of Hspa4l−/− testes contained all stages of germ cells, the number of mature sperm in the epididymis and sperm motility were drastically reduced. The reduction of the sperm count was due to the elimination of a significant number of developing germ cells via apoptosis. No defects in fertility were observed in female mutants. In addition, 12% of null mutant mice developed hydronephrosis. Concentrations of plasma and urine electrolytes in Hspa4l−/− mice were similar to wild-type values, suggesting that the renal function was not impaired. However, Hspa4l−/− animals were preferentially susceptible to osmotic stress. These results provide evidence that Hspa4l is required for normal spermatogenesis and suggest that Hspa4l plays a role in osmotolerance.

Reduction of Spermatogenesis but Not Fertility in Creb3l4-Deficient Mice

ABSTRACT Creb3l4 belongs to the CREB/ATF family of transcription factors that are involved in mediating transcription in response to intracellular signaling. This study shows that Creb3l4 is expressed at low levels in all organs and in different stages of embryogenesis but is present at very high levels in the testis, particularly in postmeiotic male germ cells. In contrast to CREB3L4 in the human prostate, of which specific expression was detected, Creb3l4 transcripts in the mouse prostate could be detected only by RT-PCR. To identify the physiological function of Creb3l4, the murine gene was inactivated by replacement with the gene encoding green fluorescent protein. Surprisingly, Creb3l4-deficient mice were born at expected ratios, were healthy, and displayed normal long-term survival rates. Despite a significant reduction in the number of spermatozoa in the epididymis of Creb3l4− / − mice, the breeding of mutant males with wild-type females was productive and the average litter size was not significantly altered in comparison to wild-type littermates. Further analyses revealed that the seminiferous tubules of Creb3l4 − / − mice contained all of the developmental stages, though there was evidence for increased apoptosis of meiotic/postmeiotic germ cells. These results suggest that Creb3l4 plays a role in male germ cell development, but its loss is insufficient to completely compromise the production of spermatozoa.

Compartmentalization and regulation of iron metabolism proteins protect male germ cells from iron overload

The universal importance of iron, its high toxicity, and complex chemistry present a challenge to biological systems in general and to protected compartments in particular. The high mitotic rate and avid mitochondriogenesis of developing male germ cells imply high iron requirements. Yet access to germ cells is tightly regulated by the blood-testis barrier that protects the meiotic and postmeiotic germ cells. To elucidate how iron is supplied to developing male germ cells, we analyzed iron deposition and iron transport proteins in testes of mice with iron overload and with genetic ablation of the iron regulators Hfe and iron regulatory protein 2. Iron accumulated mainly around seminiferous tubules, and only small amounts localized within the seminiferous tubules. The localization and regulation of proteins involved in iron import, storage, and export such as transferrin, transferrin receptor, the divalent metal transporter-1, cytosolic ferritin, and ferroportin strongly support a model of a largely autonomous iron cycle within seminiferous tubules. We show evidence that ferritin secretion from Sertoli cells may play an important role in iron acquisition of primary spermatocytes. During spermatogenic development iron is carried along from primary spermatocytes to spermatids, and from spermatids iron is recycled to the apical compartment of Sertoli cells, which traffic it back to a new generation of spermatocytes. Losses are replenished by the peripheral circulation. Such an internal iron cycle essentially detaches the iron homeostasis within the seminiferous tubule from the periphery and protects developing germ cells from iron fluctuations. This model explains how compartmentalization can optimize cellular and systemic nutrient homeostasis.

Generation and Characterization of a Transgenic Mouse with a Functional Human TSPY

Abstract To generate an animal model that is suitable for the analysis of regulation and expression of human testis-specific protein, Y-encoded TSPY, a transgenic mouse line, TgTSPY9, harboring a complete structural human TSPY gene was generated. Fluorescence in situ hybridization and Southern analyses show that approximately 50 copies of the human TSPY transgene are integrated at a single chromosomal site that maps to the distal long arm of the Y chromosome. The transgene is correctly transcribed and spliced according to the human pattern and is mainly expressed in testicular tissue, with spermatogonia and early primary spermatocytes (leptotene and zygotene) as expressing germ cells. TSPY transgenic mice are phenotypically normal, and spermatogenesis is neither impaired nor enhanced by the human transgene. The present study shows that a human TSPY gene integrated into the mouse genome follows the human expression pattern although murine tspy had lost its function in rodent evolution millions of years ago.

CFTR, SPINK1, PRSS1, and CTRC mutations are not associated with pancreatic cancer in German patients.

Objective Mutations in the cationic trypsinogen (PRSS1), cystic fibrosis transmembrane conductance regulator (CFTR), serine protease inhibitor Kazal type 1 (SPINK1), and chymotrypsin C (CTRC) genes are associated with an elevated risk for chronic pancreatitis, which is a known risk factor for pancreatic cancer (PC). Therefore, we analyzed whether PRSS1, CFTR, SPINK1, and/or CTRC mutations are associated with pancreatic adenocarcinoma. Methods The study cohort was composed of 121 PC patients, of whom 74 were classified as having chronic pancreatitis, 102 patients with idiopathic chronic pancreatitis, and 130 as healthy controls. Mutation analyses for the CFTR, SPINK1, PRSS1, and CTRC genes were performed for the presence of the most common mutations. Results The frequency of CFTR mutations in patients with PC was not significantly different in comparison with healthy controls and controls with pancreatitis. The SPINK1 mutation frequency was significantly decreased in patients with PC in comparison with patients with idiopathic pancreatitis but varied not significantly in comparison with healthy controls. None of the selected 121 PC samples showed a pancreatitis-predisposing mutation in the PRSS1 or CTRC gene. Conclusions Mutations in the genes CFTR, SPINK1, PRSS1, and CTRC do not seem to significantly increase the risk for pancreatic adenocarcinoma.

1,25-Dihydroxyvitamin D decreases HTRA1 promoter activity in the rhesus monkey--a plausible explanation for the influence of vitamin D on age-related macular degeneration?

Age-related macular degeneration is the major cause of blindness in the elderly worldwide and the risk is influenced by both environmental and genetic risk factors. One important disease-associated region in humans is located on 10q26 and includes the two candidate genes ARMS2 and HTRA1. However, determination of the causative gene has not yet been possible and examining the situation in the rhesus monkey may help understand the situation in humans. In a recent paper, we characterized the rhesus monkey 10q26-orthologue region on chromosome 9 in detail and identified the drusen-associated HTRA1 promoter SNP rs196357513 as a putative risk factor. In this study, we predicted 9 binding sites for the vitamin D-dependent transcription factor vitamin D receptor in the rhesus HTRA1 promoter, one of which is destroyed by the rs196357513-risk allele. As patients with vitamin D deficit are at increased risk for age-related macular degeneration, a luciferase assay in transiently transfected ARPE19-cells was performed to evaluate the influence of the SNP rs196357513 and of 1,25-dihydroxyvitamin D on the rhesus monkey HTRA1 promoter activity. This revealed that the luciferase activity of the promoter construct containing the rs196357513 wild type allele was significantly reduced after vitamin D stimulation. An in silico analysis and literature search imply that this regulation could also play a role in human HTRA1 expression. Moreover, HTRA1 promoter activity of the construct containing the rs196357513 risk allele appeared diminished in comparison to the construct with the wild type allele, albeit this difference was not significant. The lower promoter activity due to the rhesus monkey rs196357513 risk allele apparently contradicts the common hypothesis for the human HTRA1 promoter risk allele of SNP rs11200638, for which a higher promoter activity has been observed. Our data point to a yet unexpected effect of decreased HTRA1 expression on drusen pathogenesis. Thus not only a higher HTRA1 expression, but an imbalance of HTRA1 might be disease-relevant. Both findings require closer analysis, but if relevance for humans proves true, it would impact current age-related macular degeneration research and treatment.

Characterization of a human TSPY promoter

Human TSPY is a candidate oncogene and is supposed to function as a proliferation factor during spermatogenesis. It is the only mammalian protein-coding gene known to be organized as a tandem repeat gene family. It is expressed at highest level in spermatogonia and to a lower amount in primary spermatocytes. To characterize the human TSPY promoter we used the luciferase reporter system in a mouse spermatogonia derived cell line (GC-1spg) and in a GC-4spc cell line, that harbour prophase spermatocytes of the preleptotene and early pachytene stage. We isolated a 1303 bp fragment of the 5′-flanking region of exon 1 that shows significant promoter activity in GC-1spg and reduced activity in GC-4spc cells. In order to gain further insight into the organization of the TSPY-promoter, stepwise truncations of the putative promoter sequence were performed. The resulting fragments were cloned into the pGL3-vector and analysed for reporter gene activity in the murine germ cell lines GC-1spg and GC-4spc, leading to the characterization of a core promoter (−159 to −1), an enhancing region (−673 to −364) and a silencing region (−1262 to −669). Database research for cis-active elements yielded two putative SOX-like binding sites in the enhancing region and reporter gene activity was drastically reduced when three nucleotides of the AACAAT SOX core sequence were mutated. Our findings strongly suggest that testis-specific expression of human TSPY is mediated by Sox proteins. (Mol Cell Biochem 276: 159–167, 2005)

Expression of the human TSPY gene in the brains of transgenic mice suggests a potential role of this Y chromosome gene in neural functions

The testis specific protein Y-encoded (TSPY) is a member of TSPY/SET/NAP1 superfamily, encoded within the gonadoblastoma locus on the Y chromosome. TSPY shares a highly conserved SET/NAP-domain responsible for protein--protein interaction among TSPY/SET/NAP1 proteins. Accumulating data, so far, support the role of TSPY as the gonadoblastoma gene, involved in germ cell tumorigenesis. The X-chromosome homolog of TSPY, TSPX is expressed in various tissues at both fetal and adult stages, including the brain, and is capable of interacting with the multi-domain adapter protein CASK, thereby influencing the synaptic and transcriptional functions and developmental regulation of CASK in the brain and other neural tissues. Similar to TSPX, we demonstrated that TSPY could interact with CASK at its SET/NAP-domain in cultured cells. Transgenic mice harboring a human TSPY gene and flanking sequences showed specific expression of the human TSPY transgene in both testis and brain. The neural expression pattern of the human TSPY gene overlapped with those of the endogenous mouse Cask and Tspx gene. Similarly with TSPX, TSPY was co-localized with CASK in neuronal axon fibers in the brain, suggesting a potential role(s) of TSPY in development and/or physiology of the nervous system.

TSPY Expression Is Variably Altered in Transgenic Mice with Testicular Feminization

Abstract TSPY (testis-specific protein, Y-encoded) genes are expressed in premeiotic germ cells and round spermatids. The topology and timing of TSPY expression, and also its homology to members of the TTSN-family, suggest that TSPY is a proliferation factor for germ cells. There is also evidence for a role of TSPY in the aetiology of testis cancer. TSPY is a candidate for GBY, the elusive gonadoblastoma locus on the human Y chromosome, which is thought to predispose dysgenetic gonads of 46, XY sex-reversed females to develop gonadoblastoma. We have previously generated a TSPY transgenic mouse line (Tg(TSPY)9Jshm) that carries approximately 50 copies of the human TSPY gene on the mouse Y chromosome. In order to elucidate TSPY expression under complete androgen insensitivity and to investigate a possible role of TSPY in gonadal tumorigenesis, we have now generated sex-reversed TSPY transgenic ArTfm mice hemizygous for the X-linked testicular feminization mutation (ArTfm). We can show that the TSPY transcript is aberrantly spliced in the testes of TSPY-ArTfm mice, and that TSPY expression is upregulated by androgen insensitivity in some but not all animals. TSPY transgenic mice showed significantly increased testes weights. In one TSPY transgenic ArTfm animal, spermatogenesis proceeded beyond meiotic prophase. No tumors of germ cell origin were found in the testes of TSPY-ArTfm mice. Five out of 46 TSPY transgenic ArTfm mice, and 3 out of 31 age-related NMRI-ArTfm controls developed Leydig cell tumors, whereas none of the age-matched ArTfm mice (n = 44) on a wild type background were affected by Leydig cell tumorigenesis.

Transgenic Mouse Studies to Understand the Regulation, Expression and Function of the Testis-Specific Protein Y-Encoded (TSPY) Gene

The TSPY gene, which encodes the testis-specific protein, Y-encoded, was first discovered and characterized in humans, but orthologous genes were subsequently identified on the Y chromosome of many other placental mammals. TSPY is expressed in the testis and to a much lesser extent in the prostate gland, and it is assumed that TSPY serves function in spermatogonial proliferation and/or differentiation. It is further supposed that TSPY is involved in male infertility and exerts oncogenic effects in gonadal and prostate tumor formation. As a member of the TSPY/SET/NAP protein family, TSPY is able to bind cyclin B types, and stimulates the cyclin B1-CDK1 kinase activity, thereby accelerating the G2/M phase transition of the cell cycle of target cells. Because the laboratory mouse carries only a nonfunctional Y-chromosomal Tspy-ps pseudogene, a knockout mouse model for functional research analyses is not a feasible approach. In the last decade, three classical transgenic mouse models have been developed to contribute to our understanding of TSPY regulation, expression and function. The different transgenic mouse approaches and their relevance for studying TSPY regulation, expression and function are discussed in this review.