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Impaired spermatogenesis and fertility in mice carrying a mutation in the Spink2 gene expressed predominantly in testes.

ABSTRACT: Spermatogenesis is a complex process involving an intrinsic genetic program composed of germ cell-specific and -predominant genes. In this study, we investigated the mouse Spink2 (serine protease inhibitor Kazal-type 2) gene, which belongs to the SPINK family of proteins characterized by the presence of a Kazal-type serine protease inhibitor-pancreatic secretory trypsin inhibitor domain. We showed that recombinant mouse SPINK2 has trypsin-inhibitory activity. Distribution analyses revealed that Spink2 is transcribed strongly in the testis and weakly in the epididymis, but is not detected in other mouse tissues. Expression of Spink2 is specific to germ cells in the testis and is first evident at the pachytene spermatocyte stage. Immunoblot analyses demonstrated that SPINK2 protein is present in male germ cells at all developmental stages, including in testicular spermatogenic cells, testicular sperm, and mature sperm. To elucidate the functional role of SPINK2 in vivo, we generated mutant mice with diminished levels of SPINK2 using a gene trap mutagenesis approach. Mutant male mice exhibit significantly impaired fertility; further phenotypic analyses revealed that testicular integrity is disrupted, resulting in a reduction in sperm number. Moreover, we found that testes from mutant mice exhibit abnormal spermatogenesis and germ cell apoptosis accompanied by elevated serine protease activity. Our studies thus provide the first demonstration that SPINK2 is required for maintaining normal spermatogenesis and potentially regulates serine protease-mediated apoptosis in male germ cells.

SUBMITTER: Lee B

PROVIDER: S-EPMC3190718 | biostudies-other | 2011 Aug

REPOSITORIES: biostudies-other

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Impaired spermatogenesis and fertility in mice carrying a mutation in the Spink2 gene expressed predominantly in testes.

Lee Boyeon B Park Inju I Jin Sora S Choi Heejin H Kwon Jun Tae JT Kim Jihye J Jeong Juri J Cho Byung-Nam BN Eddy Edward M EM Cho Chunghee C

The Journal of biological chemistry 20110624 33

Spermatogenesis is a complex process involving an intrinsic genetic program composed of germ cell-specific and -predominant genes. In this study, we investigated the mouse Spink2 (serine protease inhibitor Kazal-type 2) gene, which belongs to the SPINK family of proteins characterized by the presence of a Kazal-type serine protease inhibitor-pancreatic secretory trypsin inhibitor domain. We showed that recombinant mouse SPINK2 has trypsin-inhibitory activity. Distribution analyses revealed that ...[more]

PMID: 21705336

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Project description:BACKGROUND:Previously, we have demonstrated that genes involved in ovarian function are highly conserved throughout evolution. In this study, we aimed to document the conservation of genes involved in spermatogenesis from flies to vertebrates and their expression profiles in vertebrates. RESULTS:We retrieved 379 Drosophila melanogaster genes that are functionally involved in male reproduction according to their mutant phenotypes and listed their vertebrate orthologs. 83% of the fly genes have at least one vertebrate ortholog for a total of 625 mouse orthologs. This conservation percentage is almost twice as high as the 42% rate for the whole fly genome and is similar to that previously found for genes preferentially expressed in ovaries. Of the 625 mouse orthologs, we selected 68 mouse genes of interest, 42 of which exhibited a predominant relative expression in testes and 26 were their paralogs. These 68 mouse genes exhibited 144 and 60 orthologs in chicken and zebrafish, respectively, gathered in 28 groups of paralogs. Almost two thirds of the chicken orthologs and half of the zebrafish orthologs exhibited a relative expression ?50% in testis. Finally, our focus on functional in silico data demonstrated that most of these genes were involved in the germ cell process, primarily in structure elaboration/maintenance and in acid nucleic metabolism. CONCLUSION:Our work confirms that the genes involved in germ cell development are highly conserved across evolution in vertebrates and invertebrates and display a high rate of conservation of preferential testicular expression among vertebrates. Among the genes highlighted in this study, three mouse genes (Lrrc46, Pabpc6 and Pkd2l1) have not previously been described in the testes, neither their zebrafish nor chicken orthologs. The phylogenetic approach developed in this study finally allows considering new testicular genes for further fundamental studies in vertebrates, including model species (mouse and zebrafish).

Project description:The role of cytokines in testicular function under normal conditions has not been completely understood. Here, we evaluated testicular macrophages (TM), steroidogenesis by Leydig cells (LC) and seminiferous tubules integrity in cytokines-deficient rat testes induced by diacerein, an anti-inflammatory drug that inhibits interleukin-1 (IL-1) and tumor necrosis factor-alpha (TNF-α). Male rats received daily 100 mg/kg of diacerein (DIAG; n = 8) or saline (CG; n = 8) for 30 days. Serum testosterone (T) levels were measured and the seminiferous tubule (ST) area, epithelial area (EA), frequency of damaged ST and number of Sertoli cells (SC) were evaluated. TUNEL method and immunoreactions for detection of pro-IL-1β, TNF-α, steroidogenic acute regulatory protein (StAR), 17β-hydroxysteroid dehydrogenase (17β-HSD), androgen receptor (AR) and scavenger receptor for hemoglobin-haptoglobin complexes (CD163), a TM marker, were performed. Testicular AR, 17β-HSD and IL-1β levels were detected by Western blot. Data were submitted to Student t test (p ≤ 0.05). In DIAG, T and testicular AR, 17β-HSD and IL-1β levels decreased significantly (p < 0.05). The number of TUNEL-positive interstitial cells increased and LC showed weak StAR, 17β-HSD and AR immunoexpression in association with reduced IL-1β immunoexpression and number of CD163-positive TM in the interstitial tissue from diacerein-treated rats. Numerous damaged ST were found in DIAG, and reduction in the EA were associated with germ cells death. Moreover, the number of SC reduced and weak AR and TNF-α immunoexpression was observed in SC and germ cells, respectively. The cytokines deficiency induced by diacerein impairs TM, LC and spermatogenesis, and points to a role of IL-1β in steroidogenesis under normal conditions. In the ST, the weak AR and TNF-α immunoexpression in SC and germ cells, respectively, reinforces the idea that TNF-α plays a role in the SC androgenic control.

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Impaired spermatogenesis and fertility in mice carrying a mutation in the Spink2 gene expressed predominantly in testes.

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Impaired spermatogenesis and fertility in mice carrying a mutation in the Spink2 gene expressed predominantly in testes.

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