Spermatogenesis and the regulation of Ca(2+)-calmodulin-dependent protein kinase IV localization are not dependent on calspermin

Calspermin and Ca(2+)-calmodulin-dependent protein kinase IV (CaMKIV) are two proteins encoded by the Camk4 gene. CaMKIV is found in multiple tissues, including brain, thymus, and testis, while calspermin is restricted to the testis. In the mouse testis, both proteins are expressed within elongating spermatids. We have recently shown that deletion of CaMKIV has no effect on calspermin expression but does impair spermiogenesis by disrupting the exchange of sperm basic nuclear proteins. The function of calspermin within the testis is unclear, although it has been speculated to play a role in binding and sequestering calmodulin during the development of the germ cell. To investigate the contribution of calspermin to spermatogenesis, we have used Cre/lox technology to specifically delete calspermin, while leaving kinase expression intact. We unexpectedly found that calspermin is not required for male fertility. We further demonstrate that CaMKIV expression and localization are unaffected by the absence of calspermin and that calspermin does not colocalize to the nuclear matrix with CaMKIV.     

MARCH7 E3 ubiquitin ligase is highly expressed in developing spermatids of rats and its possible involvement in head and tail formation

Spermatogenesis is a highly complicated metamorphosis process of male germ cells. Recent studies have provided evidence that the ubiquitin–proteasome system plays an important role in sperm head shaping, but the underlying mechanism is less understood. In this study, we localized membrane-associated RING-CH (MARCH)7, an E3 ubiquitin ligase, in rat testis. Northern blot analysis showed that March7 mRNA is expressed ubiquitously but highly in the testis and ovary. In situ hybridization of rat testis demonstrated that March7 mRNA is expressed weakly in spermatogonia and its level is gradually increased as they develop. Immunohistochemical analysis detected MARCH7 protein expression in spermiogenic cells from late round spermatids to elongated spermatids and in epididymal spermatozoa. Moreover, MARCH7 was found to be localized to the caudal end of the developing acrosome of late round and elongating spermatids, colocalizing with β-actin, a component of the acroplaxome. In addition, MARCH7 was also detected in the developing flagella and its expression levels were prominent in elongated spermatids. We also showed that MARCH7 catalyzes lysine 48 (K48)-linked ubiquitination. Immunolocalization studies revealed that K48-linked ubiquitin chains were detected in the heads of elongating spermatids and in the acrosome/acroplaxome, neck, midpiece and cytoplasmic lobes of elongated spermatids. These results suggest that MARCH7 is involved in spermiogenesis by regulating the structural and functional integrity of the head and tail of developing spermatids.     

Tsp57: a novel gene induced during a specific stage of spermatogenesis

Recently, we described the identification of a novel protein, nuclear receptor-associated protein 80 (RAP80), which is highly expressed in spermatocytes and appears to have a role in regulating gene expression. To identify proteins interacting with this protein, we performed yeast two-hybrid screening using full-length RAP80 as bait. This screen identified one in-frame clone encoding a novel testis-specific protein (Tsp), referred to as Tsp57. Tsp57 encodes a basic protein with a mass of 56.8 kDa. The amino acid sequence of Tsp57 is highly conserved (87%) between mouse and human. The mouse and human Tsp57 genes map to chromosomes 9A1 and 11q21, respectively. Northern blot analysis showed that the expression of Tsp57 mRNA was highly restricted to the testis and temporally regulated during testicular development. Tsp57 mRNA was greatly induced between Day 21 and Day 25 of postnatal testicular development. In situ hybridization analysis demonstrated that the hybridization signal for Tsp57 mRNA was strongest in sections of seminiferous tubules at stages VI-VIII of spermatogenesis, consistent with the conclusion that Tsp57 is most highly expressed in round spermatids. Study of Tsp57 expression in several purified subpopulations of spermatogenic cells confirmed maximum levels of expression in round spermatids. Consistently, Tsp57 expression was absent in testes from vitamin A-deficient mice, which do not have any round spermatids, and was reduced in RARalpha null mice, which have lowered numbers of round spermatids in their testes. These results indicate the possibility that Tsp57 protein plays a role in the postmeiotic phase of germ cell differentiation. Tsp57 contains two putative nuclear localization signals: NLS1 and NLS2. Examination of the cellular localization showed that the green fluorescent protein-Tsp57 fusion protein localized to both cytoplasm and nucleus. After deletion of NLS1 but not NLS2, Tsp57 localized solely to the cytoplasm, indicating a role for NLS1 in the nuclear localization of Tsp57. The localization suggests a nuclear function for Tsp57. Pull-down analysis demonstrated that Tsp57 and RAP80 form a complex in intact cells.     

Identification of a heat-shock protein Hsp40, DjB1, as an acrosome- and a tail-associated component in rodent spermatozoa

Iba1 is a 17-kDa EF-hand protein highly expressed in the cytoplasm of elongating spermatids in testis. Using Iba1 as a bait, we performed yeast Two-hybrid screening and isolated a heat-shock protein Hsp40, DjB1, from cDNA library of mouse testis. To characterize DjB1 that is encoded by Dnajb1 gene, we carried out immunoblot analyses, in situ hybridization, and immunohistochemistry. Immunoblot analyses showed that DjB1was constitutively expressed in mouse testis and that its expression level was not changed by heat shock. Dnajb1 mRNA was exclusively expressed in spermatocytes and round spermatids in mouse testis, and Dnajb1 protein DjB1 was predominantly expressed in the cytoplasm of spermatocytes, round spermatids, and elongating spermatids. In mature mouse spermatozoa, DjB1 was localized in the middle and the end pieces of flagella as well as in association with the head (acrosomal region). Association of DjB1 with the acrosomal region in sperm head was also observed in rat spermatozoa. These data suggested that DjB1, which was constitutively expressed in postmeiotic spermatogenic cells in testis, was integrated into spermatozoa as at least two components, that is, sperm head and tail of rodent spermatozoa.     

Molecular cloning of a novel putative G-protein coupled receptor expressed during rat spermiogenesis.

A cDNA clone encoding a novel putative G-protein coupled receptor has been isolated from a rat testis cDNA library using a PCR-amplified cDNA fragment as a hybridization probe. Northern blot analysis reveals that a corresponding 1.5 kb mRNA is exclusively expressed in testis. By in situ hybridization experiments this mRNA has been localized in spermatocytes and spermatids but not in spermatogonia, Leydig or sertoli cells. Ontogenic studies show that expression of the receptor-encoding mRNA and sexual maturation are correlated reaching highest levels during the second and third months. Although the ligand for this receptor has not yet been identified, this receptor may play a role during reproduction.     

Differential Expression and Sex Chromosome Association of CHD3/4 and CHD5 during Spermatogenesis

ATP-dependent nucleosome remodelers of the CHD family play important roles in chromatin regulation during development and differentiation. The ubiquitously expressed CHD3 and CHD4 proteins are essential for stem cell function and serve to orchestrate gene expression in different developmental settings. By contrast, the closely related CHD5 is predominantly expressed in neural tissue and its role is believed to be restricted to neural differentiation. Indeed, loss of CHD5 contributes to neuroblastoma. In this study, we first demonstrate that CHD5 is a nucleosome-stimulated ATPase. We then compare CHD3/4 and CHD5 expression in mouse brain and show that CHD5 expression is restricted to a subset of cortical and hippocampal neurons whereas CHD3/4 expression is more widespread. We also uncover high levels of CHD5 expression in testis. CHD5 is transiently expressed in differentiating germ cells. Expression is first detected in nuclei of post-meiotic round spermatids, reaches a maximum in stage VIII spermatids and then falls to undetectable levels in stage IX spermatids. Surprisingly, CHD3/4 and CHD5 show complementary expression patterns during spermatogenesis with CHD3/4 levels progressively decreasing as CHD5 expression increases. In spermatocytes, CHD3/4 localizes to the pseudoautosomal region, the X centromeric region and then spreads into the XY body chromatin. In postmeiotic cells, CHD5 colocalises with macroH2A1.2 in association with centromeres and part of the Y chromosome. The subnuclear localisations of CHD4 and CHD5 suggest specific roles in regulation of sex chromosome chromatin and pericentromeric chromatin structure prior to the histone-protamine switch.     

Molecular cloning sequence and distribution of rat calspermin, a high affinity calmodulin-binding protein

Calspermin is a heat-stable, acidic calmodulin-binding protein predominantly found in mammalian testis. The cDNA representing the rat form of this protein has been cloned from a rat testis lambda gt11 library. Sequence analysis of two overlapping clones revealed a 232-nucleotide 5'-nontranslated region, 510 nucleotides of open reading frame, a 148-nucleotide 3'-untranslated region, and a poly(A) tail. Authenticity of the clones was confirmed by comparison of a portion of the deduced amino acid sequence with the sequence of a tryptic peptide obtained from the rat testis protein. The lambda gt11 fusion protein was recognized by affinity purified antibodies to pig testis calspermin and bound 125I-calmodulin in a Ca2+-dependent manner. Calspermin cDNA encodes a 169-residue protein with a calculated Mr of 18,735. The putative calmodulin-binding domain is very close to the amino terminus of the protein. This region shows 46% identity with the calmodulin-binding region of rat brain Ca2+/calmodulin-dependent protein kinase II and 32% identity with the equivalent region of chicken smooth muscle myosin light chain kinase. The 5'-nontranslated region reveals significant homology with a portion of the catalytic region of the calmodulin-dependent protein kinase family. Calspermin contains a stretch of 17 contiguous glutamic acid residues in the central region of the molecule. Computer analysis predicts calspermin to be 81% alpha-helix and 14% random coil. Analysis of genomic DNA indicates calspermin to be the product of a unique gene. Northern blot analysis of rat testis RNA reveals a 1.1-kilobase mRNA. This RNA is restricted to testis among several rat tissues examined and could not be identified in total RNA isolated from testes of other mammals. Analysis of cells isolated from rat testis reveals calspermin mRNA to be predominantly expressed in postmeiotic cells indicating that it may be specific to haploid cells.     

SRG4在出生后小鼠睾丸及隐睾中的表达特性

研究小鼠生精新基因SRG4在出生后小鼠睾丸及手术隐睾中的表达特性, 为了解SRG4在精子发生中的作用奠定基础。取出生后1, 3, 12 w小鼠睾丸进行免疫组化检测, 观察SRG4蛋白在出生后小鼠不同发育阶段睾丸中的表达; 制备单侧手术隐睾模型, 取术后0~18 d 的隐睾组织进行半定量RT-PCR检测, 观察SRG4 mRNA在隐睾病变过程中的表达变化, 并对隐睾术后18 d 睾丸进行组织原位杂交分析。免疫组化分析结果表明, SRG4蛋白在出生1 w的小鼠睾丸中几乎检测不到, 在出生3 w的小鼠睾丸中有明显表达, 在出生12 w的小鼠中大量表达, 主要分布在精母细胞和圆形精子细胞胞浆及胞膜, 呈不均匀分布。半定量RT-PCR结果发现, SRG4 mRNA在小鼠隐睾术后0~6 d表达没有明显下调, 9 d 开始表达下调, 第18 d表达最低。组织原位杂交结果表明, 术后18 d隐睾睾丸生殖细胞大量凋亡, 精曲小管中仅见到个别的SRG4阳性信号, 而对照则不受影响。上述结果说明, SRG4蛋白表达受小鼠生长发育调控; 隐睾模型中, 随着生殖细胞的大量凋亡, SRG4基因表达下调, 提示SRG4基因可作为一个精子发生特定阶段的分子标记用以研究精子发生过程。     

Expression of the Centrosomal Colon Cancer Autoantigen Gene during Spermatogenesis in the Maturing Rat Testis

We analyzed the gene and protein expression of serologically defined colon cancer antigen 8. Gene expression was upregulated in the maturing rat testis, and was localized to the spermatocytes. Protein was detected in the spermatids and at the sites of mRNA expression. Specific expression of colon cancer antigen 8 was observed in the maturing rat testis.