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PubMed2002

Molecular cloning and characterization of a complementary DNA encoding sperm tail protein SHIPPO 1.

Formation of the tail in developing sperm is a complex process involving the organization of the axoneme, transport of periaxonemal proteins from the cytoplasm to the tail, and assembly of the outer dense fibers and fibrous sheath. Although detailed morphological descriptions of these events are available, the molecular mechanisms remain to be fully elucidated. We have isolated a new gene, named shippo 1, from a haploid germ cell-specific cDNA library of mouse testis, and also its human orthologue (h-shippo 1). The isolated cDNA is 1.2 kilobases long, carrying a 762-base pair open reading frame that encodes SHIPPO 1, a sperm protein predicted to consist of 254 amino acids. The amino acid sequence includes 6 Pro-Gly-Pro repeats, which are also present in the human orthologue protein (hSHIPPO 1) as well as in 2 other newly reported proteins of Drosophila melanogaster. Transcription of shippo 1 is exclusively observed in haploid germ cells. Antibody raised against SHIPPO 1 identified a testis-specific M(r) 32 x 10(-3) band in Western blot analysis. The protein was further localized in the flagella of the elongated spermatids and along the entire length of the tail in mature sperm. SHIPPO 1 in sperm is resistant to treatment with nonionic detergents and coextracted with the cytoskeletal core proteins of the mouse sperm tail.

Amino Acid Sequence↗
PubMed1996

Expression of IL-3 receptor in testis.

Interleukin-3 (IL-3) is a potent growth factor for hematopoietic cells. IL-3 transmits its signal through a specific cell surface receptor which consists of a primary binding alpha subunit (IL-3R alpha) and a signaling beta subunit that is shared by the IL-5 and GM-CSF receptors. Here, we describe the expression of the alpha and beta subunit of the IL-3 receptor in mouse testis and testicular cell lines both at nucleic acid and protein levels. Our data indicate that IL-3R alpha is expressed on the cell surface of testicular Leydig cells and a Leydig cell tumor cell line I-10. Expression of the beta subunit mRNA was only faintly detected in testis, but not in I-10 cells. Immunoelectron microscopy has revealed that IL-3R alpha is present on cell membrane and microvilli.

Animals↗
PubMed2000

Molecular cloning of testis-abundant finger Protein/Ring finger protein 23 (RNF23), a novel RING-B box-coiled coil-B30.2 protein on the class I region of the human MHC.

We have identified a genomic DNA fragment, using the PCR method with degenerate oligonucleotide primers which contain the conserved sequence of the RING finger domain. Using the DNA fragment as a probe, a novel cDNA was cloned from human and mouse testis. The cDNA had a domain structure of the typical RING-B box-coiled coil (RBCC)-B30.2 domain and therefore was named testis-abundant finger protein (tfp). Indeed, the transcript was highly expressed in the testis, although it was also found ubiquitously in various organs by Northern blot analysis. The tfp gene was mapped at the class I region of the human MHC (major histocompatibility complex), within which some known RBCC-B30.2 proteins such as RFP, RFB30/HERF1, AFP, and HZF had been localized. These findings demonstrate that several RBCC-B30.2 proteins including tfp, which are non-HLA proteins, are clustered within the class I region of the human MHC.

Amino Acid Sequence↗
PubMed2001

Transient disruption of spermatogenesis by deregulated expression of neurturin in testis.

Two related ligands, glial cell line-derived neurotrophic factor (GDNF) and neurturin (NRTN), are expressed by Sertoli cells, but their cognate ligand-binding co-receptors, GDNF family receptor alpha1 and alpha2, are displayed by different germ cells suggesting different targets for the ligands. GDNF regulates cell fate decision of undifferentiated spermatogonia 'Science 287 (2000) 1489'. The role of NRTN was now approached by targeted overexpression in mouse testis. Between 3 and 5 weeks of age, transient degeneration of spermatogenic cells was observed in approximately 20% of all five transgenic lines generated. Spermatids and pachytene spermatocytes underwent segmental degeneration, if the rete testis was undilated. When it was dilated, the spermatids and spermatocytes were more generally depleted. After 5 weeks of age, spermatogenic defects were no more observed and the NRTN overexpressing mice were fertile. The data suggest that NRTN might regulate survival and differentiation of spermatocytes and spermatids, but the low penetrance indicates that either the transgene expression has not been high enough or NRTN is not as essential as GDNF for spermatogenesis.

Animals↗
PubMed2005

Phospholipase Cdelta4 associates with glutamate receptor interacting protein 1 in testis.

We reported previously that phospholipase C (PLC) delta4 is required for calcium mobilization in the zona pellucida-induced acrosome reaction in sperm. Here we focused on the function of the C2 domain of PLCdelta4 and report that glutamate receptor-interacting protein1 (GRIP1) was identified as a binding protein of the PLCdelta4-C2 domain on yeast two-hybrid screening. Physiological interaction of GRIP1 with PLCdelta4 in mouse testis was confirmed by immunoprecipitation with anti-PLCdelta4 antibodies and the association seemed to correlate with the maturation stage of sperm. We also determined that a PDZ-binding motif at the C-terminus of the PLCdelta4-C2 domain is responsible for GRIP1 binding, whereas the sixth or seventh PDZ domain of GRIP1 is essential and sufficient for association with the PLCdelta4-C2 domain. These results indicate that PLCdelta4 binds via its C2 domain to the PDZ6 or PDZ7 domain of GRIP1, and that this association may play a role in spermatogenesis.

Adaptor Proteins, Signal Transducing↗
PubMed2000

Ropporin, a sperm-specific binding protein of rhophilin, that is localized in the fibrous sheath of sperm flagella.

The small GTPase Rho; functions as a molecular switch that regulates various cellular processes such as cell adhesion, motility, gene expression and cytokinesis. We previously isolated several putative Rho; targets including rhophilin which bound selectively to the GTP-bound form of Rho;. Rhophilin is expressed highly in testis and is localized specifically in sperm flagella. The presence of a PDZ domain at the carboxy terminus of rhophilin suggested that rhophilin works as an adaptor molecule. To test this hypothesis, we employed a yeast two hybrid system using the rhophilin PDZ domain as a bait, and screened a mouse testis cDNA library. We isolated several positive clones containing the same insert. The open reading frame of the cDNA encoded a novel protein of 212 amino acids designated as ropporin from a Japanese word 'oppo' (the tail). The amino-terminal 40 amino acid sequence of ropporin showed high homology to that of the regulatory subunit of type II cAMP-dependent protein kinase, which is involved in dimerization and binding to A-kinase anchoring proteins. Consistently, a yeast two hybrid assay and gel filtration of recombinant ropporin indicated that ropporin dimerizes through this domain. Deletion analysis indicated that the carboxy-terminal four amino acids are essential for binding of ropporin to rhophilin, and ropporin and RhoV14 coprecipitated in the presence of rhophilin in vitro. Northern blot analysis showed that ropporin is exclusively expressed in testis, and induced at the late stage of spermatogenesis. This induction paralleled that of rhophilin. Immunocytochemistry using anti-ropporin antibody showed that ropporin is localized in the principal piece and the end piece of sperm flagella. Electronmicroscopy revealed that ropporin is mostly localized in the inner surface of the fibrous sheath while rhophilin is present in the outer surface of the outer dense fiber. These results suggest that rhophilin and ropporin may form a complex in sperm flagella.

Adaptor Proteins, Signal Transducing↗
PubMed1976

Demonstration of a pituitary gonadotrophin hormone activity in the male foetal mouse.

The testosterone production by 18-days-old foetal mouse testis was measured in an organ culture system, by RIA in the culture medium. This production was time-dependent, and could be stimulated by ovine LH and age-matched foetal pituitary. The gonadotrophin activity derived from foetal pituitary appeared to be released into the culture medium as a limited reserve. These data clearly show that a biologically active gonadotrophin material is present in the pituitary of the 18-days-old mouse foetus.

Adrenocorticotropic Hormone↗
PubMed2003

Mouse round spermatids developed in vitro from preexisting spermatocytes can produce normal offspring by nuclear injection into in vivo-developed mature oocytes.

It has been shown that mature oocytes injected with nuclei from round spermatids collected from mouse testis can generate normal offspring and that round spermatids can develop in vitro. An undetermined issue is whether spermatids developed in vitro are capable of generating fertile offspring by nuclear injection into oocytes. Herein, we report the production of normal and fertile offspring by nuclear injection using haploid spermatid donors derived from mouse primary spermatocyte precursors cocultured with Sertoli cells. Cocultured spermatogonia and spermatocytes were characterized by their nuclear immunoreactive patterns determined by an antibody to phosphorylated histone H2AX (gamma-H2AX), a marker for DNA double-strand breaks. Cocultured round spermatid progenies display more than one motile flagellum, whose axonemes were recognized by antitubulin immunostaining. Flagellar wavelike movement and flagellar-driven propulsion of round spermatids developed in vitro were documented by videomicroscopy (http://www.sci.ccny.cuny.edu/ approximately kier). We also show that breeding of male and female mouse offspring generated by spermatid nuclear injection produced fertile offspring. In addition to their capacity to produce fertile offspring, cocultured, flagellated round spermatids can facilitate the analysis of the mechanisms of centriolar polarity, duplication, assembly, and flagellar growth, including the intraflagellar transport of cargo proteins.

Animals↗
PubMed1999

Regulation by pH of the alternative splicing of the stem cell factor pre-mRNA in the testis.

Proliferation and differentiation of progenitor stem cells are mainly controlled by diffusible and adhesion molecules. Stem cell factor (SCF), an essential regulator of spermatogenesis produced by Sertoli cells, utilize both modes of cell to cell communication. Indeed, SCF exists in soluble (SCFs) and membrane-bound (SCFm) forms, which are required for a complete spermatogenesis, and are generated by alternative splicing of optional exon 6, encoding sites of proteolysis. We show that in the mouse testis, the alternative splicing of SCF is developmentally regulated. SCFs predominates in fetal and neonatal gonads and is then replaced by SCFm in the prepubertal and adult gonads. By sequencing SCF exon 6, we show that the flanking intronic sequences perfectly follow the gt-at rule, suggesting that the basal splicing machinery might not be responsible by itself for exon 6 skipping. Moreover, freshly isolated Sertoli cells mainly express SCFm, but a switch to SCFs occurs after 48 h of culture. We found that this change can be prevented by acidification of the culture medium at pH 6.3 or by addition of lactate. The sustained synthesis of SCFm at low pH was no longer observed in the presence of cycloheximide, suggesting that SCF exon 6 skipping requires de novo protein synthesis. Accordingly, UV cross-linking experiments show that nuclear Sertoli cell protein(s) bind in a sequence-specific manner to exon 6. Together, our data allow the proposal of an integrated mechanism in which the synthesis of lactate by Sertoli cells is used in the same time as an energetic substrate for germ cells and as a promoter of their survival/proliferation through the production of SCFm.

Alternative Splicing↗
PubMed1976

Properties of the testicular lactate dehydrogenase isoenzyme.

1. Studies were carried out with pure lactate dehydrogenase isoenzymes C4 (LDH isoenzyme X), B4, (LDH isoenzyme 1) and A4 (LDH isoenzyme 5) isolated from mouse testis, heart and muscle tissue respectively; with LDH isoenzyme X purified from pigeon testes and with crude lysates of spermatozoa from man, bull and rabbit. 2. LDH isoenzyme X from all species showed greater ability than the other isoenzymes to catalyse the NAD+-linked interconversions of 2-oxobutanoate into 2-hydroxybutanoate and of 2-oxopentanoate into 2-hydroxypentanoate. 3. Mouse LDH isoenzyme X presented the broadest spectrum of substrate specificity. It exhibited very similar Km values for a variety of 2-oxo acids: 2-oxopropanoate (pyruvate), 2-oxobutanoate, 2-oxo-3-methylbutanoate, 2-oxopentanoate, 2-oxo-3-methylpentanoate, 2-oxo-4-methylpentanoate, 2-oxohexanoate and 2-oxo-3-phenylpropanoate (phenylpyruvate). The corresponding 2-hydroxy acids were also readily utilized in the reverse reaction. A strong inhibition by substrate and product was demonstrated for the direct reaction. 4. Intracellular distribution of LDH isoenzyme X was investigated in mouse testes. LDH isoenzyme X activity was located in the fraction of "heavy mitochondria" and in the soluble phase. 5. A possible functional role for LDH isoenzyme X is proposed: the redox couple-2-oxo acid-2-hydroxy acid could integrate a shuttle system transferring reducing equivalents from cytoplasm to mitochondria.

Animals↗
PubMed2003

Ataxia telangiectasia mutated expression and activation in the testis.

Ionizing radiation (IR) and consequent induction of DNA double-strand breaks (DSBs) causes activation of the protein ataxia telangiectasia mutated (ATM). Normally, ATM is present as inactive dimers; however, in response to DSBs, the ATM dimer partners cross-phosphorylate each other on serine 1981, and kinase active ATM monomers are subsequently released. We have studied the presence of both nonphosphorylated as well as active serine 1981 phosphorylated ATM (pS1981-ATM) in the mouse testis. In the nonirradiated testis, ATM was present in spermatogonia and spermatocytes until stage VII of the cycle of the seminiferous epithelium, whereas pS1981-ATM was found only to be present in the sex body of pachytene spermatocytes. In response to IR, ATM became activated by pS1981 cross-phosphorylation in spermatogonia and Sertoli cells. Despite the occurrence of endogenous programmed DSBs during the first meiotic prophase and the presence of ATM in both spermatogonia and spermatocytes, pS1981 phosphorylated ATM did not appear in spermatocytes after treatment with IR. These results show that spermatogonial ATM and ATM in the spermatocytes are differentially regulated. In the mitotically dividing spermatogonia, ATM is activated by cross-phosphorylation, whereas during meiosis nonphosphorylated ATM or differently phosphorylated ATM is already active. ATM has been shown to be present at the synapsed axes of the meiotic chromosomes, and in the ATM knock-out mice spermatogenesis stops at pachytene stage IV of the seminiferous epithelium, indicating that indeed nonphosphorylated ATM is functional during meiosis. Additionally, ATM is constitutively phosphorylated in the sex body where its continued presence remains an enigma.

Animals↗
PubMed1987

Quantitation of sperm disposal and phagocytic cells in the tract of short- and long-term vasectomized mice.

By calculating the number of spermatozoa produced by the mouse testis after vasectomy, and actually counting the number of spermatozoa present in the epididymides and vasa deferentia, the number of spermatozoa resorbed at different times was quantified. The contributions of sperm phagocytosis and intraluminal dissolution of spermatozoa (separate sperm heads and tails) in sperm disposal were examined. Sperm resorption was clearly demonstrated, with about 100 X 10(6) spermatozoa and 426 X 10(6) spermatozoa having been resorbed by 6 weeks and 6 months after vasectomy, respectively. A characteristic of the vasectomized tract was the high proportion of degenerating spermatozoa, and small lymphocytes, but very few intraluminal phagocytes were observed. The results suggest that spermatozoa are resorbed after vasectomy and that intraluminal sperm dissolution, rather than phagocytosis, is a prominent mechanism of sperm disposal in the tract of the vasectomized mouse.

Animals↗
PubMed1983

Steroid secretion by mouse testes perfused in vitro.

We determined the concentration of steroids in blood plasma of CBF1 male mice and the steroidogenic potential of the mouse testis. Steroid secretion rates are based on measuring selected C18, C19, and C21 steroids in the venous effluent of testes perfused with a defined medium containing luteinizing hormone. Steroids were isolated by thin-layer and/or column chromatography and quantified by radioimmunoassay. These include pregnenolone, 17 alpha-hydroxypregnenolone, dehydroepiandrosterone, androstenediol, progesterone, 17 alpha-hydroxyprogesterone, androstenedione, testosterone, dihydrotestosterone, 3 alpha-androstanediol, and 3 beta-androstanediol. Testosterone is the primary steroid secreted by mouse testes perfused in vitro and is the chief androgen present in blood plasma. Pregnenolone, an obligatory intermediate in steroid synthesis, is converted to testosterone via two separate steroidogenic pathways in approximately equal proportions. This is unlike other species in which testosterone biosynthesis proceeds preferentially via either the delta 4 or the delta 5 pathway. Our results, taken together, provide the first comprehensive assessment of Leydig cell steroidogenic activity in the mouse, demonstrate putative enzymatic pathways subserving androgen biosynthesis, and establish the predominant steroids in the peripheral circulation of adult mice.

Androgens↗
PubMed1994

Identification and characterization of a sperm peptide antigen recognized by a monoclonal antisperm autoantibody derived from a vasectomized mouse.

A monoclonal autoantibody (MAb) to mouse sperm was established from a vasectomized mouse. The MAb, termed 4A11, reacted exclusively to mouse and rat live sperm tail, without any cross-reaction with sperm of other mammalian species or any somatic organ tissues in the mouse. MAb 4A11 significantly inhibited in vitro fertilization of mouse ova. Western blotting showed a single band of 67 kDa as the corresponding antigen protein of MAb 4A11. By screening a mouse testis cDNA library with MAb 4A11, one positive clone (964bp) was obtained. DNA sequence of the clone showed no homology with any previously reported mammalian genes, but showed 54% homology with a bacterial gene of sucrose-specific phosphotransferase enzyme II. The rabbit antisera raised to the recombinant protein of the 4A11 cDNA gene showed reaction to mouse, rat, hamster and guinea-pig sperm.

Amino Acid Sequence↗
PubMed2001

GLI1 localization in the germinal epithelial cells alternates between cytoplasm and nucleus: upregulation in transgenic mice blocks spermatogenesis in pachytene.

The zinc finger transcription factor GLI1 is the mediator of signaling by members of the Hedgehog (Hh) family. Male mice in which Desert hedgehog (Dhh), an Hh homologue expressed in Sertoli cells of the testis, was knocked out are sterile, suggesting that the Dhh/GLI1 pathway plays a role in spermatogenesis. Using an antiserum raised against human GLI1, we found that during the first round of spermatogenesis, GLI1 expression is initially cytoplasmic, then shifts to the nuclei of Sertoli and germ cells, and finally shifts back to the cytoplasm. In the adult mouse testis, GLI1 expression localized to the nuclei of germ cells, beginning with pachytene cells and persisting through round spermatids. Localization of GLI1 in elongating spermatids shifted from the nucleus to the cytoplasm and became associated with microtubules. We also examined a line of transgenic mice that overexpressed human GLI1. Male mice in this line were sterile. Spermatogenesis was blocked at the pachytene stage, and a subset of the morphologically indistinguishable pachytene cells underwent apoptosis. Patched-2, which is a Dhh receptor, and Fused, another component of the signal transduction pathway, are expressed in Leydig cells and in primary and secondary spermatocytes. Expression of GLI1 in the same cell types as Patched-2 and Fused and the disruption of spermatogenesis by GLI1 overexpression suggest that GLI1 is the mediator of the Dhh signal in the testis, and that it may be a regulator of spermatogenesis.

Animals↗
PubMed2006

The multi PDZ domain protein MUPP1 as a putative scaffolding protein for organizing signaling complexes in the acrosome of mammalian spermatozoa.

Spermatozoa undergo complex sequences of precisely timed events during the process of fertilization. These priming events, which comprise capacitation, egg recognition, acrosome reaction, and sperm-oocyte fusion, are regulated by the activation of different intracellular signaling pathways. The efficacy and accuracy of signal transduction pathways often depend on the assembly of multiprotein signaling complexes, thereby coordinating and guiding the flow of regulatory information. To address the question whether PDZ-domain proteins, the most abundant protein interaction modules involved in the assembly of supramolecular signaling complexes, are present in rodent sperm, homologue of the RT-PCR approaches were performed with specific primer pairs for the vertebrate INAD-like PDZ domain protein MUPP1. The results revealed that this scaffolding protein, which comprises 13 different PDZ domains, is expressed in mouse testis. To obtain further support for the expression of the multi-PDZ domain protein MUPP1 in testicular tissue, immunohistochemical as well as immunocytochemical experiments were performed using a MUPP1-specific antibody. Detailed analyses of the spatial MUPP1-expression profile revealed that immunoreactivity is concentrated within the acrosomal region of round as well as elongated mouse spermatozoa. These results were confirmed in experimental approaches demonstrating that MUPP1 immunofluorescence was shed off from the acrosome region after acrosome reaction. To examine whether MUPP1 is also present in other mammalian sperm, immunocytochemical approaches were performed with isolated bovine as well as human sperm. The results revealed prominent MUPP1 expression which was restricted to the apical acrosomal region and, most notably, to the equatorial segment of the acrosome. The predominant expression profile of MUPP1 in sperm of different mammalian species suggests that this PDZ-domain protein may be involved in organizing signaling molecules mediating primary reactions of fertilization.

Acrosome↗
PubMed2006

FNDC3A is required for adhesion between spermatids and Sertoli cells.

Symplastic spermatids (sys) male mice are sterile due to a recessive mutation that causes defective adhesion between spermatids and Sertoli cells within the seminiferous epithelium. We show that the mutation in sys mice involves a deletion of 1.24 Mb of chromosome 14. Comparative genomic analysis suggests that this region contains only one gene, Fndc3a. A genetic complementation analysis using mice with a specific mutation within Fndc3a verifies that mutation of Fndc3a is the cause of male sterility in sys mice. Fndc3a is a member of a three-gene family in mice. Fndc3a, which is expressed in several tissues including testis, encodes a novel protein composed of a proline-rich amino-terminus, nine fibronectin type-III domains, and a hydrophobic carboxy-terminus. The proline-rich region of each family member contains conserved amino acids that include a PPGY consensus binding site for type I WW domain containing proteins. The hydrophobic carboxy-terminus is similar to that found in 'tail-anchored' proteins, integral membrane proteins that are localized to the cytosolic face of the endoplasmic reticulum. Immunohistochemical staining indicated that FNDC3A localizes to the acrosome of spermatids, as well as to Leydig cells in the mouse testis. Acrosomal localization of FNDC3A is observed in spermatids between step 2 and step 10 inclusive. In step 12 spermatids, FNDC3A is largely absent from the acrosomal region with immunostaining being localized to vesicular structures located within the cytoplasm of elongate spermatids. Models are presented for the function of FNDC3A in mediating spermatid-Sertoli adhesion during mouse spermatogenesis.

Alleles↗
PubMed2002

Identification of Gasz, an evolutionarily conserved gene expressed exclusively in germ cells and encoding a protein with four ankyrin repeats, a sterile-alpha motif, and a basic leucine zipper.

To discover causes of infertility and potential contraceptive targets, we used in silico subtraction and genomic database mining to identify conserved genes with germ cell-specific expression. In silico subtraction identified an expressed sequence tag (EST) present exclusively in a newborn mouse ovary library. The full-length cDNA sequence corresponding to this EST encodes a novel protein containing four ankyrin (ANK) repeats, a sterile-alpha motif (SAM), and a putative basic leucine zipper (bZIP) domain. Northern blot and semiquantitative RT-PCR analyses demonstrated that the mRNA is exclusively expressed in the mouse testis and ovary. The expression sites were localized by in situ hybridization to pachytene spermatocytes in the testis and oocytes in the ovary. Immunohistochemistry showed that the novel protein is localized to the cytoplasm in pachytene spermatocytes and early spermatids, oocytes at all stages of oogenesis, and in early preimplantation embryos. Based on its germ cell-specific expression and the presence of ANK, SAM, and basic leucine zipper domains, we have termed this novel protein GASZ. The mouse Gasz gene, which consists of 13 exons and spans 60 kb, is located on chromosome 6 between the Wnt2 and cystic fibrosis transmembrane conductance regulator (Cftr) genes. Using genomic database mining, orthologous genes encoding GASZ were identified in the rat, cow, baboon, chimpanzee, and human. Phylogenetic analyses reveal that the GASZ proteins are highly conserved among these species. Human and mouse GASZ proteins share 85.3% amino acid identity, and human and chimpanzee GASZ proteins differ by only 3 out of 475 amino acids. In humans, the GASZ gene resides on chromosome 7 and is similarly composed of 13 exons. Because both ANK repeats and the SAM domain function as protein-protein interaction modules that mediate signal transduction cascades in some systems, GASZ may represent an important cytoplasmic signal transducer that mediates protein-protein interactions during germ cell maturation in both males and females and during preimplantation embryogenesis.

Adaptor Proteins, Signal Transducing↗