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Defining the spermatogonial stem cell.

Through the use of donor cells from transgenic rats expressing GFP exclusively in the germline, we have defined culture conditions where male germ cells lose (on STO cells) or maintain (on MSC-1 cells) stem cell activity. A cadre of germ cell transcripts strikingly decrease in relative abundance as a function of testis age or culture time on STO cells, but only a subset of these transcripts (approximately 248) remain elevated when cultured on MSC-1 cells. If specific gene expression regulates stem cell activity, some or all of these transcripts are candidates as such regulators. We establish a spermatogonial stem cell index (SSCI) that reliably predicts relative stem cell activity in rat or mouse testis cell cultures, and through the use of an antibody to a robust signal (Egr3) within the index find intense signals in single or paired cells. As germ cells form longer interconnected chains (incomplete cytokinesis), the Egr3 signal disappears coincident with a loss of stem cell activity. Thus, molecular markers specific for spermatogonial stem cells establish a reliable and rapid means by which to define these cells in culture and alleviate the need for laborious testicular transfers in initial cell culture studies.

Animals↗

Genomic organization of the mouse Msh4 gene producing bicistronic, chimeric and antisense mRNA.

By degenerate PCR and screening of mouse testis cDNA library, we have identified seven cDNAs from the meiotic recombination gene Msh4. Variant alpha and probably beta are likely involved in meiotic DNA recombination. Other variants have distinctive structures; variant epsilon, theta; and iota form a bicistronic operon, while variant delta contains antisense RNA for the endoplasmic reticulum (ER) chaperon Hspa5 gene and small open reading frame (ORF) identical to epsilon ORF2. Analysis of the exon-intron structures revealed unusual genomic organizations: the first three exons of delta and the first exon of epsilon are respectively mapped to the Hspa5 locus on chromosome 2 and the Pcbp3 locus on chromosome 10; the remaining exons of both variants are mapped to the Msh4 locus on chromosome 3. The first exon of variant beta is located on chromosome 16, while the others are located on chromosome 3. Synthesis of these mRNAs is assumed to require interchromosomal trans-splicing alone (beta and epsilon) or in combination with converse-splicing (delta). Most Msh4 variant mRNAs are mainly expressed in testis, but a small amount of each variant except for epsilon is also expressed in brain, heart, thymus, ovary and embryonic head. Enhanced green fluorescent protein (EGFP) fusion experiments showed that all the ORFs are translated, and most of those proteins are localized to a particular subcellular compartment. It also appeared that expression of variant delta induces cell death. This study suggests that the dynamic interchromosomal (intergenic) trans-splicing generates functional diversity of the mouse Msh4 gene.

Alternative Splicing↗

Molecular cloning and characterization of the clock gene period2 in the testis of lizard Podarcis sicula and its expression during seasonal reproductive cycle.

Clock genes are known to oscillate with circadian rhythmicity in the central clock structure, the suprachiasmatic nucleus of the hypothalamus, and also in peripheral tissues. Reproduction is a peripheral activity that is strongly influenced by a circadian clock in many organisms. Most mammals that exhibit a seasonal cycle are able to decode the daily changes in light across the year and to translate these in hormonal signals that regulate reproductive cycles. Expression of many clock genes has been revealed in mouse testis, although transcription of these genes seems to be constitutive in 24 h, suggesting that these genes may play in the testis a different role with regard to the central clockwork function. The seasonal breeding lizard Podarcis sicula represents an attractive model for studying some developmental and differentiation phenomena, such as gonadal maturation, since in the adult male the testis shows a spring full activity and a complete summer regression. Experimental data seem to suggest that in lizard the environmental factors, as photoperiod and temperature, affect the endogenous elements, although the interaction mechanisms are unknown. It is known that temperature signals have a direct influence on clock processes such as transcription, translation, protein phosphorylation and degradation. In addition, most data show that the expression of circadian clock genes, such as period2, is affected by length of photoperiod. In this way, the core clockwork may also decode seasonal information. Here we report the cloning, sequencing and bioinformatic analysis of period2 gene, isolated from the testis of lizard P. sicula, and its expression both in the testis and in other tissues during the different phases of the seasonal cycle. RT-PCR assays enlighten the presence of transcript in testis, brain, heart, liver and kidney in all the phases analysed. Moreover, real time quantitative PCR assays detect a peak of per2 testicular expression during gonadal regression. Our preliminary results cannot clearly demonstrate the involvement of per2 gene in seasonal reproductive cycle of male lizard P. sicula, but its presence in the testis may suggest a role of this gene during spermatogenesis. Besides, our work can provide numerous starting points to clarify the role of per2 during seasonal reproductive cycle.

Amino Acid Sequence↗

CLIP-50 immunolocalization during mouse spermiogenesis suggests a role in shaping the sperm nucleus.

The spermatid nucleus and cytoplasm undergo dramatic morphological modifications during spermatid differentiation into mature sperm. Some of the external force causing this nuclear shaping is generated by a microtubular structure termed the manchette, which attaches to the perinuclear ring of the spermatid. Here, we report the isolation and characterization of a protein component of this perinuclear ring in an immunological screening of a mouse testis cDNA library. We termed this protein CLIP-50 because of its high similarity at the amino acid level to the C-terminal region of the microtubule-binding protein CLIP-170/restin. CLIP-50 lacks the characteristic microtubule-binding motif, but retains a portion of the predicted coiled-coiled domain and the metal-binding motif. The CLIP-50 transcript and protein are abundant in testis. The protein is also expressed in heart, lung, kidney, and skin. A distinct size variant exists in brain. In the spermatids, CLIP-50 protein localizes specifically to the centriolar region where the sperm tail originates and to the perinuclear ring from which the manchette emerges. CLIP-50 staining is retained in the ring throughout its migration over the surface of the nucleus which accompanies the nuclear shaping into its characteristic sperm configuration. This localization pattern indicates a very specific function for this novel CLIP derivative during mouse spermiogenesis.

Animals↗

Nuclear localization of antigens detected by a monoclonal antibody against a synthetic peptide of rfp finger protein.

A monoclonal antibody (MAb) was generated against a synthetic peptide corresponding to amino acids 148 to 163 of the rfp protein with zinc finger domains. The MAb, designated RFP-1 (IgM), which was positive with the immunizing peptide in enzyme-linked immunosorbent assay, was reactive in immunoblotting with an in vitro translated rfp product as well as with native proteins in cell extracts made from mouse testis and HL-60 human leukemia cell line, both of which were previously shown to express high levels of rfp mRNA. When HL-60 cells were fractionated into nuclear and cytoplasmic components, the protein reactive with RFP-1 MAb was detectable only in the nuclear fraction. By the avidin-biotin complex immunoperoxidase method, this MAb strongly stained over 90% of the nuclei of human and mouse spermatogenic cells, except mature spermatozoon, and of human testicular tumor cells. In other human adult tissues, up to 60% of positive cells were observed. These antibody activities were clearly absorbed by pre-incubation of RFP-1 MAb with the immunizing peptide. These results thus indicated that RFP-1 MAb recognizes a nuclear protein which is expressed at high levels in male germ cells.

Amino Acid Sequence↗

Distinct transcripts are recognized by sense and antisense riboprobes for a member of the murine HSP70 gene family, HSP70.2, in various reproductive tissues.

The expression of hsp70.2, an hsp70 gene family member, originally characterized by its high levels of expression in germ cells in the adult mouse testis, was detected in several other reproductive tissues, including epididymis, prostate, and seminal vesicles, as well as in extraembryonic tissues of mid-gestation fetuses. In addition, hybridization with RNA probes transcribed in the sense orientation surprisingly indicated the presence of slightly larger "antisense" transcripts in several tissues. The levels of antisense transcripts varied among the tissues, with the highest signal detected in the prostate and no signal being detectable in the testis. Consistent with these results, in situ hybridization analysis clearly localized the sense-orientation transcripts to pachytene spermatocytes, while no antisense-orientation transcripts were observed in adjacent sections of the same tubules. Our findings have thus shown that although hsp70.2 was expressed abundantly and in a highly stage-specific manner in the male germ line, it was also expressed in other murine tissues. Furthermore, we have made the surprising observation of antisense transcription of the hsp70.2 gene in several mouse tissues, revealing another level of complexity in the regulation and function of heat shock proteins.

Animals↗

Autoimmune-like activity of sperm specific LDH: a pathophysiological and electron microscopic study of atrophied testis and epididymis.

Hyper-immunization of male mice with human LDH-C4 evoked autoimmune reactions illustrated by the loss of LDH activity, associated histopathological changes in testes and epididymis and induction of sterility in mice. This was substantiated by the altered morphology of sperm mitochondria and plasma membrane, and by reduced number of cytoplasmic droplets as observed by electron microscopy. However, the presence of lymphoblasts and other lymphoid cells in testes indicated that the testicular damage is accentuated by activated T lymphocytes. It is concluded that immunization with human LDH-C4 produces lesions in mouse testis and epididymis, which are similar to experimentally induced autoimmune orchitis.

Animals↗

Characterization and potential function of a novel testis-specific nucleoporin BS-63.

A 1933 bp cDNA fragment, coding a truncated testis-specific novel nucleoporin, was isolated from a human testis lambdaZAPII cDNA library, designated as BS-63 and assigned GenBank accession number: U64675. By applying the methods of rapid amplification of cDNA ends (5' RACE) and PCR, a full-length BS-63 cDNA composed of 5475 bp was obtained. BS-63 cDNA contained an open reading frame consisting of 1765 codons and XFXFG or GLFG repetitive sequence motifs. These repetitive motifs are structural characteristic of nucleoporins. BS-63 cDNA has high homology with Nup358/Ran BP2. A 1599 bp fragment, corresponding to the C-terminus of BS-63 cDNA, was prepared and expressed in E. coli BL21(DE3). The recombinant product was purified by affinity chromatography and SDS-PAGE and polyclonal antibodies raised. In rat testis section, the BS-63 protein was localized at the sites of nuclear pores in spermatids by immuno-gold transmission electron microscopy and on the nuclear membrane of Triton X-treated sperm by colloidal silver immuno-gold scanning electron microscopy. The recombinant BS-63 protein can be phosphorylated in vitro with PKC and p34(cdc2). A yeast two-hybrid system was used to screen a mouse testis cDNA library to identify proteins capable of interacting with BS-63. Using the 1.6 kb cDNA fragment as bait, the following interacting proteins were identified: Ran, transportin (karyopherin beta2), two proteins related to the nucleocytoplasmic transporter and aF10 protein. The latter protein is a putative transcriptor containing a cysteine-rich N-terminus, a LAP/PHD finger, a leucine zipper domain and a glutamine-rich C-terminus. Also it is highly expressed in murine testis and is located in the cell nucleus and cytoplasm. The interaction of BS-63 with aF10 (696-1001aa) was validated by surface plasmon resonance and by affinity precipitation combined with Western blot. aF10 (696-1001aa) interacted in vitro with BS-63 extracted from rat testis germ cells. It is hypothesized that BS-63 is a testis-specific nucleoporin and possibly acts as a docking site and a cotransporter of Ran and transportin. The complex performs the task of a carrier system in transporting aF10 into the nucleus of germ cells during spermiogenesis.

Amino Acid Sequence↗

Late arrest of spermiogenesis and germ cell apoptosis in mice lacking the TBP-like TLF/TRF2 gene.

Metazoan genomes encode two related proteins, TBP and the TBP-like factor (TLF/TRF2), sharing a highly conserved saddle-like domain. TLF is highly expressed in a finely regulated pattern in the mouse testis during spermatogenesis. The murine TLF gene has been inactivated using homologous recombination. TLF-/- mice are viable, but mutant male mice are sterile due to a late, complete arrest of spermiogenesis. In mutant animals, spermatogonia and spermatocytes develop normally, but round spermatids undergo apoptosis at step 7. Although the expression of the transcriptional activator CREM and many other postmeiotic genes was unaltered in TLF null mice, several spermiogenesis genes transcribed in late round spermatids appeared to be under TLF control. Hence, TLF is not required for embryonic development in the mouse but is essential for spermiogenesis.

Animals↗

Testosterone regulates FGF-2 expression during testis maturation by an IRES-dependent translational mechanism.

Spermatogenesis is a complex process involving cell proliferation, differentiation, and apoptosis. Fibroblast growth factor 2 (FGF-2) is involved in testicular function, but its role in spermatogenesis has not been fully documented. The control of FGF-2 expression particularly occurs at the translational level, by an internal ribosome entry site (IRES)-dependent mechanism driving the use of alternative initiation codons. To study IRES activity regulation in vivo, we have developed transgenic mice expressing a bicistronic construct coding for two luciferase genes. Here, we show that the FGF-2 IRES is age-dependently activated in mouse testis, whereas EMCV and c-myc IRESs are not. Real-time PCR confirms that this regulation is translational. By using immunohistological techniques, we demonstrate that FGF-2 IRES stimulation occurs in adult, but not in immature, type-A spermatogonias. This is correlated with activation of endogenous FGF-2 expression in spermatogonia; whereas FGF-2 mRNA transcription is known to decrease in adult testis. Interestingly, the FGF-2 IRES activation is triggered by testosterone and is partially inhibited by siRNA directed against the androgen receptor. Two-dimensional analysis of proteins bound to the FGF-2 mRNA 5'UTR after UV cross-linking reveals that testosterone treatment correlates with the binding of several proteins. These data suggest a paracrine loop where IRES-dependent FGF-2 expression, stimulated by Sertoli cells in response to testosterone produced by Leydig cells, would in turn activate Leydig function and testosterone production. In addition, nuclear FGF-2 isoforms could be involved in an intracrine function of FGF-2 in the start of spermatogenesis, mitosis, or meiosis initiation. This report demonstrates that mRNA translation regulation by an IRES-dependent mechanism participates in a physiological process.

5' Untranslated Regions↗

Molecular cloning and sequence analysis of the mouse protein C inhibitor gene.

The gene encoding mouse protein C inhibitor (mPCI) was isolated and its nucleotide sequence determined. Alignment of the genomic sequence with that of a cDNA obtained from mouse testis revealed that the mPCI gene (like the human counterpart) is composed of five exons and four introns with highly conserved exon/intron boundaries. It encodes a pre-polypeptide of 405 amino acids, which shows 63% identity with human PCI (hPCI). The putative reactive site is identical to that of hPCI from P5 to P3', suggesting a similar protease specificity. Also the putative heparin binding sites and 'hinge' regions are highly homologous in mouse and hPCI.

Amino Acid Sequence↗

Characterization of glycosylphosphatidylinositol-anchored decay accelerating factor (GPI-DAF) and transmembrane DAF gene expression in wild-type and GPI-DAF gene knockout mice using polyclonal and monoclonal antibodies with dual or single specificity.

Decay-accelerating factor (DAF, CD55) is a glycosylphosphatidylinositol (GPI)-linked membrane inhibitor of complement activation. While human and other mammalian species contain only one DAF gene, two distinct DAF genes, referred to as GPI-DAF and transmembrane (TM)-DAF, respectively, have been identified in the mouse. Using several independently generated monoclonal and polyclonal antibodies, either with dual or single specificity for GPI-DAF and TM-DAF gene products, we have examined the expression of the two DAF genes in tissues of the wild-type and a strain of knockout mouse whose GPI-DAF gene has been inactivated. By fluorescence-activated cell sorting (FACS) analysis, we found that DAF protein is present on the wild-type mouse erythrocytes and lymphocytes but no signal was detectable on the same cells of GPI-DAF gene knockout mice. Both T and B lymphocytes and splenic macrophages express the GPI-DAF gene but the expression level is higher on B lymphocytes than on T lymphocytes. Within the T cell population, both CD4+ and CD8+ T cells are positive. DAF protein was detected by immunohistochemistry at high levels on wild-type mouse spermatids and mature sperm. In contrast, only mature sperm stained positive in the GPI-DAF gene knockout mouse testis, suggesting that GPI-DAF but not the TM-DAF gene is expressed on spermatids. Examination of the fetoplacental unit at the day 7.5 stage revealed that GPI-DAF but not the TM-DAF gene is expressed in the maternal decidua cells surrounding the trophoectoderm of the embryo. No DAF expression was detected on trophoblast or the embryo proper. These findings suggest that although the TM-DAF gene is irrelevant on mouse blood cells, the two DAF genes may have different roles in germ cell development and/or mature sperm function. Because complement receptor 1-related gene/protein y (Crry) has been shown to be expressed on early mouse embryos, the complete lack of GPI-DAF and TM-DAF gene expression in early mouse development may explain the observed sensitivity of Crry-deficient embryos to maternal complement attack.

Animals↗

Testis-specific expression of the rat histone Hlt gene in transgenic mice.

The testis-specific histone Hlt gene is transcribed only in testis. The appearance of testis-specific nuclear proteins that bind to a unique promoter sequence element designated Hlt/TE located between the H1/AC box and the H1/CCAAT box correlates with the onset of transcription of the Hlt gene during the meiotic cell cycle. In order to determine whether sequences flanking the rat Hlt gene are sufficient to confer tissue-specific expression in vivo, a 6859 bp EcoRI restriction fragment of genomic DNA containing the rat histone Hlt gene has been microinjected into mouse embryos. S1 nuclease protection analysis has shown that the descendants of the resulting transgenic mice express the rat gene in the proper tissue and at the proper meiotic cell cycle stage. Furthermore, when populations of mouse testis cells were prepared by centrifugal elutriation, only the fraction enriched in pachytene primary spermatocytes had a significant steady-state level of rat Hlt mRNA. Although the copy-number of the transgene was variable in these animals, rat Hlt mRNA levels in high copy-number animals never exceeded 2.6 times the level in normal rat testes. The appearance of appropriate meiotic cell cycle-specific transcription indicates the importance of the conserved promoter sequence elements between the two species.

Animals↗

Methylation-dependent silencing of the testis-specific Pdha-2 basal promoter occurs through selective targeting of an activating transcription factor/cAMP-responsive element-binding site.

In this study, we demonstrate that methylation-dependent repression of the Pdha-2 core promoter is mediated regionally through a consensus activating transcription factor/cAMP-responsive element-binding site located between nucleotides -54 and -62 upstream of the major transcriptional start site. Targeting of the CpG dinucleotide within this cis-element significantly disrupts the ability of this basal promoter to activate gene expression in vitro and completely abolishes promoter activity in vivo. DNase I footprinting experiments indicated that availability of the nuclear factor(s) binding this element is limiting in sexually immature mouse testis, and as such, these factors may play an important role in the coordinate activation of early spermatogenic gene expression. Interestingly, CpG dinucleotides associated with the hypersensitive region flanking the activating transcription factor/cAMP-responsive element-binding site appear to confer some conformational structure on the promoter since mutations at these specific CpG dinucleotides result in elevated basal levels of transcription. This raises the possibility of a potential bifunctional role for CpG dinucleotides in either methylation-dependent or -independent processes. Our data support the notion that hypomethylation and transcription factor recruitment are necessary events that precede gene activation at the early stages of spermatogenesis.

3T3 Cells↗

Distribution of dynamins in testis and their possible relation to spermatogenesis.

Dynamin 2 and dynamin 3 are highly expressed in testis. However, their functions in the tissue remain unclear. Considering that dynamin 1, neuron-specific isoform of dynamin, plays a pivotal role in endocytosis, functions of dynamin 2 and dynamin 3 in testis must be essential. Cellular expression and subcellular localization of dynamin 2 and dynamin 3 in testis were investigated. Dynamin 2 and dynamin 3 were highly expressed in germ cells and Sertoli cells, constituents of seminiferous tubules. By immunofluorescence it was revealed that dynamin 2 colocalizes with clathrin both at the plasmamembrane and at Golgi in a cell line of Sertoli cells. Immunoreactivity for dynamin 3, on the other hand, appeared as finer puncta, which did not colocalize with clathrin, suggesting that these two dynamins have distinct functions in Sertoli cells. In the klotho deficient mouse testis, which demonstrates disorder in spermatogenesis, expression of dynamin 2 and dynamin 3 was drastically reduced indicating possible association of these proteins with spermatogenesis.

Animals↗

Identification of a new variant of PDE1A calmodulin-stimulated cyclic nucleotide phosphodiesterase expressed in mouse sperm.

In mature sperm, cAMP plays an important role as a second messenger regulating functions that include capacitation, the acrosome reaction, motility, and, in some cases, chemosensing. We have cloned from mouse testis a novel calmodulin-stimulated cyclic nucleotide phosphodiesterase 1A isoform, Pde1a_v7 (mmPDE1A7), which arises from an alternative transcription start in the cyclic nucleotide phosphodiesterase 1A gene. The open reading frame is predicted to encode a polypeptide with a molecular mass of 52 kDa. Two further variants of this form, which contain two additional new exons, arise from alternative splicing. Analysis of testis cDNA by real-time polymerase chain reaction (PCR) indicates that the Pde1A_v7 transcript variant is the most abundant. The PDE1A_v7 protein uniquely lacks the first amino-terminal calmodulin-binding domain, but does possess an inhibitory domain and a second calmodulin-binding site shared with other variants. In vitro translation of the corresponding Pde1a_v7 cDNA produced a 52-kDa polypeptide having cyclic nucleotide hydrolytic activity, which was stimulated threefold by calcium-bound calmodulin. Immunoprecipitation of cyclic nucleotide phosphodiesterase 1 activity from detergent extracts of mouse sperm revealed a major protein of the size expected for PDE1A_v7, and the immunocytochemical staining for cyclic nucleotide phosphodiesterase 1A in mouse sperm showed intense immunoreactivity in the tail only. These observations, along with the PCR data, strongly suggest that this new variant PDE1A_v7 is the major form of cyclic nucleotide phosphodiesterase 1A expressed in mature sperm and is therefore likely to play an important role in cyclic nucleotide regulation of mature sperm function.

Alternative Splicing↗

Rapid cDNA sequencing in combination with RNA expression studies in mice identifies a large number of male germ cell-specific sequence tags.

A large number of cDNA clones were isolated from an adult mouse testis cDNA library and partially sequenced. Sequence comparisons revealed that many of them displayed similarities to genes previously identified only in invertebrates and lower eukaryotes, including, e.g., one cDNA clone related to the Drosophila melanogaster suppressor of forked gene. Other cDNA clones were found to be related, but not identical, to rodent genes involved in a variety of cellular activities, e.g., signal transduction and tumor development. The RNA expression patterns of 258 cDNA clones were analyzed through use of Northern blot methodology. Forty-two novel cDNA clones were found to be expressed only in male germ cells, the majority of them during spermiogenesis. One male germ cell-specific mouse cDNA clone was found to be similar to tektin A1, a protein known to interact with the flagellar microtubules of sea urchin sperm.

Amino Acid Sequence↗

Defining the cellular and molecular mechanisms of toxicant action in the testis.

A symposium was held at the 41st annual meeting of the Society of Toxicology with presentations that emphasized novel molecular and cellular pathways that modulate the response to testicular toxicants. The first two presentations described cellular alterations after exposure to the Sertoli cell toxicant, mono-(2-ethylhexyl) phthalate (MEHP). The expression of flamingo1, a G protei coupled receptor family member that may couple cell-cell adhesion to G protein-dependent signaling in Sertoli cells, was rapidly altered after MEHP exposure. Sertoli cell associated flamingo1 immunostaining was redistributed early (within 2 h) after MEHP exposure and disappeared by 12 h, suggesting that flamingo1 is a proximal phthalate target. MEHP was also described to alter the expression and activity of the newly identified death receptors DR4, 5 and 6 in the testis. The differential cellular changes in the levels of DR4, 5 and 6 after phthalate exposure suggested that they may act as surrogates or in concert with the widely described Fas-signaling pathway in the initiation of germ cell apoptosis after MEHP exposure. The next two presentations focused on revealing mechanisms that may explain the persistent post-exposure testicular atrophy that is observed in rodents after a variety of chemical or physical insults (radiation, chemotherapeutics, toxicants) and possible strategies to re-initiate spermatogenesis in the atrophic testis. Hormonal manipulations that lower testosterone and serum FSH levels allow for re-initiation of spermatogonial development. Recent investigation of additional models of persistent atrophy such as mutant mice, the aged Brown Norway rat, EDS-induced Leydig cell deficient rat, and primates, have broadened insight into the mechanisms responsible for persistent atrophy. The last presentation described the use of cDNA arrays in the investigation of cellular elements and mechanisms responsible for disruption of spermatogenesis by the drinking water disinfectant bromochloroacetic acid (BCA). A custom mouse testis cDNA array interrogating 950 genes was used for analysis of testis mRNA. BCA altered the expression of 53 of the 950 genes, including two encoding sperm proteins known to be significant for male fertility, and other genes involved in spermatogenesis, stress response, and cell communication/adhesion. These observations strengthen the hypothesis that BCA disrupts spermatogenesis by altering the process of spermiogenesis.

Animals↗