Isolation of rat testis histone TH2B-x, and interaction of TH2B-x antiserum with histones and mononucleosomes

A method is reported for the isolation of histone TH2B-x from rat testis by affinity chromatography on an agarose-p-chloromercurianilino column. This purified TH2B-x was used to raise antibodies in the rabbit, and the antiserum was assayed by an enzyme-linked double-antibody procedure. At low concentration the antiserum cross-reacts with histone H2B and with histones TH1-x + H1 to the extent of 11-14% of the interaction with TH2B-x. Antiserum preincubated in three successive H2B-coated tubes still retains 80-89% of the original anti-TH2B-x activity when assayed subsequently in TH2B-x-coated tubes, but cross-reaction with H2B is practically zero. The anti-TH2B-x antibodies also interact with tubes coated with mononucleosomes isolated from nuclei of seminiferous epithelial cells (SEC) of rat testis, but the interaction with mononucleosomes from rat liver nuclei is almost zero. The data suggest that in nucleosomes some of the antigenic determinants which are unique to TH2B-x are accessible, while those determinants which are common to H2B and TH2B-x are not accessible for interaction with antibodies. Competition by mononucleosomes, both from rat testis SEC and rat liver (to a lesser degree), in solution is detected by the reduction of binding of enzyme-labeled IgG to TH2B-x-coated tubes. However, an attempted competition by histones TH2B-x or H2B in solution resulted in an increase in the binding of the enzyme-labeled IgG to the mononucleosome-coated tubes. The interpretation of this type of competition assay is complicated by possible interaction of added histones with the coating mononucleosomes, followed by binding of antibodies to the histones. This TH2B-x antibody should be useful in studying changes in structure and function of chromatin during spermatogenesis and in the isolation of TH2B-x mRNA.     

Identification, cloning, and initial characterization of a novel mouse testicular germ cell-specific antigen

A monoclonal antibody, designated TES101, was raised by immunizing BALB/c mice with an allogenic mouse testicular homogenate followed by immunohistochemical selection as the initial screening method. By searching the expressed sequence tag (EST) database with the N-terminal amino acid sequence of TES101 reactive protein, we found that the predicted amino acid sequence encoded by a mouse testicular EST clone matched the TES101 protein sequence. Sequence analysis of the clone revealed no homologous molecule in the DNA/protein database. Based on data obtained from N-terminal amino acid analysis of the TES101 protein, the derived amino acid sequence contained a signal peptide region of 25 amino acids and a mature protein region of 225 amino acids, which translated into a protein with a molecular weight of 24 093. Northern blot analysis showed that mRNA of the TES101 protein was found in testis but not in any other mouse tissues examined. Western blot analysis revealed that TES101 reacted with a 38-kDa band on SDS-PAGE under nonreducing conditions, and this reactivity was abrogated under reducing conditions. Immunoelectron microscopic studies demonstrated that the molecule was predominantly located on the plasma membrane of spermatocytes and spermatids but not in Sertoli cells or interstitial cells, including Leydig cells. Thus, the TES101 protein is a novel molecule present primarily on the surface of developing male germ cells. TES101 protein may play a role in the processes underlying male germ cell formation.     

Identification and characterization of a novel testicular germ cell-specific gene Ggnbp1

A novel gene Ggnbp1 was identified during yeast two-hybrid screening of gametogenetin protein 1 (GGN1)-interacting proteins. Ggnbp1 gene was found in mouse, rat, and human genomes but not in sequenced yeast, worms, fly, or fish genomes. Northern blotting analysis revealed that the gene was specifically expressed in the testis but not expressed in the other tissues. In situ hybridization showed that it was testicular germ cell-specific and was specifically expressed in later primary spermatocytes, meiotic cells, and early round spermatids. Western blotting analysis detected a protein of expected size in and only in the testis. By making membrane and cytosolic fractions of germ cells, we were able to show that GGNBP1 associated with the membrane. The identification and characterization of a novel germ cell-specific gene Ggnbp1 is the first step toward the defining of the functions of Ggnbp1 in spermatogenesis.     

Effects of a murine germ cell-specific knockout of Connexin 43 on Connexin expression in testis and fertility

Connexin 43 (Cx 43)—expressed by germ cells (GC), Sertoli cells (SC) and Leydig cells—is one of at least eleven Cx in the murine testis. A general knockout (KO) of Cx 43 in mice results in perinatal death and a SC-specific KO of Cx 43 (SCCx43KO) causes infertility of male mice by preventing the initiation of spermatogenesis. To further elucidate the role of Cx 43 in the testis, a new mouse model with a GC-specific KO of Cx 43 (GCCx43KO) was created by using the Cre/loxP recombination system. A transgenic mouse line expressing the Cre gene under the tissue non-specific alkaline phosphatase promoter and a transgenic floxed Cx 43-LacZ mouse line were mated. The resulting F1-generation was backcrossed with homozygous Cx 43 floxed mice, and offspring was genotyped. Immunohistochemical analysis of testes of different aged homozygous mice revealed normal spermatogenesis and reduced Cx 43 immunoreactions. RT-qPCR and Western blots showed a downregulation of Cx 43 mRNA and protein, and a nearly unchanged mRNA expression of Cx 26, Cx 33 and Cx 45 in pubertal and adult KO mice. Western blots revealed considerable immunoreactive bands for Cx 26 and Cx 45. Male and female homozygous GCCx43KO mice were viable and fertile. Our data suggest, in contrast to inter SC and inter SC–GC cross talk in SCCx43KO mice which depends selectively on Cx 43 expression, that Cx 43 in GC seems not to be essential in GC–SC communication, when other Cx persist to be expressed.     

Isolation and characterization of a haploid germ cell-specific novel complementary deoxyribonucleic acid; testis-specific homologue of succinyl CoA:3-Oxo acid CoA transferase

We have isolated a cDNA clone encoding a mouse haploid germ cell-specific protein from a subtracted cDNA library. Sequence analysis of the cDNA revealed high homology with pig and human heart succinyl CoA:3-oxo acid CoA transferase (EC 2.8.3.5), which is a key enzyme for energy metabolism of ketone bodies. The deduced protein consists of 520 amino acid residues, including glutamate 344, known to be the catalytic residue in the active site of pig heart CoA transferase and the expected mitochondrial targeting sequence enriched with Arg, Leu, and Ser in the N-terminal region. Thus, we termed this gene scot-t (testis-specific succinyl CoA:3-oxo acid CoA transferase). Northern blot analysis, in situ hybridization, and Western blot analysis demonstrated a unique expression pattern of the mRNA with rapid translation exclusively in late spermatids. The scot-t protein was detected first in elongated spermatids at step 8 or 9 as faint signals and gradually accumulated during spermiogenesis. It was also detected in the midpiece of spermatozoa by immunohistochemistry. The results suggest that the scot-t protein plays important roles in the energy metabolism of spermatozoa.     

Partial characterization of a histone acetyltransferase from trout testis.

Histone acetyltransferase activity of trout testis was studied both in intact nuclei, and in high salt nuclear extracts. With intact nuclei, the distribution of incorporated [14C]acetate in the various histones was similar to that observed in vivo; the arginine-rich histones H3 and H4 showed the highest specific activities, and lower amounts of label were detected in histones H2a and H2b. Histone H1 incorporated little or no label. Acetyltransferase activity could be detected in purified, sheared chromatin after the addition of MgCl2 or KCl, suggesting that the enzyme is bound to chromatin. Treatment of nuclei with 0.4 M NaCl caused the dissociation of acetyltransferase activity. Most of this solubilized activity failed to bind to DEAE Sephadex and behaved as a high molecular weight heterogeneous complex on Sephadex G-100, suggesting that the enzyme is present as an aggregate with other proteins in the extract. The pH optimum of this preparation was approximately 8.5, and the enzyme showed a preference for histones H3 and H4 as substrates.     

Identification and characterization of a novel splice variant of beta3 subunit of GABA(A) receptor in rat testis and spermatozoa

Gamma-aminobutyric acid type A (GABA(A)) receptors are the major sites of inhibitory action of fast synaptic neurotransmission in the brain. Their receptors are also widely distributed in peripheral and endocrine tissues. A full-length cDNA encoding a novel splice variant of beta3 subunit of GABA(A) receptor, designated as beta3t, was identified in rat testis. This isoform contains a segment, having identical amino acid sequence as the beta3 subunit of neuronal GABA(A) receptors except for a section composed of 25 different amino acid sequence in the N-terminus. Northern blot shows that this isoform is found in rat testis. The beta3t isoform mRNA was detected in germ cells in the late step of spermatogenesis by in situ hybridization assay. Results of immunohistochemical and immunocytochemical assays indicate that the beta3t isoform is expressed in rat testis and spermatozoa. To determine a possible function of the N-terminal 25 amino acid segment, a recombinant plasmid of beta3t-EGFPC was constructed by fusing green fluorescent protein to the C-terminus of the beta3t isoform. The chimera product failed to be translocated unto the cell surface when expressed in HEK 293 cells; whereas, the beta3 subunit of rat brain is incorporated into the plasma membrane. In conclusion, the present results show that one variant of beta3 subunit of GABA(A) receptor, designated as beta3t, is found in germ cells of rat testis and sperm. The inability of the beta3t variant to target into the plasma membrane maybe a consequence of the unique 25 amino acid segment in the N-terminus.     

Isolation and characterization of histone H3 lysine 4 demethylase-containing complexes

Histone methylation is involved in the regulation of many cellular processes. In the past 2 years, several histone demethylases including BHC110/LSD1 have been characterized. BHC110, the first known histone lysine demethylase, removes methyl groups from methylated histone H3 lysine 4 and has been found in many multi-protein complexes. Using one-step affinity purification, we have isolated enzymatically active BHC110-containing complexes. Here, we detail the methods used for the isolation and characterization of these histone demethylase complexes from a human stable cell line.     

猪原始生殖细胞的分离、培养与鉴定

Embryonic germ cells (EG cells) are pluripotential undifferentiated stem cells isolated from cultured primordial germ cells (PGCs). Like ES cells, EG cells are of importance for gene targeting, therapeutical cloning and organ trans-plantation. The aim of this study was to isolate and characterize EG cells from porcine PGCs. The genital ridges from 24- 26 days old porcine embryos were treated in 0.02% EDTA for 20 min and pricked with a needle to release PGCs. The isolated PGCs were cultured on a SNL feeder layer in an EG cell medium. The EG cell medium consisted of Dulbecco‘‘s modified Eagle‘‘ s medium (DMEM) supplemented with 20 % Buffalo rat lever (BRL) cell-conditioned medium, 15 % fe-tal bovine serum, 1 mmol L-glutamine, 0.1 mol nonessential amino acids, 10 μmol β-mercaptoethanol and antibiotics.The freshly isolated PGCs were positive for alkaline phosphatase activity and Periodic acid-Schiff‘‘ s staining. Under this culture regime, PGCs could be maintained in an undifferentiated state and used for further cultures. One strain of the cul-tured PGCs was cultured 8 times, and alkaline phosphatase activity was detected in the colony formed from this strain.These cultured PGCs could spontaneously differentiate into fibroblast-like cells. These data suggested that we had success-fully isolated EG-like cells from oorcine PGCs.     

Cellular localization of mRNAs for retinoic acid receptor-alpha, cellular retinol-binding protein, and cellular retinoic acid-binding protein in rat testis: evidence for germ cell-specific mRNAs

In the present study we have examined the cellular localization and developmental changes of mRNAs for retinoid-binding proteins in rat testis. We demonstrate that mRNA (0.7 kb) for cellular retinol-binding protein (CRBP) is expressed only in Sertoli cells and peritubular cells. The mRNA for CRBP could not be detected in other testicular cells. In contrast, mRNA for cellular retinoic acid-binding protein (CRABP) was detected primarily in germ cells and to a small extent in tumor Leydig cells. The mRNA for CRABP in germ cells revealed distinct size heterogeneity and three distinct mRNA species were observed (1.0, 1.8, and 1.9 kb), in contrast to previous data for somatic cells where only the 1.0-kb mRNA has been reported. Messenger RNAs for retinoic acid receptor-alpha (RAR alpha) were detected in both somatic and haploid germ cells. The highest level of RAR alpha was seen in Sertoli cells, round spermatids, and tumor Leydig cells. Lower, but distinct, levels were observed in peritubular cells. Furthermore, we observed germ cell-specific species of RAR alpha mRNA (4 kb and approximately 7 kb). The smallest mRNA for RAR alpha (2.7 kb) in somatic cells was absent in germ cells. The levels of mRNAs for the various retinoid-binding proteins in whole testis obtained from rats of various ages confirmed this cellular localization. The mRNAs for CRBP, the small molecular size (2.7 kb) mRNA for RAR alpha (localized to somatic cells), and the 1-kb mRNA for CRABP showed an age-dependent decrease.(ABSTRACT TRUNCATED AT 250 WORDS)     

Genomic structure and promoter activity of the testis haploid germ cell-specific intronless genes,Tact1 and Tact2

The Tact1 and Tact2 genes, each of which encodes an actin-like protein, are exclusively expressed and translated in haploid germ cells in testis. To characterize the haploid germ cell-specific gene structure, a mouse genomic library was screened with a Tact1 cDNA as a probe, and four independent phage clones containing the Tact1 gene were isolated. Southern hybridization and sequencing analyses revealed that Tact1 and Tact2 were single copy genes contained on a common fragment in a head-to-head orientation, and that the distance between these genes was less than 2 kb. Comparison of the nucleotide sequences of genomic DNA and cDNA demonstrated that Tact1 and Tact2 lack introns, although all known actin or actin-related genes in mammals contain introns. Human Tact orthologues also lack introns and are located within 6.4 kb in a head-to-head orientation. These findings indicate that Tact1 and Tact2 or one of these genes arose by retroposition of a spliced mRNA transcribed from an actin progenitor gene prior to the divergence of rodents and primates. The Tact1 and Tact2 genes are unusual retroposons in that they have retained an open reading frame and are expressed in testicular germ cells, because almost all retroposons become pseudogenes. It was revealed that a 2kb sequence between the two genes bidirectionally controls haploid germ-cell specific expression by analyzing transgenic mice. Comparison of the murine Tact genes with their human orthologues showed a high level of identity between the two species in the 5'-upstream and non-coding sequences as well as in the coding region, indicating that conserved elements in these regions may be involved in the regulation of haploid germ cell-specific expression. The promoter region contains no TATA-, CCAAT- or GC-boxes, although there are potential cAMP response element (CRE)-like motifs in the 5'-upstream region and the 5'-untranslated region in Tact1 and Tact2, respectively. Transient promoter analyses indicate that CREMtau may activate Tact1 and Tact2 expression in germ cells.     

Molecular cloning of haploid germ cell-specific tektin cDNA and analysis of the protein in mouse testis.

Tektins are a class of proteins that form filamentous polymers in the walls of ciliary and flagellar microtubules. We report here the molecular cloning of a new member of the tektin family, tektin-t, identified from a mouse haploid germ cell-specific cDNA library. Tektin-t mRNA encodes a protein of 430 deduced amino acids possessing RSNVELCRD, the conserved sequence of tektin family proteins. Western blotting showed a single band having a molecular weight of 86 kDa in the mouse testis. Immunohistochemistry of the testis showed that tektin-t is localized in the flagella of elongating spermatids from developmental step 15 to maturity.