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Molecular cloning and characterization of fox testis kinectin.

Kinectin was isolated and characterized from a fox testis cDNA library using a monoclonal antibody (FTA-1) raised against testis surface proteins. The cDNA sequence of 4,479 nucleotides encodes an ORF of 1,330 amino acids (aa) with high homology to mouse, human, and chicken kinectins (GenBank Accession Number AF095786). Southern analysis was used to show that genes homologous to kinectin are present in several mammal species and in at least one marsupial, but not in bacteria. Alternatively spliced forms of fox kinectin were identified, and one of these is uniquely expressed in brain and spleen tissues. Kinectin expression was highest in testis relative to other tissues examined. Sequence analysis and comparisons between species revealed that kinectin encodes multiple alpha-helical coiled coils predicted to form dimers, and is, therefore, likely to exist as a dimer. The results presented in this article suggest that kinectin is required for spermatogenesis, but is not a likely candidate for use in immunocontraceptive vaccines.

Alternative Splicing↗

Translational regulation of a novel testis-specific RNF4 transcript.

The RING-finger protein SNURF/RNF4, a modulator of both steroid receptor dependent and basal transcription, is expressed at very high levels in testis and at much lower levels in several other tissues. In somatic tissues, the RNF4 gene is expressed as a 3-kb transcript while an additional shorter sized transcript (1.6 kb) was found in mouse testis. In murine germ cells, RNF4 protein expression is strongly modulated during progression of spermatogonia to spermatids, with a peak in spermatocytes. The expression of 3-kb transcript correlated with protein levels in the different germ cell populations. Conversely, the 1.6-kb transcript was abundantly and specifically expressed in spermatids, in which RNF4 protein was detected at very low levels. We have then examined possible mechanisms underlying this discrepancy. Primer extension and RNase protection analyses demonstrated that the 1.6- and 3.0-kb transcripts originate from the same promoter, encode for the same protein and differ in the 3' UTR. In vitro assays showed that protein degradation is not involved in the regulation of RNF4 protein level. Finally, polysome analysis revealed that only a slight fraction of the testis-specific transcript is engaged in translation, thus providing a feasible mechanism for the quantitative differences of RNF4 mRNA and protein levels. Present results demonstrate that RNF4 short transcript is poorly translated suggesting that this mechanism could be essential for normal spermatogenesis.

Alternative Splicing↗

Cloning and sequencing of cDNA encoding for a novel human testis-specific contraceptive vaccinogen: role in immunocontraception.

Sperm-specific antigens are attractive candidates for the development of a contraceptive vaccine. Using the subtractive cDNA hybridization technology, the present study was conducted to obtain a human sperm-specific antigen. The 32P-labeled single stranded cDNA of human testis, subtracted with poly(A)+ RNA of human peripheral white blood cells, was used to screen the human testis cDNA-ZAP II library. The putative positive clones were further screened for binding with the solubilized human oocyte zona pellucida preparation (HZP). After screening 10(7) colonies, one positive clone, designated contraceptive vaccinogen (CV), was obtained. It had an insert of approximately 1.3 kb, that was cloned and sequenced. The sense strand was identified by using the in vitro transcription and translation procedures, and the full-length sequence was obtained by using the 5' rapid amplification of 5' -cDNA ends (5'-RACE) procedure. The full-length CV cDNA has an ORF of 312 amino acids (aa) with the first ATG Met start codon at nucleotide (nt) 35 and the stop codon TAA, at nt 959. The translated protein has a calculated molecular mass of 35.3 kD and four potential N-linked glycosylation and several phosphorylation sites. Hydropathy plot generated from the deduced aa sequence showed it to be a membrane-anchored peptide. Extensive computer search in the database did not find any homology of existing sequences with CV both for nt and aa. Northern blot analysis indicated the human testis-specific expression of CV antigen. The coding region of CV cDNA was subcloned into pET22b(+) vector and expressed. The expressed recombinant (r)CV protein had a molecular size of approximately 44 kD, and it specifically reacted with the ZP3 component of HZP. Rabbit rCV antibodies recognized the rCV, and a cognate antigen of approximately 64 kD in the human sperm extract. The antibodies showed binding with the live and methanol-fixed human sperm, and significantly (P < 0.001) inhibited human sperm penetration of zona-free hamster oocytes, as well as human sperm binding to human oocyte zona pellucida. These findings indicate that the testis/sperm- specific CV antigen has a role in human sperm function and may find clinical applications in the contraceptive vaccine development and in the specific diagnosis and treatment of male infertility.

Adult↗

Detection of transgene in progeny at different developmental stages following testis-mediated gene transfer.

We recently reported that exogenous DNA injected into testis as a liposome complex can be transferred into the egg via sperm by natural mating and integrated in the genome (testis-mediated gene transfer: TMGT). Here, we studied the efficiency of each of the several liposomes in associating foreign DNA with sperm, the expression of an introduced gene in early embryos, and the presence of the DNA in fetuses and pups at different ages. The CMV/beta-actin/EGFP fusion gene, encapsulated with different liposomes, was injected into rat testis, and spermatozoa in the cauda epididymis were obtained 1, 4, and 14 days after injection. We tested each of the 8 liposomes, and found that only 2, DMRIE-C and SuperFect, led to the detection of foreign DNA on all of the days examined, with relatively higher ratios of rats having positive sperm. By means of TMGT using either of those two liposomes, more than 80% of morula-stage embryos expressed EGFP, as observed by fluorescence microscopy. Then we detected introduced DNA in the progeny by PCR and Southern dot blot, and found that the ratio of animals carrying the foreign DNA decreased as they developed, and that only a part of postpartum progeny were foreign-DNA-positive with high incidence of mosaicism. These results suggest that, although, the success rate is still limited, foreign DNA could be integrated into the genome of the progeny by TMGT at least under specific experimental conditions, the efficiency of which depends largely on the characteristics of the liposome. The results also suggest that TMGT could be applicable to fetal gene therapy as well as to the generation of transgenic animals.

Animals↗

Analysis of the expression of a large number of novel genes isolated from mouse prepubertal testis.

The analysis of genes expressed in a restricted temporal and spatial manner during spermatogenesis has given insights into different gene-regulatory mechanisms active in germ cells. However, very few genes have so far been described that are predominantly active in spermatogonia and the early meiotic cell types of testis. To isolate a battery of such genes, more than 100 different mRNA molecules were isolated from a mouse prepubertal testicular cDNA library, and their expression patterns in different tissues analyzed. Thirty mRNAs, 26 of them previously not described in the literature, were found to be predominantly expressed in mouse testis. A detailed analysis of their expression patterns identified a number of mRNA molecules differentially expressed in testicular cell types, including both germ cells and somatic cell types. Characterization of these mRNAs also revealed five distinct temporal phases of gene expression during prepubertal germ cell development. Three different genes, mainly active in the spermatogonial and the early meiotic cell types of testis, were isolated and will be used to characterize further stage-specific gene expression during germ cell differentiation.

Animals↗

Direct evidence that the mouse sex-determining gene Sry is expressed in the somatic cells of male fetal gonads and in the germ cell line in the adult testis.

Reverse transcriptase polymerase chain reaction (RT-PCR) analysis shows that Sry mRNA is expressed in male fetal urogenital ridges from 12.5 day p.c. embryos, but not in enriched populations of primordial germ cells from the same embryos, indicating that Sry is expressed in the somatic cells of the embryonal gonad at the time of testis determination. We also show that, in the adult male mouse testis, Sry mRNA is expressed at high levels in meiotic and postmeiotic germ cells and, at much lower levels, also in Sertoli cells. Treatment with cyclic adenosine monophosphate (cAMP) analogs of cultured Sertoli cells from postnatal testis completely abolishes Sry mRNA expression.

Animals↗

Increased accessibility of the N-terminus of testis-specific histone TH2B to antibodies in elongating spermatids.

Changes in chromatin structure during spermatogenesis were investigated using a monoclonal antibody that immunoreacts with the N-terminus of the testis-specific histone TH2B. This monoclonal antibody, which had been raised against rat tyrosine hydroxylase (TH), cross-reacted with TH2B because of sequence homology at the N-termini of TH and TH2B. The epitope was localized to the N-terminus of TH2B as trypsin-digested chromatin which lacked the N-terminal tail did not react with anti-TH and preincubating anti-TH with a synthetic peptide made from the homologous sequence between TH2B and TH inhibited its binding to TH and TH2B. In histological sections of rat testis, the primary spermatocytes and round spermatids immunoreacted weakly, whereas elongating spermatids at steps 10-12 immunoreacted intensely with anti-TH. Increased staining of elongating spermatids was also observed in mouse and hamster by immunohistochemistry. However, immunoblotting proteins extracted from separated rat testis cells showed no increase in the TH2B content of these late steps of spermatids. The apparent increase in the immunohistochemical staining corresponds to increased accessibility of the epitope in the elongating spermatids. This indicated that the N-terminus of TH2B is less tightly bound to DNA or to other proteins at this time in preparation for the removal of TH2B and other histones.

Amino Acid Sequence↗

Proteome analysis of rat spermatogonia: reinvestigation of stathmin spatio-temporal expression within the testis.

Stathmin is a protein known to be involved in various cell processes including cell proliferation and differentiation. It has already been described in the testis but its recent identification using a proteomic approach in mitotic spermatogenetic stem cells named spermatogonia (Guillaume et al., 2000) has lead us to reinvestigate its expression within the testis. Stathmin and its mRNAs were studied in isolated cells by Western and Northern blots and in situ using immunohistochemistry. We demonstrated that stathmin is indeed expressed in spermatogonia, and that it is also intensively expressed in the meiotic spermatocytes and in the first generations of spermatids. Furthermore, we showed aggregations of the protein in the cytoplasm of the later generations of spermatids preceding its elimination at the time of spermiation. Our Northern blots reveal the presence of two stathmin transcripts of 1.1 and 3.2 kb within the testis from the fetal stage onwards, in spermatogonia, spermatocytes, and spermatids. However, the 3.2 kb RNA transcript was barely detectable in the spermatids. Stathmin expression is known to be associated with microtubule dynamics. Therefore, its expression in the germ line is most probably related to the extremely complex structural cellular rearrangements occurring in germ cells during spermatogenesis. However, the exact role of stathmin and the reason of the existence of two transcripts in the male germ lineage awaits further investigation.

Animals↗

Germ-cell specific protein gametogenetin protein 2 (GGN2), expression in the testis, and association with intracellular membrane.

Gametogenetin (Ggn) is a germ cell-specific gene with multiple splicing variants giving rise to three predicted protein products, gametogenetin protein 1 (GGN1), gametogenetin protein 2 (GGN2), and gametogenetin protein 3 (GGN3). GGN1 and GGN3 were reported to interact with Fanconi anemia complementation group L (FANCL) per proliferation of germ cells (POG), a ubiquitin E3 ligase involved in germ-cell-deficient (gcd) mutation. While GGN2, another protein from Ggn by alternative splicing did not interact with FANCL/POG since it lacked the domain mediating the interaction. Little is known about the expression and function of GGN2. Here through Northern blotting experiment we showed that Ggn was mainly expressed in the testis but hardly detectable in the ovary or the somatic tissues. By preparing GGN2-specific antibody we showed that GGN2 was detectable and only detectable in the testis. By comparing the expression of Ggn mRNA and GGN2 protein in developing mouse testis, we showed that there was no evident delay of the translation of Ggn mRNA after their transcription. Both the subcellular localization study and the germ cell membrane protein fractionation implied that GGN2 associated with the intracellular membrane system. Co-fractionation on Superdex and yeast two-hybrids suggested that like GGN1, GGN2 was also a potential interaction partner of gametogenetin binding protein 1 (GGNBP1). Our data suggested that gametogenetin proteins were mainly involved in male germ cell development and GGN2 was also a possible interaction partner of GGNBP1. Like GGN1, GGN2 was also possibly involved in cell trafficking. The possible involvement of GGN2 in acrosome biogenesis was proposed.

Acrosome↗

A mouse gene encoding a novel member of the WD family of proteins is highly conserved and predominantly expressed in the testis (Wdr13).

Wdr13, a novel member of the WD family of proteins and the mouse homolog of WDR13 is localized to the locus XA1.1 and is predominantly expressed in the testis. The expression begins at the early stages of gonadal development and is maintained throughout the adult life with a predominant expression in the germ cells of adult testis. RNA in situ hybridization on the testis and brain sections indicated a cytoplasmic expression of the transcript. The alternatively spliced transcripts of the gene are generated by different methods and showed a differential pattern of expression, suggesting functional diversity. The expression of the gene in the unfertilized egg and in the neural stem cells indicated the functional significance of the gene from the early stages of development. The nuclear localization of the mouse WDR13 protein suggested a regulatory function. Evolutionary analysis of the gene indicated an extensive functional conservation across diverse species. Comparison of the genomic organization of the different homologs revealed a varied organization in the invertebrate homolog and the retention of the functionally significant introns in the same.

Alternative Splicing↗

Analysis of the mRNA expression of the TGF-Beta family in testicular cells and localization of the splice variant TGF-beta2B in testis.

The transforming growth factors (TGF)-beta, TGF-beta1, TGF-beta2, and TGF-beta3, and their receptors [TbetaRI, TbetaRII, TbetaRIII (betaglycan)] elicit many functions in the testis, for example, they perturb the blood testis barrier (BTB). Although expression of the ligands and receptors have been investigated, the alternative splice variants are incompletely examined. We therefore have analyzed all ligands, the receptors, and the splice variants TbetaRIB, TbetaRIIB, and TGF-beta2B in testicular cells from rat and mouse. In mouse, the novel transcript variant TGF-beta2B was identified and was found in Leydig cells, spermatogonia, pachytene spermatocytes, and in the apical regions of the Sertoli cells in adult testis. Even though expression of the splice variant TbetaRIB could be shown in mouse and rat, we never found the isoform TbetaRIIB in the rat cell lines studied. Whereas in all testicular cells expression of all TGF-beta ligands could be shown, receptor mRNA expression was slightly more diverse. Furthermore, expression pattern of the splice variants was more heterogeneous, for example, TbetaRIB was not detectable in adult Sertoli cells, primary peritubular cells, and immortalized peritubular cells. The heterogeneous expression of the receptors and especially of the splice variants might provide possible clues for the different functions of the TGF-beta ligands in testicular cells.

Alternative Splicing↗

Noninvasive analysis of aldose reductase activities in rat testis: 3-FDG NMR spectroscopy and imaging.

Noninvasive investigation of aldose reductase activities in rat testis was performed using 3-fluoro-3-deoxy-D-glucose (3-FDG) and nuclear magnetic resonance (NMR) spectroscopy/imaging. Quantitative determination of testis aldose reductase activities, expressed as the sorbitol index, showed a value similar to that of brain. Sorbitol imaging demonstrated aldose reductase activities in testis to be confined primarily to the central region of this organ. The 3-FDG 19F NMR method appears to have clinical potential in the evaluation of testicular function, especially that of spermatogenesis, noninvasively.

Aldehyde Reductase↗

The testis in mice infected with Mycobacterium leprae.

Following inoculation either locally or intravenously with Mycobact. leprae of human origin, the histopathology and bacteriology of the testis in experimental mice is described. Normal mice, and mice rendered immunologically deficient by thymectomy and whole-body irradiation, were studied. Attention is drawn to a heavy bacillation of the testis in mice from both groups. Bacilli were found in and beneath the tunica albuginea, but mainly in interstitial cells and in macrophages surrounding the tubules. The percentage of solidly staining bacilli was high, and globi were frequent. The study showed that the testis in mice is particularly favourable for the lodgement and multiplication of Mycobact. laprae following either local or intravenous inoculation. The significance of this in relation to the metabolism of the leprosy bacillus and to the frequent occurrence of testicular damage in the lepromatous male patient is discussed. This work was supported by grants to A. G. M. Weddell and A. C. McDougall from the Medical Research Council and the British Leprosy Relief Association (LEPRA).

Animals↗

Non-androgenic role of testis in enhancing ventral prostate growth in rats.

This study was conducted to investigate whether the testis, aside from its ability to secrete androgen, is able to promote prostatic growth in rats. Increasing quantities of silastic capsules filled with crystalline dihydrotestosterone (DHT) were implanted subcutaneously into adult Sprague-Dawley rats at the time of bilateral epididymo-orchiectomy or sham operation on the testes. Control animals received empty capsules. Twenty-eight days later, the growth of the ventral prostate as measured by wet weight, DNA, and protein content per prostate was significantly greater in rats with intact testes than in orchiectomized rats. An overall increased growth was noted at all doses of exogenous DHT administered. Serum levels of luteinizing hormone in animals treated with DHT were undetectable. Serum levels of testosterone in intact rats treated with DHT were not significantly different from those in castrated rats. These observations suggest a non-androgenic role for the testis and, perhaps, epididymis in promoting prostatic growth in rats, and are consistent with the concept that a non-androgenic substance, produced from the testis and/or epididymis, is able to enhance prostate growth induced by androgen stimulation. The possibility that this phenomenon may play a role in the benign growth of the prostate observed in aging human males with decreased blood levels of androgen warrants consideration.

Analysis of Variance↗

Ultrastructural changes in the testis and epididymis of rats following treatment with the benzene chromatographic fraction of the chloroform extract of the seeds of Carica papaya.

The benzene chromatographic fraction of the chloroform extract of the seeds of Carica papaya at a dose of 10 mg/rat/day for 150 days, which has shown a total inhibition of motility, reduced sperm count and infertility, was tested to de fi ne the mode of action at the subcellular level in the testis and epididymis. The ultrastructure of the testis of the treated animals revealed no appreciable changes in the subcellular characteristics. The mechanism of protein synthesis as well as steroidogenesis were evident in the Sertoli cells while the spermatogonia, spermatocytes and spermatids, both round and elongated, depicted a prominent nucleus, distinct nuclear membrane and cytoplasmic characteristics indicating normal germ cell differentiation. The principal cells of the cauda epididymis were characterized by the presence of well-de fi ned rough endoplasmic reticulum, mitochondria, Golgi bodies and secretary granules, suggesting active secretory functions. The absorptive function of the cauda epididymis was evidenced by the presence of numerous vesicles and multivesicular bodies adjacent to stereocilia. It is concluded that the inhibition of sperm motility by the drug could be due to other epididymal factors rather than the subcellular characteristics of testis and epididymis.

Animals↗

Localization of D-aspartic acid in elongate spermatids in rat testis.

In the current study, localization of D-aspartic acid (D-Asp) in rat testis was studied by immunohistochemical and biochemical techniques. Immunohistochemical staining of this tissue using specific polyclonal antibody to D-Asp revealed D-Asp immunoreactivity (IR) in the cytoplasm of germ cells, especially around the region rich in elongate spermatids, the most mature of the germ cells. Weak IR was also noted in cytoplasm of spermatocytes and round spermatids; however, it was negligible in interstitial cells and Sertoli cells. The intensity of immunostaining in each seminiferous tubule differed according to its distinct germ cell composition. In testis of young rats, seminiferous tubules lack elongated spermatids, and D-Asp was found to be localized in spermatocytes, the most mature population of germ cells at that age. We used various toxicants to destroy specific testicular cell populations and to confirm the localization of D-Asp in rat testis. Administration of ethane dimethane sulfonate induced a selective destruction of all Leydig cells in this tissue. This resulted in a significant decrease in the D-Asp level, which was probably due to a drop in testosterone brought about by this treatment, and this was followed by a modulation of spermatogenesis. Three days after treatment with methoxyacetic acid (MAA), many seminiferous tubules were found to lack or to have severe depletions of pachytene spermatocytes, but not of elongate spermatids. This caused reductions in protein content and in the total amount of L-Asp, but not that of D-Asp. Twenty days after treatment with MAA, the depleted population of germ cells progressed through the spermatogenic cycle from pachytene spermatocytes to elongate spermatids. At this time, the level of D-Asp decreased significantly, as did that of L-Asp and protein, consistent with D-Asp localization in elongate spermatids. This decrease in the D-Asp level was also seen with immunostaining.

Acetates↗

Chicken lacks the testis specific isozyme of angiotensin converting enzyme found in mammals.

Chicken angiotensin converting enzyme (ACE) cDNA was cloned based on homology to the mouse ACE sequence. The chicken ACE protein is highly homologous to the somatic isozyme of ACE found in human, mouse, bovine, and rabbit. Like the mammalian somatic forms, the chicken enzyme consists of two putative zinc binding sites at the center of two homologous domains. All known functional residues are absolutely conserved. Unlike the mammals, no evidence for a single domain, testis specific form of ACE was found in the chicken testis by either Northern blot or enzyme assay. This result is unexpected since the adult mammalian testis expresses one of the highest tissue levels of ACE.

Amino Acid Sequence↗

p53 is associated with the nuclear envelope in mouse testis.

p53 has been postulated to play a role in meiosis as well as in the regulation of germ cell numbers by apoptosis. This study investigated the subcellular localization of p53 in the testis, including conditions known to induce germ cell apoptosis. Western blot analysis showed that p53 was enriched in the nuclear envelope fraction, and confocal microscopy confirmed that p53 was associated with the nuclear envelope of germ cells. Exposure of the testis to heat stress induced translocation of p53 into the nucleus. Nuclear envelope binding provides an optimal site for rapid entry of p53 into the nucleus, where it may act as a DNA-binding protein to induce apoptosis or cell cycle arrest in response to appropriate stimuli. The nuclear envelope sequestration of p53 also provides a framework to understand how mitosis and meiosis in the testis may proceed despite high intracellular concentration of p53.

Animals↗

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