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Cloning of the gene encoding the murine orphan receptor TAK1 and cell-type-specific expression in testis.

We have cloned the gene encoding the mouse homologue of the orphan receptor, TAK1, a member of the nuclear receptor superfamily, from a mouse testis cDNA library. The amino acid sequence of mouse TAK1 (mTAK1) is highly homologous to that of human TAK1, with an overall identity of 98%. Northern blot analysis using RNA from different testicular cell types showed that the mTAK1 transcript is predominantly expressed in pachytene spermatocytes at a low level in round spermatids, but not in germ cells at earlier phases of spermatogenesis or in Sertoli cells. Southern analysis using genomic DNA prepared from a panel of hamster/human and mouse/human hybrid cell lines indicated that the TAK1 gene is located on human chromosome 3.

Amino Acid Sequence↗

Comparative squalene synthase gene expression in mouse liver and testis.

RNA from various mouse organs was analyzed by Northern hybridization to determine the response of squalene synthase (SQS) mRNA to dietary cholesterol, or lovastatin and cholestyramine, administration. Two size-classes of highly abundant mouse SQS (mSQS) mRNAs of approximately 1.9 and 2.0 kb were found in testis. These transcripts were unresponsive to sterol regulation. A single size-class of liver mSQS mRNA of approximately 1.9 kb was sterol-regulated. Studies using primer extension and 5' rapid amplification of cDNA ends (RACE) indicated that the size differences in liver and testis mSQS transcripts were due to variations in the lengths of the 5' untranslated regions (UTRs). The longest testis 5' UTR extended approximately 106 nt 5' of the primary transcription initiation site in liver of mice fed lovastatin and cholestyramine. These results suggest that tissue-specific promoter elements control the transcriptional regulation of the promoters for the mSQS gene in liver and testis.

5' Untranslated Regions↗

Ultrastructural observations of spermatogenesis following transplantation of rat testis cells into mouse seminiferous tubules.

The testes of busulfan-treated immunodeficient mice receiving seminiferous tubule injections of testis cells from rats were examined by light and electron microscopy. The presence of active rat spermatogenesis was verified by criteria that are known to characterize spermatogenic cells of this species. In addition, spermatogenesis from the mouse was identified as taking place in some seminiferous tubules as the result of reinitiation of spermatogenesis after busulfan treatment. Rat spermatogenesis in mouse seminiferous tubules showed the generally recognized associations of cells known to characterize stages of spermatogenesis of the rat. The Sertoli cells associated with rat spermatogenesis were identified ultrastructurally as being of mouse origin. Thus, rat spermatogenesis, which has a cycle length that is 50% longer than mouse spermatogenesis, can proceed among mouse Sertoli cells, which supposedly exert much shorter cyclic influences in concert with mouse germ cell development. Studies are needed to determine if the timing of rat spermatogenesis is controlled by the germ cells or the Sertoli cells. These observations are considered preliminary since a thorough study of somatic-germ cell relationships was not undertaken. It is concluded that a mouse Sertoli cell in the environment provided by the mouse testis can produce both mouse and rat gametes.

Acrosome↗

Zfp-37, a new murine zinc finger encoding gene, is expressed in a developmentally regulated pattern in the male germ line.

To begin to examine the function in the mouse testis of genes containing the zinc finger motif, we have screened an adult mouse total testis cDNA library with probes to a conserved region of zinc fingers. We have isolated cDNAs for a new murine zinc finger encoding gene that has been designated Zfp-37. Northern blot hybridization analysis revealed Zfp-37 transcripts at high levels in the testis, the only adult tissue in which Zfp-37 expression was observed. Zfp-37 was also expressed at lower levels in the mid-gestation embryo and placenta. The major testicular transcripts are 2.3 and 2.6 kb. A 4.0 kb transcript was detected at lower levels in the testis as well as in embryo and placenta. Northern blot and in situ hybridization analysis revealed that expression of Zfp-37 was most abundant in germ cells which have completed meiosis and are undergoing the complex morphogenetic changes of spermiogenesis. The pattern of expression of Zfp-37 and the presence of the zinc finger domain suggest that Zfp-37 may have a role in regulating spermiogenesis.

Amino Acid Sequence↗

Cloning, expression analysis, and tissue distribution of esp-1/testisin, a membrane-type serine protease from the rat.

Esp-1/testisin, a serine protease abundantly expressed in human and mouse testis, is presumed to play an important role in the process of spermatogenesis and fertilization. In this study, we cloned an esp-1/testisin cDNA from rats, and analyzed its expression and tissue distribution. The isolated cDNA consisted of 1099 nucleotides with a single open reading frame encoding 328 amino acids and an expected molecular mass of 36.6 kDa. The deduced amino acid sequence of rat Esp-1/Testisin had 89% and 62% identity with its murine and human counterparts, respectively, and appeared to be a trypsin-type serine protease with a hydrophobic region at the C-terminus. By quantitative real-time polymerase chain reaction analysis, rat esp-1/testisin mRNA was predominantly expressed in testis, as in human and mouse. However, its immunohistochemical distribution was predominantly in the elongated spermatids at steps 12 to 19, and not in the primary spermatocytes and round spermatids. This different distribution profile suggests that Esp-1/Testisin plays a role in species-specific proteolytic events during spermatogenesis and fertilization.

Amino Acid Sequence↗

Identification of a novel protein p59(scr), which is expressed at specific stages of mouse spermatogenesis.

A novel cDNA-encoding polypeptide of 545 amino acid residues was identified from a mouse testis cDNA library. The transcript of this gene, p59(scr), was predominantly expressed in the testis and was developmentally regulated during spermatogenesis. The encoded protein was expressed in spermatocytes and round spermatids within seminiferous tubules of the adult testis. Using an adult-mouse model of experimental unilateral cryptorchidism, it was observed that the expression of the p59(scr) mRNA was reduced in the cryptorchid testes in association with destruction of spermatogenesis. In vitro heat stress experiment further demonstrated that p59(scr) mRNA was more sensitive to heat stress than the other mRNA species, such as germ-cell-specific meiosis-activating kinase (mak) gene and a housekeeping beta-actin gene. Our results suggest that this novel p59(scr) gene is involved in spermatogenesis and may play an important role in development of testicular germ cells.

Amino Acid Sequence↗

Immunochemical and functional characterization of a polyclonal antibody to human sperm antigen.

A polyclonal antibody was raised against a 16 kDa human sperm protein identified by a monoclonal antibody to human sperm. The antibody showed significant reactivity with mouse spermatozoa as seen by ELISA. Immunohistochemical analysis showed that the antibody reacted with antigens from mouse testis, prostate as well as seminal vesicle. In both mouse and human testis the antibody localized antigens in round as well as elongated spermatids and mature spermatozoa. By SDS-PAGE and Western blot analysis the antibody reacted with a 16 kDa protein in the testis and seminal vesicle, whereas in the prostate it identified two proteins, one at 20 kDa and another at 25 kDa. Immunofluorescent localization by the antibody showed reactivity with acrosomal and/equatorial and midpiece region of human spermatozoa. The antibody showed extensive agglutination both in mouse and human spermatozoa. The results indicate that the antigen may be a conserved antigen. Cross reactivity of the antibody with mouse spermatozoa enabled us to carry out antifertility trials. Passive immunization of female mice with this antibody caused 67% reduction in fertility. It is likely that the antifertility effect could be partly due to agglutinating nature of the antibody which may have caused inhibition of all processes that depend on forward motility such as cervical mucus penetration and possibly preventing sperm egg interaction. Such well characterized and functionally relevant antibodies will enable to identify sperm antigens relevant for fertility. Identification of such antigens may also help in diagnosis of immuno infertility.

Animals↗

Activating signal cointegrator 1 is highly expressed in murine testicular Leydig cells and enhances the ligand-dependent transactivation of androgen receptor.

Activating signal cointegrator 1 (ASC-1) has been recently reported as a coactivator of some nuclear receptors. In the present study, we have analyzed the expression of ASC-1 in the mouse testis and investigated its capacity to modulate the transcriptional activity of androgen receptor (AR). We found that although ASC-1 mRNA was ubiquitously expressed at a low level in mouse tissues, a couple of testis-specific mRNAs were expressed in the adult testis. Cloning of one testis-specific variant revealed that the ubiquitous and testis-specific transcripts of ASC-1 share at least the same open reading frame. The expression of the testis-specific ASC-1 mRNAs was developmentally regulated, and the onset of their expression coincided with the initiation of spermatogenesis. In situ hybridization of mouse testis with ASC-1 antisense probe demonstrated predominant expression of ASC-1 in the interstitial Leydig cells that express AR. Moreover, yeast two-hybrid tests and glutathione S-transferase pull-down assays revealed that ASC-1 associates directly with AR and that the hinge domain of AR and a putative zinc-finger motif of ASC-1 are major determinants for their interaction. Transient transfection assays performed by expressing ASC-1 in combination with AR and an androgen-responsive reporter gene showed that ASC-1 moderately alters the induction of the reporter gene. Taken together, these results suggest that ASC-1 may function as an AR coregulator and have a role in testicular functions.

Animals↗

Insulin-like 6 immunoreactivity in the mouse brain and testis.

Insulin-like 6 immunoreactivity (irINSL6) was detected in Leydig cells of the mouse testis. In the brain, labeled somata were detected mainly in the caudal hypothalamus and midbrain. Double labeling the brainstem sections revealed that irINSL6 somata were 5-hydroxytryptamine (5-HT) positive. The presence of irINSL6 in discrete populations of hypothalamic and brainstem neurons and in Leydig cells of the testis suggests a diverse biological function of this novel peptide.

Animals↗

Characterization of spermatogonial stem cell maturation and differentiation in neonatal mice.

Initiation of the first wave of spermatogenesis in the neonatal mouse testis is characterized by the differentiation of a transient population of germ cells called gonocytes found in the center of the seminiferous tubule. The fate of gonocytes depends upon these cells resuming mitosis and developing the capacity to migrate from the center of the seminiferous tubule to the basement membrane. This process begins approximately Day 3 postpartum in the mouse, and by Day 6 postpartum differentiated type A spermatogonia first appear. It is essential for continual spermatogenesis in adults that some gonocytes differentiate into spermatogonial stem cells, which give rise to all differentiating germ cells in the testis, during this neonatal period. The presence of spermatogonial stem cells in a population of cells can be assessed with the use of the spermatogonial stem cell transplantation technique. Using this assay, we found that germ cells from the testis of Day 0-3 mouse pups can colonize recipient testes but do not proliferate and establish donor-derived spermatogenesis. However, germ cells from testes of Day 4-5 postpartum mice colonize recipient testes and generate large areas of donor-derived spermatogenesis. Likewise, germ cells from Day 10, 12, and 28 postpartum animals and adult animals colonize and establish donor-derived spermatogenesis, but a dramatic reduction in the number of colonies and the extent of colonization occurs from germ cell donors Days 12-28 postpartum that continues in adult donors. These results suggest spermatogonial stem cells are not present or not capable of initiating donor-derived spermatogenesis until Days 3-4 postpartum. The analysis of germ cell development during this time frame of development and spermatogonial stem cell transplantation provides a unique system to investigate the establishment of the stem cell niche within the mouse testis.

Animals↗

Impaired reproduction in transgenic mice overexpressing Gamma-aminobutyric acid transporter I (GAT1).

It is well documented that g-aminobutyric acid (GABA) system existed in reproductive organs. Recent researches showed that GABAA and GABAB receptors were present in testis and sperm, and might mediate the acrosome reaction induced by GABA and progesterone. GABA transporter I (GAT1) also existed in testis and sperm, but its physiological function was unknown. In the present study, we used GAT1 overexpressing mice to explore GAT1 function in male reproductive system. We found that the expression level of GAT1 continuously increased in wild-type mouse testis from 1 month to 2 months after birth. GAT1 overexpression in mouse affected testis development, which embodied reduced testis mass and slowed spermatogenesis in transgenic mice. Moreover, transgenic mice showed increase of the percentage of broken sperm. The further study revealed that the reproductive capacity was impaired in GAT1 overexpressing mice. In addition, testosterone level was significantly low in transgenic mice compared with that in wild-type mice. Our findings provided the first evidence that abnormal expression of GAT1 could result in dysgenesis, and indicated that GAT1 might be therapeutically targeted for contraception or dysgenesis treatment.

Animals↗

Expression of antimicrobial defensins in the male reproductive tract of rats, mice, and humans.

Defensins are antimicrobial peptides that play a major role in innate immunity. Using reverse transcriptase-polymerase chain reaction, immunochemistry, or both, we performed a search of all presently known defensins in rat testis, epididymis, and isolated testicular cells; in mouse testis and epididymis; and in human testis and ejaculates. In the rat, all alpha- and beta-defensins except RNP-4 were expressed within the testis, whereas alpha-defensins RNP1-2, RNP-4, and beta-defensins RBD-1 and RBD-2 were present within the epididymis. In the mouse, the cryptdin transcripts CRS1C, mBD-1, and mBD-2 were detected in the testis and epididymis, whereas mBD-3 and mBD-4 were expressed only in the epididymis, and CRS4C was absent in both organs. In the human testis, transcripts for four known defensins were expressed with the consistent exception of HBD-2 and HBD-3. In rat interstitial tissue, resident macrophages expressed most of the defensins studied, whereas Leydig cells produced only RBD-2. In contrast, all studied defensins except RNP-4 were present in the seminiferous tubules. Within these tubules, peritubular and Sertoli cells expressed most of the studied alpha- and beta-defensins, whereas spermatogonia displayed only alpha-defensins, but at relatively high levels. Meiotic pachytene spermatocytes expressed only beta-defensins, whereas postmeiotic spermatids and their cytoplasmic lobes displayed both types. In humans, the HBD-1 peptide was expressed mainly in the germ line from pachytene spermatocytes to late spermatids. The peptide was also present in ejaculated spermatozoa and seminal plasma, where multiple soluble forms were present. Finally, high salt concentration or dithiothreitol-sensitive cationic extracts from human seminal plasma were indeed found to display antimicrobial activity. We conclude that the male reproductive tract produces defensins that most probably assume an important, innate organ defense system against pathogens.

Animals↗

Refinement of the expression pattern of a mouse homologue of the porcine 135-kDa alpha-d-mannosidase (MAN2B2).

In the article by Hiramoto et al. (1997) (Stage-specific expression of a mouse homologue of the porcine 135 kDa alpha-d-mannosidase (MAN2B2) in type A spermatogonia, Biochem. Biophys. Res. Commun. 241, 439-445) a new homologue to the porcine epididymis-specific 135 kDa alpha-d-mannosidase (MAN2B2) was cloned from a mouse testis cDNA library. Northern blot analysis with RNA of different tissues showed a testis-specific expression by using a mouse MAN2B2 oligonucleotide. In situ hybridization revealed that the mouse MAN2B2 transcript is localized exclusively in spermatogonia. In consequence to this it was proposed by Hiramoto et al. that the mouse MAN2B2 homologue could serve as a marker for spermatogonia. By repeating the published experiments we observed a different expression pattern of the mouse MAN2B2 gene. Northern blot analysis with either testicular RNA from prepubertal males or with testicular RNA from the W/W(v)-mutant mouse which lacks germ cells showed an expression of the MAN2B2 gene. Furthermore, Northern blot analysis with RNA from different somatic tissues revealed that the gene is ubiquitously expressed. Therefore, our refinement clearly demonstrates that the MAN2B2 mouse homologue is not specifically expressed in spermatogonia.

Aging↗

Spermatogonial stem cell transplantation, cryopreservation and culture.

Testis cells of a fertile male mouse can be transplanted to the seminiferous tubules of an infertile male, where the donor spermatogonial stem cells will establish spermatogenesis and produce spermatozoa that transmit the donor haplotype to progeny. In addition, stem cells can be cryopreserved for long periods, thereby making male germ lines immortal. Recently, mouse testis cells have been cultured for longer than 3 months and, following transplantation, produced spermatogenesis. These techniques are likely to be applicable to many species, since rat testis cells can be cryopreserved and generate spermatogenesis in the seminiferous tubules of immunodeficient mice.

Animals↗

A scanning electron microscope study of the tunica vaginalis testis in the mouse.

On the visceral layer of the mouse tunica vaginalis testis are two populations of mesothelial cells: one carrying numerous surface microvillia and the other with fewer microvilli thus presenting almost bare areas. Intercellular stomata and smaller gaps are seen only between the cells of the bare areas. Underlying the stomata the surface of a second cell, which probably represents the endothelial lining of submesothelial lymph vessels, is often seen. Mesothelial cells of the parietal layer lack microvilli, stomata and gaps but show numerous long filamentous processes on the apical cell surface and at the lateral cell borders where they connect neighbouring cells as they overlie unusually wide intercellular clefts.

Animals↗

Effects of in-utero exposure to zeranol or diethylstilboestrol on morphological development of the fetal testis in mice.

The morphological development of the fetal mouse testis exposed to alpha-zearalanol (zeranol) or diethylstilboestrol (DES) was evaluated as part of an examination of the effects of transplacental exposure to non-steroid oestrogens on susceptible tissues. On days 9 and 10 of gestation, pregnant NMRI mice were given subcutaneous injections of ethyl oleate alone (0.1 ml) or zeranol or DES (150 micrograms/kg body weight) in ethyl oleate. The mice were killed from days 12 to 18 of gestation and the male fetuses were examined. Microscopical examination of the gonads indicated that the onset of testicular differentiation was earlier in the oestrogen-treated fetuses than in controls. Abnormal differentiation of gonocytes and foci of hyperplasia of fetal Leydig cells were observed in the oestrogen-treated mice. Male fetuses from female mice treated with DES showed a delay in testicular descent and progressive decrease in reactivity for cytokeratin (CK) 8 in fetal Sertoli cells. These morphological findings suggest that prenatal exposure to zeranol or DES induces abnormal testicular differentiation in the mouse.

Age Factors↗

Molecular cloning of mouse allantoicase cDNA.

The uric acid degradation pathway is progressively lost during vertebrate evolution. In mammals, the end product of this catabolic pathway is allantoin and, therefore, no allantoicase should be present in mouse tissues. Surprisingly, we have found an expressed sequence tag (EST) from mouse testis with high similarity to allantoicase. To characterize this transcript, we have completely sequenced the corresponding EST clone insert and found a 1495 bp long cDNA coding for a 414 amino acid long protein. Identities of mouse versus microorganism allantoicases range from 25 to 30%. Identity reaches 54% when compared to Xenopus allantoicase. Among the tested tissues, only testis possesses the allantoicase transcript. Although no deleterious mutations were found in the coding region, no allantoicase activity could be detected in mouse testis.

Amino Acid Sequence↗

Cloning and expression of activator of CREM in testis in human testicular tissue.

Activator of cAMP-responsive element modulator (CREM) in testis (ACT) has recently been found in the mouse testis where it activates CREM, a transcription factor essential for the differentiation of spermatids into mature spermatozoa. The importance of CREM in human spermatogenesis prompted us to examine whether ACT was also present in the human testis. Western blot analysis, performed with an anti-mouse ACT serum, showed the presence of a single immunoreactive band of a size similar to murine ACT. A library screening resulted in the isolation and characterization of the complete cDNA which showed 88% homology with the mouse counterpart. The human ACT gene is composed of five coding exons, being the first untranslated, and the mRNA spans 835 nucleotides coding for a 284 amino acid protein. Expression studies by RT-PCR confirmed that ACT is present in normal human testis. The human ACT gene is localized on the chromosome 6.

Amino Acid Sequence↗