Health status of the lizard Podarcis siculus (Rafinesque-Schmaltz, 1810) subject to different anthropogenic pressures

The terrestrial lizard Podarcis siculus is the most abundant reptile in Italy, where is considered a ubiquitous species. This lizard is widely distributed from the islands to the Apennines, from cultivated fields to anthropized areas such as gardens and city parks. For this reason, these animals are exposed to extensive physical and chemical stresses, as well as to the possibility of coming into contact with industrial pollutants and substances used in agricultural practices. Here, we review the health status of lizard specimens inhabiting natural non-anthropized areas and fields devoted to organic farming, considering the condition of (1) liver, representing the main detoxifying organ, directly influenced by feeding, and (2) gonads, essential for reproduction and, therefore, for the survival of the species. The morphological and biomolecular condition of these organs was then compared with those obtained from lizard specimens experimentally treated with nonylphenol, a co-formulant of many insecticides and plant protection products widely used in conventional farming, known to have harmful estrogenic effects. Taken together, data demonstrate that lizards inhabiting manured soil are in good health status and show a regular morphology of liver, testis, and ovary. Animals are found to be less exposed to the toxic heavy metals cadmium and lead if compared with specimens collected in areas not devoted to agriculture, but probably more exposed to vehicular traffic. However, manure, as well as nonylphenol, exerts a xeno-estrogenic effect, particularly evident in male specimens, more sensitive to estrogenic contamination.     

Cloning and expression profiling of testis-expressed microRNAs

Using a new small RNA cloning method, we identified 141 miRNAs from the mouse testis, of which 29 were novel. The 141 miRNAs were mapped onto all chromosomes except the Y chromosome and 2/3 of these miRNA genes exist as clusters. ∼ 70% of these miRNA genes were located in intronic or intergenic regions, whereas the remaining miRNAs were derived from exonic sequences. We further validated these cloned miRNAs by examining their expression in multiple mouse organs including developing testes and also in purified spermatogenic cells using semi-quantitative PCR analyses. Our expression profiling assays revealed that 60% of the testis-expressed miRNAs were ubiquitously expressed and the remaining are either preferentially (35%) or exclusively (5%) expressed in the testis. We also observed a lack of strand selection during testicular miRNA biogenesis, characterized by paired expression of both the 5′ strands and 3′ strands derived from the same precursor miRNAs. The present work identified numerous miRNAs preferentially or exclusively expressed in the testis, which would be interesting targets for further functional studies.     

Haploinsufficiency of Bcl-x leads to male-specific defects in fetal germ cells: differential regulation of germ cell apoptosis between the sexes

In germ cells, the function of which is to form the next generation, apoptotic cell death occurs during development, as in the case of somatic cells. In this study, we show that Bcl-x knockout heterozygous (Bcl-x(+/-)) mice exhibit severe defects in male germ cells during development. A substantial increase in apoptosis of male germ cells occurs at around embryonic day 13.5 (E13.5) in Bcl-x(+/-) embryos, leading to hypoplasia of postnatal testes and reduced fertility. On the other hand, female germ cells at the same stages do not show discernible differences between wild-type and Bcl-x(+/-) embryos. This phenotype of Bcl-x haploinsufficiency shows that regulation of apoptosis becomes different between the sexes at around the onset of sex differentiation. Through this study, we found that, in wild-type embryos, (1) apoptosis is much more frequent (approximately 10 times) in the male than in female germ cells, and (2) expression of Bcl-xL, but not that of Bax, is higher in female than in male germ cells, at around E13.5. Male fetal germ cells, cultured with gonadal somatic cells in vitro, showed higher frequencies of apoptosis than those cultured without gonadal somatic cells. On the other hand, in the absence of gonadal somatic cells, both male and female fetal germ cells in vitro showed similar frequencies of apoptosis to female fetal germ cells in vivo. Therefore, male germ cell apoptosis, of which the default pathway is similar to that of the female, is likely to be influenced by male gonadal environments.     

Expression of a novel HsMCAK mRNA splice variant,tsMCAK gene,in human testis

Identification of specifically expressed genes in the adult or fetal testis is very important for the study of genes related to the development and function of the testis. In this study, a human adult testis cDNA microarray was constructed and hybridized with 33P-labeled human adult and embryo testis cDNA probes, respectively. After differential display analyzing, a number of new genes related to the development of testis and spermatogenesis had been identified. One of these new genes is tsMCAK. tsMCAK was expressed 2.62 folds more in human adult testis than fetal testis. The full length of tsMCAK is 2401 bp and contains a 2013 bp open reading frame, encoding a 671-amino-acid protein. Sequence analysis showed that it has a central kinesin motor domain and is homologous to HsMCAK gene of the somatic cells. Blasting human genome database localized tsMCAK to human chromosome 1P34 and further investigation showed that it is a splice variant of HsMCAK. The tissue distribution of tsMCAK was determined by RT-PCR and it is expressed highly and specifically in the testis. Southern blot studies of its expression in patients with infertility indicated its specific expression in spermatogenic cells and its correlation with male infertility. The above results suggested that tsMCAK is a candidate gene for the testis-specific KRPs and its specific expression in the testis was correlated with spermatogenesis and may be correlated with male infertility.     

Diethylstilbestrol attenuates antioxidant activities in testis from male mice

It has been reported that acute exposure to diethylstilbestrol (DES) induces apoptosis in the testis, and antioxidants play a role in preventing DES-induced tissue damage. In this study, the effect of chronic exposure to DES on the antioxidants was examined in the testis and liver. Eight-week old male ICR mice were treated subcutaneously with various doses of DES for 20 days. Morphologically apparent apoptotic changes, 4-hydroxy-2-nonenal-positive cells and TUNEL-positive DNA-fragmentation, were demonstrated in the testis, but were minimal in the liver. Activities of antioxidants such as glutathione (GSH) peroxidase and GSH S -transferase decreased in both the liver and testis. The activity of Mn-superoxide dismutase (SOD) decreased in the liver but increased in the testis. The activity of Cu, Zn-SOD decreased in the liver but was unchanged in the testis. On Western and Northern blots, gamma-glutamylcysteine synthetase ( γ-GCS), a rate limiting enzyme of GSH synthesis, was increased in the liver dependent on the dose of DES. However, the expression of γ-GCS was reduced in the testis. Since quinones, metabolites of DES, generate reactive oxygen species, which damage DNA, antioxidants are important to prevent the damage. The data suggest that antioxidant activities are impaired by DES, and the levels of GSH are related to DES-induced apoptosis in the testis.     

Identification and characterization of two novel low-molecular-weight dual specificity phosphatases

We have cloned and characterized two novel human low molecular weight dual specificity phosphatases (LMW-DSPs). Both genes are expressed exclusively in the testis, but are not altered in any of several disease states examined. Transfection into COS cells indicates that both proteins are expressed in the nucleus and the cytoplasm. Both proteins are able to dephosphorylate the phosphotyrosine analog pNPP in vitro and can be inhibited by sodium orthovanadate. In vitro experiments also demonstrate that both DSPs can dephosphorylate single and diphosphorylated synthetic MAPK peptides, with preference for the phosphotyrosine and diphosphorylated forms over phosphothreonine. However, when co-transfected with MAPKs into COS cells, the novel DSPs exhibited no detectable in vivo activity against MAPKs under our conditions. Our data suggest that these novel LMW-DSPs might belong to a new subclass of testis-specific proteins that act independently of the MAPK signal transduction cascade and do not depend on N-terminal docking regions for substrate binding.     

The expression pattern of a mouse doublesex-related gene is consistent with a role in gonadal differentiation

The signal for somatic sex determination in mammals, Caenorhabditis elegans and Drosophila melanogaster is chromosomal, but the overall mechanisms do not appear to be conserved between the phyla. However it has been found quite recently that the C. elegans sex-determining gene Mab-3 contains a domain highly homologous to the Drosophila sex-determining gene doublesex (dsx) and shares a similar role. These data suggest that at least some aspects of the regulation of sex determination might be conserved. In humans, a doublesex-related gene (DMRT1) was identified at less than 30 kb from the critical region for sex reversal on chromosome 9p24 (TD9). In order to get insights into the role of DMRT1 in sex determination/differentiation, we have isolated DMRT1 mouse homologue (Dmrt1) and analysed its expression pattern. The gene is expressed in the genital ridges of both sexes during the sex-determining switch and it shows male/female dimorphism at late stages of sex differentiation.     

Spermatogonia-specific proteins expressed in prepubertal buffalo (Bubalus bubalis) testis and their utilization for isolation and in vitro cultivation of spermatogonia

Buffalo is an economically important livestock species in Asia. Little is known about male germ line technology owing to lack of sufficient understanding regarding expression of germ- and somatic-cell specific-proteins in the testis. In this study, we identified UCHL-1 (PGP 9.5) and lectin- Dolichos biflorus agglutinin (DBA) as specific markers for spermatogonia in buffalo testis. Expression of germ-cell and pluripotency-specific proteins such as DDX4 (VASA) and POU5F1 (OCT3/4) were also present in spermatogonia. Interestingly, the expression of somatic cell-specific proteins such as VIMENTIN and GATA4 were also detected in germ cells. Using two-step enzymatic digestion followed by differential plating and Percoll density-gradient centrifugation, an approximately 55% spermatogonia-enriched cell population could be obtained from the prepubertal buffalo testis. Isolated spermatogonia could survive and proliferate in vitro in DMEM/F12 medium containing 10% fetal bovine serum in the absence of any specific growth factors for a week. Cultured spermatogonia showed DBA affinity and expressed DDX4 and POU5F1. These results may help to establish a long-term culture system for buffalo spermatogonia.     

Spermatogenesis and distinctive mature sperm in the giant freshwater prawn, Macrobrachium rosenbergii (De Man, 1879)

The structures of differentiating male germ cells in the testis of the giant freshwater prawn, Macrobrachium rosenbergii, were studied by light and electron microscopy. Based on ultrastructural characteristics, the developing male germ cells are classified into 12 stages, including spermatogonia, six phases of primary spermatocytes (leptotene, zygotene, pachytene, diplotene, diakinesis and metaphase), secondary spermatocyte, three stages of spermatids and mature sperm. During spermatogenesis, the differentiating germ cells have characteristics similar to those of other invertebrates, but they exhibit some unique characteristics during spermiogenesis. In particular, an early spermatid has a round nucleus with highly condensed heterochromatin, appearing as thick interconnecting cords throughout the nucleus. In contrast to most invertebrates and vertebrates, the chromatin begins to decondense in one-half of the nucleus at the mid spermatid stage. In the late spermatid, the chromatin becomes almost entirely decondensed with only a small crescent-shaped heterochromatin patch remaining at the anterior pole of the nucleus. Mature sperm possess an everted umbrella-shaped plate with a spike covering the anterior pole of the nucleus, whose chromatin is totally decondensed as only small traces of histones H3 and H2B remain. The acrosome appears at the ruffled border of the spike plate as small sac-like structures. Few mitochondria remain in the cytoplasm at the posterior pole.     

Rat organic solute carrier protein 1 (rOscp1) mediated the transport of organic solutes in Xenopus laevis oocytes: isolation and pharmacological characterization of rOscp1

Rat organic solute carrier protein 1 (rOscp1) was isolated from a rat testis cDNA library. Isolated rOscp1 cDNA consisted of 1089 base pairs that encoded a 363-amino acid protein, and the amino acid sequence was 88% and 93% identical to that of human OSCP1 (hOSCP1) and mouse Oscp1 (mOscp1), respectively. The message for rOscp1 is highly detected in rat testis. When expressed in X. oocytes, rOscp1 mediated the high affinity transport of p-aminohippurate (PAH) with a Km value of 15.7+/-1.9 microM, and rOscp1-mediated organic solutes were exhibited in time- and Na+-independent manners. rOscp1 also transported various structurally heterogenous compounds such as testosterone, dehydroepiandrosterone sulfate (DHEA-S), and taurocholate with some differences in substrate specificity compared with hOSCP1. Immunohistochemical analysis revealed that the rOscp1 protein is localized in the basal membrane side of Sertoli cells as observed in mouse testis [Kobayashi et al., 2007; Kobayashi, Y., Tsuchiya, A., Hayashi, T., Kohyama, N., Ohbayashi, M., Yamamoto, T., 2007. Isolation and characterization of polyspecific mouse organic solute carrier protein 1 (mOscp1). Drug Metabolism and Disposition 35 (7), 1239-1245]. Thus, the present results indicate that a newly isolated cDNA clone, rOscp1, is a polyspecific organic solute carrier protein with some differences in substrate specificity compared with human and mouse OSCP1.