GPX5 orthologs of the mouse epididymis-restricted and sperm-bound selenium-independent glutathione peroxidase are not expressed with the same quantitative and spatial characteristics in large domestic animals.

We report here on the cloning of cDNAs coding bovine and equine orthologs of mouse epididymis-restricted and sperm-bound glutathione peroxidase 5 (GPX5), a selenium-independent member of the multigenic GPX family in mammals. The complete sequence of bovine GPX5 as well as a partial sequence of the equine GPX5 were characterized, conceptually translated and aligned with other known mammalian GPX5 proteins. Using Northern blotting assays, we show that the level of expression of GPX5 is high in bovine but low in equine and that in both species the regionalization of GPX5 expression in epididymis is not totally identical to what was reported for rodent mouse GPX5. An antibody was produced against GPX5 and used in Western blot assays as well as in immunohistochemistry assays on bovine epididymis sections. It shows that the protein is essentially present in the cytoplasmic compartment of the caput segment 2 epithelium of the bovine epididymis. Unlike in the mouse model, bovine GPX5 seems to be poorly secreted and does not seem to be present on cauda epididymal spermatozoa.

Characterization and expression pattern of zebrafish Anti-Müllerian hormone (Amh) relative to sox9a, sox9b, and cyp19a1a, during gonad development.

The role of Anti-Müllerian hormone (Amh) during gonad development has been studied extensively in mammals, but is less well understood in other vertebrates. In male mammalian embryos, Sox9 activates expression of Amh, which initiates the regression of the Mullerian ducts and inhibits the expression of aromatase (Cyp19a1), the enzyme that converts androgens to estrogens. To better understand shared features of vertebrate gonadogenesis, we cloned amh cDNA from zebrafish, characterized its genomic structure, mapped it, analyzed conserved syntenies, studied its expression pattern in embryos, larvae, juveniles, and adults, and compared it to the expression patterns of sox9a, sox9b and cyp19a1a. We found that the onset of amh expression occurred while gonads were still undifferentiated and sox9a and cyp19a1a were already expressed. In differentiated gonads of juveniles, amh showed a sexually dimorphic expression pattern. In 31 days post-fertilization juveniles, testes expressed amh and sox9a, but not cyp19a1a, while ovaries expressed cyp19a1a and sox9b, but not amh. In adult testes, amh and sox9a were expressed in presumptive Sertoli cells. In adult ovaries, amh and cyp19a1a were expressed in granulosa cells surrounding the oocytes, and sox9b was expressed in a complementary fashion in the ooplasm of oocytes. The observed expression patterns of amh, sox9a, sox9b, and cyp19a1a in zebrafish correspond to the patterns expected if their regulatory interactions have been conserved with mammals. The finding that zebrafish sox9b and sox8 were not co-expressed with amh in oocytes excludes the possibility that amh expression in zebrafish granulosa cells is directly regulated by either of these two genes.

Molecular cloning of testican-2: defining a novel calcium-binding proteoglycan family expressed in brain.

We have screened a human cDNA library using an expressed sequence tag related to the BM-40/secreted protein, acidic and rich in cysteine (SPARC)/osteonectin family of proteins and isolated a novel cDNA. It encodes a protein precursor of 424 amino acids that consists of a signal peptide, a follistatin-like domain, a Ca2+-binding domain, a thyroglobulin-like domain, and a C-terminal region with two putative glycosaminoglycan attachment sites. The protein is homologous to testican-1 and was termed testican-2. Testican-1 is a proteoglycan originally isolated from human seminal plasma that is also expressed in brain. Northern blot hybridization of testican-2 showed a 6.1-kb mRNA expressed mainly in CNS but also found in lung and testis. A widespread expression in multiple neuronal cell types in olfactory bulb, cerebral cortex, thalamus, hippocampus, cerebellum, and medulla was detected by in situ hybridization. A recombinant fragment consisting of the Ca2+-binding EF-hand domain and the thyroglobulin-like domain of testican-2 showed a reversible Ca2+-dependent conformational change in circular dichroism studies. Testican-1 and -2 form a novel Ca2+-binding proteoglycan family built of modular domains with the potential to participate in diverse steps of neurogenesis.

Temperature-dependent sex determination in the red-eared slider turtle, Trachemys scripta.

Temperature-dependent sex determination (TSD) in the red-eared slider turtle, Trachemys scripta, has been the subject of a variety of past studies. Incubation temperature appears to affect sex determination in a dose-dependent fashion. This suggests that temperature could be affecting a dosage-sensitive element in the sex-determination cascade. Sex determination in T. scripta is sensitive to estrogen, and data from many studies support the hypothesis that endogenous estrogen production may be involved in female sex determination. However, this hypothesis has not yet been evaluated through aromatase expression studies in this species. Several recent studies have cloned cDNAs for genes that could be involved in sex determination and/or sex differentiation. The cDNAs for SF-1 and MIS have been cloned in T. scripta, indicating that these may represent conserved elements in the sex-determination/sex-differentiation cascade of reptiles. The SOX9 cDNA also has been cloned in T. scripta (Spotila et al., '98), and it shows a sex-specific expression pattern. Future studies targeted at aromatase expression as well as the expression of factors such as SOX9, SF-1, and MIS will begin to provide a more comprehensive picture of the events involved in TSD in T. scripta. Further, such studies could help pinpoint the temperature-sensitive element(s).

SRY recognizes conserved DNA sites in sex-specific promoters.

Formation of male-specific structures and regression of female primordia are regulated in early male embryogenesis by SRY, a single-copy gene on the Y chromosome. Assignment of SRY as the testis-determining factor in eutherian mammals is supported by molecular analysis of cytogenetic sex reversal (i.e., XX males and XY females) and by complementary studies of transgenic murine models. Here we characterize the putative DNA-binding domain of SRY, which contains a conserved sequence motif shared by high-mobility group nuclear proteins and a newly recognized class of transcription factors. The SRY DNA-binding domain specifically recognizes with nanomolar affinity proximal upstream elements (designated SRYe) in the promoters of the sex-specific genes encoding P450 aromatase and Mullerian inhibiting substance (MIS). P450 aromatase catalyzes the conversion of testosterone to estradiol, and in the male embryo its expression is down-regulated. Conversely, MIS is expressed in the male embryo to induce testicular differentiation and regression of female reproductive ducts. SRYe-binding activity is observed in nuclear extracts obtained from embryonic urogenital ridge immediately preceding morphologic testicular differentiation. Our results support the hypothesis that SRY directly controls male development through sequence-specific regulation of target genes.

Sequence homology of androgen-regulated epididymal proteins with glutathione peroxidase in mice.

The M53 cDNA for Mr 24,000 androgen-regulated secretory proteins of the mouse caput epididymidis previously reported has been sequenced. This clone presents a 5'-incomplete open reading frame of 525 base pairs. The 3'-untranslated region of 946 base pairs contains a repetitive DNA element of the rodent B1 family just between two canonical polyadenylation signals AATAAA, upstream of the poly(A) track. The deduced amino acid sequence for the Mr 24,000 proteins reveals significant homologies of 66.6, 66, 65.2, 63.1 and 64.5% with mouse, rat, man, bull and rabbit cloned selenium-dependent glutathione peroxidases, respectively. The present results emphasize previous studies performed in numerous laboratories suggesting that the major protective system against oxidative damage in mouse spermatozoa could be an enzyme similar to glutathione peroxidase.