Lower Levels In Testis Research Articles

Molecular cloning and characterization of amh and dax1 genes and their expression during sex inversion in rice-field eel Monopterus albus.

The full-length cDNAs of amh and dax1 in the hermaphrodite, rice-field eel (Monopterus albus), were cloned and characterized in this study. Multiple sequence alignment revealed Dax1 was well conserved among vertebrates, whereas Amh had a low degree of similarity between different vertebrates. Their expression profiles in gonads during the course of sex inversion and tissues were investigated. The tissue distribution indicated amh was expressed mostly in gonads and was scarcely detectable in other tissues, whereas the expression of dax1 was widespread among the different tissues, especially liver and gonads. amh was scarcely detectable in ovaries whereas it was abundantly expressed in both ovotestis and testis. By contrast, dax1 was highly expressed in ovaries, especially in ♀IV (ovaries in IV stage), but it was decreased significantly in ♀/♂I (ovotestis in I stage). Its expression was increased again in ♀/♂III (ovotestis in III stage), and then decreased to a low level in testis. These significant different expression patterns of amh and dax1 suggest the increase of amh expression and the decline of dax1 expression are important for the activation of testis development, and the high level of amh and a low level of dax1 expression are necessary for maintenance of testis function.

Molecular identification and expression of the Foxl2 gene during gonadal sex differentiation in northern snakehead Channa argus.

Channa argus is one of the most commercially important fish species in China. Studies show that males of C. argus grow faster than females at the same age. In order to explore the sex differentiation mechanism of C. argus, we isolated the full length of the sex-related gene Foxl2 cDNA and analysed its expression patterns during gonadal sex differentiation. Alignment of known Foxl2 amino acid sequences from vertebrates confirmed the conservation of the Foxl2 open reading frame, especially the forkhead domain and C-terminal region. Quantitative RT-PCR revealed that Foxl2 is predominantly expressed in brain, pituitary, gill and ovary, with its highest level in ovary but low levels in testis and other tissues, reflecting a potential role for Foxl2 in the brain-pituitary-gonad axis in C. argus. Our ontogenetic stage data showed that C. argus Foxl2 expression was significantly upregulated from 1 to 11 days posthatching (dph) and that the initiation of expression preceded the first anatomical ovarian differentiation (27 dph), suggesting that Foxl2 might play a potential role in early gonadal sex differentiation in C. argus. In addition, the Foxl2 protein was primarily located in granulosa cells surrounding the oocytes of mature C. argus, implying that Foxl2 may have a basic function in granulosa cell differentiation and the maintenance of oocytes.

Dynamics of DNA hydroxymethylation in zebrafish.

During embryonic development in mammals, most of the methylated cytosines in the paternal genome are converted to 5-hydroxymethyldeoxycytidine (hmC), as part of DNA methylation reprogramming. Recent data also suggest tissue-specific functional roles of hmC, perhaps as an epigenetic mark. However, limited data are available on the levels and tissue distribution in zebrafish. In this study, we used high-performance liquid chromatography mass spectrometry to quantify hmC and 5-methyldeoxycytidine (mC) in zebrafish during development and in different tissues of the adult fish. Low levels of mC were found at 0.5 hours postfertilization (hpf) (1-2 cell stage) (1.9%), and increased to 8.4% by 96 hpf, with similar levels observed in different adult tissues. No hmC was detected up to 12 hpf, but levels increased during development from 24 up to 96 hpf (0.23%). In tissues, the highest levels of hmC were found in the brain (0.49%), intermediate levels in muscle (0.13%), liver (0.08%), and intestine (0.06%) and low levels in testis (0.01%), with an inverse correlation between hmC and mC. Our results indicate similar tissue distribution and levels of hmC between zebrafish and mammals, but distinct differences during embryonic development. Although more research is needed, these results support the use of zebrafish as an alternative model in the elucidation of tissue-specific functions of hmC.

Disruption of a Spermatogenic Cell-Specific Mouse Enolase 4 (Eno4) Gene Causes Sperm Structural Defects and Male Infertility1

Sperm utilize glycolysis to generate ATP required for motility, and several spermatogenic cell-specific glycolytic isozymes are associated with the fibrous sheath (FS) in the principal piece of the sperm flagellum. We used proteomics and molecular biology approaches to confirm earlier reports that a novel enolase is present in mouse sperm. We then found that a pan-enolase antibody, but not antibodies to ENO2 and ENO3, recognized a protein in the principal piece of the mouse sperm flagellum. Database analyses identified two previously uncharacterized enolase family-like candidate genes, 64306537H0Rik and Gm5506. Northern analysis indicated that 64306537H0Rik (renamed Eno4) was transcribed in testes of mice by Postnatal Day 12. To determine the role of ENO4, we generated mice using embryonic stem cells in which an Eno4 allele was disrupted by a gene trap containing a beta galactosidase (beta-gal) reporter (Eno4(+/Gt)). Expression of beta-gal occurred in the testis, and male mice homozygous for the gene trap allele (Eno4(Gt/Gt)) were infertile. Epididymal sperm numbers were 2-fold lower and sperm motility was reduced substantially in Eno4(Gt/Gt) mice compared to wild-type mice. Sperm from Eno4(Gt/Gt) mice had a coiled flagellum and a disorganized FS. The Gm5506 gene encodes a protein identical to ENO1 and also is transcribed at a low level in testis. We conclude that ENO4 is required for normal assembly of the FS and provides most of the enolase activity in sperm and that Eno1 and/or Gm5506 may encode a minor portion of the enolase activity in sperm.

Developmental expression of steroidogenic factor-1, cyp19a1a and cyp19a1b from common carp ( Cyprinus carpio)

Steroidogenic factor-1 (SF-1), cyp19a1a and cyp19a1b play pivotal roles in vertebrate steroidogenesis and reproduction. In this study, a SF-1 cDNA ( EU022463) was cloned from common carp ( Cyprinus carpio). The transcript contains a 1509 base pair (bp) open reading frame (ORF) encoding a 503 amino acid sequence. Comparisons of deduced amino acid sequences demonstrated that carp SF-1 is highly homologous with those of other vertebrates. Tissue specific expressions of SF-1, cyp19a1a and cyp19a1b mRNA were analyzed in 10-month-old carp. SF-1 was abundant in the hypothalamus, pituitary, gonad, spleen and liver (females only). Cyp19a1b was preferentially expressed in the brain of both sexes but also was present at much lower levels in testis, ovary and kidney (females only). Although cyp19a1a expression was preferentially expressed in ovaries, it was also present at much lower levels in brain, testis, kidney and spleen (males only). Northern blot analysis revealed that testes and brains of both sexes expressed a transcript of about 2.8 kb in size. The expression pattern of SF-1, cyp19a1a and cyp19a1b in carp gonads suggested their involvement in sexual development. In 3-month-old carp, SF-1 and cyp19a1b were expressed highly in testes but were at much lower levels in ovaries, while the opposite pattern was observed with cyp19a1a expression. In 10-month-old carp, SF-1 expression was much higher in testes than in ovaries, while the opposite pattern was observed with cyp19a1a expression. These developmental expression patterns in carp gonads suggest important roles of SF-1 and cyp19a1b in testis development and of cyp19a1a in ovary development.

Expression of melatonin (MT1, MT2) and melatonin-related receptors in the adult rat testes and during development

It is well known that melatonin provokes reproductive alterations in response to changes in hours of daylight in seasonally breeding mammals, exerting a regulatory role at different levels of the hypothalamic-pituitary-gonadal axis. Although it has also been demonstrated that melatonin may affect testicular activity in vertebrates, until now, very few data support the hypothesis of a local action of melatonin in the male gonads. The aim of this study was to investigate whether MT1, MT2 melatonin receptors and the H9 melatonin-related receptor, are expressed in the adult rat testes and during development. A semi-quantitative RT-PCR method was used to analyse the expression of MT1, MT2 and H9 receptors mRNAs in several rat tissues, mainly focusing on testes during development and adult life. Our results provide molecular evidences of the presence of both MT1 and, for the first time, MT2 melatonin receptors as well as of the H9 melatonin-related receptor in the examined tissues, including adult testes. During development MT1 and MT2 transcripts are expressed at lower levels in testes of rats from 1 day to 1 week of age, lightly increased at 2 weeks of age and remained permanently expressed throughout development until 6 months. These data strongly support the hypothesis that melatonin acts directly in male vertebrate gonads suggesting that rat testes may be a suitable model to verify the role of indolamine in vertebrate testicular activity.

Pancreatic lipase-related protein (PY–PLRP) highly expressed in the vitellogenic ovary of the scallop, Patinopecten yessoensis

A cDNA (1206 bp) encoding a pancreatic lipase-related protein (PY–PLRP) was obtained from the ovary of the scallop, Patinopecten yessoensis, using a differentially expressed gene system. The open reading frame specified a protein containing 353 amino acids (~ 38 kDa). The N-terminal catalytic domain, which contained the catalytic triad of serine, aspartate, and histidine residues, 10 cysteine residues involved in disulfide bridges, and the conserved lid domain, indicated that the protein would be catalytically active. However, PY–PLRP lacked the C-terminal colipase-binding domain present in mammalian PLRPs. Sequence analysis of the catalytic domains of PY–PLRP with members of the pancreatic triglyceride lipase (PTL) family suggested that PY–PLRP was related to mammalian PLRP1, PLRP2, and PTL. End-point reverse transcriptase-polymerase chain reaction (RT-PCR) after 25 cycles showed that PY–PLRP was expressed at a high level in the ovary and at low levels in testis and mantle; expression was not detected in gill, digestive gland, and adductor muscle. Quantitative PCR of RNA from ovaries at different stages of development showed that PY–PLRP was expressed at a significantly higher level in the late growth stage than in the ripe and spent stages. These data suggest that PY–PLRP plays a role in lipid metabolism associated with oocyte maturation and vitellogenesis that occurs during ovarian growth.

Characterization of the genomic region containing the Shadow of Prion Protein (SPRN) gene in sheep

BackgroundTSEs are a group of fatal neurodegenerative diseases occurring in man and animals. They are caused by prions, alternatively folded forms of the endogenous prion protein, encoded by PRNP. Since differences in the sequence of PRNP can not explain all variation in TSE susceptibility, there is growing interest in other genes that might have an influence on this susceptibility. One of these genes is SPRN, a gene coding for a protein showing remarkable similarities with the prion protein. Until now, SPRN has not been described in sheep, a highly relevant species in prion matters.ResultsIn order to characterize the genomic region containing SPRN in sheep, a BAC mini-contig was built, covering approximately 200,000 bp and containing the genes ECHS1, PAOX, MTG1, SPRN, LOC619207, CYP2E1 and at least partially SYCE1. FISH mapping of the two most exterior BAC clones of the contig positioned this contig on Oari22q24. A fragment of 4,544 bp was also sequenced, covering the entire SPRN gene and 1206 bp of the promoter region. In addition, the transcription profile of SPRN in 21 tissues was determined by RT-PCR, showing high levels in cerebrum and cerebellum, and low levels in testis, lymph node, jejunum, ileum, colon and rectum.ConclusionAnnotation of a mini-contig including SPRN suggests conserved linkage between Oari22q24 and Hsap10q26. The ovine SPRN sequence, described for the first time, shows a high level of homology with the bovine, and to a lesser extent with the human SPRN sequence. In addition, transcription profiling in sheep reveals main expression of SPRN in brain tissue, as in rat, cow, man and mouse.

Cloning, sequence analysis, tissue distribution, and sex-specific expression of the neural form of P450 aromatase in juvenile sea bass ( Dicentrarchus labrax)

The teleost brain is characterized by exceptionally high levels of aromatase, the enzyme that converts androgens into estrogens, and by its continuous growth throughout life. Gonadal estrogens have been implicated in sex differentiation and the control of reproduction in adult fish, but the role of neural estrogens during early development is far from clear. The present study describes the isolation and characterization of the cDNA sequence from brain aromatase (P450aromB) in the European sea bass ( Dicentrarchus labrax L.), a well established model for neuroendocrine research in fish. P450aromB was cloned from a brain cDNA library and encoded a predicted protein of 505 residues, with a calculated molecular weight of 57.2 kDa. Comparisons of the deduced amino acid sequence to that of the ovarian aromatase (P450aromA) in the same species revealed 62% identity, lower than the 84% identity shared between sea bass and tilapia brain aromatases. Phylogenetic analysis showed the occurrence of a gene duplication for P450arom in the teleost lineage after its divergence from the tetrapods. Moreover, the low percentage of identity between brain and ovarian forms within the same species suggests that both genes evolved separately right after the appearance of the teleosts. Tissue-specific expression of P450aromA and P450aromB mRNA was studied in adult sea bass. P450aromB was preferentially expressed in brain of both males and females but also present at much lower levels in testis, ovary and head kidney, an organ known for its steroidogenic capabilities in fish. However, P450aromA expression was restricted to testis and ovary. A semiquantitative PCR was developed to measure P450aromB mRNA levels. Analysis of the expression of P450aromB in the brain of juvenile sea bass showed that females exhibited higher mRNA levels than males at 200 days post fertilization (dpf), by the time of gonadal sex differentiation. A switch in expression occurred thereafter, between 200 and 250 dpf, with males exhibiting higher levels than females. This situation was maintained by 300 dpf and is in agreement with measured levels of enzymatic activity in adults. These changes and sex-related differences in expression profiles may imply differences in the functionality of the enzyme between males and females, suggesting an important role for P450aromB in sea bass sex differentiation. However, due to the continuous growth of the teleost brain throughout life, a role in neurogenesis for brain aromatase should also be considered.

Characterization, expression pattern and chromosomal localization of the spermatogenesis associated 6 gene (Spata6).

We report the cloning and characterization of the spermatogenesis associated 6 gene (Spata6) encoding a predicted protein of 488 amino acids. It exhibits similarity with the motor domain of kinesin related proteins and with the Caenorhabditis elegans neural calcium sensor protein (NCS-2). The gene encodes three mRNAs of approximately 2.6, approximately 1.8 and approximately 1.2 kb. The expression of the 2.6 kb mRNA is detected at low levels in testis, ovary, thymus and placenta, while the 1.8 and 1.2 kb transcripts are exclusively expressed in testis. The 1.8 and 1.2 kb transcripts are specifically expressed in haploid germ cells. Data from in situ hybridization experiments suggested that mRNA expression of Spata6 in spermatids is higher than in spermatocytes and spermatogonia. RT-PCR analysis and whole mount in situ hybridization demonstrate that the Spata6 transcript is expressed during embryonic development and is localized in neural tube, somites and limb buds of mouse embryo. The Spata6 gene consists of 15 exons ranging in size between 40 and 596 bp. The 2.6 and 1.8 kb transcripts have different 5' untranslated sequences but have the same translational initiation site and therefore may encode the same protein with a predicted molecular weight of 49.7 kDa. The 1.2 kb transcript is derived from a proximal promoter between exons 7 and 8, and contains a translation initiation codon AUG, which is in frame with initiator AUG codon of the 2.6 and 1.8 kb transcripts. Therefore, the 1.2 kb transcript may code for a truncated protein of 32 kDa. Western blot analysis with the antiserum raised against a synthetic peptide from the C-terminal of the deduced Spata6 protein detects only a single protein of 53 kDa in all tissues studied. The Spata6 gene was localized to chromosome 5, region q34-35 in the rat and to chromosome 1, region p32-35 in the human. In an effort to determine the function of Spata6, we inactivated the mouse gene in embryonic stem cells through homologous recombination. Although the heterozygous mutant cells were able to generate low coat colour chimeric mice, all chimeras did not transmit the targeted allele to their progeny suggesting that a high contribution of Spata6(+/-) cells lead to the lethality of the chimeric embryos.

Identification of Glis1, a Novel Gli-related, Krüppel-like Zinc Finger Protein Containing Transactivation and Repressor Functions

In this study, we describe the identification and characterization of a novel Krüppel-like protein named Gli-similar 1 (Glis1). The Glis1 gene encodes an 84.3-kDa proline-rich protein. Its five tandem zinc finger motifs exhibit highest homology with those of members of the Gli and Zic subfamilies of Krüppel-like proteins. Glis1 was mapped to mouse chromosome 4C6. Northern blot analysis showed that expression of the 3.3-kb Glis1 mRNA is most abundant in placenta and adult kidney and expressed at lower levels in testis. Whole mount in situ hybridization on mouse embryos demonstrated that Glis1 is expressed in a temporal and spatial manner during development; expression was most prominent in several defined structures of mesodermal lineage, including craniofacial regions, branchial arches, somites, vibrissal and hair follicles, limb buds, and myotomes. Confocal microscopic analysis showed that Glis1 is localized to the nucleus. The zinc finger region plays an important role in the nuclear localization of Glis1. Electrophoretic mobility shift assays demonstrated that Glis1 is able to bind oligonucleotides containing the Gli-binding site consensus sequence GACCACCCAC. Although monohybrid analysis showed that in several cell types Glis1 was unable to induce transcription of a reporter, deletion mutant analysis revealed the presence of a strong activation function at the carboxyl terminus of Glis1. The activation through this activation function was totally suppressed by a repressor domain at its amino terminus. Constitutively active Ca(2+)-dependent calmodulin kinase IV enhanced Glis1-mediated transcriptional activation about 4-fold and may be mediated by phosphorylation/activation of a co-activator. Our results suggest that Glis1 may play a critical role in the control of gene expression during specific stages of embryonic development.

Extensive vascularization of developing mouse ovaries revealed by caveolin-1 expression.

Expression screening for genes preferentially expressed in mouse fetal ovaries relative to testes identified Cav-1 as a candidate female-specific gene. Cav-1 encodes caveolin-1, a component of the cell membrane invaginations known as caveolae, which are involved in lipid regulation and signal transduction. In situ hybridization revealed high levels of Cav-1 mRNA in developing ovaries, compared with moderate or low levels in testes. Analysis of caveolin-1 protein distribution by immunofluorescence showed this difference to be due to the development of a dense and complex vascular network in the developing ovary. These observations point to a higher degree of differentiation and organization of the early stage mammalian ovary than previously suspected.

TheKLHLI-antisense transcript (KLHLIAS) is evolutionarily conserved

Spinocerebellar ataxia type 8 (SCA8) is caused by a CTG expansion in an untranslated, endogenous antisense RNA that overlaps theKelch-like I (KLHLI) gene. The normal function of this transcript is currently unknown. We have now identified the promoter region for theKLHL1-antisense (KLHLIAS) RNA and report that aKlhl1as transcript is present in the mouse as well. Human and mouseKlhl1AS are transcribed from homologous promoter regions in the first intron ofKLHLI and extend through the transcription and translation start sites as well as the first splice donor sequence ofKLHL1. We found that the mouseKlhlIas RNA is not spliced and terminates in a polyadenylation site in theKlhlI promoter region, whereas both the present and previous work show that humanKLHLIAS is highly variably spliced into processed transcripts that contain up to six exons. MouseKlhlIas transcript was detected in RNA isolated from the cerebellum and from total adult brain and total fetal tissue, and at a low level in testis and ovary. Similarly, humanKLHL1AS is expressed in various brain tissues, including the cerebellum, the tissue most affected by SCA8, and was detected at low levels in testis and kidney. The evolutionary conservation of this antisense/sense transcriptional organization strongly indicates thatKLHLIAS transcripts play a significant biological role in both human and mouse, presumably as a regulator ofKLHLI expression.

Molecular Cloning and Expression Analysis of RatRgs12andRgs14

We report the cloning of two novel rat regulators of G-protein signaling (RGS) cDNAs using a degenerate PCR strategy. TherRgs12andrRgs14cDNAs encode predicted polypeptides of 1387 and 544 amino acids, respectively. We have also identified the human orthologue ofrRgs12by alignment of cosmid sequences in the database which map the humanRGS12gene to chromosome 4p16.3. Furthermore, we identified human ESTs with high homology torRgs14which map to human chromosome 5qter. Northern blot analysis indicates thatrRgs14is expressed at high levels in brain, lung, and spleen, whereasrRgs12is expressed at high levels in brain and lung and lower levels in testis, heart, and spleen. Analysis of the predicted rRGS12 and rRGS14 polypeptides indicates that they are closely related and possess regions of homology outside of the conserved RGS domain. We have also identified conserved regions in RGS12 which are similar to protein domains found in mouse rhophilin and coiled-coil proteins suggesting possible interactions with ras-like G-proteins.

Isolation, mapping, and regulated expression of the gene encoding mouse C-type natriuretic peptide.

Genomic sequences encoding mouse C-type natriuretic peptide (CNP) were isolated from bacteriophage libraries and characterized by restriction enzyme and sequence analysis. The mouse CNP gene (Nppc) comprised at least two exons and one intron and included several cis-regulatory elements in the 5'-flanking sequence. The deduced amino acid sequence of mouse CNP-22 was identical to other mammalian CNPs. Analysis of allele distributions in interspecific back-cross and recombinant inbred strains assigned Nppc to chromosome 1. CNP transcripts were detected by ribonuclease protection analysis in brain, ovary, and uterus, with lower levels in testes and epididymus. Uterine CNP transcripts and protein were low in sexually immature mice and adults at estrus and increased at proestrus, but similar variations in ovarian CNP expression were not statistically significant. Atrial natriuretic peptide and B-type natriuretic peptide transcripts were not detected in mouse ovary or uterus. Thus CNP gene expression is regulated by tissue-specific and inducible mechanisms in female reproductive organs. Correlations between CNP expression and uterine fluid content suggest that CNP may regulate uterine fluid balance in mice and other mammals.

Expression and Regulation by Insulin of Glut 3 in UMR 106-01, a Clonal Rat Osteosarcoma Cell Line

Expression of the glucose transporter GLUT 3 is mainly restricted to neuronal tissues, with lower levels in testis and placenta. In addition, GLUT 3 has recently been reported in neonatal rat calvaria byin situhybridisation. We report the co-expression of GLUT 1 and GLUT 3 mRNA and protein in UMR 106-01, a clonal osteosarcoma cell line. By semi-quantitative analysis we show that GLUT 3 protein is expressed at levels comparable to GLUT 1. Insulin stimulates glucose uptake in UMR 106-01 cells. GLUT 3 mRNA and protein are increased by chronic (16 h) treatment with insulin, and the increase in GLUT 3 mRNA is not inhibited by cycloheximide. Regulation of GLUT 3 mRNA by insulin has not been previously shown. UMR 106-01 represents a useful model for investigating regulation of GLUT 3 expression.

Inactivation of mammalian X-chromosome during spermatogenesis: Temporal expression of genes in the laboratory mouse

At zygotene/pachytene stage of me iosis in mammalian testis, the X—Y heterobivalent is sequesterd into a heterochromatinized body whose genetic inactivity is shown by lack of uridine incorporation. For the genic level evaluation of the X-inactivation, activities of three X-linked genes were assayed in testicular cell types in the laboratory mouse. While hypoxanthine phosphoribosyl transferase is functional at least up to pachytene stage of primary spermatocytes, glucose-6-phosphate dehydrogenase appears to be active only in the Sertoli cells. No transcript of the muscle specific muscular dystrophin gene was obtained from its regular 5' promoter. Thus, inactivation of X-linked genes in testis occurs at different stages during spermatogenesis, independently of heterochrornatinizati on of the XY-body. We propose that Xist transcript, the putative regulator of X-inactivation in female soma, is also the regulator in testis. However, due to its extremely low level in testis the transcript may regulate by "spreading" in a gradient and affect the genes in a temporal order. Thus, besides other factors, physical proximity of the genes to Xist may determine the stage of their inactivation.

Isolation and Characterization of cDNA for an Androgen-Regulated mRNA in the Flank Organ of Hamsters

Flank organs of hamster are useful for studying androgen-dependent growth of hair follicles and sebaceous glands. To elucidate the mechanism of gene expression regulated by androgen, we constructed a cDNA library from flank organs of male hamsters and screened by a differential hybridization method using cDNA probes from normal and castrated males. We isolated a cDNA clone, termed FAR-17a, whose expression was found to be highly sensitive to androgen. FAR-17a mRNA of 1.8 kb was reduced after castration and reappeared after testosterone treatments. Among several tissues examined, FAR-17a gene was expressed at a high level in flank organ and a low level in testis and earlobe. FAR-17a probe detected a few fragments in genomic DNA of hamster, mouse, suncus, pig, and human, suggesting that this gene is phylogenetically conserved. The sequence of FAR-17a cDNA predicts a protein of 231 amino acids (27,216 daltons) having basic properties. The deduced protein has no significant homologies to proteins previously described.

Gonadal steroid levels in the turtle Emys orbicularis L.: A preliminary study in embryos, hatchlings, and young as a function of the incubation temperature of eggs

Eggs of Emys orbicularis L. were incubated at either 25° (100% male-producing temperature) or at 30° (100% female-producing temperature). Gonadal levels of seven steroids were subsequently measured in embryos (stage 24), hatchlings, and young (3 and 9 months). According to the stage, 22–80 gonads were first removed and cultured in synthetic medium for 2 hr. They were then pooled and homogenized in this medium for steroid assay (RIA). The total steroid levels were weak (10–30 pg/gonad) in embryos and hatchlings and increased in the young turtles. In embryos, total steroid level was higher in males than in females, while at 9 months it was higher in females. As for the different steroids, the main following results were obtained. In embryos, progesterone (Prog), dehydroepiandrosterone (DHA), and testosterone (Testo) were not detected in either testes at 25° or ovaries at 30°, while Δ 4-androstenedione (Δ4A), dihydrotestosterone (DHT), and estrone (E1) were found in higher levels in testes (chiefly E1). Low levels in testes or traces in ovaries of estradiol (E2) were present. At hatching, E2 was no longer detectable, while DHA in both sexes and Testo in males were measurable. At this time, the other hormones displayed either minor variations in females or were decreased in males. At 3 months, Prog and E2 were still missing and only traces of E1 were detected. DHA decreased in males, while it increased in females. Levels of the other steroids, mainly Testo (in both sexes) and DHT (in males) increased. At 9 months, all steroids except E2 were detected. The total steroid content increased markedly, with the highest levels being found for Prog and Δ4A, chiefly in ovaries. This latter increase could be related to the posthatching differentiation of interstitial gland (Leydig cells) in testes and to follicle growth in ovaries.