Oestrogens and temperature-dependent sex determination in reptiles: all is in the gonads.

In many species of oviparous reptiles, the first steps of gonadal sex differentiation depend on the incubation temperature of the eggs. Feminization of gonads by exogenous oestrogens at a male-producing temperature and masculinization of gonads by antioestrogens and aromatase inhibitors at a female-producing temperature have irrefutably demonstrated the involvement of oestrogens in ovarian differentiation. Nevertheless, several studies performed on the entire gonad/adrenal/mesonephros complex failed to find differences between male- and female-producing temperatures in oestrogen content, aromatase activity and aromatase gene expression during the thermosensitive period for sex determination. Thus, the key role of aromatase and oestrogens in the first steps of ovarian differentiation has been questioned, and extragonadal organs or tissues, such as adrenal, mesonephros, brain or yolk, were considered as possible targets of temperature and sources of the oestrogens acting on gonadal sex differentiation. In disagreement with this view, experiments and assays carried out on the gonads alone, i.e. separated from the adrenal/mesonephros, provide evidence that the gonads themselves respond to temperature shifts by modifying their sexual differentiation and are the site of aromatase activity and oestrogen synthesis during the thermosensitive period. Oestrogens act locally on both the cortical and the medullary part of the gonad to direct ovarian differentiation. We have concluded that there is no objective reason to search for the implication of other organs in the phenomenon of temperature-dependent sex determination in reptiles. From the comparison with data obtained in other vertebrates, we propose two main directions for future research: to examine how transcription of the aromatase gene is regulated and to identify molecular and cellular targets of oestrogens in gonads during sex differentiation, in species with strict genotypic sex determination and species with temperature-dependent sex determination.

Degree of sex reversal as related to plasma steroid levels in genetic female chickens (Gallus domesticus) treated with Fadrozole.

The objectives of this work were to determine whether or not plasma levels of testosterone and estradiol reflect the various grades of sex reversal in genetic female chickens treated with Fadrozole (CGS 16949 A), a nonsteroidal aromatase inhibitor, and whether gonadal aromatase activity and plasma levels of testosterone and estradiol in treated females can or not be modified by post-hatch treatments with Fadrozole or Fadrozole + testosterone. Eggs were injected with 1 mg Fadrozole on day 4 of incubation. In females having developed sex-reversed gonads, endocrine parameters (estradiol and testosterone) at and after 13 weeks of age were indicative of the degree of sex reversal, with, for example, sex-reversed females with two testes having the highest levels of testosterone and the lowest levels of estradiol. Among these females, eight (from a total of 13) produced ejaculates with scarce and abnormal spermatozoa. Some motility was observable in the ejaculates from five of them. None of the post-hatch treatments had a significant effect on plasma levels of testosterone or estradiol (measured at 3-week intervals from week 4 to week 28 post-hatch) or on gonadal aromatase activity (measured at 12 and 28 weeks). In conclusion, these results indicate that plasma levels of testosterone and estradiol at and after 13 weeks of age are valuable indicators of the degree of sex reversal in female chickens treated with Fadrozole prior to gonadal sex differentiation. In pre-cited conditions, post-natal treatments with either Fadrozole or Fadrozole + testosterone had no apparent effect on the degree of sex reversal in these birds. Finally, the occurrence of ejaculates with motile although scarce and abnormal spermatozoa, revealed that epididymes and ducti deferens can develop and become functional in sex-reversed female chickens.

Sex reversal and aromatase in chicken.

Aromatase inhibitors administered before sexual differentiation of the gonads can induce sex reversal in female chickens. To analyze the process of sex reversal, we have followed for several months the changes induced by Fadrozole, a nonsteroidal aromatase inhibitor, in gonadal aromatase activity and in morphology and structure of the female genital system. Fadrozole was injected into eggs on day four of incubation, and its effects were examined during the embryonic development and for eight months after hatching. In control females, aromatase activity in the right and the left gonad was high in the middle third of embryonic development, and then decreased up to hatching. After hatching, aromatase activity increased in the left ovary, in particular during folliculogenesis, whereas in the right regressing gonad, it continued to decrease to reach testicular levels at one month. In treated females, masculinization of the genital system was characterized by the maintenance of the right gonad and its differentiation into a testis, and by the differentiation of the left gonad into an ovotestis or a testis; however, in all individuals, the left Müllerian duct and the posterior part of the right Müllerian duct were maintained. In testes and ovotestes, aromatase activity was lower than in gonads of control females (except in the right gonad as of one month after hatching) but remained higher than in testes of control and treated males. Moreover, in ovotestes, aromatase activity was higher in parts displaying follicles than in parts devoid of follicles. The main structural changes in the gonads during sex reversal were partial (in ovotestes) or complete (in testes) degeneration of the cortex in the left gonad, and formation of an albuginea and differentiation of testicular cords/tubes in the two gonads. Testicular cords/tubes transdifferentiated from ovarian medullary cords and lacunae whose epithelium thickened and became Sertolian. Transdifferentiation occurred all along embryonic and postnatal development; thus, new testicular cords/tubes were continuously formed while others degenerated. The sex reversed gonads were also characterized by an abundant fibrous interstitial tissue and abnormal medullary condensations of lymphoid-like cells; in the persisting testicular cords/tubes, spermatogenesis was delayed and impaired. Related to aromatase activity, persistence of too high levels of estrogens can explain the presence of oviducts, gonadal abnormalities and infertility in sex reversed females.

Expression of AMH, SF1, and SOX9 in gonads of genetic female chickens during sex reversal induced by an aromatase inhibitor.

Aromatase inhibitors administered prior to histological signs of gonadal sex differentiation can induce sex reversal of genetic female chickens. Under the effects of Fadrozole (CGS 16949A), a nonsteroidal aromatase inhibitor, the right gonad generally becomes a testis, and the left gonad a testis or an ovotestis. We have compared the expression pattern of the genes encoding AMH (the anti-Müllerian hormone), SF1 (steroidogenic factor 1), and SOX9 (a transcription factor related to SRY) in these sex-reversed gonads with that in control testes and ovaries, using in situ hybridization with riboprobes on gonadal sections. In control males, the three genes are expressed in Sertoli cells of testicular cords; however, only SOX9 is male specific, since as observed previously AMH and SF1 but not SOX9 are expressed in the control female gonads. In addition to testicular-like cords, sex-reversed gonads present many lacunae with a composite, thick and flat epithelium. We show that during embryonic and postnatal development, AMH, SF1 and SOX9 are expressed in the epithelium of testicular-like cords and in the thickened part but not in the flattened part of the epithelium of composite lacunae. AMH and SF1 but not SOX9 are expressed in follicular cells of ovotestes. Coexpression of the three genes, of which SOX9 is a specific Sertoli-cell marker, provides strong evidence for the transdifferentiation of ovarian into testicular epithelium in gonads of female chickens treated with Fadrozole.

Sex reversal and aromatase in the European pond turtle: treatment with letrozole after the thermosensitive period for sex determination.

In the European pond turtle (Emys orbicularis), gonadal sex differentiation is temperature-dependent. The temperature sensitive period (TSP) of gonadogenesis lies between stages 16 and 22 of embryonic development. Previous studies have shown that embryos incubated at 30 degrees C, a temperature yielding 100% phenotypic females, can be sex reversed by treatments with an aromatase inhibitor administered during TSP or even somewhat after TSP (as of stage 22+). The goal of the present study was to determine whether the ovary still retains male potential at later stages of embryonic development and whether the induced male characters persist after hatching. For this purpose, eggs of E. orbicularis were treated with letrozole, a nonsteroidal aromatase inhibitor, at or as of stages 23, 24 or 25, then gonadal aromatase activity in each individual and the related gonadal structure were studied at hatching (stage 26) and for one year after hatching. Two kinds of treatments were carried out: 1) repeated applications of 10 microg of letrozole in ethanolic solution onto the eggshell; and 2) a single injection of 10 microg of letrozole in olive oil. Similar results were obtained with either application or injection of the aromatase inhibitor. In treatments as of or at stage 23, individuals with gonadal aromatase activity lower than 20 fmoles/hour/gonad had ovotestes, i.e., 22% of the treated individuals. At hatching, the inner part of these ovotestes contained testicular cords and also mixed lacunae presenting various degrees of transdifferentiation of the epithelium into a Sertolian epithelium. The cortex was maintained, although some germ cells degenerated within it. These processes continued after hatching. However, at 12 months, gonads were still ovotestes displaying some follicles with a growing oocyte in the remaining parts of the cortex. In treatments as of or at stages 24 or 25, only a few individuals were masculinized. One had ovotestes; in others, the cortex was absent in some parts and when it was present oocytes were degenerating. These results show that in the European pond turtle, differentiation of ovotestes from ovaries can be induced by treatment with an aromatase inhibitor starting at late stages of embryonic development (between the end of TSP and hatching), although such differentiation is less frequent as embryonic development proceeds. Sex reversal persists for at least one year after hatching. J. Exp. Zool. 290:490-497, 2001.

Temperature-dependent sex determination and gonadal differentiation in reptiles.

In many reptile species, sexual differentiation of gonads is sensitive to temperature (temperature-dependent sex determination, TSD) during a critical period of embryonic development (thermosensitive period, TSP). Experiments carried out with different models including turtles, crocodilians and lizards have demonstrated the implication of estrogens and the key role played by aromatase (the enzyme complex that converts androgens to estrogens) in ovary differentiation during TSP and in maintenance of the ovarian structure after TSP. In some of these experiments, the occurrence of various degrees of gonadal intersexuality is related to weak differences in aromatase activity, suggesting subtle regulations of the aromatase gene at the transcription level. Temperature could intervene in these regulations. Studies presently under way deal with cloning (cDNAs) and expression (mRNAs) of genes that have been shown, or are expected, to be involved in gonadal formation and/or differentiation in mammals. Preliminary results show that homologues of the WT1, SF1, SOX9, DAX1 and AMH genes exist in TSD reptiles. However, the expression patterns of these genes during gonadal differentiation may be different between mammals and TSD reptiles and also between different reptile species. How these genes could interact with aromatase is being examined.

Reelin mRNA expression during embryonic brain development in the turtle Emys orbicularis.

The expression of reelin messenger ribonucleic acid (mRNA) was studied during embryonic brain development in the turtle Emys orbicularis, by using radioactive in situ hybridization. A high expression was consistently found in the olfactory bulb and in a few neurons in the marginal zone and, to a lesser extent, in the subplate of the dorsal and medial cortical sectors. In the diencephalon, the ventral division of lateral geniculate nuclei and the prospective reticular thalamic nuclei were strongly positive. High reelin signal was also associated with some layers of the tectum and with the external granule cell layer of the cerebellum. A more moderate signal was detected in the septal nuclei, striatum, dorsal ventricular ridge, retina, habenular nuclei, and hypothalamus, and in some reticular nuclei of the midbrain and hindbrain and in ventral spinal cord. The cortical plate, basal forebrain, amygdala, and tegmentum were weakly labeled. When they are compared to reelin expression during mammalian brain development, our data reveal an evolutionarily conserved canvas of reelin expression and significant differences, particularly in developing cortical fields. Most significantly, the developing turtle cortex does not display the heavy reelin expression in subpial Cajal-Retzius cells that is so typical of its mammalian counterpart. Given the key role of reelin in laminar cortical development, our data suggest that the increase in the number of reelin-producing cells and/or the amplification of reelin expression in the cortical marginal zone might have been a driving factor during the evolution of the laminated cerebral cortex from stem reptiles to mammals, as indicated in previous comparative analyses.

Temperature-dependent sex determination and gonadal differentiation in reptiles.

In many reptile species, sexual differentiation of gonads is sensitive to temperature during a critical period of embryonic development (thermosensitive period, TSP). Experiments carried out with different models among which turtles, crocodilians and lizards have demonstrated the implication of estrogens and the key role played by aromatase (the enzyme complex that converts androgens to estrogens) in ovary differentiation during TSP and in maintenance of the ovarian structure after TSP. In some of these experiments, the occurrence of various degrees of gonadal intersexuality is related to weak differences in aromatase activity, suggesting subtle regulations of the aromatase gene at the transcription level. Temperature could intervene in these regulations. Present studies deal with cloning (complementary DNAs) and expression (messenger RNAs) of genes that have been shown, or are expected, to be involved in gonadal formation and/or differentiation in mammals. Preliminary results indicate that homologues of AMH, DAX1, SF1, SOX9 and WT1 genes with the same function(s) as in mammals exist in reptiles. How these genes could interact with aromatase is being examined.

Early expression of AMH in chicken embryonic gonads precedes testicular SOX9 expression.

In mammals, anti-Müllerian hormone (AMH) is produced by Sertoli cells from the onset of testicular differentiation and by granulosa cells only after birth. SOX9, a transcription factor related to the testis-determining factor SRY, is expressed in mouse testis 1 day before AMH. To determine the relationship between AMH and SOX9 in birds, we cloned the AMH promoter in search of SOX9 response elements, and we compared the expression of AMH and SOX9 in the gonads of chick embryos using in situ hybridization. Potential SOX response elements were found in the AMH promoter; however, AMH is expressed in both sexes at stage 25, 1 day before the first SOX9 transcripts appear in the male gonads. SOX9 is never expressed in the female. These results do not support the hypothesis that SOX9 could trigger the expression of testicular AMH in the chick but does not exclude a later role in testis development.

Sexual differentiation of gonads as a function of temperature in the turtle Emys orbicularis: endocrine function, intersexuality and growth.

Emys orbicularis is a freshwater turtle with temperature-dependent sex determination. Estrogens play a major role in gonadal differentiation; when they are produced at high levels during the thermosensitive period (TSP), ovaries differentiate; when their synthesis is very low, testes differentiate. Estrogens are synthesized from androgens through the activity of aromatase. We examine here two aspects of gonadal differentiation, intersexuality and growth, in E. orbicularis. For gonadal intersexuality, we studied the relationship between gonadal aromatase activity and gonadal structure at 28.5 degrees C (pivotal temperature), from the beginning of TSP to hatching, and compared results to those obtained at 30 degrees C (producing 100% females) and 25 degrees C (producing 100% males). At 28.5 degrees C, both males and females are obtained. However, histological differentiation of gonads is delayed compared to that at 25 degrees C and 30 degrees C, and an ovarian-like cortex of various thicknesses often develops at the surface of the male gonads; thus, several individuals display ovotestes at hatching. Despite important individual variations, the aromatase activity in ovaries differentiating at 28.5 degrees C increases during development as in ovaries differentiating at 30 degrees C. In most cases, however, activity is slightly lower than at 30 degrees C, and at the end of embryonic life, it becomes similar to that at 30 degrees C. In testes or ovotestes differentiating at 28.5 degrees C, aromatase activity remains low but is generally slightly higher than in testes at 25 degrees C; however, at the end of embryonic development, it becomes similar to that at 25 degrees C. Oocytes in the cortex of ovotestes begin to degenerate around hatching and continue to degenerate after hatching. Therefore, ovotestes evolve as testes. However, some oocytes may persist at the surface of testes up to the adult age. To estimate gonadal growth, the protein content was measured at different embryonic stages at 25 degrees C and at 30 degrees C. Testis growth is fast during TSP, somewhat slower after TSP, and decreases around hatching. Ovary growth is much slower than testis growth during TSP and then accelerates up to the end of embryonic development. This differential growth is well correlated with gonadal aromatase activity--much higher at 30 degrees C than at 25 degrees C--and can be explained by the fact that during TSP, testicular cords develop at 25 degrees C whereas they are inhibited at 30 degrees C; the ovarian cortex begins to form during this period but grows chiefly after TSP. Both inhibition of testicular cord development and stimulation of cortex development are under the control of endogenous estrogens. In the case of ovotestes, slight increases in estrogen synthesis, compared to that in typical testes, are sufficient to induce the transient formation of an ovarian-like cortex although they do not inhibit the development of testicular cords.

Expression of the Emx-1 and Dlx-1 homeobox genes define three molecularly distinct domains in the telencephalon of mouse, chick, turtle and frog embryos: implications for the evolution of telencephalic subdivisions in amniotes.

Homologies between vertebrate forebrain subdivisions are still uncertain. In particular the identification of homologs of the mammalian neocortex or the dorsal ventricular ridge (DVR) of birds and reptiles is still a matter of dispute. To get insight about the organization of the primordia of the main telencephalic subdivisions along the anteroposterior axis of the neural tube, a fate map of the dorsal prosencephalon was obtained in avian chimeras at the 8- to 9-somite stage. At this stage, the primordia of the pallium, DVR and striatum were located on the dorsal aspect of the prosencephalon and ordered caudorostrally along the longitudinal axis of the brain. Expression of homeobox-containing genes of the Emx, Dlx and Pax families were used as markers of anteroposterior developmental subdivisions of the forebrain in mouse, chick, turtle and frog. Their expression domains delineated three main telencephalic subdivisions in all species at the onset of neurogenesis: the pallial, intermediate and striatal neuroepithelial domains. The fate of the intermediate subdivisions diverged, however, between species at later stages of development. Homologies between forebrain subdivisions are proposed based on the conservation and divergence of these gene expression patterns.

There is no highly conserved embryonic stage in the vertebrates: implications for current theories of evolution and development.

Embryos of different species of vertebrate share a common organisation and often look similar. Adult differences among species become more apparent through divergence at later stages. Some authors have suggested that members of most or all vertebrate clades pass through a virtually identical, conserved stage. This idea was promoted by Haeckel, and has recently been revived in the context of claims regarding the universality of developmental mechanisms. Thus embryonic resemblance at the tailbud stage has been linked with a conserved pattern of developmental gene expression - the zootype. Haeckel's drawings of the external morphology of various vertebrates remain the most comprehensive comparative data purporting to show a conserved stage. However, their accuracy has been questioned and only a narrow range of species was illustrated. In view of the current widespread interest in evolutionary developmental biology, and especially in the conservation of developmental mechanisms, re-examination of the extent of variation in vertebrate embryos is long overdue. We present here the first review of the external morphology of tailbud embryos, illustrated with original specimens from a wide range of vertebrate groups. We find that embryos at the tailbud stage - thought to correspond to a conserved stage - show variations in form due to allometry, heterochrony, and differences in body plan and somite number. These variations foreshadow important differences in adult body form. Contrary to recent claims that all vertebrate embryos pass through a stage when they are the same size, we find a greater than 10-fold variation in greatest length at the tailbud stage. Our survey seriously undermines the credibility of Haeckel's drawings, which depict not a conserved stage for vertebrates, but a stylised amniote embryo. In fact, the taxonomic level of greatest resemblance among vertebrate embryos is below the subphylum. The wide variation in morphology among vertebrate embryos is difficult to reconcile with the idea of a phyogenetically-conserved tailbud stage, and suggests that at least some developmental mechanisms are not highly constrained by the zootype. Our study also highlights the dangers of drawing general conclusions about vertebrate development from studies of gene expression in a small number of laboratory species.

The ovary retains male potential after the thermosensitive period for sex determination in the turtle Emys orbicularis.

Emys orbicularis is a turtle with temperature-dependent sex determination. The thermosensitive period (TSP) lies between embryonic stages 16 and 22. Gonadal differentiation begins during this period involving oestrogens. Treatment with oestrogens during TSP results in the differentiation of ovaries at a male-producing temperature (25 degrees C), whereas treatment with an antioestrogen (tamoxifen) or with nonsteroidal aromatase inhibitors results in gonadal masculinization at a female-producing temperature (30 degrees C). The present study examines the effects on the ovary of inhibiting aromatase activity after TSP. Eggs of E. orbicularis incubated at 30 degrees C were given five or seven applications of 10 micrograms aromatase inhibitor Letrozole (CGS 20267) in ethanol, between stages 22+ and 24-25 when ovarian aromatase activity strongly increases. Individuals which received five applications were sacrificed at stages 24(+)-25. Those which received seven applications were sacrificed either at stage 25+ (close to hatching), or 34-36 days after hatching. Gonadal aromatase activity and related gonadal structure were studied in each individual. In the three series, the gonadal aromatase activity in individuals treated with Letrozole varied from similar or close to that in controls to much lower, and the gonadal structure varied from ovary-like to ovotestis. Ovotestes had the lowest levels of aromatase activity, under 4 fmoles/h/gonad, close to testis levels. They were found in 7 out of 26 individuals given Letrozole. Besides ovotestes, gonads presenting various degrees of masculinization, with enlarged epithelial cords and lacunae in the medulla, were found. Therefore, by inhibiting aromatase activity and thus estrogen synthesis, we were able to obtain the differentiation of testis-like cords or tubes in ovaries of E. orbicularis, after the period of temperature sensitivity. These results show that the ovary retains male potential after this period. Thus, besides their implication during the critical embryonic period for gonadal sex differentiation, oestrogens play a role in maintaining the ovarian structure after this period. A decrease in oestrogen levels could explain some other cases of ovarian masculinization known in vertebrates.

Cloning and expression of the chick anti-Müllerian hormone gene.

Müllerian duct regression in male embryos is due to early production by fetal Sertoli cells of anti-Müllerian hormone, a homodimeric protein of the transforming growth factor- beta superfamily. In mammals, both female Müllerian ducts develop into the uterus and Fallopian tubes, whereas in birds, the right oviduct does not develop. To gain insight into sex differentiation in birds, we have cloned the cDNA for chick anti-Müllerian hormone using antibodies raised against the partially purified protein. Expression cloning was required because of the lack of cross-hybridization between mammalian and chick anti-Müllerian hormone DNA. The chick DNA and protein are significantly longer, due to insertions that abolish nucleotide homology, except in the cDNA coding for the C-terminal, bioactive part of the protein. Nevertheless, the general structure of the gene, sequenced from the transcription initiation to the polyadenylation site, and the main features of the protein are conserved between the chick and mammals. The chick anti-Müllerian hormone gene is expressed at high levels in Sertoli cells of the embryonic testes and in lower amounts in both ovaries, higher levels being reached on the left side after 10 days of incubation.

Endocrine sex reversal of gonads by the aromatase inhibitor Letrozole (CGS 20267) in Emys orbicularis, a turtle with temperature-dependent sex determination.

In embryos of Emys orbicularis, the sexual differentiation of gonads is influenced by the incubation temperature of eggs. Estrogens administered during the thermosensitive period result in the feminization of gonads at 25 degrees (male-producing temperature), whereas an antiestrogen or aromatase inhibitors masculinize the gonads at 30 degrees (female-producing temperature). The nonsteroidal aromatase inhibitor Letrozole induces gonads with different degrees of masculinization, from ovary-like to testis-like. The present study examines the endocrine function of such masculinized gonads, at the end of embryonic life. Aromatase activity (which is involved in estrogen synthesis in ovary) and the status of Müllerian ducts (the regression of which reflects the secretion of a putative anti-Müllerian hormone by the Sertoli cells) were examined. One month after treatment with Letrozole, the gonads of embryos presented various levels of aromatase activity. There was a strong correlation among aromatase activity, gonadal structure, and Müllerian duct status; high levels of aromatase (similar or close to those in control females) were found in ovary-like gonads; intermediate levels were found in gonads (masculinized ovaries or ovotestes?) exhibiting a cortex and a composite medulla containing a mixture of ovarian lacunae and testicular cord-like structures; low levels (similar or close to those in control males) were found in strongly masculinized gonads (testis-like or ovotestes). Müllerian ducts were regressing in the majority of embryos with gonads containing low levels of aromatase activity. In these individuals, gonads functioned as embryonic testes. These results confirm the implication of estrogens in gonadal differentiation. The origin of these hormones is controversial, so that the aromatase activity was compared in gonads, in the undissociated adrenal-mesonephric complex (AM), and in different parts of this complex during the thermosensitive period. At the female-producing temperature, the aromatase activity per unit of tissue increased in differentiating ovaries but it was low in AM and similar to that found in AM at male-producing temperature. In embryos whose gonads had been masculinized by early treatment with Letrozole, aromatase activity was unchanged in AM. These results suggest that the main source of estrogens involved in ovarian differentiation is the gonad itself.

Aromatase activity in larval gonads of Pleurodeles waltl (Urodele Amphibia) during normal sex differentiation and during sex reversal by thermal treatment effect.

Estrogens are involved in gonadal differentiation in birds and in reptiles with temperature-dependent sex determination, suggesting a key role for aromatase, the enzyme complex converting androgens to estrogens. Pleurodeles waltl is an amphibian with ZZ/ZW genotypic sex determination but gonadal differentiation is thermosensitive. The sexual phenotype is in conformity with the sexual genotype at ambient temperature (20 +/- 2 degrees), but ZW animals are sex-reversed in functional males when larvae are reared at 32 degrees from stage 42 to stage 54 (thermosensitive period). Histological sexual differentiation of gonads begins at stage 53. Aromatase activity was measured either in the gonadmesonephric complexes (stages 47 to 52) or in the gonads (stages 53 to 56) of ZZ and ZW larvae reared at ambient temperature or at 32 degrees and of ZW larvae shifted at stages 53, 55, or 56 from ambient temperature to 32 degrees for 48 hr. At ambient temperature, aromatase activity was detected, at low levels, in gonad-mesonephric complexes of both ZZ and ZW larvae at stages 47 and 50. At stage 52, it was significantly higher in ZW individuals than in ZZ ones. Then it remained low in gonads of ZZ males, whereas it markedly increased in gonads of ZW females. At 32 degrees, the gonad-mesonephric complexes (stage 52) and the gonads (stages 53 to 56) of ZW larvae had low aromatase activity similar to that in ZZ individuals. The exposure to 32 degrees for 48 hr of ZW individuals significantly decreased gonadal aromatase activity during the thermosensitive period (shift at stage 53) but not after the thermosensitive period (shifts at stages 55 and 56).(ABSTRACT TRUNCATED AT 250 WORDS)

Early development of GABA-like immunoreactive cells in the retina of turtle embryos.

Gamma aminobutyric acid (GABA) is one of the earliest neuroactive substances appearing in the developing central nervous system. The distribution and the time course of the appearance of GABA-like immunoreactivity in the retina of the turtle Emys orbicularis were investigated from embryonic stage 13 to hatching. The first GABA-like immunoreactive cells were observed at stage 14. These cells were located in both the scleral third of the neuroblastic layer and the inner layers of the retina. They were identified as presumptive immature horizontal cells and amacrine cells, respectively. The observation of numerous labelled fibers in the nerve fiber layer suggests that some of the GABA-like immunoreactive cells in the layers were ganglion cells. The development of GABA-like immunoreactive cells followed a gradient of maturation from central to peripheral retina. At hatching, the central retina appeared nearly morphologically mature. In conclusion, GABA is present before the morphofunctional maturation of the retina and this precocious existence supports the idea of its involvement in a neurotrophic role preceding the establishment of synaptic connections and neurotransmitter function.

Sensitive stages for the effects of temperature on gonadal aromatase activity in embryos of the marine turtle Dermochelys coriacea.

Many data suggest an involvement of estrogens in gonadal differentiation in reptiles with temperature-dependent sex determination (TSD). However, the site of estrogen synthesis in two species of freshwater turtles is unclear. In Emys orbicularis, estrogens were shown to be produced by the gonads, whereas in Trachemys scripta, gonadal steroids were not detected. The marine turtle Dermochelys coriacea exhibits TSD but in gonadal development, ovarian differentiation is delayed. Gonadal aromatase activity and estrogen content in this species were measured in embryos incubated at 27 degrees and in embryos incubated at 30.5 degrees, respectively, masculinizing and feminizing temperatures within the range of temperatures found in natural nests. At all stages studied, aromatase activity was present and found to be higher at 30.5 degrees than at 27 degrees. Estrogens were only found at 30.5 degrees. The effects of temperature shifts on gonadal aromatase activity were then examined. Eggs were shifted from 27 to 35 degrees (feminizing temperature) at different embryonic stages and exposed to 35 degrees for 6 days. An increase in gonadal aromatase activity, although with significant individual variations, was seen only when eggs were shifted between stages 23 and 27. These stages are in the range of the thermosensitive stages for sexual differentiation of the gonads determined in other turtles. These results are similar to those previously obtained in E. orbicularis and agree with a key role for endogenous estrogens in gonadal differentiation of reptiles with TSD.(ABSTRACT TRUNCATED AT 250 WORDS)