Claude Pieau

Transitional range of temperature, pivotal temperatures and thermosensitive stages for sex determination in reptiles

The function relating phenotypic sex ratio to incubation temperature in reptiles can vary in a number of ways in addition to simple differences in the temperatures giving 50% of each sex. This paper offers terminology and definitions for describing these relationships. These definitions accomodate interactions between genetic and environmental effects on sexual differentiation, and variability within populations. The paper also discusses the concept of a sensitive stage/period within incubation during which temperature can affect the direction of sexual differentiation. Thermosensitive period has previously been assessed in a variety of ways. A suggestion for a more general way of defining thermosensitive stage/period is made.

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. Dev. Dyn. 1998;212:522–532.

TEMPERATURE-DEPENDENT SEX DETERMINATION AND GONADAL DIFFERENTIATION IN REPTILES

Abstract. 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.

Determination of temperature sensitive stages for sexual differentiation of the gonads in embryos of the turtle, Emys orbicularis

In order to determine the temperature sensitive stages for sexual differentiation of the gonads in Emys orbicularis, eggs of this turtle were shifted at different stages of embryonic development from the male‐producing temperature of 25°C to the female‐producing temperature of 30°C and reciprocally. Based on the series of developmental stages described by Yntema (′68) for Chelydra serpentina, temperature begins to influence sexual differentiation of Emys orbicularis at stage 16, a stage in which the gonads are still histologically undifferentiated. Its action lasts over the first steps of histological differentiation of the gonads. The minimal exposure at 25°C required for male differentiation of all individuals extends from stage 16 to somewhat before stage 21. For 100% female differentiation, incubation at 30°C must be longer, from stage 16 to somewhat before stage 22. Shorter exposures at 25°C or 30°C during these periods result in different percentages of males, females, and intersexes. Our results show that there is a critical stage (stage 16) which is the same for both male and female differentiation of the gonads. The thermosensensitive periods are rather long, corresponding to 11–12 days at 25°C and 30°C.

Aromatase activity in gonads of turtle embryos as a function of the incubation temperature of eggs.

In embryos of the European pond turtle, sexual differentiation of gonads is temperature-dependent. Production of oestrogens appears to play a key role in this phenomenon. Gonadal aromatase activity was measured in embryos incubated at 25 degrees C (masculinizing temperature) and at 30 degrees C (feminizing temperature). At the beginning of the thermosensitive period, the aromatase activity was low at both temperatures but was somewhat higher at 30 than at 25 degrees C. Afterwards, it remained low in differentiating testes at 25 degrees C, whereas it increased in differentiating ovaries at 30 degrees C to form a marked peak when germ cells underwent meiotic prophase. Eggs were shifted either from 25 to 30 degrees C (highly feminizing) or from 30 to 35 degrees C for 6 days at different stages of embryonic development. The 25-35 degrees C shifts performed during the thermosensitive period strongly increased the aromatase activity but were ineffective after this period. The 30-35 degrees C shifts increased the aromatase activity at all stages. Altogether, results indicate that, in differentiating gonads of turtle embryos, temperature acts on the regulation of synthesis (and therefore activity) of cytochrome P-450 aromatase (P-450-aro). The expression of the P-450-aro gene itself could be temperature-dependent. However, temperature could also act upon the expression of another gene involved in P-450-aro regulation.

Forelimb-hindlimb developmental timing changes across tetrapod phylogeny

BackgroundTetrapods exhibit great diversity in limb structures among species and also between forelimbs and hindlimbs within species, diversity which frequently correlates with locomotor modes and life history. We aim to examine the potential relation of changes in developmental timing (heterochrony) to the origin of limb morphological diversity in an explicit comparative and quantitative framework. In particular, we studied the relative time sequence of development of the forelimbs versus the hindlimbs in 138 embryos of 14 tetrapod species spanning a diverse taxonomic, ecomorphological and life-history breadth. Whole-mounts and histological sections were used to code the appearance of 10 developmental events comprising landmarks of development from the early bud stage to late chondrogenesis in the forelimb and the corresponding serial homologues in the hindlimb.ResultsAn overall pattern of change across tetrapods can be discerned and appears to be relatively clade-specific. In the primitive condition, as seen in Chondrichthyes and Osteichthyes, the forelimb/pectoral fin develops earlier than the hindlimb/pelvic fin. This pattern is either retained or re-evolved in eulipotyphlan insectivores (= shrews, moles, hedgehogs, and solenodons) and taken to its extreme in marsupials. Although exceptions are known, the two anurans we examined reversed the pattern and displayed a significant advance in hindlimb development. All other species examined, including a bat with its greatly enlarged forelimbs modified as wings in the adult, showed near synchrony in the development of the fore and hindlimbs.ConclusionMajor heterochronic changes in early limb development and chondrogenesis were absent within major clades except Lissamphibia, and their presence across vertebrate phylogeny are not easily correlated with adaptive phenomena related to morphological differences in the adult fore- and hindlimbs. The apparently conservative nature of this trait means that changes in chondrogenetic patterns may serve as useful phylogenetic characters at higher taxonomic levels in tetrapods. Our results highlight the more important role generally played by allometric heterochrony in this instance to shape adult morphology.

Conserved pattern of tangential neuronal migration during forebrain development.

Origin, timing and direction of neuronal migration during brain development determine the distinct organization of adult structures. Changes in these processes might have driven the evolution of the forebrain in vertebrates. GABAergic neurons originate from the ganglionic eminence in mammals and migrate tangentially to the cortex. We are interested in differences and similarities in tangential migration patterns across corresponding telencephalic territories in mammals and reptiles. Using morphological criteria and expression patterns of Darpp-32, Tbr1, Nkx2.1 and Pax6 genes, we show in slice cultures of turtle embryos that early cohorts of tangentially migrating cells are released from the medial ganglionic eminence between stages 14 and 18. Additional populations migrate tangentially from the dorsal subpallium. Large cohorts of tangentially migrating neurons originate ventral to the dorsal ventricular ridge at stage 14 and from the lateral ganglionic eminence from stage 15. Release of GABAergic cells from these regions was investigated further in explant cultures. Tangential migration in turtle proceeds in a fashion similar to mammals. In chimeric slice culture and in ovo graft experiments, the tangentially migrating cells behaved according to the host environment - turtle cells responded to the available cues in mouse slices and mouse cells assumed characteristic migratory routes in turtle brains, indicating highly conserved embryonic signals between these distant species. Our study contributes to the evaluation of theories on the origin of the dorsal cortex and indicates that tangential migration is universal in mammals and sauropsids.

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.

Sexual differentiation as a function of the incubation temperature of eggs in the sea-turtle Dermochelys coriacea (Vandelli, 1761)

Eggs of Dermochelys coriacea were incubated in the laboratory at different constant temperatures between 27 and 32°C up to hatching. The genital system of 72 neonates and 11 embryos was dissected and processed for histological study. All the individuals from the 27, 28 and 28.75°C incubations were phenotypic males. In the testes, the medulla was voluminous and very dense, composed of many epithelial cords, some enclosing germ cells, whereas the surface was covered by a thin layer of epithelial cells between which germ cells were still visible. The Mullerian ducts were in the process of regression. In all the individuals from eggs incubated at 29.75, 30.5 and 32°C, the gonads were somewhat longer than testes but were considerably reduced in width and thickness. The medulla was strongly inhibited, showing similar epithelial cords but much less numerous than in testes. The epithelium of the gonadal surface was pseudo-stratified and enclosed germ cells which had not enter meiotic prophase as in typical ovarian cortex. The Mullerian ducts were complete. These individuals were classified as potential females. These results show that temperature influences sexual differentiation of the gonads of Dermochelys coriacea and indicate that the threshold temperature (or pivotal temperature) lies between 28.75 and 29.75°C. They also reinforce fears about masculinising turtle populations by incubating eggs at cool temperatures in artificial hatcheries.