Sex determination in reptiles

Developmental Biology. 6th edition.

Temperature-dependent sex determination (TSD) is a type of environmental sex determination in which the temperatures experienced during embryonic/larval development determine the sex of the offspring. It is only observed in reptiles and teleost fish. The sex-determining mechanisms of squamate reptiles are poorly known, relative to the large number of species in the group (2, snakes,. 3, lizards. Sex-determining mechanisms in reptiles are broadly divided into two main categories: genotypic sex determination (GSD) and.

PDF | In many egg-laying reptiles, the incubation temperature of the egg determines the sex of the offspring, a process known as. Sex-determining mechanisms in reptiles are broadly divided into two main categories: genotypic sex determination (GSD) and. The sex-determining mechanisms of squamate reptiles are poorly known, relative to the large number of species in the group (2, snakes,. 3, lizards.

The sex of a reptile embryo partly results from the production of sex hormones during development, and one such process to produce those. Two factors in reptile sex determination have been studied: (1) the presence or Temperature-dependent sex determination (TSD) is common in turtles and has. Sex determination and sex differentiation are two separate but related in the embryos of reptiles with genetic or environmental sex determination. Con-.

Alex Quinn, a Ph. Sex-determining mechanisms in reptiles are broadly divided into two main categories: genotypic sex determination GSD and temperature-dependent sex determination TSD. Species in the genotypic group, like mammals and birds, have sex chromosomes, which in sex come in two major types.

Many species—such as several species of turtle and lizards, like the green iguana—have X and Y sex chromosomes again, like mammalswith females being "homogametic," that is, having two identical X chromosomes.

Males, on the other hand, are "heterogametic," with one X chromosome and one Y chromosome. Other reptiles governed by GSD have a system, similar to one found in birds, with Z and W sex chromosomes.

In this case—which governs all snake species—males are the homogametic sex ZZ and females are the heterogametic sex ZW. In temperature-dependent sex determination, however, it is the environmental temperature during sex critical period of embryonic development that determines whether an egg develops as male or female. This reptiles period occurs sex the egg determination been determination, so sex determination in these reptiles is at the mercy of the ambient conditions affecting egg clutches in nests.

For example, in many turtle species, eggs from cooler nests hatch determination all males, and eggs from warmer nests hatch as all females. In crocodilian species—the most studied of which is the American alligator— both low and high temperatures reptiles in females and intermediate temperatures select for males.

A widely held view is that temperature-dependent and genotypic sex sex are mutually exclusive, incompatible mechanisms—in other words, a reptile's sex is never under the influence of both sex chromosomes and environmental sex. This model indicates that there reptiles no genetic predisposition for the embryo of a temperature-sensitive reptile determination develop as either male or female, so the early embryo does not have a "sex" until it enters the thermosensitive period of its development.

This paradigm, though, has been determination challenged, with new evidence now emerging that there may indeed be both sex chromosomes and reptiles involved in the reptiles determination of some reptile species. Apparently, in animals determination both occur, certain incubation temperatures can "reverse" the genotypic sex of an embryo.

For example, there is an Australian skink lizard that is genotypically governed by X and Y sex chromosomes. A low incubation temperature during the development of this lizard's egg reverses some determination females XX into "phenotypic" determination that they have only functioning male reproductive organs. A slightly different example of this temperature-induced sex reversal is found in an Australian dragon lizard, which has the ZW system of sex chromosomes.

In this species, high incubation reptiles during sex development reverses genotypic males ZZ into phenotypic females; so females can be ZZ or ZW, but males are always ZZ.

Reptiles in which both reptiles temperature and sex chromosomes interact to determine sex may represent "transitional" evolutionary states between two end points: complete GSD and complete TSD. It is quite possible that determination are other species of reptiles with more complicated scenarios of temperature reversal of chromosomal sex.

There are certainly many known examples of fish and amphibians with GSD, in which both high and low incubation temperatures can cause sex reversal. You have free article s left. Already a subscriber? Sign reptiles. See Subscription Options. Get smart.

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Next, we present some approaches that have the potential to accomplish these aims. While it certainly makes sense to study homologs of genes first identified in mammals, researchers investigating sex determination in reptiles should not limit themselves to this approach.

First, there is no guarantee that the primary sex-determining genes in reptiles will be orthologous to sex-determining genes in mammals. Such genes could be entirely unique to reptiles. This scenario would be analogous to SRY evolution in therian mammals [ Wallis et al. Alternatively, the primary sex-determining genes in reptiles could have mammalian orthologs, but those genes may play no part in sex determination in mammals.

Thus, it is possible that we would never discover a primary sex-determining gene in reptiles. Second, by waiting for the discovery of bona fide sex-determining genes in mammals, we limit the rate of discovery of candidate sex-determining genes in reptiles.

Wilhelm et al. Functional characterization of all these genes via reverse genetics in mice will be a very slow process. As a result, identification of new sex-determining genes in mammals and reptiles will be sporadic.

Fortunately, there are a large number of techniques for unbiased screening of the transcriptome and proteome. Older methods for identifying differentially expressed genes are relatively inexpensive and simple to use i. There are advantages and disadvantages to each method beyond expense and technical difficulty, but we leave it to the reader to explore these by reading the literature and approaching colleagues that use or have used these methods.

Here we provide examples of novel candidates that were discovered via unbiased screens. We have used differential display PCR to identify genes that are differentially expressed in gonads early in the temperature-sensitive period in the common snapping turtle.

Eggs were collected and incubated as previously described [ Rhen et al. In brief, we dissected gonads from embryos incubated at We sampled embryos from both thermal regimes on days 2, 3, 4, and 5 of the shift and stored gonads in RNA later solution Ambion, Austin, Tex. We extracted and analyzed total RNA from gonad pairs isolated from individual embryos as previously described [ Rhen et al.

We used univariate and multivariate ANOVA to identify bands that were differentially expressed between temperatures. Differentially expressed bands were cut out of the gel, extracted, and re-amplified as per the manufacturer's instructions RNAimage Kit. Bands were cloned and sequenced as previously described [ Rhen et al. This approach is significantly more rigorous than most differential display PCR studies because prior studies did not use biological replicates i.

We have confirmed that temperature influences expression of these genes using independent biological samples and quantitative PCR using the methods described in Rhen et al. Such genes may play a conserved role in gonad development and may have evolved sex linkage in mammals or birds as a result of this role.

A few other genes also deserve mention. Calreticulin expression was significantly higher at a male-versus a female-determining temperature, which is consistent with a putative role in spermatogenesis in rats [ Nakamura et al. It is even more significant that calreticulin is expressed in sperm in C. Altered Psmc3 expression in mouse testes is associated with male infertility [ Rockett et al.

Expression of the turtle ribosomal protein L13a also differed between temperatures. This gene is involved in the regulation of translation [ Mazumder et al. This is significant because CIRBP expression is influenced by temperature in other species and because it regulates expression of other genes via translational repression [ Nishiyama et al. Hence, CIRBP may regulate expression or activity of other sex-determining genes in the snapping turtle.

We are conducting additional studies of this gene to explore its potential role in TSD in the snapping turtle. One group of eggs was incubated at Another group of eggs was incubated at Our finding that 2 translational regulators, ribosomal protein L13a and CIRBP were differentially expressed at male-versus female-determining temperatures is particularly interesting in light of an early report by Harry et al.

These authors used antibodies to study temperature-dependent expression of heat shock protein 70 and heat shock protein 90 in the loggerhead sea turtle, a TSD species. Expression of these proteins was similar in gonads from embryos incubated at male- and female-determining temperatures. However, antibodies cross-reacted with other proteins that were differentially expressed between incubation temperatures.

The authors went on to identify these proteins as heterogeneous nuclear ribonucleoprotein particles hnRNPs. Although Harry et al. The list of candidate sex-determining genes in reptiles is growing and will lengthen with the application of genome-wide screens.

Genes that are differentially expressed at male-versus female-producing temperatures are likely to be involved in TSD, either as the thermosensitive gene itself, as direct targets of such a gene, or as downstream components of a larger gene regulatory network.

However, some candidate genes may not be involved in TSD per se. We must therefore distinguish genes that are causally related to TSD from genes whose expression is only correlated with TSD. Several approaches could be used to distinguish these genes, including linkage studies, gene perturbation studies i. Unfortunately, maps of molecular markers or visible phenotypes have not been constructed in any reptile. Long generation times hamper such studies in turtles, snakes, and crocodilians, although some lizards mature in their first year.

A careful screening of such species for other key traits like high fecundity, small body size, and ease of captive breeding may eventually lead to the first model species for reptile genetics. In the short term, however, the most promising route for functional studies of specific genes is to manipulate their expression via RNA interference and transfection of expression vectors in cultured embryonic gonads i.

Methods are available for studying fetal and neonatal mouse gonads in culture [ Eppig and O'Brien, ; Livera et al. These methods have been extended to the study of TSD in gonads from sea turtle embryos [ Moreno-Mendoza et al. Yao et al. Using procedures similar to Moreno-Mendoza et al. Such studies suggest that we may be able to carry out functional studies of candidate TSD genes.

At this time, however, we are not able to induce gonad differentiation in culture. Development of such an in vitro system will require extensive optimization. Nevertheless, these studies highlight the potential power of studying gonad development in organ culture.

An older but powerful technique is pharmacological manipulation of signaling pathways in ovo. One can treat individual embryos with almost any small molecule by dissolving it in the appropriate solvent and placing a drop of the solvent i. The compound is presumably carried through the shell, absorbed by the chorioallantoic vasculature, and transported to the embryo.

Crews and colleagues [] pioneered this procedure to administer estrogens to the developing embryo. Many researchers subsequently used the technique to study the impact of other steroids, steroid receptor antagonists, and steroidogenic enzyme inhibitors on sex determination. This approach has recently been used to deliver busulfan to ablate germ cells within the developing gonad [ Dinapoli and Capel, ]. A general caveat for any study using pharmacological agonists and inhibitors is that these compounds may activate or inhibit related signaling pathways.

Extra care should be taken in the design of experimental controls when compounds developed for mammals are used in reptiles. Finally, one should validate the assumption that compounds reach the embryo at concentrations that reflect the amount applied to the eggshell [ Muller et al.

Functional characterization of individual genes is clearly one of the biggest challenges facing researchers that study reptiles. An exciting new approach that deserves special consideration is modeling of gene regulatory networks. The most complete models integrate a vast array of data, including a list of all genes expressed in the cell or tissue of interest, the molecular function of those genes, information on cis regulatory elements and trans regulatory factors, and gene expression profiles after numerous gene perturbation experiments [ Bolouri and Davidson, ; Friedman, ].

However, it is also possible to develop models from time series data for a smaller number of genes with fewer experimental perturbations [ Bansal et al. A variety of statistical techniques, including Bayesian analyses and structural equation modeling, are used to develop explicit models of gene regulatory networks [ Rockman, ].

Here, we use temperature-dependent expression profiles for 3 genes and a simple statistical method to illustrate one method for modeling gene regulatory networks.

We previously observed that aromatase and FOXL2 expression increased in snapping turtle embryos shifted from a male- to a female-determining temperature [ Rhen et al. Androgen receptor AR expression also increased in embryos shifted to the female-determining temperature, but the increase was delayed one day relative to the increase in aromatase and FOXL2 expression [ Rhen et al.

These patterns suggest that aromatase and FOXL2 might regulate each other and that one or both genes might be upstream of AR in the ovary-determining pathway. We tested this hypothesis by incubating snapping turtle eggs at a temperature We used the same procedures for collecting and incubating eggs and administering hormones as described in Rhen and Lang []. In brief, equal or approximately equal numbers of viable eggs from each clutch were assigned to one of 4 hormone treatments to control for clutch differences.

Eggs were placed in containers filled with moist vermiculite and then randomly positioned within foam box incubators set at A few eggs were sampled periodically to monitor development and make sure treatments were applied at the correct stage. Upon reaching stage 15, eggs received one of 4 treatments.

As described above, this is a well-established method of delivering hormones to embryos that develop inside eggs. Gonads from equal numbers of embryos were collected at stage 15, 16, 17, and 18 and placed in RNA later solution Ambion, Austin, Tex. In addition, a subset of eggs was allowed to hatch to determine the effect of hormone treatments on sex ratios.

Flutamide feminized sex ratio in some families but masculinized sex ratio in others, suggesting this compound can act as an AR agonist or antagonist in the snapping turtle. In fact, flutamide acted as a partial agonist to weakly induce aromatase mRNA fig. It is noteworthy that dihydrotestosterone and flutamide did not influence gene expression until stage 18, which corresponds to the stage when AR levels increased in embryos shifted to a female-producing temperature [ Rhen et al.

These findings demonstrate that androgens and AR regulate aromatase and FOXL2 expression in embryonic gonads and that correlated expression of aromatase and FOXL2 is due, in part, to the pleiotropic effects of AR a transcription factor. Androgens have been shown to regulate aromatase in other species, but the androgen-AR-FOXL2 pathway is a novel regulatory interaction that has never been described in any species.

Yet, observations in mice suggest that this may be a conserved regulatory module: inactivation of AR leads to premature ovarian failure in female mice, as does inactivation of FOXL2 i. Embryos were allocated to 1 of 4 groups at stage 15, including non-treated and vehicle-treated controls, a dihydrotestosterone- DHT treated group, a flutamide- Flut treated group, and a group treated with both DHT and Flut.

Embryos were sampled at developmental stages 15—18 after treatment. Asterisks indicate a significant difference between the treated versus control groups. Although this was a very simple example of how to model a gene regulatory network, it highlights the potential for techniques like path analysis and structural equation modeling to reveal novel regulatory relationships among sex-determining genes.

However, it is important to note that these gene regulatory models are hypothetical and that they require experimental testing before firm conclusions are drawn. Fundamental similarities in gonad development are evident at the genetic, molecular, cellular, developmental, and physiological levels in all vertebrates.

Many, but not all, genes appear to play a conserved role in sex determination and morphogenesis of testes and ovaries. It is therefore reasonable to hypothesize that additional features of sex determination are conserved and that further study of orthologs of mammalian sex-determining genes are in order.

However, researchers studying sex determination in reptiles should not be limited to this approach. We present a variety of methods that can be used to discover novel candidate genes at a genome-wide scale. We present specific examples of novel genes that we discovered using an unbiased screen for genes that are differentially expressed between male- and female-producing temperatures.

Researchers should also endeavor to test the hypothesis that these genes are involved in sex determination. We present several experimental techniques that can be used to test gene function and to develop explicit models of the gene regulatory networks underlying testis and ovary formation. We must become more adventurous if we truly hope to identify primary sex-determining genes in reptiles with TSD or GSD.

We also wish to thank the Minnesota Department of Natural Resources for providing special permits for collection of snapping turtle eggs.

National Center for Biotechnology Information , U. Sex Dev. Published online Feb 9. Author information Article notes Copyright and License information Disclaimer. Received May 28; Accepted Aug Karger AG, Basel. This article has been cited by other articles in PMC. Abstract Charles Darwin first provided a lucid explanation of how gender differences evolve nearly years ago. Key Words: Genotypic sex determination, Gonad, Molecular genetics, Temperature-dependent sex determination.

Introduction Individuals come in one of two distinct forms, male or female, in most metazoans. Sex Determination Although the specific molecular mechanism that determines sex has not been revealed in any reptilian species, general modes of sex determination can be described [ Bull, ; Janzen and Paukstis, ].

Gonad Differentiation The basic pattern of gonad development is conserved in reptiles [ Raynaud and Pieau, ; Morrish and Sinclair, ]. Putative Sex-Determining Genes Virtually all studies of the molecular biology of sex determination in reptiles have examined homologs of sex-determining genes first identified in mammals. Table 1 Reptilian orthologs of mammalian sex-determining genes.

Ramsey and Crews, ; Rhen et al. Open in a separate window. Identification of New Candidate Sex-Determining Genes While it certainly makes sense to study homologs of genes first identified in mammals, researchers investigating sex determination in reptiles should not limit themselves to this approach.

Functional Analysis of Sex-Determining Genes The list of candidate sex-determining genes in reptiles is growing and will lengthen with the application of genome-wide screens. Modeling the Gene Regulatory Network for Sex Determination Functional characterization of individual genes is clearly one of the biggest challenges facing researchers that study reptiles.

Conclusions Fundamental similarities in gonad development are evident at the genetic, molecular, cellular, developmental, and physiological levels in all vertebrates. Androgen excess fetal programming of female reproduction: A developmental aetiology for polycystic ovary syndrome? Hum Reprod Update. Multiple alternative splicing of Dmrt1 during gonadogenesis in Indian mugger, a species exhibiting temperature-dependent sex determination. A genomic analysis of Drosophila somatic sexual differentiation and its regulation.

Extended production of the Mullerian duct regressor in the American alligator. Gen Comp Endocrinol. Inference of gene regulatory networks and compound mode of action from time course gene expression profiles. How to infer gene networks from expression profiles.

Mol Syst Biol. Blurring the edges in vertebrate sex determination. Curr Opin Genet Dev. Cloning and in situ hybridization analysis of estrogen receptor in the developing gonad of the red-eared slider turtle, a species with temperature-dependent sex determination. Dev Growth Differ.

Modeling transcriptional regulatory networks. Genes Dev. Sex determination in reptiles. Recent studies Bergeron et al. This knowledge may have important consequences in environmental conservation efforts to protect endangered turtle species. As mentioned in Chapter 3, the sex of the echiuroid worm Bonellia depends on where a larva settles. If a Bonellia larva lands on the ocean floor, it develops into a cm-long female. If the larva is attracted to a female's proboscis, it travels along the tube until it enters the female's body.

Therein it differentiates into a minute 1—3-mm-long male that is essentially a sperm-producing symbiont of the female see Figure 3. Another example in which sex determination is affected by the location of the organism is the case of the slipper snail Crepidula fornicata. In this species, individuals pile up on top of one another to form a mound Figure Young individuals are always male. This phase is followed by the degeneration of the male reproductive system and a period of lability.

The next phase can be either male or female, depending on the animal's position in the mound. If the snail is attached to a female, it will become male. If such a snail is removed from its attachment, it will become female. Similarly, the presence of large numbers of males will cause some of the males to become females. However, once an individual becomes female, it will not revert to being male Coe More examples of context-dependent sex determination will be studied in Chapter Cluster of Crepidula snails.

Two individuals are changing from male to female. After these molluscs become female, they will be fertilized by the male above them. After Coe Nature has provided many variations on her masterpiece. In some species, including most mammals and insects, sex is determined solely by chromosomes; in other species, sex is a matter of environmental conditions.

We are finally beginning to understand the mechanisms by which this masterpiece is created. By agreement with the publisher, this book is accessible by the search feature, but cannot be browsed. Turn recording back on.

High levels of aromatase activity increase the production of female hormones , resulting in the development of female characteristics. While aromatase activity remains low for much of development in individuals that exhibit TSD, during the thermosensitive period, variations in temperature increase the activity of aromatase. This increase in aromatase enables individuals to develop into males or females depending on the temperatures experienced.

Although other environmental influences can have similar effects, temperature is the most wide-spread factor that alters aromatase activity and sex determination. Charnier observed that temperature affected sex ratios, which are the number of females versus males in a population or a single clutch of eggs, of the rainbow Agama lizard, Agama agama. Charnier published her results in the meeting records of the local Society of Biology in West Africa, a journal with limited distribution, and her efforts were not widely recognized for several years.

In the late s, James J. Although few previous studies had supported the theory in reptiles, Pieau proposed TSD as an alternative to genotypic sex determination.

At this point, little evidence supported TSD as a possible mode of sex determination. In researchers established the existence of genotypic sex determination among turtles , a result that weakened support for TSD in reptiles and in vertebrates. When Bull and Eric Charnov, at the University of Utah, in Salt Lake City, Utah, proposed a model for the evolution of environmental sex determination in , they only suggested applying the model to plants and invertebrates, and not to vertebrates.

This evolutionary model, called the Charnov-Bull model, outlines the conditions under which the evolution of environmental sex determination occurs, and scientists later applied it to vertebrates with TSD.

The results, which found evidence of TSD in four out of five species, confirmed that some vertebrate species exhibit TSD.

As of , sixty-five of seventy-nine tested species of turtles were found to exhibit TSD. Over the next two decades, scientists worked to test mechanisms of sex differentiation in more species and to pinpoint pivotal temperatures, which are species-specific temperature ranges in which males and females are produced in equal number. Pieau and his colleagues focused on defining the TSD thermosensitive period, or the time of development during which changes in temperature can alter sexual organ growth.

Turtle species that display TSD are thought to follow one of two patterns of temperature dependence. In some species, low temperatures produce mainly females, and high temperatures produce mostly males.

Other species show disproportionately high female production at both high and low temperatures, with intermediate temperatures causing mostly male development. In the s, David Crews, a biologist at the University of Texas at Austin , and colleagues found that some environmental pollutants caused sex-altering effects in turtles and other reptiles.