The influence of thermal signals during embryonic development on intrasexual and sexually dimorphic gene expression and circulating steroid hormones in American alligator hatchlings (Alligator mississippiensis)
Introduction
The bipotential embryonic gonad possesses the capacity to develop as either a testis or an ovary, an attribute that sets the stage for one of the most crucial events in vertebrate development. The process of sex determination bears significant fitness consequences for progeny via its influence on the development of secondary sex characteristics and varying levels of biological organization, extending from molecular and cellular dynamics to morphology and behavior. Many reptiles, including all crocodilians studied, employ a form of sex determination in which thermal cues experienced during embryonic development serve as the primary determinant of sex (temperature-dependent sex determination, TSD) (Charnier, 1966, Ferguson and Joanen, 1982, Lang and Andrews, 1994). For the American alligator, egg incubation treatments and assessments of resulting sex ratios identified the pattern of TSD as female-male-female (FMF) with extreme temperatures on either end of this spectrum producing mostly females and intermediate temperatures resulting in the development of primarily males (Lang and Andrews, 1994). Further characterization of the alligator TSD system indicates that embryos display thermosensitivity through a discrete period of embryogenesis (stage 21–24,∼30–45 days post-oviposition), which occurs during the middle third of development (Lang and Andrews, 1994). However, supplementary thermal shift designs conducted by our laboratory indicate that temperatures experienced prior to this discrete window also affect sex ratios in this species (McCoy et al., 2015).
Inherent in the documented FMF pattern of alligator sex determination are multiple incubation temperatures expected to yield both males and females. Consequently, the alligator sex determination system provides a natural framework for exploring how environmental factors, such as temperature, influence sexual variation at two levels. First, by comparing individuals of the same sex, but derived from different incubation temperatures, linkages between embryonic incubation temperature and intrasexual variation can be assessed. Second, for traits that display sex biased expression patterns, we are able to evaluate how a specific temperature influences the magnitude of sexual dimorphism. We address each of these questions and document how embryonic incubation temperatures impact the expression patterns of sex biased genes with upstream roles in sex development and circulating sex hormones.
The contribution of the embryonic environment to intrasexual variation is exemplified by the intrauterine position phenomenon noted for litter-bearing eutherian mammals (Vom Saal, 1983, Vom Saal and Bronson, 1978). In this context, the uterine positioning of an embryo relative to its male and female littermates significantly shapes the hormone milieu of that embryo and influences individual reproductive characteristics including gonadal morphology, reproductive behavior and success. Female rodents that develop between two males appear masculinized with increased anogenital distance (a marker for of prenatal androgen exposure), elevated aggressive behaviors in adulthood, and a shortened estrous cycle relative to female embryos that are positioned between two females during development (Clemens et al., 1978, Vom Saal, 1983). These findings provide evidence that slight modifications to the hormonal environment impact secondary sex differentiation within one sex. However, it is not known if intrasexual variation in upstream genetic pathways (e.g., hormone synthesis) might lead to effects on secondary sex differentiation at the individual level.
It is clear that thermal features experienced before hatch promote persistent effects beyond the period of sex determination in TSD species (Crews et al., 1998, Fisher et al., 2014, Joanen et al., 1987). Here we utilize the American alligator as a model TSD species to investigate potential underpinnings of adult intrasexual variation and test the hypothesis that individuals of the same sex, yet derived from different incubation temperatures, show temperature-sensitive variation in the expression of sex biased genes and circulating hormone profiles. This hypothesis is supported by observations in turtles showing differential responsiveness to the feminizing effects of exogenous estradiol. Male embryos from incubation temperatures near the female-producing end of the temperature spectrum are feminized with lower doses of estradiol relative to males derived from temperatures closer to the male producing temperature (Crews et al., 1991, Wibbels et al., 1991). Changes in incubation temperature may therefore influence similar quantitative alterations in the expression of genes important for propagating the temperature signal within individuals of the same sex. Furthermore, findings by our laboratory indicate that the developmental timing of exposure to a feminizing temperature during development significantly influences the mRNA abundance of aromatase, a terminal enzyme in estrogen biosynthesis (McCoy et al., 2015). It is conceivable that these temperature-induced variations in gene expression may contribute to long-term functional consequences and represent important sources of intrasexual variation.
Investigating temperature induced variation within an ovary or a testis with respect to sex biased transcript abundance and steroidogenic capacity offers unique insights into the biological foundation of sexual dimorphism along with previously unappreciated sources of variation. Most of the efforts aimed at assessing sexual dimorphism, for example, ascertain the degree of sexual dimorphism by broadly contrasting male and female traits (Allsteadt and Lang, 1995, Gunderson et al., 2001, Milnes et al., 2008). This approach is useful for identifying and categorizing the general differences between the sexes, but likely obscures detection of the variation existing within a single sex and how this variation contributes to overall measures of sexual dimorphism. Furthermore, the identification of temperature induced changes within a particular sex may provide an added layer of information regarding how environmentally vulnerable organisms will respond to threats such as modern climate change and exposure to endocrine active compounds that are found in the environment.
Section snippets
Egg collection, incubation treatments, and hatchling measures
The Institutional Animal Care and Use Committee at the Medical University of South Carolina approved all experiments reported here (protocol AR#3069). Fieldwork was conducted under permits from the Florida Fish and Wildlife Conservation Commission and the U.S. Fish and Wildlife Service.
On June 21, 2013, seven clutches of eggs were collected from nest sites at Lake Apopka, Florida and securely transported to our laboratory in Charleston, South Carolina. Within 24 h of arrival, all eggs were
Observed sex ratios
Experimental incubation conditions generated mixed sex ratios with the exception of the 30 °C treatment, which produced exclusively females (Table 2). Female biased sex ratios were observed at lower incubation temperatures (30 °C and 32 °C), while male biased sex ratios characterized those groups incubated at higher temperatures (33.5 °C and 34.5 °C). Contrary to expectations, a shift from male to female biased ratios was not detected at temperatures above 33.5 °C (Table 2).
Intrasexual patterns of sex biased gonadal genes
We quantified expression
Discussion
Sex is broadly considered a binary trait. However, this generalized view may obscure an underlying continuum of putative epigenetic modifications and gene expression profiles that together contribute to overall bimodal phenotypes. Previous study of TSD reptiles demonstrates that certain epigenetic modifications (e.g., DNA methylation) display temperature sensitive sexually dimorphic patterning (Matsumoto et al., 2014, Parrott et al., 2013). The sensitivity of the mechanisms governing this
Acknowledgments
This manuscript is dedicated to the memory of Dr. Louis J. Guillette, Jr., who believed in basic science and repeated often that we must seek to understand “normal” before we can understand “abnormal”. We are indebted to the members of the Florida Fish and Wildlife Commission for their continued support of our research endeavors. We thank Roger Newman and Brenna Doheny for their constructive feedback regarding the organization and content of this manuscript. The experiments described here were
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Mechanisms related to sexual determination by temperature in reptiles
2019, Journal of Thermal BiologyCitation Excerpt :An interesting thing about this research is that they studied the sensitivity of the reproductive system, when encountering different thermal signals during development, because they wanted to know if there were differences between the transcripts and hormones of the gonad and brain, related to the different temperatures that generate the same sex of individual (see Fig. 2). No differences were found that can be explained by the intensity of embryonic incubation temperatures, except for the magnitude of dimorphic expression in Amh and Sox9 genes, which seems to be exclusively related to the incubation temperature in the gonad, but not in the brain (Gabriel et al., 2001; McCoy et al., 2016). In contrast, it is thought that the nerve endings within the gonad are able to perceive the incubation temperature spatially and temporally (during the TSP) and release some chemical messenger that influences the sexual determination of the individual (Gutiérrez-Ospina et al., 1999; Seebacher and Craig, 2005; Seebacher and Murray, 2007).