Incubation temperature alters stripe formation and head colouration in American alligator hatchlings and is unaffected by estradiol-induced sex reversal.

Considerations of the impact climate change has on reptiles are typically focused on habitat change or loss, range shifts and skewed sex ratios in species with temperature-dependent sex determination. Here, we show that incubation temperature alters stripe number and head colouration of hatchling American alligators (Alligator mississippiensis). Animals incubated at higher temperatures (33.5°C) had, on average, one more stripe than those at lower temperatures (29.5°C), and also had significantly lighter heads. These patterns were not affected by estradiol-induced sex reversal, suggesting independence from hatchling sex. Therefore, increases in nest temperatures as a result of climate change have the potential to alter pigmentation patterning, which may have implications for offspring fitness.

Exposure to ionizing radiation disrupts normal epigenetic aging in Japanese medaka.

Alterations to the epigenome are a hallmark of biological aging and age-dependent patterning of the DNA methylome ("epigenetic aging") can be modeled to produce epigenetic age predictors. Rates of epigenetic aging vary amongst individuals and correlate to the onset of age-related disease and all-cause mortality. Yet, the origins of epigenetic-to-chronological age discordance are not empirically resolved. Here, we investigate the relationship between aging, DNA methylation, and environmental exposures in Japanese medaka (Oryzias latipes). We find age-associated DNA methylation patterning enriched in genomic regions of low CpG density and that, similar to mammals, most age-related changes occur during early life. We construct an epigenetic clock capable of predicting chronological age with a mean error of 61.1 days (~8.4% of average lifespan). To test the role of environmental factors in driving epigenetic age variation, we exposed medaka to chronic, environmentally relevant doses of ionizing radiation. Because most organisms share an evolutionary history with ionizing radiation, we hypothesized that exposure would reveal fundamental insights into environment-by-epigenetic aging interactions. Radiation exposure disrupted epigenetic aging by accelerating and decelerating normal age-associated patterning and was most pronounced in cytosines that were moderately associated with age. These findings empirically demonstrate the role of DNA methylation in integrating environmental factors into aging trajectories.

Assessing the Ability of Developmentally Precocious Estrogen Signaling to Recapitulate Ovarian Transcriptomes and Follicle Dynamics in Alligators from a Contaminated Lake.

BACKGROUND: Concern has grown in recent decades over anthropogenic contaminants that interfere with the functioning of endocrine hormones. However, mechanisms connecting developmental processes to pathologies associated with endocrine-disrupting chemical (EDC) exposure are poorly understood in naturally exposed populations. OBJECTIVES: We sought to a) characterize divergence in ovarian transcriptomic and follicular profiles between alligators originating from a historically EDC-contaminated site, Lake Apopka, and a reference site; b) test the ability of developmentally precocious estrogen exposure to recapitulate site-associated patterns of divergence; and c) test whether treatment with exogenous follicle-stimulating hormone (FSH) is capable of rescuing phenotypes associated with contaminant exposure and/or embryonic estrogen treatment. METHODS: Alligators eggs were collected from a contaminated site and a reference site, and a subset of eggs from the reference site were treated with estradiol (E2) during embryonic development prior to gonadal differentiation. After hatching, alligators were raised under controlled laboratory settings for 5 months. Juveniles from both sites were divided and treated with exogenous FSH. Histological analyses and RNA-sequencing were conducted to characterize divergence in ovarian follicle dynamics and transcriptomes between sites, between reference and E2-treated animals, and between FSH-treated and nontreated animals. RESULTS: We observed broad site-of-origin divergence in ovarian transcriptomes and reductions in ovarian follicle density between juvenile alligators from Lake Apopka and the reference site. Treating embryos from the reference site with E2 overwhelmingly recapitulated transcriptional and histological alterations observed in Lake Apopka juveniles. Ovarian phenotypes observed in Lake Apopka alligators or resulting from estrogen treatment were only partially rescued by treatment with exogenous FSH. DISCUSSION: Recapitulation of ovarian abnormalities by precocious E2 revealed a relatively simple mechanism underlying contaminant-induced pathologies in a historical example of environmental endocrine disruption. Findings reported here support a model where the developmental timing of estrogen signaling has the potential to permanently alter ovarian organization and function. https://doi.org/10.1289/EHP6627.

Assessing impacts of precocious steroid exposure on thyroid physiology and gene expression patterns in the American alligator (Alligator mississippiensis).

The thyroid gland is sensitive to steroid hormone signaling, and many thyroid disrupting contaminants also disrupt steroid hormone homeostasis, presenting the possibility that thyroid disruption may occur through altered steroid hormone signaling. To examine this possibility, we studied short-term and persistent impacts of embryonic sex steroid exposure on thyroid physiology in the American alligator. Alligators from a lake contaminated with endocrine disrupting contaminants (Lake Apopka, FL, USA) have been shown to display characteristics of thyroid and steroid hormone disruption. Previous studies suggest these alterations arise during development and raise the possibility that exposure to maternally deposited contaminants might underlie persistent organizational changes in both thyroidal and reproductive function. Thus, this population provides a system to investigate contaminant-mediated organizational thyroid disruption in an environmentally-relevant context. We assess the developmental expression of genetic pathways involved in thyroid hormone biosynthesis and find that expression of these genes increases prior to hatching. Further, we show that nuclear steroid hormone receptors are also expressed during this period, indicating the developing thyroid is potentially responsive to steroid hormone signaling. We then explore functional roles of steroid signaling during development on subsequent thyroid function in juvenile alligators. We exposed alligator eggs collected from both Lake Apopka and a reference site to 17ß-estradiol and a non-aromatizable androgen during embryonic development, and investigated effects of exposure on hatchling morphometrics and thyroidal gene expression profiles at 5 months of age. Steroid hormone treatment did not impact the timing of hatching or hatchling size. Furthermore, treatment with steroid hormones did not result in detectable impacts on thyroid transcriptional programs, suggesting that precocious or excess estrogen and androgen exposure does not influence immediate or long-term thyroidal physiology.

Embryonic estrogen exposure recapitulates persistent ovarian transcriptional programs in a model of environmental endocrine disruption.

Estrogens regulate key aspects of sexual determination and differentiation, and exposure to exogenous estrogens can alter ovarian development. Alligators inhabiting Lake Apopka, FL, are historically exposed to estrogenic endocrine disrupting contaminants and are characterized by a suite of reproductive abnormalities, including altered ovarian gene expression and abated transcriptional responses to follicle stimulating hormone. Here, we test the hypothesis that disrupting estrogen signaling during gonadal differentiation results in persistent alterations to ovarian gene expression that mirror alterations observed in alligators from Lake Apopka. Alligator embryos collected from a reference site lacking environmental contamination were exposed to estradiol-17 beta or a nonaromatizable androgen in ovo and raised to the juvenile stage. Changes in basal and gonadotropin-challenged ovarian gene expression were then compared to Apopka juveniles raised under identical conditions. Assessing basal transcription in untreated reference and Apopka animals revealed a consistent pattern of differential expression of key ovarian genes. For each gene where basal expression differed across sites, in ovo estradiol treatment in reference individuals recapitulated patterns observed in Apopka alligators. Among those genes affected by site and estradiol treatment were three aryl hydrocarbon receptor (AHR) isoforms, suggesting that developmental estrogen signaling might program sensitivity to AHR ligands later in life. Treatment with gonadotropins stimulated strong ovarian transcriptional responses; however, the magnitude of responses was not strongly affected by steroid hormone treatment. Collectively, these findings demonstrate that precocious estrogen signaling in the developing ovary likely underlies altered transcriptional profiles observed in a natural population exposed to endocrine disrupting contaminants.

In ovo treatment with an estrogen receptor alpha selective agonist causes precocious development of the female reproductive tract of the American alligator (Alligator mississippiensis).

The molecular signaling processes involved the differentiation of the Müllerian duct (MD) into the female reproductive tract, or oviduct, in non-mammalian vertebrates are not well understood. Studies in mammals and birds indicate that steroid hormones play a role in this process, as the embryonic MD has been shown to be vulnerable to exogenous estrogens and progestins and environmental endocrine disrupting contaminants. In a previous study, developmental treatment with an estrogen receptor α (ERα) agonist, 4,4',4″-(4-propyl-[1H]-pyrazole-1,3,5-triyl)trisphenol (PPT), induced significant enlargement of the MD in alligator embryos incubated at a male-producing temperature, which was not observed in embryos treated with an estrogen receptor ß (ERß) agonist, 7-bromo-2-(4-hydroxyphenyl)-1,3-benzoxazol-5-ol (WAY 200070), or with 17ß-estradiol (E2). In order to understand the role of estrogen signaling in female alligator oviduct development, we incubated eggs at a female-producing temperature and treated them with E2 and these ER selective agonists, PPT and WAY 200070, just prior to the thermosensitive window of sex determination. At stage 27, one stage prior to hatching, PPT induced significant enlargement of the MD with precocious development of secretory glands and connective tissue differentiation similar to characteristics of mature adult oviduct. PPT treatment in ovo increased mRNA expression of ERß, progesterone receptor, androgen receptor and insulin-like growth factor 1 in MD at stage 27, while expression of ERα was decreased. Neither WAY 200070 nor E2 treatment induced these effects seen in PPT-treated MD. The results of this study provide insight into the critical factors for healthy reproductive system formation in this sentinel species, although further investigation is needed to determine whether the observed phenomena are directly due to selective stimulation of ERα or related to some other aspect of PPT treatment.

A temporal signature of epidermal growth factor signaling regulates the differentiation of germline cells in testes of Drosophila melanogaster.

Tissue replenishment from stem cells follows a precise cascade of events, during which stem cell daughters first proliferate by mitotic transit amplifying divisions and then enter terminal differentiation. Here we address how stem cell daughters are guided through the early steps of development. In Drosophila testes, somatic cyst cells enclose the proliferating and differentiating germline cells and the units of germline and surrounding cyst cells are commonly referred to as cysts. By characterizing flies with reduced or increased Epidermal Growth Factor (EGF) signaling we show that EGF triggers different responses in the cysts dependent on its dose. In addition to the previously reported requirement for EGF signaling in cyst formation, a low dose of EGF signaling is required for the progression of the germline cells through transit amplifying divisions, and a high dose of EGF signaling promotes terminal differentiation. Terminal differentiation was promoted in testes expressing a constitutively active EGF Receptor (EGFR) and in testes expressing both a secreted EGF and the EGFR in the cyst cells, but not in testes expressing either only EGF or only EGFR. We propose that as the cysts develop, a temporal signature of EGF signaling is created by the coordinated increase of both the production of active ligands by the germline cells and the amount of available receptor molecules on the cyst cells.

Control of germline stem cell division frequency--a novel, developmentally regulated role for epidermal growth factor signaling.

Exploring adult stem cell dynamics in normal and disease states is crucial to both better understanding their in vivo role and better realizing their therapeutic potential. Here we address the division frequency of Germline Stem Cells (GSCs) in testes of Drosophila melanogaster. We show that GSC division frequency is under genetic control of the highly conserved Epidermal Growth Factor (EGF) signaling pathway. When EGF signaling was attenuated, we detected a two-fold increase in the percentage of GSCs in mitotic division compared to GSCs in control animals. Ex vivo and in vivo experiments using a marker for cells in S-phase of the cell cycle showed that the GSCs in EGF mutant testes divide faster than GSCs in control testes. The increased mitotic activity of GSCs in EGF mutants was rescued by restoring EGF signaling in the GSCs, and reproduced in testes from animals with soma-depleted EGF-Receptor (EGFR). Interestingly, EGF attenuation specifically increased the GSC division frequency in adult testes, but not in larval testes. Furthermore, GSCs in testes with tumors resulting from the perturbation of other conserved signaling pathways divided at normal frequencies. We conclude that EGF signaling from the GSCs to the CySCs normally regulates GSC division frequency. The EGF signaling pathway is bifurcated and acts differently in adult compared to larval testes. In addition, regulation of GSC division frequency is a specific role for EGF signaling as it is not affected in all tumor models. These data advance our understanding concerning stem cell dynamics in normal tissues and in a tumor model.

Nucleoporin98-96 function is required for transit amplification divisions in the germ line of Drosophila melanogaster.

Production of specialized cells from precursors depends on a tightly regulated sequence of proliferation and differentiation steps. In the gonad of Drosophila melanogaster, the daughters of germ line stem cells (GSC) go through precisely four rounds of transit amplification divisions to produce clusters of 16 interconnected germ line cells before entering a stereotypic differentiation cascade. Here we show that animals harbouring a transposon insertion in the center of the complex nucleoporin98-96 (nup98-96) locus had severe defects in the early steps of this developmental program, ultimately leading to germ cell loss and sterility. A phenotypic analysis indicated that flies carrying the transposon insertion, designated nup98-96(2288), had dramatically reduced numbers of germ line cells. In contrast to controls, mutant testes contained many solitary germ line cells that had committed to differentiation as well as abnormally small clusters of two, four or eight differentiating germ line cells. This indicates that mutant GSCs rather differentiated than self-renewed, and that these GSCs and their daughters initiated the differentiation cascade after zero, or less than four rounds of amplification divisions. This phenotype remained unaffected by hyper-activation of signalling pathways that normally result in excessive proliferation of GSCs and their daughters. Expression of wildtype nup98-96 specifically in the germ line cells of mutant animals fully restored development of the GSC lineage, demonstrating that the effect of the mutation is cell-autonomous. Nucleoporins are the structural components of the nucleopore and have also been implicated in transcriptional regulation of specific target genes. The nuclear envelopes of germ cells and general nucleocytoplasmic transport in nup98-96 mutant animals appeared normal, leading us to propose that Drosophila nup98-96 mediates the transport or transcription of targets required for the developmental timing between amplification and differentiation.