Neural control of reproduction in reptiles.

Reptiles display considerable diversity in reproductive behavior, making them great models to study the neuroendocrine control of reproductive behavior. Many reptile species are seasonally breeding, such that they become reproductively active during their breeding season and regress to a nonreproductive state during their nonbreeding season, with this transition often prompted by environmental cues. In this review, we will focus on summarizing the neural and neuroendocrine mechanisms controlling reproductive behavior. Three major areas of the brain are involved in reproductive behavior: the preoptic area (POA), amygdala, and ventromedial hypothalamus (VMH). The POA and VMH are sexually dimorphic areas, regulating behaviors in males and females respectively, and all three areas display seasonal plasticity. Lesions to these areas disrupt the onset and maintenance of reproductive behaviors, but the exact roles of these regions vary between sexes and species. Different hormones influence these regions to elicit seasonal transitions. Circulating testosterone (T) and estradiol (E2) peak during the breeding season and their influence on reproduction is well-documented across vertebrates. The conversion of T into E2 and 5α-dihydrotestosterone can also affect behavior. Melatonin and corticosterone have generally inhibitory effects on reproductive behavior, while serotonin and other neurohormones seem to stimulate it. In general, there is relatively little information on the neuroendocrine control of reproduction in reptiles compared to other vertebrate groups. This review highlights areas that should be considered for future areas of research.

SARS-CoV-2 active infection prevalence and seroprevalence in the adult population of St. Louis County.

BACKGROUND: The true prevalence of COVID-19 is difficult to estimate due to the absence of random population-based testing. To estimate current and past COVID-19 infection prevalence in a large urban area, we conducted a population-based survey in St. Louis County, Missouri. METHODS: The population-based survey of active infection (PCR) and seroprevalence (IgG antibodies) of adults (≥18 years) was conducted through random-digit dialing and targeted sampling of St. Louis County residents with oversampling of Black residents. Infection prevalence of residents was estimated using design-based and raking weighting. RESULTS: Between August 17 and October 24, 2020, 1245 residents completed a survey and underwent PCR testing; 1073 residents completed a survey and underwent PCR and IgG testing or self-reported results. Weighted prevalence estimates of residents with active infection were 1.9% (95% CI, 0.4%-3.3%) and 5.6% were ever infected (95% CI, 3.3%-8.0%). Overall infection hospitalization and fatality ratios were 4.9% and 1.4%, respectively. CONCLUSIONS: Through October 2020, the percentage of residents that had ever been infected was relatively low. A markedly higher percentage of Black and other minorities compared to White residents were infected with COVID-19. The St. Louis region remained highly vulnerable to widespread infection in late 2020.

Characterizing seasonal transitions: Breeding-like morphology and behavior during the late non-breeding season in green anole lizards.

Seasonally breeding animals, such as green anole lizards (Anolis carolinensis), allow for the examination of the control of reproduction during different reproductive states. During the breeding season, the gonads are large and reproductively active. Following the breeding season, gonads regress and become less active, and the lizards enter a refractory period where breeding is inhibited. After this stage, a post-refractory period occurs during which the lizards are still in a non-breeding state, but environmental changes can trigger the onset of breeding. However, it is unclear what causes these changes in reproductive state and we hypothesized that this may be due to alterations in gonadotropin-releasing hormone (GnRH) signaling. The present study aimed to identify morphological and behavioral differences in GnRH- and saline-injected refractory and post-refractory male anoles when housed under the same non-breeding environmental conditions. We found that post-refractory anoles had increased testicular weight, recrudescence, sperm presence, and reproductive behavior, with no impact of GnRH injection. Renal sex segment size and steroidogenic acute regulatory protein (StAR) mRNA levels did not differ among groups, indicating that testosterone levels likely had not increased in post-refractory lizards. Post-refractory anoles in this study were beginning to transition towards a breeding state without exposure to changing environmental conditions, and GnRH was not necessary for these changes. These data reveal a complex interaction between the activation of breeding, changing environmental conditions, and the underlying physiology regulating reproduction in seasonally breeding lizards. Future studies are needed to further elucidate the mechanisms that regulate this relationship.

FAM171B is a novel polyglutamine protein widely expressed in the mammalian brain.

Mutation in proteins containing polyglutamine (polyQ) tracts has been shown to underlie a number of severe human neurodegenerative disorders such as Huntington's Disease and Spinocerebellar Ataxia. In this study, we identify and describe FAM171B as a novel polyQ protein containing fourteen consecutive glutamine residues in its National Center for Biotechnology Information (NCBI) referenced sequence. Utilizing western blotting, in situ hybridization, and immunohistochemistry, we demonstrate that FAM171B is widely expressed in mouse brain with pronounced localization in the hippocampus, cerebellum, and cerebral cortex. Furthermore, immunofluorescence experiments reveal that FAM171B predominantly localizes to vesicle-like structures in the cytoplasm of neurons. Finally, bioinformatic analysis suggests that FAM171B is robustly expressed in human brain, and (similar to other polyQ disease genes) its polyQ tract is polymorphic within the general human population. Thus, as a polyQ protein that is expressed in brain, FAM171B should be considered a candidate gene for an as yet molecularly uncharacterized neurodegenerative disease.

All Hands on Deck: Local Public Health Agencies Leveraging the Incident Command System During Crises.

The COVID-19 pandemic has stretched limited public health resources beyond measures, particularly at the local level. What started as an interesting report of pneumonia of unknown etiology in late December 2019 in Wuhan, China, bloomed into an international crisis by mid-January 2020. However, it was not until late January, when the first case was reported in the United States, that a new reality took shape for US public health agencies. After all, severe acute respiratory syndrome never made it to this country, and the only 2 cases of Middle East respiratory syndrome here were imported and never spread. Local public health agencies are notoriously short-staffed and underfunded. Therefore, when a crisis looms, personnel from a multitude of areas within the agencies are called upon to help out. Under its innovative and forward-thinking leadership, the St. Louis County Department of Health internally implemented the Incident Command System, a component of the National Incident Management System. While reassignment of individuals to new responsibilities under a new and temporary reporting structure did not always go perfectly, Incident Command System kept its promise to be adaptable to a fast-evolving situation, to clearly outline needed areas of responsibility, and to provide scaffolding that kept the Department of Health functional in chaotic times. It was able to be implemented quickly within hours of the first confirmed COVID-19 case in St. Louis County and enhanced the quality and timeliness of the public health response. This experience is being shared to provide a model of how organizations with limited personnel can use the Incident Command System to reorganize and meet unexpected challenges with increased success.

The hypothalamic-pituitary-gonadal axis and thyroid hormone regulation interact to influence seasonal breeding in green anole lizards (Anolis carolinensis).

Reproductive physiology and behavior is mainly regulated by the hypothalamus-pituitary-gonad (HPG) axis, although abnormal thyroid hormone (TH) levels alter HPG axis activity. Seasonally breeding animals, such as green anole lizards (Anolis carolinensis), undergo drastic hormonal and behavioral changes between breeding and non-breeding seasons, with increased sex steroid hormones, larger gonads and increased reproductive behaviors during the breeding compared to non-breeding seasons. Relatively less is known regarding the regulation of gonadal TH in seasonal reproduction. We examined whether the gonadal expression of enzymes involved in TH activation are altered in concert with seasonal reproduction. Type 2 deiodinase (Dio2) mRNA, the TH activating enzyme, was upregulated in breeding compared to non-breeding testes, while type 3 deiodinase (Dio3) mRNA, the TH deactivating enzyme, was upregulated in breeding ovaries. To study the association between the HPG axis and local activation of TH, we manipulated the HPG axis during the non-breeding season by subcutaneously injecting luteinizing hormone (LH) and follicle stimulating hormone (FSH) in male lizards. We found that acute LH and FSH injections induced many aspects of breeding, with increased testes size and testosterone levels. Surprisingly, Dio3 was upregulated in the testes after LH and FSH injections, while Dio2 mRNA levels were unchanged. These results suggest that there might be different roles for local TH activation in developing and maintaining fully mature and functional gonads. Our findings continue to support the role for TH in regulating reproduction.

Seasonal regulation of steroidogenic enzyme expression within the green anole lizard (Anolis carolinensis) brain and gonad.

Steroid hormones, such as testosterone and estradiol, are necessary for reproductive behavior. Seasonally breeding animals have increased sex steroid hormone levels during the breeding compared to non-breeding season, with increased reproductive behaviors and altered brain morphology in breeding individuals. Similar to other seasonally breeding animals, green anole lizards (Anolis carolinensis) have high sex steroid hormone levels and increased reproductive behaviors in the breeding season. Relatively less is known regarding the regulation of steroidogenesis in reptiles and this experiment examined whether enzymes involved in sex steroid hormone synthesis vary seasonally within the brain and gonads in wild-caught anole lizards. Specifically, we examined mRNA expression of steroidogenic acute regulatory protein (StAR), P450 17α-hydroxylase/C17-20lyase (Cyp17α1), 17 beta-hydroxysteroid dehydrogenase type 3 (17ßHSD 3), and aromatase (Cyp19α1). We found that the mRNA for each of these genes was expressed in the lizard brain. Interestingly, Cyp19α1 mRNA expression in the brain was increased during the non-breeding season, potentially revealing a role for aromatase expression in the non-breeding brain. In the anole gonads, StAR mRNA expression levels were increased in both males and females during the breeding season, while the mRNA expression levels of CYP17α1 and 17ßHSD 3 are increased when StAR mRNA expression was decreased, suggesting that the enzymes in the steroidogenic pathway are potentially regulated independently of StAR. This work reveals the seasonal regulation of steroidogenesis in the reptilian brain and gonad, although more work is necessary to determine the regulatory mechanisms that control these expression patterns.

Adult Neurogenesis Leads to the Functional Reconstruction of a Telencephalic Neural Circuit.

UNLABELLED: Seasonally breeding songbirds exhibit pronounced annual changes in song behavior, and in the morphology and physiology of the telencephalic neural circuit underlying production of learned song. Each breeding season, new adult-born neurons are added to the pallial nucleus HVC in response to seasonal changes in steroid hormone levels, and send long axonal projections to their target nucleus, the robust nucleus of the arcopallium (RA). We investigated the role that adult neurogenesis plays in the seasonal reconstruction of this circuit. We labeled newborn HVC neurons with BrdU, and RA-projecting HVC neurons (HVCRA) with retrograde tracer injected in RA of adult male white-crowned sparrows (Zonotrichia leucophrys gambelii) in breeding or nonbreeding conditions. We found that there were many more HVCRA neurons in breeding than nonbreeding birds. Furthermore, we observed that more newborn HVC neurons were back-filled by the tracer in breeding animals. Behaviorally, song structure degraded as the HVC-RA circuit degenerated, and recovered as the circuit regenerated, in close correlation with the number of new HVCRA neurons. These results support the hypothesis that the HVC-RA circuit degenerates in nonbreeding birds, and that newborn neurons reconstruct the circuit in breeding birds, leading to functional recovery of song behavior. SIGNIFICANCE STATEMENT: We investigated the role that adult neurogenesis plays in the seasonal reconstruction of a telencephalic neural circuit that controls song behavior in white-crowned sparrows. We showed that nonbreeding birds had a 36%-49% reduction in the number of projection neurons compared with breeding birds, and the regeneration of the circuit in the breeding season is due to the integration of adult-born projection neurons. Additionally, song structure degraded as the circuit degenerated and recovered as the circuit regenerated, in close correlation with new projection neuron number. This study demonstrates that steroid hormones can help reestablish functional neuronal circuits following degeneration in the adult brain and shows non-injury-induced degeneration and reconstruction of a neural circuit critical for producing a learned behavior.

Aromatase and 5α-reductase type 2 mRNA in the green anole forebrain: an investigation of the effects of sex, season and testosterone manipulation.

Aromatase and 5α-reductase (5αR) catalyze the synthesis of testosterone (T) metabolites: estradiol and 5α-dihydrotestosterone, respectively. These enzymes are important in controlling sexual behaviors in male and female vertebrates. To investigate factors contributing to their regulation in reptiles, male and female green anole lizards were gonadectomized during the breeding and non-breeding seasons and treated with a T-filled or blank capsule. In situ hybridization was used to examine main effects of and interactions among sex, season, and T on expression of aromatase and one isozyme of 5αR (5αR2) in three brain regions that control reproductive behaviors: the preoptic area, ventromedial nucleus of the amygdala and ventromedial hypothalamus (VMH). Patterns of mRNA generally paralleled previous evaluations of intact animals. Although no main effects of T were detected, interactions were present in the VMH. Specifically, the density of 5αR2 expressing cells was greater in T-treated than control females in this region, regardless of season. Among breeding males, blank-treated males had a denser population of 5αR2 positive cells than T-treated males. Overall, T appears to have less of a role in the regulation of these enzymes than in other vertebrate groups, which is consistent with the primary role of T (rather than its metabolites) in regulation of reproductive behaviors in lizards. However, further investigation of protein and enzyme activity levels are needed before specific conclusions can be drawn.

Amygdala recruitment in schizophrenia in response to aversive emotional material: a meta-analysis of neuroimaging studies.

Emotional dysfunction has long been established as a critical clinical feature of schizophrenia. In the past decade, there has been extensive work examining the potential contribution of abnormal amygdala activation to this dysfunction in patients with schizophrenia. A number of studies have demonstrated under-recruitment of the amygdala in response to emotional stimuli, while others have shown intact recruitment of this region. To date, there have been few attempts to synthesize this literature using quantitative criteria or to use a formal meta-analytic approach to examine which variables may moderate the magnitude of between-group differences in amygdala activation in response to aversive emotional stimuli. We conducted a meta-analysis of amygdala activation in patients with schizophrenia, using a bootstrapping approach to investigate: (a) evidence for amygdala under-recruitment in schizophrenia and (b) variables that may moderate the magnitude of between-group differences in amygdala activation. We demonstrate that patients with schizophrenia show statistically significant, but modest, under-recruitment of bilateral amygdala (mean effect size = -0.20 SD). However, present findings indicate that this under-recruitment is dependent on the use of a neutral vs emotion interaction contrast and is not apparent if amygdala activation by patients and controls is evaluated in a negative emotional condition only.

The distribution of estrogen receptor ß mRNA in male and female green anole lizards.

Estrogens are critical for a variety of aspects of brain development and adult processes. These steroids act via receptors within specific tissues. Several estrogen receptors (ER) are thought to exist, including ERα and ERß, which function via classical, genomic mechanisms. These two ERs are found in a variety of species and are critical to diverse functions, including reproductive behaviors. ERß was discovered more recently than ERα, and very little work has been done on this receptor in reptiles. Currently no data are available on its distribution in the brain in this vertebrate group. Here, we have cloned ERß in the green anole lizard, mapped its distribution using in situ hybridization, and quantified expression in three brain areas controlling reproductive behaviors-the preoptic area, ventromedial amygdala (AMY), and ventromedial hypothalamus (VMH). ERß was detected in discrete areas throughout the anole brain, with high levels in limbic regions and motor nuclei in the brainstem. Females had a greater density of ERß positive cells in the AMY and VMH than males. While the functional consequences of these differences are not clear, they may result in an increased ability to respond to local levels of estradiol. The present work documents that neural ERß is distributed similarly in reptilian, rodent and avian species, suggesting that it may perform similar roles. However, more work is necessary to elucidate the function of ERß in this group.

Testing alternative hypotheses for evolutionary diversification in an African songbird: rainforest refugia versus ecological gradients.

Geographic isolation in rainforest refugia and local adaptation to ecological gradients may both be important drivers of evolutionary diversification. However, their relative importance and the underlying mechanisms of these processes remain poorly understood because few empirical studies address both putative processes in a single system. A key question is to what extent is divergence in signals that are important in mate and species recognition driven by isolation in rainforest refugia or by divergent selection across ecological gradients? We studied the little greenbul, Andropadus virens, an African songbird, in Cameroon and Uganda, to determine whether refugial isolation or ecological gradients better explain existing song variation. We then tested whether song variation attributable to refugial or ecological divergence was biologically meaningful using reciprocal playback experiments to territorial males. We found that much of the existing song variation can be explained by both geographic isolation and ecological gradients, but that divergence across the gradient, and not geographic isolation, affects male response levels. These data suggest that ecologically divergent traits, independent of historical isolation during glacial cycles, can promote reproductive isolation. Our study provides further support for the importance of ecology in explaining patterns of evolutionary diversification in ecologically diverse regions of the planet.

Relationships among reproductive morphology, behavior, and testosterone in a natural population of green anole lizards.

Laboratory studies of reproductive systems have long supported the idea that neural and/or muscular structures used frequently are often enhanced in size. However, field studies integrating behavioral, morphological, and hormonal data are needed to better understand relationships in natural environments. We examined a natural population of green anole lizards (Anolis carolinensis) to determine whether variation in reproductive morphology both within and between the sexes paralleled differences in courtship and copulatory behaviors and circulating testosterone levels. Display rate in males was positively correlated with the sizes of the cartilage supporting the dewlap (a throat fan used in courtship and aggression) and renal sex segments (portions of the kidney that function similarly to the mammalian prostate), but correlated negatively with seminiferous tubule size. Plasma testosterone in males was negatively correlated with display behavior and was not correlated with any measures of morphology. Females, which display rarely, exhibited no relationships between morphology and frequency of behavior. Comparisons between the sexes show that males have consistently larger courtship and copulation morphologies than females, even when accounting for sex differences in body size. The results not only support the idea of relationships between increased function and enhanced structures, but also show the complexity of mechanistic interactions associated with reproductive behavior in wild animals.

Distribution of two isozymes of 5α-reductase in the brains of adult male and female green anole lizards.

The 5α-reductase (5αR) enzyme converts testosterone to 5α-dihydrotestosterone. This local metabolism within the brain is important for the full expression of male sexual behavior in many species, including green anole lizards. Two isozymes of 5αR exist and little is known about their specific distributions. We conducted in situ hybridization for both isozymes in intact male and female green anole brains during the breeding (BS) and non-breeding (NBS) seasons. 5αR1 mRNA was only detected in the brainstem, while 5αR2 was expressed in specific areas throughout the brain. As our primary interest was evaluating the potential role of 5αR in forebrain regulation of reproductive behavior, we quantified 5αR2 expression in the preoptic area, amygdala (AMY), and ventromedial hypothalamus (VMH). More 5αR2 cells were detected during the NBS than BS in the AMY, and the density of these cells was greater in females than males. In the VMH, the right side contained more 5αR2 cells than the left, an effect driven by a lateralized increase in the NBS. These data expand understanding of the distribution and potential roles of both isozymes in the adult brain, and differences in expression patterns between mammals and birds suggest that they may have been co-opted for different functions later in evolution.

Newly deposited maternal hormones can be detected in the yolks of oviductal eggs in the green anole lizard.

Studies often examine egg yolks after oviposition with the goal of drawing conclusions about maternal allocation of gonadal steroid hormones and how it may affect offspring development. However, these hormones might originate from a few sources, including the ovary, blood plasma, or the embryo itself. The goal of this study was to investigate whether maternal steroids can enter oviductal eggs. In Experiment 1, gravid female green anole lizards were injected with 1 microCi 3H-T. Plasma, ovarian follicles (separated into yolking and non-yolking samples), and shelled oviductal eggs were collected at times ranging from 15 min to 24 hr after treatment. Main effects of tissue, time, and an interaction between them all existed on recovered 3H-hormone corrected for tissue mass. Of particular interest, there was a decrease in plasma with coincident increase in eggs. In Experiment 2, females were injected with doses ranging from 0.01 to 0.45 microCi of 3H-T per gram body weight. Across tissues, 3H-hormone levels corrected for mass were greater with increasing doses. Values also differed among tissues and an interaction was detected. Within each dose, plasma and non-yolking follicles generally had higher concentrations of 3H-hormone than did yolking follicles and oviductal eggs. However, at and after 6 hr, eggs had higher total radioactivity levels than both yolking and non-yolking follicles had (not corrected for mass). The results indicate that steroids can cross through relatively well-formed shells before oviposition, suggesting a way in which maternal hormones might influence developmental factors after yolk deposition.

Testosterone selectively affects aromatase and 5alpha-reductase activities in the green anole lizard brain.

Testosterone (T) and its metabolites are important in the regulation of reproductive behavior in males of a variety of vertebrate species. Aromatase converts T to estradiol and 5alpha-reductase converts T to 5alpha-dihydrotestosterone (DHT). Male green anole reproduction depends on androgens, yet 5alpha-reductase in the brain is not sexually dimorphic and does not vary with season. In contrast, aromatase activity in the male brain is increased during the breeding compared to non-breeding season, and males have higher levels than females during the breeding season. Aromatase is important for female, but not male, sexual behaviors. The present experiment was conducted to determine whether 5alpha-reductase and aromatase are regulated by T. Enzyme activity was quantified in whole brain homogenates in both the breeding and non-breeding seasons in males and females that had been treated with either a T or blank implant. In males only, T increased 5alpha-reductase activity regardless of season and up-regulated aromatase during the breeding season specifically. Thus, regulation of both enzymes occurs in males, whereas females do not show parallel sensitivity to T. When considered with previous results, the data suggest that aromatase might influence a male function associated with the breeding season other than sexual behavior. 5alpha-Reductase can be mediated by T availability, but this regulation may not serve a sex- or season-specific purpose.