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Animals face unpredictable challenges that require rapid, facultative physiological reactions to support survival but may compromise reproduction. Bats have a long-standing reputation for being highly sensitive to stressors, with sensitivity and resilience varying both within and among species, yet little is known about how stress affects the signaling that regulates reproductive physiology. Here, we provide the first description of the molecular response of the hypothalamic-pituitary-gonadal (HPG) axis of male big brown bats (Eptesicus fuscus) in response to short-term stress using a standardized restraint manipulation. This acute stressor was sufficient to upregulate plasma corticosterone and resulted in a rapid decrease in circulating testosterone. While we did not find differences in the mRNA expression of key steroidogenic enzymes (StAR, aromatase, 5-alpha reductase), seminiferous tubule diameter was reduced in stressed bats coupled with a 5-fold increase in glucocorticoid receptor (GR) mRNA expression in the testes. These changes, in part, may be mediated by RFamide-related peptide (RFRP) because fewer immunoreactive cell bodies were detected in the brains of stressed bats compared with controls - suggesting a possible increase in secretion - and increased RFRP expression locally in the gonads. The rapid sensitivity of the bat testes to stress may be connected to deleterious impacts on tissue health and function as supported by significant transcriptional upregulation of key pro-apoptotic signaling molecules (Bax, cytochrome c). Experiments like this broadly contribute to our understanding of the stronger ecological predictions regarding physiological responses of bats within the context of stress, which may impact decisions surrounding animal handling and conservation approaches., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2023. Published by The Company of Biologists Ltd.)

Comparative analyses of bats indicate that hibernation is associated with increased longevity among species. However, it is not yet known if hibernation affects biological ageing of individuals. Here, we use DNA methylation (DNAm) as an epigenetic biomarker of ageing to determine the effect of hibernation on the big brown bat, Eptesicus fuscus . First, we compare epigenetic age, as predicted by a multi-species epigenetic clock, between hibernating and non-hibernating animals and find that hibernation is associated with epigenetic age. Second, we identify genomic sites that exhibit hibernation-associated change in DNAm, independent of age, by comparing samples taken from the same individual in hibernating and active seasons. This paired comparison identified over 3000 differentially methylated positions (DMPs) in the genome. Genome-wide association comparisons to tissue-specific functional elements reveals that DMPs with elevated DNAm during winter occur at sites enriched for quiescent chromatin states, whereas DMPs with reduced DNAm during winter occur at sites enriched for transcription enhancers. Furthermore, genes nearest DMPs are involved in regulation of metabolic processes and innate immunity. Finally, significant overlap exists between genes nearest hibernation DMPs and genes nearest previously identified longevity DMPs. Taken together, these results are consistent with hibernation influencing ageing and longevity in bats.

Gonadotropin-inhibitory hormone (GnIH, also known RFRP-3 in mammals) is an important regulator of the hypothalamic-pituitary-gonadal axis and downstream reproductive physiology. Substantial species differences exist in the localization of cell bodies producing RFRP-3 and patterns of fiber immunoreactivity in the brain, raising the question of functional differences. Many temperate bat species exhibit unusual annual reproductive patterns. Male bats upregulate spermatogenesis in late spring which is asynchronous with periods of mating in the fall, while females have the physiological capacity to delay their reproductive investment over winter via sperm storage or delayed ovulation/fertilization. Neuroendocrine mechanisms regulating reproductive timing in male and female bats are not well-studied. We provide the first description of RFRP-precursor peptide of GnIH -expression and localization in the brain of any bat using a widespread temperate species (Eptesicus fuscus, big brown bat) as a model. RFRP mRNA expression was detected in the hypothalamus, testes, and ovaries of big brown bats. Cellular RFRP-immunoreactivity was observed within the periventricular nuclei, dorsomedial nucleus of the hypothalamus, arcuate nucleus (Arc), and median eminence (ME). As in other vertebrates, RFRP fiber immunoreactivity was widespread, with the greatest density observed in the hypothalamus, preoptic area, Arc, ME, midbrain, and thalamic nuclei. Putative interactions between RFRP-ir fibers and gonadotropin-releasing hormone (GnRH) cell bodies were observed in 16% of GnRH-immunoreactive cells, suggesting direct regulation of GnRH via RFRP signaling. This characterization of RFRP distribution contributes to a deeper understanding of bat neuroendocrinology, which serves as foundation for manipulative approaches examining changes in reproductive neuropeptide signaling in response to environmental and physiological challenges within, and among, bat species., (© 2021 Wiley Periodicals LLC.)

AbstractBats (order Chiroptera) are the second largest group of mammals, diverging ~52.5 million years ago. Many species exhibit an unusual reproductive cycle and extreme longevity without reproductive senescence, yet steroid profiles exist for few bats. Big brown bats ( Eptesicus fuscus ) are temperate insectivores found throughout North America. They mate promiscuously in fall, store sperm during winter hibernation, and have delayed ovulation and fertilization in spring. Here, we report the first urinary steroid profile in bats by quantifying 17β-estradiol (E 2 ) in captive male and female E. fuscus across their reproductive cycle. Male bats had higher urinary E 2 levels than females, and adults had higher levels than yearlings following creatinine adjustment for hydration. In nonpregnant females, several seasonal differences in creatinine-adjusted and unadjusted urinary E 2 levels were observed. Urinary E 2 was higher in males than females in winter for both conditions and in autumn for creatinine-adjusted levels. We quantified progesterone (P 4 ) in a subset of females. In nonpregnant females, urinary P 4 was constant across seasons except for unadjusted levels, which were highest in the summer. In pregnant females, urinary E 2 and P 4 levels peaked beginning ~20 d before parturition, with both steroids returning to baseline in the following weeks. Knowing how urinary steroid levels fluctuate with age and sex and across the annual season is key to understanding reproductive cycling in bats. Our research furthers the potential for bats as a model for medical reproductive research. Moreover, it complements previous studies on the potential role of steroids in primer pheromonal effects in bats.

Exceptionally long-lived species, including many bats, rarely show overt signs of aging, making it difficult to determine why species differ in lifespan. Here, we use DNA methylation (DNAm) profiles from 712 known-age bats, representing 26 species, to identify epigenetic changes associated with age and longevity. We demonstrate that DNAm accurately predicts chronological age. Across species, longevity is negatively associated with the rate of DNAm change at age-associated sites. Furthermore, analysis of several bat genomes reveals that hypermethylated age- and longevity-associated sites are disproportionately located in promoter regions of key transcription factors (TF) and enriched for histone and chromatin features associated with transcriptional regulation. Predicted TF binding site motifs and enrichment analyses indicate that age-related methylation change is influenced by developmental processes, while longevity-related DNAm change is associated with innate immunity or tumorigenesis genes, suggesting that bat longevity results from augmented immune response and cancer suppression.

Growing evidence shows that sex steroids not only act within the individual whose glands produce them; they can also act on proximate conspecifics. Previous studies show that exogenous 17β-estradiol (E 2 ) can be absorbed both nasally and percutaneously, arriving in blood, neural, reproductive, and peripheral tissues. When male bats were injected with radiolabeled E 2 ( 3 H-E 2 ) and housed with females during the mating season, radioactivity was reliably measured in the females' tissues. The present study was designed to compare E 2 transfer from male to female bats at three time points in the annual reproductive cycle: spring (ovulation and fertilization), summer (maternal season), and autumn (mating season). Pairs of mature female bats were housed with a mature 3 H-E 2 -treated male (50 μCi). Following 48 h of communal housing, radioactivity was measured in the tissues of female bats. Higher levels of radioactivity were present in the uterus and other tissues during the spring and autumn seasons compared to the summer season. We also measured natural levels of bioactive, unconjugated E 2 in the urine of male bats using enzyme immunoassays, and found that it was present in all three seasons but at lower levels during the summer. Male-excreted E 2 could transfer to females within the close confines of a roost, potentially influencing their reproductive physiology and behavior. These results suggest increased E 2 transfer coincides with female reproduction, with urine as a likely vector. We suggest that sex steroid transfer among interacting individuals may explain several mammalian phenomena historically viewed as "pheromonal"., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Experiments using female mice and bats have demonstrated that tritium-labeled 17β-estradiol ( 3 H-E 2 ) can be absorbed via cutaneous and intranasal routes and distributed to reproductive and neural tissues. Radioactivity has also been measured in tissues of untreated females after 48h cohabitation with 3 H-E 2 injected males. The present study was designed to quantify steroid transfer among female bats. Radioactive quantification via liquid scintillation counting revealed absorption of tritium-labeled progesterone ( 3 H-P 4 ) in adult females 1h after cutaneous and intranasal application (10μCi). Subsequently, pairs of mature females were each housed for 48h with a single mature female that had been administered 3 H-P 4 (50μCi) via intraperitoneal injection. Radioactivity was observed in all collected tissues of all non-injected females at levels significantly greater than the control group. Following the same paradigm, radioactivity was not observed in the tissues of untreated female bats that were housed with stimulus females treated with 3 H-E 2 (50μCi). Enzyme immunoassays revealed measurable levels of unconjugated progesterone and estradiol in the urine of female bats, suggesting urine as a vector for steroid transfer. Given that bats of this species live in predominantly female roosts in very close contact, progesterone transfer among individuals is likely to occur in natural roosts., (Copyright © 2017 Elsevier Inc. All rights reserved.)

The powerful estrogen, 17β-estradiol, has been found to pass from male excretions to the reproductive organs, brain, and other tissues of cohabiting females in laboratory mice. The current studies were designed to examine whether this phenomenon also occurs in big brown bats (Eptesicus fuscus), a mammal appropriate for testing cross-species generality because of its phylogenetic distance from mice. When tritiated estradiol ((3)H-E2) was administered directly on the nasal area of adult female bats, radioactivity was reliably observed in the uterus and ovaries, and also in the brain and other tissues. When (3)H-E2 was applied to the skin, radioactivity was observed in reproductive and other peripheral tissues. We injected male bats with minute quantities of (3)H-E2 and housed each of them directly with groups of adult females for 48h. We then measured radioactivity in male and female bat tissues. In each of several replications of one male housed with three females, radioactivity was reliably observed in the uterus of all females, and in many other tissues in almost every female. Measurement in the organs of males directly exposed to (3)H-E2 showed high levels of radioactivity in the testes and especially the epididymides. These data indicate that estradiol is transferred from males to females, likely via absorptions from males' excretions and potentially also via intravaginal exposure during mating. Given the potency of estradiol in regulating female reproductive physiology and behavior, our data strongly suggest the potential for pheromonal action whereby male mammals induce sexual receptivity and ovulation in females., (Copyright © 2014 Elsevier Inc. All rights reserved.)