Broad-Scale Assessment of Methylmercury in Adult Amphibians.

Mercury (Hg) is a toxic contaminant that has been mobilized and distributed worldwide and is a threat to many wildlife species. Amphibians are facing unprecedented global declines due to many threats including contaminants. While the biphasic life history of many amphibians creates a potential nexus for methylmercury (MeHg) exposure in aquatic habitats and subsequent health effects, the broad-scale distribution of MeHg exposure in amphibians remains unknown. We used nonlethal sampling to assess MeHg bioaccumulation in 3,241 juvenile and adult amphibians during 2017-2021. We sampled 26 populations (14 species) across 11 states in the United States, including several imperiled species that could not have been sampled by traditional lethal methods. We examined whether life history traits of species and whether the concentration of total mercury in sediment or dragonflies could be used as indicators of MeHg bioaccumulation in amphibians. Methylmercury contamination was widespread, with a 33-fold difference in concentrations across sites. Variation among years and clustered subsites was less than variation across sites. Life history characteristics such as size, sex, and whether the amphibian was a frog, toad, newt, or other salamander were the factors most strongly associated with bioaccumulation. Total Hg in dragonflies was a reliable indicator of bioaccumulation of MeHg in amphibians (R2 ≥ 0.67), whereas total Hg in sediment was not (R2 ≤ 0.04). Our study, the largest broad-scale assessment of MeHg bioaccumulation in amphibians, highlights methodological advances that allow for nonlethal sampling of rare species and reveals immense variation among species, life histories, and sites. Our findings can help identify sensitive populations and provide environmentally relevant concentrations for future studies to better quantify the potential threats of MeHg to amphibians.

Monitoring wetland water quality related to livestock grazing in amphibian habitats.

Land use alteration such as livestock grazing can affect water quality in habitats of at-risk wildlife species. Data from managed wetlands are needed to understand levels of exposure for aquatic life stages and monitor grazing-related changes afield. We quantified spatial and temporal variation in water quality in wetlands occupied by threatened Oregon spotted frog (Rana pretiosa) at Klamath Marsh National Wildlife Refuge in Oregon, United States (US). We used analyses for censored data to evaluate the importance of habitat type and grazing history in predicting concentrations of nutrients, turbidity, fecal indicator bacteria (FIB; total coliforms, Escherichia coli (E. coli), and enterococci), and estrogenicity, an indicator of estrogenic activity. Nutrients (orthophosphate and ammonia) and enterococci varied over time and space, while E. coli, total coliforms, turbidity, and estrogenicity were more strongly associated with local livestock grazing metrics. Turbidity was correlated with several grazing-related constituents and may be particularly useful for monitoring water quality in landscapes with livestock use. Concentrations of orthophosphate and estrogenicity were elevated at several sites relative to published health benchmarks, and their potential effects on Rana pretiosa warrant further investigation. Our data provided an initial assessment of potential exposure of amphibians to grazing-related constituents in western US wetlands. Increased monitoring of surface water quality and amphibian population status in combination with controlled laboratory toxicity studies could help inform future research and targeted management strategies for wetlands with both grazing and amphibians of conservation concern.

Regional variation in drivers of connectivity for two frog species (Rana pretiosa and R. luteiventris) from the U.S. Pacific Northwest.

Comparative landscape genetics has uncovered high levels of variability in which landscape factors affect connectivity among species and regions. However, the relative importance of species traits versus environmental variation for predicting landscape patterns of connectivity is unresolved. We provide evidence from a landscape genetics study of two sister taxa of frogs, the Oregon spotted frog (Rana pretiosa) and the Columbia spotted frog (Rana luteiventris) in Oregon and Idaho, USA. Rana pretiosa is relatively more dependent on moisture for dispersal than R. luteiventris, so if species traits influence connectivity, we predicted that connectivity among R. pretiosa populations would be more positively associated with moisture than R. luteiventris. However, if environmental differences are important drivers of gene flow, we predicted that connectivity would be more positively related to moisture in arid regions. We tested these predictions using eight microsatellite loci and gravity models in two R. pretiosa regions and four R. luteiventris regions (n = 1,168 frogs). In R. pretiosa, but not R. luteiventris, connectivity was positively related to mean annual precipitation, supporting our first prediction. In contrast, connectivity was not more positively related to moisture in more arid regions. Various temperature metrics were important predictors for both species and in all regions, but the directionality of their effects varied. Therefore, the pattern of variation in drivers of connectivity was consistent with predictions based on species traits rather than on environmental variation.

A new parameterization for integrated population models to document amphibian reintroductions.

Managers are increasingly implementing reintroduction programs as part of a global effort to alleviate amphibian declines. Given uncertainty in factors affecting populations and a need to make recurring decisions to achieve objectives, adaptive management is a useful component of these efforts. A major impediment to the estimation of demographic rates often used to parameterize and refine decision-support models is that life-stage-specific monitoring data are frequently sparse for amphibians. We developed a new parameterization for integrated population models to match the ecology of amphibians and capitalize on relatively inexpensive monitoring data to document amphibian reintroductions. We evaluate the capability of this model by fitting it to Oregon spotted frog (Rana pretiosa) monitoring data collected from 2007 to 2014 following their reintroduction within the Klamath Basin, Oregon, USA. The number of egg masses encountered and the estimated adult and metamorph abundances generally increased following reintroduction. We found that survival probability from egg to metamorph ranged from 0.01 in 2008 to 0.09 in 2009 and was not related to minimum spring temperatures, metamorph survival probability ranged from 0.13 in 2010-2011 to 0.86 in 2012-2013 and was positively related to mean monthly temperatures (logit-scale slope = 2.37), adult survival probability was lower for founders (0.40) than individuals recruited after reintroduction (0.56), and the mean number of egg masses per adult female was 0.74. Our study is the first to test hypotheses concerning Oregon spotted frog egg-to-metamorph and metamorph-to-adult transition probabilities in the wild and document their response at multiple life stages following reintroduction. Furthermore, we provide an example to illustrate how the structure of our integrated population model serves as a useful foundation for amphibian decision-support models within adaptive management programs. The integration of multiple, but related, data sets has an advantage of being able to estimate complex ecological relationships across multiple life stages, offering a modeling framework that accommodates uncertainty, enforces parsimony, and ensures all model parameters can be confronted with monitoring data.

Redirecting intracellular trafficking and the secretion pattern of FSH dramatically enhances ovarian function in mice.

FSH and luteinizing hormone (LH) are secreted constitutively or in pulses, respectively, from pituitary gonadotropes in many vertebrates, and regulate ovarian function. The molecular basis for this evolutionarily conserved gonadotropin-specific secretion pattern is not understood. Here, we show that the carboxyterminal heptapeptide in LH is a gonadotropin-sorting determinant in vivo that directs pulsatile secretion. FSH containing this heptapeptide enters the regulated pathway in gonadotropes of transgenic mice, and is released in response to gonadotropin-releasing hormone, similar to LH. FSH released from the LH secretory pathway rescued ovarian defects in Fshb-null mice as efficiently as constitutively secreted FSH. Interestingly, the rerouted FSH enhanced ovarian follicle survival, caused a dramatic increase in number of ovulations, and prolonged female reproductive lifespan. Furthermore, the rerouted FSH vastly improved the in vivo fertilization competency of eggs, their subsequent development in vitro and when transplanted, the ability to produce offspring. Our study demonstrates the feasibility to fine-tune the target tissue responses by modifying the intracellular trafficking and secretory fate of a pituitary trophic hormone. The approach to interconvert the secretory fate of proteins in vivo has pathophysiological significance, and could explain the etiology of several hormone hyperstimulation and resistance syndromes.

Relationships between host body condition and immunocompetence, not host sex, best predict parasite burden in a bat-helminth system.

Sex-biased parasitism highlights potentially divergent approaches to parasite resistance resulting in differing energetic trade-offs for males and females; however, trade-offs between immunity and self-maintenance could also depend on host body condition. We investigated these relationships in the big brown bat, Eptesicus fuscus, to determine if host sex or body condition better predicted parasite resistance, if testosterone levels predicted male parasite burdens, and if immune parameters could predict male testosterone levels. We found that male and female hosts had similar parasite burdens and female bats scored higher than males in only one immunological measure. Top models of helminth burden revealed interactions between body condition index and agglutination score as well as between agglutination score and host sex. Additionally, the strength of the relationships between sex, agglutination, and helminth burden is affected by body condition. Models of male parasite burden provided no support for testosterone predicting helminthiasis. Models that best predicted testosterone levels did not include parasite burden but instead consistently included month of capture and agglutination score. Thus, in our system, body condition was a more important predictor of immunity and worm burden than host sex.

Immunolocalization of G protein-coupled estrogen receptor in the rat epididymis.

BACKGROUND: Estrogen plays an important role in male reproduction, and males lacking estrogen signaling in the reproductive tissues are infertile. Estrogen signaling is mediated via two nuclear receptors, ERα and ERß, but it was recently found that a G protein-coupled estrogen receptor (GPER) is present in the testis. It is believed that GPER is a membrane form of the estrogen receptor and mediates non-classical estrogen signaling. However, the cellular localization of GPER in the epididymis is unknown. Therefore, the objective of this study was to determine the cellular and regional expression of GPER in the rat epididymis. FINDINGS: To localize expression, immunohistochemistry (IHC) was performed using fixed epididymal tissue. Three strains and ages of rats were used to identify whether GPER expression is strain or age specific. Our results are the first to demonstrate immunostaining of GPER in epididymal epithelial cells. Expression was highest near the apical membrane followed by the cytoplasm, consistent with a membrane bound receptor. The highest expression in adult rats was observed in corpus followed by cauda. Western blotting analysis of epididymal tissues from Sprague Dawley rats confirmed specificity of the antibody and regional expression. CONCLUSIONS: Expression of GPER in the corpus and cauda suggests a role for non-classical estrogen signaling in sperm maturation in the corpus, and sperm protection/storage in the cauda. GPER expression pre-pubertally suggests that estrogen may have a role in epithelial cell development in addition to regulation of adult function.

Roles of patch characteristics, drought frequency, and restoration in long-term trends of a widespread amphibian.

Despite the high profile of amphibian declines and the increasing threat of drought and fragmentation to aquatic ecosystems, few studies have examined long-term rates of change for a single species across a large geographic area. We analyzed growth in annual egg-mass counts of the Columbia spotted frog (Rana luteiventris) across the northwestern United States, an area encompassing 3 genetic clades. On the basis of data collected by multiple partners from 98 water bodies between 1991 and 2011, we used state-space and linear-regression models to measure effects of patch characteristics, frequency of summer drought, and wetland restoration on population growth. Abundance increased in the 2 clades with greatest decline history, but declined where populations are considered most secure. Population growth was negatively associated with temporary hydroperiods and landscape modification (measured by the human footprint index), but was similar in modified and natural water bodies. The effect of drought was mediated by the size of the water body: populations in large water bodies maintained positive growth despite drought, whereas drought magnified declines in small water bodies. Rapid growth in restored wetlands in areas of historical population declines provided strong evidence of successful management. Our results highlight the importance of maintaining large areas of habitat and underscore the greater vulnerability of small areas of habitat to environmental stochasticity. Similar long-term growth rates in modified and natural water bodies and rapid, positive responses to restoration suggest pond construction and other forms of management can effectively increase population growth. These tools are likely to become increasingly important to mitigate effects of increased drought expected from global climate change. Papeles de las Características del Fragmento, Frecuencia de Sequía y Restauración en las Tendencias a Largo Plazo de un Anfibio Ampliamente Distribuido.

In vitro expression of androgen and estrogen receptors in prepubertal and adult rat epididymis.

Androgens and estrogens regulate epididymal function but the mechanisms by which these hormones act is not fully understood. Epididymal culture systems have been described but none of these identify if AR, ERα and ERß are expressed concurrently under identical culture conditions. Presumably, the actions of androgens and estrogens require their receptors and our results demonstrate for the first time that rat epididymal cell cultures express AR, ERα and ERß protein under identical culture conditions. Furthermore, we demonstrate that the expression of these receptors in vitro mirrors normal in vivo expression patterns, a key finding for past and future studies. An epididymal culture system that maintains expression of androgen and estrogen receptors will allow for future investigations into the regulation and function of the epididymis. Previous studies showing prepubertal expression of ERα, did not find ERα expression in adult animals, making our study the first to demonstrate both prepubertal and adult expression of ERα. Additionally, species differences have been suggested to exist with regards to epididymal expression of ERα. Our results are the first to experimentally compare ERα expression in two different rat species and show that expression is similar between the two species. The expression of ERα and ERß protein prior to puberty and into adulthood provides further supports for the hypothesis that the epididymis may be influenced by estrogens, in addition to androgens, during development and mature function.

Using occupancy models to understand the distribution of an amphibian pathogen, Batrachochytrium dendrobatidis.

Batrachochytrium dendrobatidis is a fungal pathogen that is receiving attention around the world for its role in amphibian declines. Study of its occurrence patterns is hampered by false negatives: the failure to detect the pathogen when it is present. Occupancy models are a useful but currently underutilized tool for analyzing detection data when the probability of detecting a species is <1. We use occupancy models to evaluate hypotheses concerning the occurrence and prevalence of B. dendrobatidis and discuss how this application differs from a conventional occupancy approach. We found that the probability of detecting the pathogen, conditional on presence of the pathogen in the anuran population, was related to amphibian development stage, day of the year, elevation, and human activities. Batrachochytrium dendrobatidis was found throughout our study area but was only estimated to occur in 53.4% of 78 populations of native amphibians and 66.4% of 40 populations of nonnative Rana catesbeiana tested. We found little evidence to support any spatial hypotheses concerning the probability that the pathogen occurs in a population, but did find evidence of some taxonomic variation. We discuss the interpretation of occupancy model parameters, when, unlike a conventional occupancy application, the number of potential samples or observations is finite.

Batrachochytrium salamandrivorans (Bsal) not detected in an intensive survey of wild North American amphibians.

The salamander chytrid fungus (Batrachochytrium salamandrivorans [Bsal]) is causing massive mortality of salamanders in Europe. The potential for spread via international trade into North America and the high diversity of salamanders has catalyzed concern about Bsal in the U.S. Surveillance programs for invading pathogens must initially meet challenges that include low rates of occurrence on the landscape, low prevalence at a site, and imperfect detection of the diagnostic tests. We implemented a large-scale survey to determine if Bsal was present in North America designed to target taxa and localities where Bsal was determined highest risk to be present based on species susceptibility and geography. Our analysis included a Bayesian model to estimate the probability of occurrence of Bsal given our prior knowledge of the occurrence and prevalence of the pathogen. We failed to detect Bsal in any of 11,189 samples from 594 sites in 223 counties within 35 U.S. states and one site in Mexico. Our modeling indicates that Bsal is highly unlikely to occur within wild amphibians in the U.S. and suggests that the best proactive response is to continue mitigation efforts against the introduction and establishment of the disease and to develop plans to reduce impacts should Bsal establish.

Effects of Estrogen Treatment on Aging in the Rat Epididymis.

Previous studies from our laboratory demonstrated that estrogen signaling in the testis contributes to maintaining spermatogenesis in adult rats, and that estrogen treatment attenuated the age-associated decline in sperm production. The purpose of this study was to determine if epididymal function is also altered with age, and what effects estrogen treatment may have on the epididymis during aging. We compared untreated rats at 3 and 15 months of age to 18-month-old vehicle-treated and estrogen treated rats. In all four groups, tubule and lumen diameter of the cauda was significantly larger than more proximal regions of the epididymis. In the 3-, 15-, and 18-month-old treated animals, the epithelial cell height of the cauda was significantly shorter than that of more proximal regions. The caput cell height was shorter at 18 months compared to 3 months but this was not seen in estrogen treated animals. Thus, estrogen appears able to prevent some age related changes in epididymal morphology. Sperm transit time through the distal cauda was significantly delayed with aging. Estrogen treatment prevented this delay, indicating that sperm transit through the epididymis is an estrogen regulated function. Differences in estradiol and testosterone concentrations were observed between 3- and 15-month-old animals, but no further differences were noted between treated or untreated animals at 18 months. Interestingly, expression of androgen receptor and estrogen receptor alpha were similar between ages and treatments. Collectively, these results suggest epididymal morphology and function are affected by aging and estrogen treatment. Anat Rec, 302:1447-1457, 2019. © 2018 Wiley Periodicals, Inc.

Range-wide phylogeographic analysis of the spotted frog complex (Rana luteiventris and Rana pretiosa) in northwestern North America.

The dynamic geological and climatic history of northwestern North America has made it a focal region for phylogeography. We conducted a range-wide phylogeographic analysis of the spotted frog complex (Rana luteiventris and Rana pretiosa) across its range in northwestern North America to understand its evolutionary history and the distribution of clades to inform conservation of R. pretiosa and Great Basin R. luteiventris, candidates for listing under the US Endangered Species Act. Mitochondrial DNA sequence data from a segment of the cytochrome b gene were obtained from 308 R. luteiventris and R. pretiosa from 96 sites. Phylogenetic analysis revealed one main R. pretiosa clade and three main R. luteiventris clades, two of which overlapped in southeastern Oregon. The three R. luteiventris clades were separated from each other by high levels of sequence divergence (average of 4.75-4.97%). Two divergent clades were also uncovered within the Great Basin. Low genetic variation in R. pretiosa and the southeastern Oregon clade of R. luteiventris suggests concern about their vulnerability to extinction.

Expression of lactoferrin in the boar epididymis: effects of reduced estrogen.

Lactoferrin is regulated by estrogen in the female reproductive tract and evidence in immature mice suggests that it may be estrogen regulated in males as well. The estrogen regulation of lactoferrin in the epididymis of the boar, a high estrogen-producing male, is unknown. This study was designed to test the hypothesis that lactoferrin expression in the boar epididymis is regulated by estrogen. Twenty-one littermate pairs of boars were treated with vehicle or Letrozole, an aromatase inhibitor, from 1 week of age until castration at 2 through 8 months. Epididymal tissue was collected at castration and fixed for immunolocalization of lactoferrin. Epididymal and testicular tissues were also collected from five mature boars (1-2.5 years) and fixed for immunocytochemistry (ICC). Lactoferrin was localized in the principal cell cytoplasm of the caput, corpus and cauda of developing boars but only in the corpus and cauda of mature boars. Basal cells were negative for lactoferrin. Sperm in the corpus and cauda was also positive for lactoferrin. The efferent ducts and testes were negative for lactoferrin. Intensity of lactoferrin immunostaining increased with age in the corpus and cauda regardless of treatment. Reduced endogenous estrogen in the epididymis during development did not affect the intensity of immunostaining between control and Letrozole-treated animals. Lactoferrin expression in the epididymis of the developing boar does not appear to be regulated by estrogen.

Saprolegniaceae identified on amphibian eggs throughout the Pacific Northwest, USA, by internal transcribed spacer sequences and phylogenetic analysis.

We assessed the diversity and phylogeny of Saprolegniaceae on amphibian eggs from the Pacific Northwest, with particular focus on Saprolegnia ferax, a species implicated in high egg mortality. We identified isolates from eggs of six amphibians with the internal transcribed spacer (ITS) and 5.8S gene regions and BLAST of the GenBank database. We identified 68 sequences as Saprolegniaceae and 43 sequences as true fungi from at least nine genera. Our phylogenetic analysis of the Saprolegniaceae included isolates within the genera Saprolegnia, Achlya and Leptolegnia. Our phylogeny grouped S. semihypogyna with Achlya rather than with the Saprolegnia reference sequences. We found only one isolate that grouped closely with S. ferax, and this came from a hatchery-raised salmon (Idaho) that we sampled opportunistically. We had representatives of 7-12 species and three genera of Saprolegniaceae on our amphibian eggs. Further work on the ecological roles of different species of Saprolegniaceae is needed to clarify their potential importance in amphibian egg mortality and potential links to population declines.

Quantifying climate sensitivity and climate-driven change in North American amphibian communities.

Changing climate will impact species' ranges only when environmental variability directly impacts the demography of local populations. However, measurement of demographic responses to climate change has largely been limited to single species and locations. Here we show that amphibian communities are responsive to climatic variability, using >500,000 time-series observations for 81 species across 86 North American study areas. The effect of climate on local colonization and persistence probabilities varies among eco-regions and depends on local climate, species life-histories, and taxonomic classification. We found that local species richness is most sensitive to changes in water availability during breeding and changes in winter conditions. Based on the relationships we measure, recent changes in climate cannot explain why local species richness of North American amphibians has rapidly declined. However, changing climate does explain why some populations are declining faster than others. Our results provide important insights into how amphibians respond to climate and a general framework for measuring climate impacts on species richness.

Estrogen and androgen receptor expression in relation to steroid concentrations in the adult boar epididymis.

The steroid hormone regulation of the epididymis in a high estrogen producing animal like the boar is not currently understood. To test the hypothesis that the boar epididymis is an estrogen and androgen responsive tissue, the presence of estrogen and androgen receptors, in conjunction with steroid hormone concentrations were investigated in the boar epididymis. Epididymal (caput, corpus, cauda) and testicular samples of boars (1-2.5 years; n=5) were collected for immunolocalization of estrogen receptor alpha (ERalpha), estrogen receptor beta (ERbeta) and androgen receptor (AR). Concentrations of testosterone, estradiol and estrogen conjugates (EC) in the tissue were also determined. AR and ERbeta were localized in the principal and basal cells of all three epididymal regions. ERalpha was localized in the principal cells of the caput, some cells of the corpus and was not present in the cauda. Testosterone (p<0.0001), estradiol (p<0.0001) and EC (p<0.005) were significantly lower in the epididymis compared with the testis. The epididymal regions were not significantly different from each other for testosterone (p>0.15) or estradiol (p>0.09). EC were significantly higher in the corpus than either the caput (p=0.003) or cauda (p=0.002). These results suggest that the boar epididymis is responsive to both estrogens and androgens and that both steroid hormones are important for proper epididymal function. Since testosterone and estradiol concentrations are similar throughout the epididymis, regional differences in steroid hormone regulation are likely due to differences in receptor expression.

Ontogeny of androgen and estrogen receptor expression in porcine testis: effect of reducing testicular estrogen synthesis.

Reducing endogenous estrogen leads to increased proliferation of porcine Sertoli cells during the first 2 months of life. The resulting increase in porcine Sertoli cell numbers is maintained through puberty. The reduced estrogen appears to be the direct hormonal mediator because essentially no changes are observed in other hormones. However, the mechanism for this effect on Sertoli cell proliferation is unknown. The objective of these studies was to evaluate estrogen receptors alpha and beta (ESR1 and ESR2) in conjunction with androgen receptor (AR) on Sertoli cells and other testicular cell types, as an initial step toward understanding how reduced estrogen leads to increased Sertoli cell numbers. Testis sections from treated animals (aromatase inhibition to decrease endogenous estrogen beginning at 1 week of age) and from littermate controls treated with vehicle were subjected to immunocytochemical labeling for ESR1, ESR2, and AR. Three observers scored Sertoli cells, interstitial cells, peritubular myoid cells, and germ cells for intensity of labeling (0: absent; 1+: weak; 2+: moderate; or 3+: strong labeling). AR in Sertoli cells was readily detected at 1 week of age, was very faint in 2-month vehicle controls, and labeling appeared to increase in 3-month vehicle controls. AR in Sertoli cells, interstitial cells, and apparently germ cells was increased in treated animals at 2 months of age compared with the vehicle controls. This increase was confirmed in western blots. ESR1 and ESR 2 were clearly present in Sertoli cells from 1-week-old animals; ESR in Sertoli cells generally decreased with age with the decrease more apparent for ESR2. ESR1 in Sertoli cells and peritubular myoid cells exhibited some treatment-related effects but reduction of endogenous estrogen did not appear to affect ESR2 in the boar testis. The observed alterations in AR and ESR1 may mediate the increases in Sertoli cell proliferation following inhibition of endogenous estrogen production or may reflect the altered function of the Sertoli cells and peritubular myoid cells.

Immunolocalization of estrogen and androgen receptors and steroid concentrations in the stallion epididymis.

The presence of steroids and their receptors throughout development, specifically androgen receptor (AR), estrogen receptor alpha (ERalpha) and estrogen receptor beta (ERbeta), in the epididymis of a high estrogen producing species like the stallion has not been determined. Epididymal and testicular samples were collected for analysis of testosterone and estradiol-17beta (E(2)) concentrations and for immunolocalization of AR, ERalpha and ERbeta. The concentration of testosterone in the testis and epididymis were not different among age groups (P>0.05). AR was localized in the principal cells of the caput, corpus and cauda in all four age groups. This lack of change in testosterone concentration and receptor localization suggests that testosterone is important for both development and maintenance of epididymal function. There was an age-related increase in E(2) concentrations in all regions of the epididymis (P<0.05), suggesting that E(2) is also important for adult function. ERbeta was localized in the principal cells of the caput, corpus and cauda in all four age groups, but the localization of ERalpha was regional and age dependent. In peri-pubertal animals, ERalpha immunostaining was most prominent and estradiol was similarly present in all three epididymal regions; this suggests that estradiol also plays a key role in the maturation of the stallion epididymis during the pubertal transition when sperm first arrive in the epididymis. In conclusion, these results suggest that the stallion epididymis is regulated by both androgens and estrogens throughout development and that estradiol is more important to epididymal function in the stallion than previously believed.

Lactoferrin expression and secretion in the stallion epididymis.

Lactoferrin is one of the most abundant proteins secreted by the stallion epididymis, but its cellular localization and regulation remain unknown. This study was designed to address the following objectives: (1) identify the epididymal cell types producing lactoferrin in pre-pubertal, peri-pubertal and post-pubertal animals; (2) demonstrate that lactoferrin binds to stallion sperm; and (3) determine if testosterone and estradiol regulate lactoferrin secretion in vitro. Using an immunohistochemical method, lactoferrin was localized in the cytoplasm of principal cells in the corpus and cauda of peri- and post-pubertal animals. The epididymis of pre-pubertal animals did not express lactoferrin. Immunolabeling of lactoferrin was also observed on the mid-piece and tail of the sperm. The role of estradiol and testosterone in regulating secretion of lactoferrin in the post-pubertal epididymis was investigated using tissue culture methods. Lactoferrin concentration in the culture media was determined by validated enzyme-linked immunosorbent assays (ELISA). Testosterone did not increase the concentration of lactoferrin in the media in any epididymal region. In contrast, estradiol-17ß significantly increased the concentration of lactoferrin in the media containing tissue from the cauda. In conclusion, the expression of lactoferrin was found in the cytoplasm of principal cells in the corpus and cauda of the epididymis in peri- and post-pubertal stallions but not pre-pubertal stallions. Furthermore, lactoferrin binds to sperm, suggesting a biological role for protection or regulation of sperm in the corpus and cauda. In addition, estrogen appears to regulate lactoferrin secretion in the cauda of the epididymis in post-pubertal stallions.