Dietary Vitamin D Mitigates Coronavirus-Induced Lung Inflammation and Damage in Mice.

The COVID-19 pandemic caused by the SARS-CoV-2 (ß-CoV) betacoronavirus has posed a significant threat to global health. Despite the availability of vaccines, the virus continues to spread, and there is a need for alternative strategies to alleviate its impact. Vitamin D, a secosteroid hormone best known for its role in bone health, exhibits immunomodulatory effects in certain viral infections. Here, we have shown that bioactive vitamin D (calcitriol) limits in vitro replication of SARS-CoV-2 and murine coronaviruses MHV-3 and MHV-A59. Comparative studies involving wild-type mice intranasally infected with MHV-3, a model for studying ß-CoV respiratory infections, confirmed the protective effect of vitamin D in vivo. Accordingly, mice fed a standard diet rapidly succumbed to MHV-3 infection, whereas those on a vitamin D-rich diet (10,000 IU of Vitamin D3/kg) displayed increased resistance to acute respiratory damage and systemic complications. Consistent with these findings, the vitamin D-supplemented group exhibited lower viral titers in their lungs and reduced levels of TNF, IL-6, IL-1ß, and IFN-γ, alongside an enhanced type I interferon response. Altogether, our findings suggest vitamin D supplementation ameliorates ß-CoV-triggered respiratory illness and systemic complications in mice, likely via modulation of the host's immune response to the virus.

Ion Pair Strategy in Solid Lipid Nanoparticles: a Targeted Approach to Improve Epidermal Targeting with Controlled Adapalene Release, Resulting Reduced Skin Irritation.

PURPOSE: Adapalene (AD) is one of the main retinoids used in the topical therapy of acne, an extremely common skin disease usually associated with psychological morbidity. However, like other retinoids, AD is frequently associated with skin irritation. To overcome the skin irritation, we proposed the encapsulation of AD in solid lipid nanoparticles (SLNs) using the ion pair strategy. METHODS: The developed SLN-AD was characterized by high-performance liquid chromatography, differential scanning calorimetry, X-ray diffraction, synchrotron small-angle X-ray scattering, and transmission electron microscopy. In vitro permeation tests using porcine skin and in vivo mice skin irritation test were performed to evaluate, respectively, the drug's skin distribution and the skin irritation. RESULTS: The characterization studies were able to demonstrate that the proposed strategy effectively provided high AD encapsulation in SLNs and its incorporation into a hydrophilic gel. Sustained release, epidermal targeting, and less skin irritation were observed for SLN-AD gel in comparison to the marketed AD gel. CONCLUSIONS: The studies demonstrated that the encapsulation of AD in SLNs through the formation of an ion pair is a valuable alternative to diminish the adverse skin reactions caused by AD and can optimize patient adherence to treatment.

Tumor-Associated Macrophages (TAM) are recruited to the aging prostate epithelial lesions and become intermingled with basal cells.

BACKGROUND: Prostate cancer remains one of the most common cancers in men. Macrophages are thought to be important regulators in cancers, and their potential involvement in prostate cancer should not be overlooked. Therefore, the association between macrophages and the pre-tumorous changes in prostate epithelium during aging deserves further investigation. OBJECTIVES: We sought to investigate whether macrophages would be recruited into the prostate epithelium that display pathological lesions commonly found during aging. MATERIALS AND METHODS: Prostates of aging rats, with and without treatment with a combination of testosterone and estradiol, were examined for premalignant and malignant epithelial lesions. For comparison, prostates of castrated rats were also investigated. RESULTS: Intraepithelial macrophages were found restricted to areas of premalignant and malignant lesions. An unprecedented interaction between macrophages and basal cells was observed in the aging pathological lesions. The intraepithelial macrophages were associated with autophagy, in contrast to those found after castration. In prostate lesions, the intraepithelial macrophages had TAM phenotype (CD68+/iNOS+/CD206+/ARG+), denoting a possible involvement in cancer progression. However, M2 macrophages (CD68+/CD163+) were recruited into the epithelium after castration, possibly to phagocytize cells undergoing apoptosis. DISCUSSION AND CONCLUSION: In conclusion, macrophages were recruited into the prostate epithelium and presented diverse phenotypes and morphology, consistent with changes reflected in the hormonal environment. Macrophages with the TAM phenotype were found restricted to areas of premalignant and malignant lesions in aging prostates, denoting a possible involvement in cancer progression. In contrast, M2 macrophages were found in the regressed epithelium after castration.

Retinoic acid-loaded solid lipid nanoparticles surrounded by chitosan film support diabetic wound healing in in vivo study.

Repair of tissue damaged in diabetic wounds is essential to minimize the cases of amputation of the limbs in millions of diabetic people around the world. Although the all-trans retinoic acid (ATRA) is described as a potential wound healing agent, however its effects are controversial due to adverse reactions that may impair the wound healing during the treatment schedules. Our aim was to design and characterize an ATRA-loaded solid lipid nanoparticles surrounded by chitosan film to promote an ATRA controlled release and to evaluate its effectiveness in promoting wound healing in a diabetic mouse model. The SLN-ATRA were developed using biocompatible lipids without using organic solvent. The SLN-ATRA had high drug entrapment efficiency (98.0 %) and low polydispersity index (PDI) and average diameter, respectively, 0.24 ± 0.02 and 83.0 ± 6 nm. The transmission electron microscope (TEM) image presented that the SLN-ATRA were homogeneous in size and had spherical structures. The incorporation of SLN-ATRA in the chitosan films propitiated a homogeneous distribution of the drug and a controlled drug release. Furthermore, in vivo assay proved that chitosan films containing SLN-ATRA accelerated the closure of wounds of diabetic mice when compared to the control chitosan films without ATRA. SLN-ATRA chitosan films also reduced leukocyte infiltrate in the wound bed, improved collagen deposition, and reduced scar tissue. No sign of skin irritation was observed. These results indicated that SLN-ATRA surrounded in chitosan films are a promising candidate to treat diabetic wounds, improving tissue healing.

Targeting Wistar rat as a model for studying benign, premalignant and malignant lesions of the prostate.

AIMS: The purpose of this study was to describe a suitable experimental model for studying aging-related prostate disorders including cancer. MATERIALS AND METHODS: 12-month old Wistar rats were kept in control conditions (n = 12) or treated (n = 16) for 6 months with Silastic implants filled with testosterone (T) and estradiol (E2). After the experiment period (at 18 months of age), animals were euthanized and the prostate and other organs were harvested, dissected, weighed, and processed for morphological, ultrastructural and molecular analyses. KEY FINDINGS: We demonstrated that male rats of Wistar strain nicely recapitulate the carcinogenesis process taking place in the aging prostate through the arising of benign, precancerous and malignant lesions, and above all yields a modest incidence of spontaneous PCa (~36%). Moreover, our results highlight that 100% incidence of PCa and precancerous lesions such as prostatic intraepithelial neoplasia and proliferative inflammatory atrophy were achieved in this rat strain after T + E2 treatment, without changing the broad spectrum of changes that naturally emerge in the prostate at advanced ages. Such enhancement of precancerous lesions and tumors was linked to a decreased expression of E-cadherin and ß-catenin in parallel with an increase in Vimentin and N-cadherin, hallmark modifications of epithelial-mesenchymal transition. SIGNIFICANCE: Our findings provide solid evidence that aged Wistar rats may be an excellent model for studies regarding human prostate biology and related disorders including cancer.

Seasonal variation of cell proliferation and apoptosis in the efferent ductules and epididymis of the Neotropical bat Artibeus lituratus (Chiroptera, Phyllostomidae).

The balance between cell proliferation and apoptosis is important for maintenance of male fertility, being influenced by a variety of stimuli including androgens and estrogens. However, studies concerning regulation of these processes along the male reproductive tract under physiological conditions are scarce. Therefore, in this study, we investigated the profile of cell proliferation and apoptosis in the efferent ductules and epididymis of the Neotropical bat Artibeus lituratus, a seasonal breeder that presents natural variation in components of the androgen and estrogen responsive systems along the circannual cycle. Low rates of cell proliferation and apoptosis were found in the efferent ductules and epididymis of A. lituratus during the reproductive period, as few epithelial cells were positive for MCM7 (proliferation marker) and cleaved caspase-3 or TUNEL (apoptosis markers). In contrast, during the regressive period, the rate of both proliferating and apoptotic cells was significantly higher in the epithelium lining the efferent ductules as well as throughout the epididymis. The increased proliferative activity at this phase was positively correlated with the expression of estrogen receptor alpha (ERα), whereas the variation in apoptosis appears to be unrelated to the local expression of androgen and estrogen receptors. Together, these data suggest that cell proliferation and apoptosis are differentially modulated in the efferent ductules and epididymis of A. lituratus during the annual reproductive cycle, and support the hypothesis that ERα may be important in preparing the male reproductive tract for sexual recrudescence.

Reduced vitamin D receptor (VDR) expression and plasma vitamin D levels are associated with aging-related prostate lesions.

BACKGROUND: Protective roles have been proposed for vitamin D in prostate cancer, which has the advanced age as the major risk factor. However, little is known about the expression of the vitamin D receptor (VDR) in the aging prostate and its association with the development of epithelial lesions that affect tissue homeostasis and may precede prostate tumors. METHODS: VDR expression in the prostatic complex of young adults to senile Wistar rats, a natural model to study age-related prostatic disorders, was evaluated by immunohistochemical, Western blotting, and image-assisted analyzes. Results were correlated with the plasma levels of vitamin D and testosterone, the occurrence of punctual histopathological changes in the aging prostate, and the expression of retinoid X receptors (RXR). RESULTS: VDR was widely distributed in the prostatic complex at all ages analyzed, with the highest immunoexpression found in basal epithelial cells. As the animals aged, VDR levels increased, except in punctual areas with intraepithelial proliferation, metaplasia, or proliferative inflammatory atrophy, which had reduced expression of this receptor concomitantly with increased cell proliferation. Interestingly, RXR expression in the aging prostate was similar to that found for its partner VDR, indicating that components of the VDR/RXR complex required for vitamin D signaling are affected in aging-related prostatic lesions. Moreover, plasma vitamin D levels declined at the same ages when prostatic alterations appeared. Although circulating levels of testosterone also decreased with aging, the changes observed in the components of the vitamin D system were not correlated with androgens. CONCLUSIONS: Our data indicate that the aging prostate suffers from an imbalance on the intricate mechanism of tissue regulation by the vitamin D responsive system. We argue that the status of VDR expression might be determinant for the development of histopathological alterations in the aging prostate, which include premalignant lesions.

Basal Cells Show Increased Expression of Aromatase and Estrogen Receptor α in Prostate Epithelial Lesions of Male Aging Rats.

Besides androgens, estrogen signaling plays a key role in normal development and pathologies of the prostate. Irreversible synthesis of estrogens from androgens is catalyzed by aromatase. Interestingly, animals lacking aromatase do not develop cancer or prostatitis, whereas those with overexpression of aromatase and, consequently, high estrogen levels develop prostatitis and squamous metaplasia via estrogen receptor 1 (ERα). Even with this evidence, the aromatase expression in the prostate is controversial. Moreover, little is known about the occurrence of age-dependent variation of aromatase and its association with histopathological changes commonly found in advanced age, a knowledge gap that is addressed herein. For this purpose, the immunoexpression of aromatase was evaluated in the prostatic complex of young adult to senile Wistar rats. ERα was also investigated, to extend our understanding of estrogen responsiveness in the prostate. Moderate cytoplasmic immunoreactivity for aromatase was detected in the glandular epithelium. Eventually, some basal cells showed intense staining for aromatase. The expression pattern for aromatase appeared similar in the normal epithelium when young and senile rats were compared; this result was corroborated by Western blotting. Conversely, in senile rats, there was an increase in the frequency of basal cells intensely stained for aromatase, which appeared concentrated in areas of intraepithelial proliferation and prostatitis. These punctual areas also presented increased ERα positivity. Together, these findings suggest a plausible source for hormonal imbalance favoring estrogen production, which, by acting through ERα, may favor the development of prostatic lesions commonly found in advanced age.

Profile of cell proliferation and apoptosis activated by the intrinsic and extrinsic pathways in the prostate of aging rats.

BACKGROUND: Estrogens acting through the receptors ERα and ERß participate in prostate normal growth and cancer. ERß is highly expressed in the prostate epithelium, playing pro-apoptotic, anti-proliferative, and pro-differentiation roles. Apoptosis is activated by the intrinsic pathway after castration and by the extrinsic pathway after ERß agonist treatment. This differential activation of apoptotic pathways is important since a major problem in the treatment of prostate cancer is the recurrence of tumors after androgen withdrawal. However, a comprehensive study about the pattern of apoptosis in the aging prostate is lacking, a knowledge gap that we aimed to address herein. METHODS: Cellular age-related proliferative and apoptotic profiles of prostate tissue obtained from aging Wistar rats were evaluated. Cell death (caspase-3, -8, -9, TNFα) was assessed by immunohistochemistry, immunofluorescence, and TUNEL. Cell proliferation (MCM7) and cell survival factors (ERK1/2, p-ERK1/2, p-Akt, and NF-κB) were determined by immunohistochemistry. RESULTS: As the rats aged, the number of proliferating cells gradually reduced in the normal epithelium of all prostate lobes, while increasing in focal areas of intraepithelial proliferation. Interestingly, in areas of intraepithelial proliferation, we observed a reduction in the number of cells positive for caspase-3, -8, and -9. Regardless the animal's age, few prostate epithelial cells were positive for caspase-3, caspase-9, and TUNEL. In contrast, a progressive increase was seen in the positivity for caspase-8, especially in the atrophic epithelium of ventral prostate, which coincided with a reduction in TNFα immunoreaction. However, morphology of most caspase-8 positive cells suggests that they were not apoptotic. We also found reduced ERß expression in the same areas. Possibly, low levels of the pro-apoptotic inductors TNFα and ERß direct caspase-8 activity to an alternative pro-survival role in the atrophic epithelium. This hypothesis is supported by the increased expression of the key survival factors (ERK1/2, p-ERK1/2, p-Akt, and NF-κB) in these areas. CONCLUSIONS: Our findings reveal that, as the animals age, there is an increase of proliferation in restricted areas of the prostate epithelium, and a concomitant reduction of the apoptosis rate with an increase in cell survival induced by caspase-8, indicating a focused and spontaneous disruption of tissue homeostasis.

Changes in Estrogen Receptor ERß (ESR2) Expression without Changes in the Estradiol Levels in the Prostate of Aging Rats.

Although the prostate is androgen-dependent, it is also influenced by estrogens, which act via the estrogen receptors ERα and ERß. In the prostate, ERß is highly expressed in the epithelium and appears to participate in the regulation of cell proliferation, apoptosis and differentiation. Evidence shows that ERß is decreased in malignant prostate, suggesting that it plays an important role in protecting this tissue. Despite the relationship between reductions in ERß and abnormal growth of the gland, little is known about the age-dependent variation of this receptor. Therefore, we aimed to investigate ERß expression in the prostatic lobes of aging Wistar rats (3 to 24 months). Histopathological alterations, including hyperplasia, intraluminal concretions, nuclear atypia and prostate intraepithelial neoplasias (PIN), were observed in the prostates of aging rats. Epithelial proliferation led to cribriform architecture in some acini, especially in the ventral prostate (VP). In the VP, areas of epithelial atrophy were also observed. Furthermore, in the lateral prostate, there was frequent prostatitis. Immunohistochemistry revealed that the expression of ERß is reduced in specific areas related to PIN, atrophic abnormalities and cellular atypia in the prostate epithelium of senile rats. Corroborating the involvement of the receptor with proliferative activity, the punctual reduction in ERß paralleled the increase in cell proliferation especially in areas of PIN and nuclear atypies. The decrease in ERß reactivity occurred in a hormonal milieu characterized by a constant concentration of estradiol and decreased plasmatic and tissue DHT. This paper is a pioneering study that reveals focal ERß reduction in the prostate of aging rats and indicates a potential disorder in the ERß pathway. These data corroborate previous data from humans and dogs that silencing of this receptor may be associated with premalignant or malignant conditions in the prostate.

A rare case of endometrioma in a bitch.

BACKGROUND: Endometriosis is ectopic development of endometrial glands and stroma in extra-uterine sites and if the lesions occur as a well-defined mass is referred to as endometrioma. In the literature, endometrioma has been reported in only women and there are no reports of endometrioma in animals, including non-human primates. CASE PRESENTATION: A rare case of endometrioma is reported in an 11-year-old female German Shepherd with clinical signs of dehydration, anemia and prostration. Necropsy revealed ascites, generalized pallor, and a well-demarcated reddish mass adjacent to the left ovary and uterus and adherent to the retroperitoneum. The mass measured 25.0 × 20.0 cm with intermingled soft and firm areas. Upon incision, the mass was found to be solid with variable sized cystic cavities filled with coagulated blood. Microscopically, the mass was composed of cuboidal or prismatic epithelial cells arranged in tubules or acini. The epithelium of the mass had similar characteristics to the normal endometrium with PAS-positive secretions. The stroma was prominent and formed by loose connective tissue and smooth muscle fibers as confirmed by Masson trichrome. Extensive multifocal areas of hemorrhage were also observed in the stroma of the mass and in the interior of some epithelium-lined, cystic structures. Most of the epithelial cells had strong and diffuse cytokeratin expression, and some had vimentin expression. Epithelial and stromal cells also showed ERß, AR, VEGF and COX2 expression. The stroma showed areas with strong and diffuse vimentin expression. Factor VIII expression was observed only in the endothelium of blood vessels in the stroma. CONCLUSIONS: The macroscopic, microscopic and immunohistochemical findings are consistent with an endometrioma.

The insulin receptor translocates to the nucleus to regulate cell proliferation in liver.

UNLABELLED: Insulin's metabolic effects in the liver are widely appreciated, but insulin's ability to act as a hepatic mitogen is less well understood. Because the insulin receptor (IR) can traffic to the nucleus, and Ca(2+) signals within the nucleus regulate cell proliferation, we investigated whether insulin's mitogenic effects result from activation of Ca(2+)-signaling pathways by IRs within the nucleus. Insulin-induced increases in Ca(2+) and cell proliferation depended upon clathrin- and caveolin-dependent translocation of the IR to the nucleus, as well as upon formation of inositol 1,4,5,-trisphosphate (InsP3) in the nucleus, whereas insulin's metabolic effects did not depend on either of these events. Moreover, liver regeneration after partial hepatectomy also depended upon the formation of InsP3 in the nucleus, but not the cytosol, whereas hepatic glucose metabolism was not affected by buffering InsP3 in the nucleus. CONCLUSION: These findings provide evidence that insulin's mitogenic effects are mediated by a subpopulation of IRs that traffic to the nucleus to locally activate InsP3 -dependent Ca(2+)-signaling pathways. The steps along this signaling pathway reveal a number of potential targets for therapeutic modulation of liver growth in health and disease.

Differential expression and seasonal variation on aquaporins 1 and 9 in the male genital system of big fruit-eating bat Artibeus lituratus.

Efferent ductules and epididymis are involved in water and solute transport, which is indispensable for storage and maintenance of the sperm viability. The reabsorption process involves proteins such as aquaporins (AQP), which has been described in the male genital system of limited species, including primate, rodents, cats and dogs. To contribute with information about AQPs in the male system, here we investigated the distribution of AQP1 and AQP9 in the tropical bat Artibeus lituratus, along the annual reproductive cycle. A. lituratus is a seasonal breeder with natural variation in components of the androgen and estrogen responsive system, thus being a good model for exploring the AQPs modulation. AQP1 was found restricted to differentiating spermatids, efferent ductules epithelium and venular endothelia along the male tract. AQP9 was detected throughout the epididymis being more abundant in the cauda and ductus deferens, but was not found in testis, rete testis and efferent ductules. Contrasting with AQP1 which appear to be constitutively expressed, there was seasonal variation in AQP9 expression, which was reduced in regressed epididymis. The AQP9 does not appear to be modulated by estradiol or androgens, but possibly by other factor related to luminal sperm. The establishment of specific function for aquaporins in the male tract remains undetermined; however, the cellular distribution presently found are compatible with the main function of AQP1, as a selective water channel, and AQP9, which is a conduct for water and a plethora of neutral solutes present in the epididymis milieu such as glycerol and urea.

Seasonal variation in estrogen receptor ERα, but not ERß, androgen receptor and aromatase, in the efferent ductules and epididymis of the big fruit-eating bat Artibeus lituratus.

The efferent ductules (ED) are a major target for estrogens, which act via the estrogen receptors ERα (ESR1) and ERß (ESR2). ERα has been found in the ED of all species studied so far. However, in the epididymis (EP), the expression of ERα is controversial, as is data about the occurrence of aromatase in the epithelium lining the excurrent ducts. Therefore, to further investigate this estrogen-responsive system, we used a seasonal breeder, the Neotropical bat, Artibeus lituratus, in which testicular expression of androgen (AR) and estrogen (ER) receptors vary with reproductive phase. The localization of aromatase, ERα, ERß and AR in the ED and EP of A. lituratus was investigated. The results showed that aromatase, AR and ERß were distributed throughout the excurrent ducts and did not vary during the annual reproductive cycle. Conversely, ERα was detected primarily in the ED epithelium, had marked seasonal variation and was increased during regression, especially in the EP epithelium. The results suggest that ERα may be involved in preparing the male genital tract for recrudescence. Together, the data obtained under natural conditions emphasize that specific segments of the excurrent ducts downstream of the testis are the primary targets for estrogen action via ERα, which is similar to previous findings in animals lacking functional ERα.

Angiotensin-(1-7) induces ovulation and steroidogenesis in perfused rabbit ovaries.

A local renin-angiotensin system has been described in several organs, including the ovary; however, data indicating a role for angiotensin II in the induction of ovulation are controversial. We have previously shown the presence of a novel peptide, angiotensin-(1-7) [Ang-(1-7)], in the rat ovary and its effect on steroidogenesis. The objective of the present study was to determine whether Ang-(1-7) plays a role in ovulation. We first determined the presence and distribution of Ang-(1-7) and the receptor Mas in rabbit ovaries by immunohistochemistry. Angiotensin-(1-7) and Mas immunoreactivity were observed in interstitial cells and oocytes of immature ovaries. Immunoreactivity for Ang-(1-7) and Mas was also observed in theca and granulosa cells of preovulatory follicles in ovaries of gonadotrophin-stimulated rabbits. To verify the effect of Ang-(1-7) in ovulation and steroidogenesis, we used isolated ovaries from immature rabbits pretreated with equine chorionic gonadotrophin (50 i.u., 48 h before the experiment) and then perfused in vitro. The ovulatory efficiency was determined by the number of oocytes compared with the number of preovulatory follicles present in the ovary. Angiotensin-(1-7) stimulated oestradiol production and enhanced ovulatory efficiency, which was blocked by the specific Ang-(1-7) antagonist, A-779. Ovulation induced by human chorionic gonadotrophin was also antagonized by A-779. These results show, for the first time, the involvement of a novel regulatory peptide system, Ang-(1-7) and Mas, in the ovulatory process. More importantly, because A-779 antagonized hCG-induced ovulation, it may be inferred that Ang-(1-7) plays an important role in ovulation, possibly as a mediator of gonadotrophin action.

Roosters affected by epididymal lithiasis present local alteration in vitamin D3, testosterone and estradiol levels as well as estrogen receptor 2 (beta) expression.

Epididymal lithiasis is a reproductive dysfunction of roosters that is associated with loss of fertility and is characterized by the formation of calcium stones in the lumen of the efferent ductules of the epididymal region. The efferent ductules of birds are responsible for the reabsorption of the fluid coming from the testis as well as luminal calcium. It has been hypothesized that the epididymal stone formation may be related to the impairment of local fluid or calcium homeostasis, which depends on hormones such as estradiol (E(2)). Therefore, this study aimed to investigate possible alterations in the expression of ERα (ESR1) and ERß (ESR2) in the epididymal region of roosters affected by epididymal lithiasis. The study was performed by immunohistochemistry and western blotting assays. In addition, the concentrations of E(2), vitamin D3, and testosterone, which are also key hormones in maintenance of calcium homeostasis, were determined in the plasma and epididymal region, by ELISA. It was observed that ESR2 expression is increased in all segments of the epididymal region of affected roosters, whereas ESR1 levels are not altered. Moreover, the hormone concentration profiles were changed, as in the epididymal region of roosters with lithiasis the E(2) levels were increased and vitamin D3 as well as testosterone concentrations were significantly decreased. These results suggest that a hormonal imbalance may be involved with the origin and progression of the epididymal lithiasis, possibly by affecting the local fluid or calcium homeostasis.

Epididymal lithiasis in roosters: in the middle of the way there was a stone.

The epididymal region plays an important role in the reproduction of roosters, as it is the site of functions important in the maintenance of fertility, including fluid and calcium reabsorption and sperm surface modifications. About 10 years ago, a reproductive dysfunction characterized by the formation of luminal calcium stones in the epididymal region of roosters was described. This anomaly, known as epididymal lithiasis, is associated with a significant decrease in the fertility of affected roosters. This reproductive anomaly has been observed in multiple countries and is thought to negatively impact the poultry industry; however, the cause of epididymal lithiasis has not been fully determined. Several hypotheses have been proposed to explain the origin of epididymal lithiasis, including the presence of an infectious agent within the epididymal region, an autoimmune response, increased dietary calcium and vitamin D3 intake and the presence of genetic susceptibility factors; however, none of these has been proven to be the primary cause of the calcium stone formation. Nonetheless, considerable evidence suggests that regardless of the primary cause of epididymal lithiasis, this anomaly could result from a hormonal imbalance or a local impairment of calcium homeostasis in the epididymal region. The objectives of this mini-review are to 1) summarize the reproductive alterations observed in animals affected by epididymal lithiasis, 2) discuss the hypotheses proposed to explain the cause of luminal stone formation and 3) provide perspectives for future studies of this reproductive disorder.

Demasculinization and feminization of male gonads by atrazine: consistent effects across vertebrate classes.

Atrazine is the most commonly detected pesticide contaminant of ground water, surface water, and precipitation. Atrazine is also an endocrine disruptor that, among other effects, alters male reproductive tissues when animals are exposed during development. Here, we apply the nine so-called "Hill criteria" (Strength, Consistency, Specificity, Temporality, Biological Gradient, Plausibility, Coherence, Experiment, and Analogy) for establishing cause-effect relationships to examine the evidence for atrazine as an endocrine disruptor that demasculinizes and feminizes the gonads of male vertebrates. We present experimental evidence that the effects of atrazine on male development are consistent across all vertebrate classes examined and we present a state of the art summary of the mechanisms by which atrazine acts as an endocrine disruptor to produce these effects. Atrazine demasculinizes male gonads producing testicular lesions associated with reduced germ cell numbers in teleost fish, amphibians, reptiles, and mammals, and induces partial and/or complete feminization in fish, amphibians, and reptiles. These effects are strong (statistically significant), consistent across vertebrate classes, and specific. Reductions in androgen levels and the induction of estrogen synthesis - demonstrated in fish, amphibians, reptiles, and mammals - represent plausible and coherent mechanisms that explain these effects. Biological gradients are observed in several of the cited studies, although threshold doses and patterns vary among species. Given that the effects on the male gonads described in all of these experimental studies occurred only after atrazine exposure, temporality is also met here. Thus the case for atrazine as an endocrine disruptor that demasculinizes and feminizes male vertebrates meets all nine of the "Hill criteria".

Estrogen and its receptors in efferent ductules and epididymis.

Estrogens play key roles in the development and maintenance of male reproductive function and fertility. In this review, we briefly describe the localization and function of estrogen receptors ESR1 and ESR2 (also known as ERα and ERß, respectively) and the expression of G protein-coupled estrogen receptor-1 (GPER, formerly known as GPR30) in efferent ductules and epididymis. The efferent ductules present the highest levels of ESR1 and ESR2 in the male reproductive system, and represent a major target of estrogen action. In efferent ductules, ESR1 has a crucial role in the regulation of fluid reabsorption, and in the epididymis the receptor helps to maintain fluid osmolality and pH. ESR1 expression in the epididymal epithelium shows considerable variation among species, but differences in laboratory techniques may also contribute to this variation. Here we report that Esr1 mRNA and protein are higher in corpus than in other regions of the rat epididymis. The mRNA level for Gper was also higher in corpus. Although ESR1 is expressed constitutively in efferent ductules and down-regulated by estrogen, in the epididymis, both testosterone (T) and estradiol (E2) may regulate its expression. T and E2 are, respectively, higher and lower in the corpus than in the initial segment/caput and cauda regions. It is important to determine the expression of GPER, ESR1, androgen receptor, and their respective cofactors in specific cell types of this tissue, as well as the intracellular signaling pathways involved in efferent ductules and epididymis. These studies will help to explain the consequences of exposures to environmental endocrine disruptors and provide potential targets for the development of a male contraceptive.

Occurrence and cellular distribution of estrogen receptors ERα and ERß in the testis and epididymal region of roosters.

Estrogen signaling is required for the maintenance of male reproductive function and is mediated by the estrogen receptors ERα and ERß. These receptors are widely distributed in mammalian reproductive tissues, but information is limited in non-mammalian species including birds. The aim of this study was to investigate the occurrence and cellular distribution of ERα and ERß in the testis and epididymal region of roosters. The results showed for the first time that ERß was the predominant receptor detected in the testis, being expressed in the somatic and some germ cells. Within the epididymal region, ERß was strongly expressed in all segments, whereas the most intense reaction for ERα was found in the distal efferent ductules. The differential expression of ERα and ERß within the rooster testis and epididymal region suggests that these organs may be a target for different actions of estrogen.