Melatonin regulates dihydrotestosterone formation via its membrane receptor in the epididymal epithelial cells of sheep.

Both melatonin and androgen, which affect sperm fertility, are the important factors in epididymis of male animal. In the present study, we confirmed that melatonin regulates the formation of dihydrotestosterone (DHT) in sheep epididymides. Here, we investigated the localization and the expression levels of melatonin keys synthases AANAT and HIOMT, membrane receptors MT1 and MT2, and nuclear receptor RORα in sheep epididymides and testes. We also cultured epididymal epithelial cells and treated them with different concentrations of melatonin (10-11-10-7 M) and luzindole (10-5 M) and 4P-PDOT (10-5 M) to investigate whether melatonin is involved in the regulation of DHT formation and whether these effects are mediated through its receptor pathways. The results showed that AANAT, HIOMT, MT1, MT2, and RORα were differentially expressed between sheep epididymides and testes. In addition, melatonin is involved in mediating the formation of DHT in epididymal epithelial cells, and its influence on DHT is at least partially regulated by the melatonin receptor pathway. Our findings showed that melatonin regulates the functions of the testes and epididymides through an autocrine mechanism and regulates the formation of androgen in sheep epididymides via the receptor pathway. These results provide a basis for further exploring the regulatory mechanisms of melatonin in animal reproduction.

Subacute Ruminal Acidosis as a Potential Factor that Induces Endometrium Injury in Sheep.

The demand for economic benefits has led to an increase in the proportion of high-concentrate (HC) feed in the ruminant diet, resulting in an increased incidence of subacute ruminal acidosis (SARA). During SARA, a high concentration of lipopolysaccharide (LPS) translocated in the rumen induces a systemic inflammatory response. Inflammatory diseases, such as endometritis and mastitis, are often associated with SARA; however, in sheep, the mechanism of the effect of SARA on the endometrium has rarely been reported. Therefore, the aim of this study was to investigate, for the first time, the influence of LPS translocation on endometrial tight junctions (TJs) during SARA in sheep. The results showed that LPS and TNFα levels in the ruminal fluid, serum, and endometrial tissue supernatant during SARA increased, transcription levels of TLR4, NFκB, and TNFα in the endometrium increased, the protein expression level of claudin-1 in the endometrium increased, and the protein expression level of occludin decreased. 17ß-estradiol (E2) inhibits claudin-1 protein expression and promotes occludin expression, and progesterone (P4) promotes claudin-1 protein expression and inhibits occludin protein expression. E2 and P4 regulate claudin-1 and occludin protein expression through their receptor pathways. Here, we found that LPS hindered the regulatory effect of E2 and P4 on endometrial TJs by inhibiting their receptor expression. The results of this study indicate that HC feeding can cause SARA-induced LPS translocation in sheep, increase susceptibility to systemic inflammation, induce the endometrial inflammatory response, and cause endometrial epithelial TJ damage directly and/or by obstructing E2 and P4 function. LPS translocation caused by SARA has also been suggested to induce an endometrial inflammatory response, resulting in endometrial epithelial barrier damage and physiological dysfunction, which seriously affects ruminant production. Therefore, this study provides new evidence that SARA is a potential factor that induces systemic inflammation in ruminants. It provides theoretical support for research on the prevention of endometritis in ruminants.

The effect of melatonin on sheep endometrial epithelial cell apoptosis through the receptor and non-receptor pathways.

Melatonin potentially regulates the female animal reproductive function, but its regulatory mechanism in the apoptosis of sheep endometrial epithelial cells (SEECs) remains to be elucidated. In the present study, immunofluorescence staining, western blotting, and quantitative real-time polymerase chain reaction were performed to detect the distribution of melatonin receptors (MT1 and MT2) in the uterus of sheep and the effect of melatonin via the receptor and non-receptor pathways on the apoptosis of SEECs in vitro. The results showed that melatonin inhibits the apoptosis of SEECs to varying degrees to regulate the expression of estrogen receptors (ERs) and progesterone receptors (PGR) via its interaction with MT1 and MT2. In addition, the ER antagonist partially relieved the inhibitory effect of melatonin on the apoptosis of SEECs, while the PGR antagonist did not. Thus, melatonin mediates endometrial epithelial apoptosis through the MT receptors and also by regulating estrogen function. This study provides evidence of the regulatory mechanism of melatonin on the physiological function of the sheep uterus.

Regulatory role of melatonin on epidermal growth factor receptor, Type I collagen α1 chain, and caveolin 1 in granulosa cells of sheep antral follicles.

We investigated the expression of epidermal growth factor receptor (EGFR), Type I collagen α1 chain (COL1A1), and caveolin 1 (CAV1) during follicular development and examined the regulatory role of melatonin (MLT) on EGFR, COL1A1, and CAV1 in sheep antral ovaries. The expression was detected in granulosa and theca cells by immunohistochemistry. Quantitative real-time polymerase chain reaction and Western blotting were used to examine the expression levels of EGFR, COL1A1, and CAV1 in small (≤2 mm), medium (2-5 mm), and large (≥5 mm) follicles. The mRNA and protein levels of EGFR, COL1A1, and CAV1 were found to be the highest in large follicles. Furthermore, cultured granulosa cells were treated with MLT (10-7 -10-11  M), luzindole (nonselective MT1 and MT2 receptor antagonist, 10-7  M), and 4-phenyl-2-propanamide tetraldehyde (4P-PDOT, MT2 selective antagonist, 10-7  M) to detect the regulatory role of MLT on EGFR, COL1A1, and CAV1. Results indicated COL1A1 and CAV1 were at least partially regulated by MLT through MT1 and MT2 pathways, whereas EGFR was not. This study provided a reference for further studies on MLT regulatory role on EGFR, COL1A1, and CAV1 during sheep follicular development and elucidated the physiological mechanism of MLT regulator production.

Expression of dihydrotestosterone synthases and androgen receptor in sheep oviduct ampulla and its regulation by estradiol and progesterone.

Oviduct ampulla plays an important role in steroid hormone-regulated sperm-oocyte binding in female animals. Although studies have shown that androgen receptor are expressed in many species oviduct, the interaction among androgen receptor (AR), estradiol (E2) and progesterone (P4) in the sheep oviduct have rarely been reported. In this study, we evaluated the localization of two isoforms of dihydrotestosterone (DHT) sythetase enzymes 5α-reductase (5α-red1, 5α-red2) and AR in sheep oviduct ampulla by immunohistochemistry and immunofluorescence. Results showed that they were all distributed in oviduct epithelium layer. In epithelial cells, 5α-red1, 5α-red2 were expressed in cytoplast and nuclear, but AR were stained in nuclear. We also investigated their expression pattern in the sheep oviduct ampulla at different development stages of follicles (Large follicles stage; hemorrhagium, luteum and albicans of corpus stage) by molecular experiments. We found that 5α-red1, 5α-red2 and AR mRNA abundance and protein were expressed highest in corpus albicans stage and lowest in corpus hemorrhagium stage. In vitro, when sheep oviduct ampulla epithelial cells (SOAECs) were cultured and treated with different concentrations of E2/P4 (10-9-10-6 M), we found that E2 inhibited the expression of AR mRNA and protein, while P4 promoted this expression. In addition, when the SOAECs were treated with E2 (10-8 M) and/or its non-selective inhibitor ICI182780 (10-7 M) as well as with P4 (10-6 M) and/or its non-specific inhibitor RU486 (10-5 M), we found that E2 and P4 inhibited and promoted the expression of AR mRNA and proteins, respectively, via their nuclear receptor pathways. This study provides a basic insight for the further research of oviduct epithelium physiological function closely related to androgen.

ß-Estradiol inhibits melatonin synthesis and melatonin receptor expression in sheep granulosa cells.

Melatonin, an important regulator of mammalian reproduction, is mainly produced in the pineal gland, and granulosa cells (GCs), the main mammalian ovarian secretory cells, synthesize melatonin and express melatonin receptors (MRs) MT1 and MT2. However, studies on melatonin regulation in GCs are lacking in sheep. In this study, we explored the effects of ß-estradiol (E2) on melatonin production and MR expression in GCs. We cultured sheep GCs to analyze the expression of the melatonin rate-limiting enzymes AANAT and HIOMT and the effects of E2 on AANAT, HIOMT, and MR expression and melatonin synthesis. To determine whether estrogen receptors (ERs) mediated E2 action on melatonin secretion and MR expression, we assessed ERA and ERB expression in GCs and observed whether ER antagonists counterbalanced the effects of E2. GCs expressed AANAT and HIOMT mRNA, indicating that they transformed exogenous serotonin into melatonin. E2 inhibited melatonin production by downregulating AANAT, HIOMT, and MRs. GCs expressed ERA and ERB; ERA/ERB inhibitors abolished E2-mediated inhibition of melatonin secretion and MR expression. PHTPP upregulated melatonin secretion and MT1 expression in E2-treated GCs, but did not significantly affect AANAT and MT2 expression. In conclusion, melatonin secretion in GCs was inhibited by E2 through an ERA- and ERB-mediated process.

Evaluating source-oriented human health risk of potentially toxic elements: A new exploration of multiple age groups division.

Effective source-oriented human health risk assessment (HHRA) for people in different life stages will guide pollution control and risk prevention. This work integrated three receptor models of positive matrix factorization, Unmix, and factor analysis with nonnegative constraints for accurate source-oriented HHRA of potentially toxic elements in 6 age groups of populations (0-<1 year, 1-<6 years, 6-<12 years, 12-<18 years, 18-<44 years, and 44+ years). Four sources were identified. Natural source controlled As, Cr, and Ni in dust and soil as well as Pb and Zn in soil. Industrial-traffic emissions contributed most of Cd in dust and soil as well as Pb and Zn in dust. Hg in both dust and soil originated from coal combustion. Construction works contributed more to PTEs in soil than in dust. Noncarcinogenic and carcinogenic risk for both dust and soil changed in similar trends by age. The noncancer risk reduced with increasing age for people below 44 years. Carcinogenic risk of females over 44 years were the highest, while children from 0 to 1 year faced the lowest carcinogenic risk. Among the four origins of PTEs, natural sources contributed most to health risk of PTEs, followed by industrial-traffic sources, construction works, and coal combustion. Based on sequential Gaussian simulation (SGS), the susceptible population and risk areas were identified. Children from 0 to 6 years were identified as susceptible population. The areas with noncancer risk in dust were 19.15 km2 for 0-<1 year and 3.14 km2 for children from 1 to <6 years, and noncancer risk areas in soil were 30.26 km2 for 0-<1 year and 0.85 km2 for 1-<6 years. Relevant control and management works were demanded on children from 0 to 6 years and noncancer risk areas.

Assessment of progesterone synthesis and its regulation role on dihydrotestosterone secretion in sheep epididymis.

Progesterone (P4) is an anti-androgen compound whose role in sperm maturation and functionality remains unclear in sheep. Here, we aimed to investigate the regulation mechanism of P4 on the epididymal secretion of dihydrotestosterone (DHT). To this end, we performed enzyme-linked immunosorbent assays, immunohistochemical staining, western blotting, and quantitative real-time polymerase chain reaction to detect P4 concentration as well as StAR, P450scc, and 3ß-HSD expression in sheep epididymis. Besides, cauda epithelial cells were cultured at different concentrations of P4 (10-9-10-5 g ml-1) as well as with or without the P4 receptor (PGR) inhibitor RU486 (10-7 M) or the PI3K-AKT inhibitor LY294006 (10-7 M) to explore the effect of P4 on DHT secretion and the underlying regulatory mechanism. The results showed that the caput, corpus, and cauda of sheep epididymis could synthesize P4 but had different synthesis ability. The PGR expression levels were the highest in the cauda, followed by the corpus. In vitro cell culture showed that P4 inhibition of DHT secretion and 5α-reductase 1 and 2 expression in epididymal epithelial cells could be moderately mitigated by RU486 but not by LY294002. Our results indicated that the paracrine and autocrine P4 could affect the secretion of DHT in epididymal cells through PGR. Overall, this study provides new data regarding the involvement of P4 in sperm maturation and functionality in sheep.

Dihydrotestosterone regulates oestrogen secretion, oestrogen receptor expression, and apoptosis in granulosa cells during antral follicle development.

Dihydrotestosterone (DHT) is involved in the development of preantral follicles. However, the effect of DHT on the development of antral follicles has yet to be fully investigated. Herein, we used enzyme-linked immunosorbent assays, immunofluorescence assays, quantitative real time-polymerase chain reaction, immunohistochemical staining, and western blotting to investigate the effect of DHT on antral follicle development. First, we detected the concentration of DHT and the expression of the androgen receptor (AR) in different antral follicles. Second, multiple DHT concentration (10-10-10-7 M) were added to granulosa cells cultured in vitro to examine the influence of DHT on AR expression. Third, to study changes in the expression of oestrogen (E2) synthase and receptors during the development of antral follicles, we divided them according to their diameters into small (≤ 2 mm), medium (2-5 mm), and large (≥ 5 mm) groups. Fourth, we added DHT (10-8 M) and flutamide (Flu, 10-7 M) to granulosa cells to determine whether DHT regulates the expression of cytochrome P450 aromatase (CYP19A1) and the associated receptors through the AR pathway. Fifth, we tested the effect of DHT and Flu on the expression of apoptotic genes and proteins in granulosa cells. We found that AR was expressed in sheep antral follicle granulosa cells and was regulated by DHT. During antral follicle development, the concentration of E2 and the expression of CYP19A1 and E2 receptors significantly increased in granulosa cells. DHT influenced this increase, at least partially, through the AR. Moreover, DHT regulated the expression of apoptotic genes and proteins through the AR. Our study expands our knowledge on the regulatory mechanism of DHT in antral follicle development and guides further research on the androgen regulation of ovarian function.

Spatial assessment models to evaluate human health risk associated to soil potentially toxic elements.

Quantifying source apportionment of potentially toxic elements (PTEs) in soils and associated human health risk (HHR) is essential for soil environment regulation and pollution risk mitigation. For this purpose, an integrated method was proposed, and applied to a dataset consisting of As, Cd, Cr, Cu, Hg, Ni, Pb, Se, and Zn in 273 soil surface samples. Positive matrix factorization (PMF) was used to quantitatively examine sources contributions of PTEs in soils; and the HHR arising from the identified source was determined by combining source profiles and health risk assessment; at last, sequential Gaussian simulation (SGS) was used to identify the areas with high HHR. Four sources were identified by PMF. Natural and agricultural sources affected all 9 PTEs contents with contributions ranging from 19.2% to 62.9%. 41.9% of Cd, 40.8% of Pb, 58.6% of Se, and 29.8% of Zn were controlled by industrial and traffic emissions. Metals smelting and mining explained 35.5%, 30.5%, and 24.9% of Cr, Cu, and Ni variations, respectively. Hg was dominated by atmospheric deposition from coal combustion and coking (58.7%). The mean values of the total non-carcinogenic risks of PTEs were 1.55 × 10-1 and 9.40 × 10-1 for adults and children, and the total carcinogenic risk of PTEs had an average value of 8.86 × 10-5. Based on source-oriented HHR calculation, natural and agricultural sources were the most important factor influencing HHR, explaining 51.0% and 49.1% of non-carcinogenic risks for children and adults, and 44.2% of carcinogenic risk. SGS indicated that 1.1% of the total area was identified as hazardous areas with non-carcinogens risk for children.

17ß-estradiol protects sheep oviduct epithelial cells against lipopolysaccharide-induced inflammation in vitro.

Estrogen has known anti-inflammatory effects, but the mechanism whereby 17ß-estradiol (E2) protects oviduct sheep epithelial cells from inflammation remains unknown. In this study, we detected the E2 synthetase and E2 nuclear and membrane receptors in sheep oviducts, primarily in epithelial cells. Using lipopolysaccharide (LPS)-stimulated sheep oviduct epithelial cells as an in vitro inflammation model, we demonstrated that E2 attenuates the expression of inflammatory factors in a concentration-response manner. E2 also inhibited the LPS-stimulated phosphorylation of p38 MAPK and NF-κB p65 but did not reduce the phosphorylation of JNK and ERK 1/2. This attenuation was partially antagonized by an intracellular estrogen antagonist that was involved in genomic regulation and enhanced by a G protein-coupled estrogen receptor agonist that was involved in nongenomic cellular modulation. These results suggest that E2 has an inhibitory effect on LPS-induced oviduct epithelial cell inflammation in sheep, which is mediated by the downstream regulatory effects of estrogen receptors.

Androgen receptor, aromatase, oestrogen receptor α/ß and G protein-coupled receptor 30 expression in the testes and epididymides of adult sheep.

The androgen receptor (AR) plays a key role in reproduction, and aromatase (P450arom), nuclear oestrogen receptors (ERs) α and ß, and G protein-coupled receptor 30 (GPR30) are important for testicular and epididymal cell proliferation and development. In the study, we have investigated the expression and localization of AR, P450arom, ERα, ERß and GPR30 in testes and epididymides of sexually mature sheep by quantitative reverse transcription-polymerase chain reaction, Western blotting and immunohistochemistry. The results demonstrate that the AR, P450arom and ERα levels in the caput and corpus epididymis were significantly lower than those in the testis and cauda epididymis (p < .05), the ERß level in the testis was significantly higher than in the caput, corpus and cauda epididymis (p < .05), and the GPR30 level in the caput epididymis was significantly lower than in the testis and corpus and cauda epididymis (p < .05). These receptors were mainly detected in epididymal epithelial, basal, smooth muscle, Sertoli and Leydig cells, as well as in spermatozoa. Taken together, the results suggest that sheep epididymides and testes have the potential for estradiol synthesis and are the targets of both androgens and estradiol. These results provide a foundation for further studies on the mechanisms of androgens and estradiol signalling in the testes and epididymides of sheep.