Bayesian multivariate receptor model and convolutional neural network to identify quantitative sources and spatial distributions of potentially toxic elements in soils: A case study in Qingzhou City, China.

Determining sources and spatial distributions of potentially toxic elements (PTEs) is a crucial issue of soil pollution survey. However, uncertainty estimation for source contributions remains lack, and accurate spatial prediction is still challenging. Robust Bayesian multivariate receptor model (RBMRM) was applied to the soil dataset of Qingzhou City (8 PTEs in 429 samples), to calculate source contributions with uncertainties. Multi-task convolutional neural network (MTCNN) was proposed to predict spatial distributions of soil PTEs. RBMRM afforded three sources, consistent with US-EPA positive matrix factorization. Natural source dominated As, Cr, Cu, and Ni contents (78.5 %∼86.1 %), and contributed 37.1 %, 61.0 %, and 65.9 % of Cd, Pb, and Zn, exhibiting low uncertainties with uncertainty index (UI) < 26.7 %. Industrial, traffic, and agricultural sources had significant influences on Cd, Pb, and Zn (30.2 %∼61.9 %), with UI < 39.3 %. Hg originated dominantly from atmosphere deposition (99.1 %), with relatively high uncertainties (UI=87.7 %). MTCNN acquired satisfactory accuracies, with R2 of 0.357-0.896 and nRMSE of 0.092-0.366. Spatial distributions of As, Cd, Cr, Cu, Ni, Pb, and Zn were influenced by parent materials. Cd, Hg, Pb, and Zn showed significant hotspot in urban area. This work conducted a new approach exploration, and practical implications for soil pollution regulation were proposed.

Expression characteristics and in vitro antibacterial properties of C-type lysozyme in crucian carp infected with Aeromonas salmonicida.

Aeromonas salmonicida is an ancient fish pathogen. Lysozymes are important molecules in the innate immune system that fight bacterial infections. The expression characteristics of C-type lysozyme in crucian carp infected with A. salmonicida and its antibacterial effect against A. salmonicida had not been investigated. Thus, we used bioinformatics to analyze the gene and protein characteristics of C-type lysozymes in crucian carp. Changes in C-type lysozyme expression before and after crucian carp infection with A. salmonicida were detected, and the in vitro antibacterial effect of recombinant carp C-type lysozyme on A. salmonicida was validated. The results showed that the coding DNA sequence region of the lysozyme gene sequence was 438 bp long, encoding 145 amino acids and containing two conserved catalytic sites: Glu53 and Asp69. Phylogenetic analysis revealed that crucian carp C-type lysozymes clustered with Cyprinus carpio lysozyme C. After crucian carp were infected with A. salmonicida, the gene and protein expression of C-type lysozymes in the liver, spleen, kidney, and hindgut were significantly upregulated, with the liver showing the highest upregulation that was 15 times higher than that in the uninfected group. In addition, recombinant C-type lysozyme exhibited significant antibacterial activity against A. salmonicida, with an average inhibition zone radius of 0.92 cm when using 40 µg recombinant lysozyme. In conclusion, this study reveals the important role of C-type lysozymes in the innate immune response of crucian carp and provides a theoretical basis for preventing crucian carp infection with A. salmonicida.

Estimating Soil Salinity with Different Levels of Vegetation Cover by Using Hyperspectral and Non-Negative Matrix Factorization Algorithm.

Hyperspectral technology has proven to be an effective method for monitoring soil salt content (SSC). However, hyperspectral estimation capabilities are limited when the soil surface is partially vegetated. This work aimed to (1) quantify the influences of different fraction vegetation coverage (FVC) on SSC estimation by hyperspectra and (2) explore the potential for a non-negative matrix factorization algorithm (NMF) to reduce the influence of various FVCs. Nine levels of mixed hyperspectra were measured from simulated mixed scenes, which were performed by strictly controlling SSC and FVC in the laboratory. NMF was implemented to extract soil spectral signals from mixed hyperspectra. The NMF-extracted soil spectra were used to estimate SSC using partial least squares regression. Results indicate that SSC could be estimated based on the original mixed spectra within a 25.76% FVC (R2cv = 0.68, RMSEcv = 5.18 g·kg-1, RPD = 1.43). Compared with the mixed spectra, NMF extraction of soil spectrum improved the estimation accuracy. The NMF-extracted soil spectra from FVC below 63.55% of the mixed spectra provided acceptable estimation accuracies for SSC with the lowest results of determination of the estimation R2cv = 0.69, RMSEcv = 4.15 g·kg-1, and RPD = 1.8. Additionally, we proposed a strategy for the model performance investigation that combines spearman correlation analysis and model variable importance projection analysis. The NMF-extracted soil spectra retained the sensitive wavelengths that were significantly correlated with SSC and participated in the operation as important variables of the model.

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.

Regulation of progesterone during follicular development by FSH and LH in sheep.

Progesterone (P4) can participate in the development of female mammalian antral follicles through nuclear receptor (PGR). In this experiment, the differences of P4 synthesis and PGR expression in different developmental stages of sheep antral follicles (large > 5mm, medium 2-5mm, small < 2mm) were detected by enzyme-linked immunosorbent assay, immunohistochemistry, qRT-PCR and Western blotting. Secondly, sheep follicular granulosa cells were cultured in vitro. The effects of different concentrations of FSH and LH on P4 synthesis and PGR expression were studied. The results showed that acute steroid regulatory protein (StAR), cholesterol side chain lyase (P450scc) and 3ß Hydroxysteroid dehydrogenase (3ß-HSD) and PGR were expressed in antral follicles, and with the development of antral follicles in sheep, StAR, P450scc and the expression of 3ß-HSD and PGR increased significantly. In vitro experiments showed that FSH and LH alone or together treatment could regulate P4 secretion and PGR expression in sheep follicular granulosa cells to varying degrees, hint P4 and PGR by FSH and LH, and LH was the main factor. Our results supplement the effects of FSH and LH on the regulation of P4 synthesis during follicular development, which provides new data for further study of steroid synthesis and function in follicular development.

Effect of dihydrotestosterone on melatonin secretion and the expression of melatonin receptors and apoptosis-related factors in sheep epididymides.

Melatonin (MEL) is involved in homeostasis of the epididymis lumen environment. Dihydrotestosterone (DHT) partakes in the development of gonads and organs in male animals. However, whether MEL secretion, the expression of its receptors, MT1 and MT2, and sheep epididymal epithelial cell apoptosis is regulated by DHT remains unclear. In this study, we used immunohistochemical staining to detect the distribution patterns of DHT synthetases [5α-reductase (5α-red)] and its androgen receptor (AR) in sheep epididymides. 5α-red1, 5α-red2 and AR were positively expressed in sperm, epididymal epithelial cells, and the smooth muscle cells of the caput, corpus and cauda regions of the epididymis. DHT concentration and the expression levels of 5α-red and AR in the caput, corpus and cauda regions were measured by enzyme-linked immunosorbent assay, liquid chromatography-mass spectrometry, real-time quantitative polymerase chain reaction and western blot analysis. DHT concentration in the caput was significantly higher than those in corpus and cauda, probably because of the high expression of 5α-red2 in the caput and secretion and transport of DHT by the testicles. DHT inhibited MEL secretion, the expression of its membrane receptors and MEL synthetases in cultured sheep epididymal epithelial cells in vitro. In addition, the Bax/Bcl-2 ratio, ACT CASP3 and caspase-3 mRNA expression were also decreased. The decreasing effect was partially reversed after flutamide treatment. Therefore, DHT regulates sheep epididymal function by influencing MEL expression and apoptosis-related factors. This study provides basic data for further research on the reproductive physiology of male animals.

Understanding Fishermen's Willingness and Preferences for Eco-compensation of Lake Conservation Projects: A Case Study From Nansi Lake Nature Reserve, China.

Lake regions face a tradeoff between water environmental conservation and social development. Although lake conservation projects along with eco-compensation policies are considered the solutions to this problem, it is often controversial and unsustainable. Therefore, it is essential to understand the key stakeholders' willingness to participate in lake conservation projects under the different compensation schemes. In this work, we conducted a choice experiment to estimate fishermen's willingness and preference for the returning fishponds to lake (RFTL) project in the Nansi Lake Nature Reserve (NLNR). Socioeconomic, perception and spatial factors are employed to analyze the preference heterogeneity of fishermen. We employed conditional logit, mixed logit and latent class models to estimates the parameters of factor variables considering the heterogeneous preferences and choice randomness across respondents. The results indicate that on average, fishermen are unwilling to change the status quo and are most concerned about the subsidy amount and contract duration in compensation contracts. However, there is obvious spatial heterogeneity in fishermen's choice preference, mainly manifested in their habitation distance to a town and fishpond location in different zones of the NLNR. Specifically, perceptions of supply and demand for water yield and water quality services significantly influence fishermen's choice, but they have opposite effects. Moreover, we estimated marginal willingness to accept (MWTA) of attributes in compensation contract and measured compensating surplus (CS) to compare different eco-compensation policy scenarios. Our results contribute to designing targeted payment policies, and promote the effective implementation of lake conservation projects and regional sustainable development.

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.

Regulatory role of dihydrotestosterone on BMP-6 receptors in granular cells of sheep antral follicles.

Bone morphogenetic protein-6 (BMP-6) and dihydrotestosterone (DHT) affect steroid synthesis in follicles and regulate cell proliferation in the ovaries of female animals. However, little is known about granular cells (GCs) in sheep. We identified the key BMP-6 receptors, activin receptor-like kinase(ALK-6), and bone morphogenetic protein receptor type 2 (BMPRII) in sheep follicles using immunohistochemistry (IHC) and immunofluorescence (IF). Both ALK-6 and BMPRII were expressed in the GC layer, GC membranes, and cytoplasm. We evaluated ALK-6 and BMPRII expression at the follicular development stage using quantitative real-time PCR and western blotting to detect sheep GCs from large, medium, and small follicles (diameters of ≥5, 2-5, and ≤2 mm, respectively). The mRNA abundance and protein expression of ALK-6 and BMPRII were significantly higher in GCs from large follicles compared to those in GCs from small follicles (P < 0.05) and were the lowest in GCs from medium follicles. To assess whether DHT affects ALK-6 and BMPRII expression in sheep GCs, we cultured GCs from large follicles in vitro then incubated them with DHT (10-11, 10-9, 10-7 M). We found that 10-7-M DHT significantly inhibited ALK-6 and BMPRII mRNA and protein (P < 0.05). We further explored whether DHT regulates ALK-6 and BMPRII through the nuclear androgen receptor (AR) pathway and found that 10-6-M flutamide, a non-selective androgen inhibitor, partially relieved the inhibitory effect of 10-7-M DHT on ALK-6 and BMPRII expression. Thus, GCs in sheep antral follicles differentially expressed ALK-6 and BMPRII at various stages, indicating that BMP-6 plays different roles to some extent during the development of antral follicles, and that high concentrations of DHT can inhibit the expression of ALK-6 and BMPRII via the androgen receptor pathway in sheep GCs. The present study aimed to determine the expression of the main BMP-6-related main receptors, namely, ALK-6 and BMPRII, during the development of GCs in sheep antral follicles and a potential mechanism of DHT regulation in sheep GCs. Our findings lay a foundation for the further exploration of the effects of ovarian BMP-6 expression on follicular development.

Source apportionment of potentially toxic elements in soils of the Yellow River Delta Nature Reserve, China: The application of three receptor models and geostatistical independent simulation.

The Yellow River Delta (YRD) wetland, the most important estuary wetland in eastern China, has an important ecosystem service function. Rapid and intensive development has inevitably led to the accumulation of potentially toxic elements (PTEs) in soils. Therefore, identifying quantitative sources and spatial distributions of PTEs is essential for soil environmental protection in the YRD. A total of 240 topsoil samples (0-20 cm) were collected in the Yellow River Delta Nature Reserve (YRDNR) and analyzed the PTE contents. To avoid the biases of the single receptor model, positive matrix factorization, factor analysis with nonnegative constraints, and maximum likelihood principal component analysis-multivariate curve resolution-alternating least squares were used for source apportionment of soil PTEs. To promote the efficiency of multivariate geostatistical simulation, a minimum/maximum autocorrelation factor-sequential Gaussian simulation was built to map the spatial patterns of PTEs. Three factors were derived by the three receptor models, and their contributions to the source explanation were similar. As, Cr, Cu, Mn, Ni, and Zn originated from natural sources, with contributions of 85.6%-96.4 %. A total of 61.5 % of Hg was associated with atmospheric deposition of coal combustion and wastewater from upstream. Agricultural activities and oil exploitation contributed 33.5 % and 15.9 % of the Cd and Pb concentrations. Spatial distributions of soil PTEs were controlled by sedimentary grain size. A total of 47.2 % of the total study area was identified as hazardous area for Cd, 10.3 % for As, and 5.4 % for Hg. This work is expected to provide references for soil pollution assessment and management of YRDNR.

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.

Follicle-stimulating hormone and luteinizing hormone regulate the synthesis mechanism of dihydrotestosterone in sheep granulosa cells.

Steroid hormones and receptors play important roles in female reproduction, and their expression patterns affect follicular growth and development. To examine the expression of dihydrotestosterone (DHT) synthases (5α-reductases (5α-red1 and 5α-red2)) and androgen receptor (AR) during follicular development, and the regulation of DHT signalling by follicle-stimulating hormone (FSH) and luteinizing hormone (LH), we have used enzyme-linked immunosorbent assays, quantitative real-time polymerase chain reaction, immunohistochemical staining and Western blotting to examine DHT synthesis in small (≤2 mm), medium (2-5 mm) and large (≥5 mm) sheep follicles. Expression of 5α-red1, 5α-red2 and AR was observed in ovine ovaries, and with the development of follicles, the expressions of 5α-red1 and 5α-red2 mRNA and protein increased, but the levels of AR mRNA, protein and DHT level decreased. In addition, granulosa cells were treated with FSH (0.01, 0.1 and 1 international unit (IU)/ml), LH (0.01, 0.1 and 1 IU/ml) and testosterone (T, 10-7  M) to evaluate the effects of FSH and LH on DHT and oestradiol (E2) synthesis and 5α-red1, 5α-red2 and AR expression. We found that FSH and LH upregulated 5α-red1 and 5α-red2 in sheep granulosa cells, but downregulated the concentration of DHT and expression of AR. Meanwhile, FSH and LH significantly upregulated the expression of aromatase (P450arom) and secretion of E2. This result indicates that although FSH and LH promote the expression of 5α-red1 and 5α-red2, T is not transformed into DHT, but E2. This study reveals the reason why DHT concentration is downregulated in large follicles and lays a foundation for further exploring the synthesis mechanism of DHT during follicular development.

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.

The application of geostatistical analysis and receptor model for the spatial distribution and sources of potentially toxic elements in soils.

This study aimed to identify the spatial patterns of potentially toxic elements (PTEs), including the spatial distribution, spatial autocorrelation, and risk probability, and to quantify the sources of PTEs, to provide guidelines for soil management. Spatial distributions and probabilities of PTEs were determined by empirical Bayesian kriging (EBK), while spatial autocorrelation was estimated by Moran's I. Positive matrix factorization (PMF) was adopted for the quantitative source contributions of PTEs. More than 64.6% of Co, Cr, Mn, and Ni were derived from geogenic sources, with high regions and high-high clusters both correlated to sandstone. Thus, it can be deduced that parent materials dominated the spatial patterns of these PTEs. In addition, some hotspots were situated in urban areas, and the influence of human activities on these four PTEs should be considered. Industry-traffic discharge and parent materials both influenced As, Cu, Pb, and Zn. Nonetheless, the spatial patterns of As, Cu, Pb, and Zn were formed by anthropogenic emissions since hotspots and high-high clusters were contiguously situated in urban areas. 58.5% of Hg originated from atmospheric deposition related to industrial emissions, and 47.2% of Cd was controlled by the application of chemical fertilizers. High levels of Hg and Cd mainly corresponded with industrial sites and cultivated land, suggesting that industrial and geoponic production played major roles in the generation of spatial patterns for Hg and Cd, respectively. Furthermore, the Cd and Hg posed a severe risk to soils, with a high probability to surpass 1.5 times the backgrounds. The EBK, Moran's I, and PMF results showed that all ten PTEs were enriched to some degree due to natural or anthropogenic factors. The results of geostatistical analysis and the receptor model can be mutually verified, indicating the reliability of these methods.