ß-Nicotinamide mononucleotide improves chilled ram sperm quality in vitro by reducing oxidative stress damage.

OBJECTIVE: The present study aimed to investigate the effect of ß-nicotinamide mononucleotide (NMN) supplementation on ram sperm quality during storage at 4°C in vitro. METHODS: Tris-citric acid-glucose solution containing different doses of NMN (0, 30, 60, 90, and 120 µM) was used to dilute semen collected from rams and it was stored at 4°C. Sperm motility, plasma membrane integrity as well as acrosome integrity were evaluated at 0, 24, and 48 h time points after storage at 4°C. In addition, sperm mitochondrial activity, lipid peroxidation (LPO), malondialdehyde (MDA) content, reactive oxygen species (ROS) content, glutathione (GSH) content, superoxide dismutase (SOD) activity, and apoptosis were measured at 48 h time point after storage at 4°C. RESULTS: Results demonstrate that the values obtained for sperm motility, acrosome integrity, and plasma membrane integrity in the NMN treatments were significantly higher than control (p<0.05). The addition of 60 µM NMN significantly improved ram sperm mitochondrial activity and reduced LPO, MDA content, and ROS content compared to control (p<0.05). Interestingly, sperm GSH content and SOD activity for the 60 µM NMN treatment were much higher than those observed for control. NMN treatment also decreased the level of Cleaved-Caspase 3, Cleaved-Caspase 9, and Bax while increasing Bcl-2 level in sperm at 48 h time point after storage at 4°C. CONCLUSION: Ram sperm quality can be maintained during storage at 4°C with the addition of NMN at 60 µM to the semen extender. NMN also reduces oxidative stress and apoptosis. Overall, these findings suggest that NMN is efficient in improving the viability of ram sperm during storage at 4°C in vitro.

Mitochonic Acid 5 Increases Ram Sperm Quality by Improving Mitochondrial Function during Storage at 4 °C.

Semen preservation involves lengthening sperm's fertile lifespan without any detrimental effects on its biochemical, functional, and ultrastructural properties. Liquid storage at 4 °C is a ram sperm preservation method. However, this method of storage causes irreversible damage due to cold shocks, osmotic stresses, oxidative stresses, and reductions in sperm metabolism. The present study aims to investigate whether the supplementation of mitochonic acid 5 (MA-5) in a sperm extender could improve chilled ram sperm quality and elucidate its mechanism of action. Ram sperm were diluted with a tris-citrate-glucose extender containing different concentrations of MA-5 (0, 0.1, 1, 10, and 100 nM) and stored at 4 °C for up to 48 h. Sperm motility, membrane integrity, acrosome integrity, mitochondrial membrane potential, reactive oxygen species (ROS) level, ATP content, and the expression of NADPH dehydrogenase subunits 1 (MT-ND1) and NADPH dehydrogenase subunits 6 (MT-ND6) were evaluated. It was observed that compared to the control, the 10 nM MA-5 treatment significantly (p < 0.05) increased total motility (82 ± 3.5% vs. 76 ± 5.9%), progressive motility (67.6 ± 8.2% vs. 51 ± 8.3%), and other parameters (straight-line velocity (VSL), average path velocity (VAP), and curvilinear velocity (VCL)). In addition, 10 nM MA-5 supplementation also improved ram sperm membrane integrity and acrosomal integrity as well increased mitochondrial membrane potential (51.1 ± 0.7% vs. 37.7 ± 1.3%), reduced ROS levels, and elevated adenosine triphosphate (ATP) contents. Furthermore, a Western blot analysis demonstrated that the addition of MA-5 significantly (p < 0.05) increased the expression of MT-ND1 and MT-ND6 proteins in ram sperm, with the 10 nM MA-5 treatment resulting in the highest expression level. These results suggest that MA-5 improves ram sperm quality by maintaining high sperm mitochondrial function during liquid storage at 4 °C.

Pyrroloquinoline Quinone Improves Ram Sperm Quality through Its Antioxidative Ability during Storage at 4 °C.

Sperm motility is an important factor in the migration of sperm from the uterus to the oviduct. During sperm preservation in vitro, sperm generates excessive ROS that damages its function. This study aims to investigate whether the addition of pyrroloquinoline quinone (PQQ) to the diluted medium could improve chilled ram sperm quality, and then elucidates the mechanism. Ram semen was diluted with Tris-citric acid-glucose (TCG) medium containing different doses of PQQ (0 nM, 10 nM, 100 nM, 1000 nM, 10,000 nM), and stored at 4 °C. Sperm motility patterns, plasma membrane integrity, acrosome integrity, mitochondrial membrane potential, reactive oxygen species (ROS) levels, malondialdehyde (MDA) levels, superoxide dismutase (SOD) activity, and ATP levels were measured after preservation. Furthermore, the expressions of NADH dehydrogenase 1 (MT-ND1) and NADH dehydrogenase 6 (MT-ND6) in sperm were also detected by western blotting. In addition, sperm capacitation and the ability of sperm to bind to the zona pellucina were also evaluated. It was observed that the addition of PQQ significantly (p < 0.05) improved ram sperm motility, membrane integrity, and acrosome integrity during preservation. The percentage of sperm with high mitochondrial membrane potential in the PQQ treatment group was much higher than that in the control. In addition, supplementation of PQQ also decreased the sperm MDA and ROS levels, while increasing ATP levels. Interestingly, the levels of MT-ND1 and MT-ND6 protein in sperm treated with PQQ were also higher than that of the control. Furthermore, the addition of 100 nM PQQ to the medium decreased ROS damage in MT-ND1 and MT-ND6 proteins. The addition of 100 nM PQQ significantly (p < 0.05) increased protein tyrosine phosphorylation in ram sperm after induced capacitation. Furthermore, the value of the sperm-zona pellucida binding capacity in the 100 nM PQQ treatment group was also much higher than that of the control. Overall, during chilled ram- sperm preservation, PQQ protected ram sperm quality by quenching the ROS levels to reduce ROS damage and maintain sperm mitochondrial function, and preserved the sperm's high ability of fertilization.

Resveratrol Improves the Frozen-Thawed Ram Sperm Quality.

Cryopreservation generates a substantial quantity of ROS in semen, leading to a decline in sperm quality and fertilization capacity. The objective of this study was to investigate the effects of resveratrol and its optimal concentration on ram sperm quality after cryopreservation. Ram semen was diluted with a freezing medium containing different concentrations of resveratrol (0, 25, 50, 75, and 100 µM). After thawing, various sperm parameters such as total motility, progressive motility, acrosome integrity, plasma membrane integrity, mitochondrial membrane potential, glutathione (GSH) content, glutathione synthase (GPx) activity, superoxide dismutase (SOD) activity, catalase (CAT) activity, lipid peroxidation (LPO) content, malondialdehyde (MDA) content, ROS level, SIRT1 level, DNA oxidative damage, and AMPK phosphorylation level were assessed. In addition, post-thaw sperm apoptosis was evaluated. Comparatively, the addition of resveratrol up to 75 µM significantly improved the sperm motility and sperm parameters of cryopreserved ram sperm. Specifically, 50 µM resveratrol demonstrated a notable enhancement in acrosome and plasma membrane integrity, antioxidant capacity, mitochondrial membrane potential, adenosine triphosphate (ATP) content, SIRT1 level, and AMPK phosphorylation levels compared to the control group (p < 0.05). It also significantly (p < 0.05) reduced the oxidative damage to sperm DNA. However, detrimental effects of resveratrol were observed at a concentration of 100 µM resveratrol. In conclusion, the addition of 50 µM resveratrol to the cryopreservation solution is optimal for enhancing the quality of cryopreserved ram sperm.

Carboxylated ε-Poly-l-lysine Improves Post-Thaw Quality, Mitochondrial Functions and Antioxidant Defense of Goat Cryopreserved Sperm.

Carboxylated ε-poly-l-lysine (CPLL), a novel cryoprotectant, can protect the sperm membranes by inhibiting ice crystal formation during the cryopreservation process. The present study was conducted to investigate the consequence of CPLL supplementation on the post-thaw quality of cryopreserved goat sperm. For this, different doses (0, 0.5%, 1%, 1.5%, and 2%; v/v) of CPLL were added to the cryopreservation medium, and the motility, membrane and acrosome integrity, mitochondrial membrane potential (MMP), ATP level, ROS production, anti-oxidant defense system, malondialdehyde (MDA) level, and apoptosis in post-thaw sperm were evaluated. It was observed that the addition of 1% CPLL significantly (p < 0.05) increased the total motility, membrane integrity, acrosome integrity, and catalase (CAT) activity of post-thaw sperm compared to those of control and other CPLL doses. The ATP content was observed significantly (p < 0.05) higher in 0.5% and 1% CPLL, however, the SOD activity and progressive motility were significantly (p < 0.05) increased by adding CPLL at 1% and 1.5% level. Moreover, the addition of CPLL at 1% dose not only showed a lower percentage of apoptosis, but also significantly (p < 0.05) increased the MMP while reducing ROS production and MDA levels compared to those of other CPLL doses and/or control. Therefore, it is clear that the supplementation of 1% CPLL can remarkably improve the post-thaw goat sperm motility, membrane and acrosome integrity, antioxidant abundance, mitochondrial potentials, and ATP supply by protecting the sperm from cryodamage and undergoing apoptosis. These findings will provide novel insights into sperm cryobiology.

Effect of Zearalenone-Induced Ferroptosis on Mice Spermatogenesis.

Male reproductive health is critically worsening around the world. It has been reported that the mycotoxin ZEA causes reproductive toxicity to domestic animals and affects spermatogenesis, thereby inhibiting male reproductive function. Ferroptosis is a newly identified type of programmed cell death that is different from apoptosis and it depends on iron accumulation and lipid peroxidation. Whether ferroptosis is linked to ZEA's detrimental effect on spermatogenesis needs to be further explored. This study clarifies ferroptosis's involvement in ZEA-induced damage on spermatogenesis. The reproductive injury model used in this study was induced by gavaging male mice in the ZEA treatment group with 30 µg/kg of ZEA for five weeks. Results show that ZEA treatment reduced mouse sperm motility and concentration, destroyed the structure of the seminiferous tubules of the testis, damaged the antioxidant defense system, and blocked spermatogenesis. Ferrostatin-1 (Fer-1) inhibition of ferroptosis partially alleviated ZEA-induced oligozoospermia in mice. In addition, ZEA treatment was found to activate a signaling pathway associated with ferroptosis in mouse testis. ZEA also downregulated the expression of Nrf2, SLC7A11, and GPX4, and decreased the protein expression of SLC7A11 and GPX4, resulting in the accumulation of lipid peroxides and an increase in the level of 4-HNE protein in the testis. Importantly, these changes were accompanied by an increase in the relative contents of Fe2+ and Fe3+. Iron accumulation and lipid peroxidation are the causes of ferroptosis in spermatogenic cells, leading to a decrease in sperm motility and concentration. While the administration of Fer-1 at 0.5 and 1 mg/kg also increased the expression of SLC7A11 and GPX4 proteins by upregulating Nrf2 expression, reducing iron accumulation, and reversing ZEA-induced ferroptosis, Fer-1 at 1.5 mg/kg had the best repairing effect for all parameters. In conclusion, ZEA-induced ferroptosis may be mediated by a notable reduction in Nrf2, SLC7A11 and GPX4 expression levels. Overall, ferroptosis is a novel therapeutic target for mitigating ZEA-induced reproductive toxicity.

Beneficial Effect of Proline Supplementation on Goat Spermatozoa Quality during Cryopreservation.

Sperm cryopreservation contributes to the extensive utilization of artificial insemination (AI) in the daily livestock industry. However, due to the presence of few sperm with good biological function in post-thaw goat sperm, its use has been limited for AI purposes. Hence, its improvement has been the focus of many research studies. This study aimed to investigate the effects of proline supplementation of the freezing medium on goat sperm. The goat semen was cryopreserved with freezing medium supplementation of different concentrations of proline (0, 0.5, 1, 2 and 4 mM). The post-thaw sperm motility patterns, membrane integrity, acrosome integrity, lipid peroxidation (LPO) levels, malondialdehyde (MDA) levels, total antioxidant capacity (T-AOC), proline dehydrogenase (PRODH) activity, superoxide dis-mutase (SOD) activity, glutathione (GSH) levels and GSH/GSSG were evaluated. Likewise, the expression and immunofluorescent localization of PRODH in post-thaw goat sperm was also detected. It was observed that addition of 2 mM proline to the freezing medium significantly enhanced post-thaw goat sperm total motility, progressive motility, straight-linear velocity (VSL), curvilinear velocity (VCL), average path velocity (VAP), straightness (STR), linearity (LIN), membrane integrity and acrosome integrity. Interestingly, PRODH was expressed in post-thaw goat sperm, especially in the post-acrosome and sperm tail. Addition of 2 mM proline also significantly increased the post-thaw sperm PRODH activity compared to the control. Moreover, post-thaw goat sperm LPO levels and MDA levels were reduced by supplementation of 2 mM proline. Furthermore, compared to the control, the values of post-thaw goat sperm T-AOC, SOD activity, GSH level and GSH/GSSG were also significantly increased in 2 mM proline treatment. Reduction of post-thaw goat sperm apoptosis in 2 mM proline treatment was also observed as the levels of Caspase3 and Caspase9 were decreased by the supplementation with 2 mM proline. These observations suggest that the addition of 2 mM proline to the freezing medium increased post-thaw goat sperm quality by reducing oxidative stress during cryopreservation. These findings also provide novel insights into the use of proline as an efficient additive to enhance post-thaw goat sperm quality during cryopreservation.

Genome-wide profile in DNA methylation in goat ovaries of two different litter size populations.

Although some studies have investigated the DNA methylation modification in goat ovaries, it is not understood DNA methylation related to goat litter size. This investigation was designed to explore the DNA methylation status in the ovaries of high litter size and low litter size groups using whole-genome bisulfite sequencing (WGBS). We found that there was global difference on DNA methylation in high litter size and low litter size goat ovaries. Many differentially methylated region-related genes (DMGs) were found in the ovaries of these two different goat populations. Moreover, enrichment analysis discovered that many DMGs were involved in gamete development, reproductive system development, wingless-type MMTV integration site family (WNT) signalling pathways and mitogen-activated protein kinase 1 (MAPK) signalling pathways. The data indicated that DNA methylation in goat ovaries may play important roles in the folliculogenesis, the oocyte ovulation rate and finally the litter size. This study provides a comprehensive analysis of genome-wide DNA methylation patterns in ovaries of high and low litter size goat which helps the understanding of ovarian DNA methylation in relation to goat fertility capability.

Transcriptome profile of goat folliculogenesis reveals the interaction of oocyte and granulosa cell in correlation with different fertility population.

To understand the molecular and genetic mechanisms related to the litter size in one species of two different populations (high litter size and low litter size), we performed RNA-seq for the oocytes and granulosa cells (GCs) at different developmental stages of follicle, and identified the interaction of genes from both sides of follicle (oocyte and GCs) and the ligand-receptor pairs from these two sides. Our data were very comprehensive to uncover the difference between these two populations regarding the folliculogenesis. First, we identified a set of potential genes in oocyte and GCs as the marker genes which can be used to determine the goat fertility capability and ovarian reserve ability. The data showed that GRHPR, GPR84, CYB5A and ERAL1 were highly expressed in oocyte while JUNB, SCN2A, MEGE8, ZEB2, EGR1and PRRC2A were highly expressed in GCs. We found more functional genes were expressed in oocytes and GCs in high fertility group (HL) than that in low fertility group (LL). We uncovered that ligand-receptor pairs in Notch signaling pathway and transforming growth factor-ß (TGF-ß) superfamily pathways played important roles in goat folliculogenesis for the different fertility population. Moreover, we discovered that the correlations of the gene expression in oocytes and GCs at different stages in the two populations HL and LL were different, too. All the data reflected the gene expression landscape in oocytes and GCs which was correlated well with the fertility capability.

Quantitative Label-Free Proteomic Analysis of Milk Fat Globule Membrane in Donkey and Human Milk.

Previous studies have found donkey milk (DM) has the similar compositions with human milk (HM) and could be used as a potential hypoallergenic replacement diet for babies suffering from cow's milk allergy. Milk fat globule membrane (MFGM) proteins are involved in many biological functions, behaving as important indicators of the nutritional quality of milk. In this study, we used label-free proteomics to quantify the differentially expressed MFGM proteins (DEP) between DM (in 4-5 months of lactation) and HM (in 6-8 months of lactation). In total, 293 DEP were found in these two groups. Gene Ontology (GO) enrichment analysis revealed that the majority of DEP participated in regulation of immune system process, membrane invagination and lymphocyte activation. Several significant Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways were determined for the DEP, such as lysosome, galactose metabolism and peroxisome proliferator-activated receptor (PPAR) signaling pathway. Our study may provide valuable information in the composition of MFGM proteins in DM and HM, and expand our knowledge of different biological functions between DM and HM.

ß-carotene Rescues Busulfan Disrupted Spermatogenesis Through Elevation in Testicular Antioxidant Capability.

ß-carotene, precursor of vitamin A, is an excellent antioxidant with many beneficial properties. It is a lipid-soluble antioxidant and a very effective quencher of reactive oxygen species (ROS) to reduce the oxidative stress. In contrast to vitamin A, ß-carotene is not toxic even consumed in higher amount when it is delivered from natural plant products. Recently, we found that ß-carotene acts as a potential antioxidant in the oocyte to improve its quality. Even though many studies have been reported that ß-carotene has the beneficial contribution to the ovarian development and steroidogenesis, it is unknown the effects of ß-carotene on the spermatogenesis. This investigation aimed to explore the hypothesis that ß-carotene could improve spermatogenesis and the underlying mechanism. And we found that ß-carotene rescued busulfan disrupted spermatogenesis in mouse with the increase in the sperm concentration and motility. ß-carotene improved the expression of genes/proteins important for spermatogenesis, such as VASA, DAZL, SYCP3, PGK2. Moreover, ß-carotene elevated the testicular antioxidant capability by the elevation of the antioxidant glutathione and antioxidant enzymes SOD, GPX1, catalase levels. In conclusion, ß-carotene may be applied for the infertile couples by the improvement of spermatogenesis, since, worldly many couples are infertile due to the idiopathic failed gametogenesis (spermatogenesis).

scRNA-seq of ovarian follicle granulosa cells from different fertility goats reveals distinct expression patterns.

The new technology of high-throughput single-cell RNA sequencing (10 × scRNA-seq) was developed recently with many advantages. However, it was not commonly used in farm animal research. There are few reports for the gene expression of goat ovarian follicle granulosa cells (GCs) during different developmental stages. In the current investigation, the gene expression of follicle GCs at different stages from two populations of Ji'ning grey goats: high litter size (HL; ≥3/L; 2 L) and low litter size (LL; ≤2 /L; 2 L) were analysed by scRNA-seq. Many GC marker genes were identified, and the pseudo-time showed that GCs developed during the time course which reflected the follicular development and differentiation trajectory. Moreover, the gene expression difference between the two populations HL versus LL was very clear at different developmental stages. Many marker genes differentially expressed at different developmental stages. ASIP and ASPN were found to be highly expressed in the early stage of GCs, INHA, INHBA, MFGE8 and HSD17B1 were identified to be highly expressed in the growing stage of GCs, while IGFBP2, IGFBP5 and CYP11A1 were found to be highly expressed in late stage. These marker genes could be used as reference genes of goat follicle GC development. This investigation for the first time discovered the gene expression patterns in goat follicle GCs in high- or low-fertility populations (based on litter size) by scRNA-seq which may be useful for uncovering the oocyte development potential.

Pubertal exposure to low doses of zearalenone disrupting spermatogenesis through ERα related genetic and epigenetic pathways.

Endocrine disruptor zearalenone (ZEA) has been found to damage the reproductive system especially spermatogenesis. In our previous report, we have found that low dose (lower than No-Observed Effect Level, NOEL) ZEA exposure disturbed mouse spermatogenesis and diminished mouse semen quality. The purpose of current investigation was to explore the underlying mechanisms of pubertal low dose ZEA exposure upsetting spermatogenesis. And it was demonstrated that pubertal low dose ZEA exposure disrupted the meiosis process and the important genetic pathways to inhibit the spermatogenesis and even to diminish the semen quality with the decrease in spermatozoa motility and concentration. The DNA methylation markers 5mC and 5hmC were decreased, the histone methylation marker H3K27 was increased, at the same time estrogen receptor alpha was diminished in mouse testis after pubertal low dose ZEA exposure. The data indicate that the disruption in spermatogenesis by pubertal low dose ZEA exposure may be through the alterations in genetic and epigenetic pathways, and the interactions with estrogen receptor signaling pathway. Therefore, we should pay great attention on ZEA exposure to reduce its adverse impacts on male reproductive health.

ß-carotene improves oocyte development and maturation under oxidative stress in vitro.

Recently, the mean maternal age at first birth has been continuing to increase. The decline in the age-related fertility is due to the reduction in the number and the quality of the oocyte. An elevation in intra-ovarian reactive oxygen species (ROS) is correlated with the increase in maternal age, and the oxidative stress is involved in the decline in oocyte quality. Although ß-carotene, a very effective quencher of ROS, has been found to have the beneficial contribution to the ovarian development and steroidogenesis, it is unknown the effect of ß-carotene on the oocyte development especially oocyte maturation. This investigation aimed to explore the beneficial contribution of ß-carotene on oocyte maturation under oxidative stress and the underlying mechanism. We found that the oxidative stress induced by ROS reagent Rosup inhibited oocyte development/maturation and parthenogenetic activation which could be dramatically rescued by ß-carotene (57.1 ± 4.7% vs 78.9 ± 3.8%; p < 0.05) in vitro. The underlying mechanisms include that ß-carotene not only reduces ROS formation and cell apoptosis, but also it can restore actin expression, cortical granule-free domain (CGFD) formation, mitochondria homogeneous distribution, and nuclear maturation. The data suggest that ß-carotene acts as a potential antioxidant in the oocyte. Therefore, the findings from this investigation provide the fundamental 7knowledge for using ß-carotene as an antioxidant to improve the oocyte quality and even the ovarian function.

Gestational exposure to low-dose zearalenone disrupting offspring spermatogenesis might be through epigenetic modifications.

Zearalenone (ZEA), a F-2 mycotoxin produced by Fusarium, has been found to be an endocrine disruptor through oestrogen receptor signalling pathway to impair spermatogenesis. The disruption on reproductive systems by ZEA exposure might be transgenerational. In our previous report, we have found that low dose (lower than no-observed effect level, NOEL) of ZEA impaired mouse spermatogenesis and decreased mouse semen quality. The purpose of the current investigation was to explore the impacts of low-dose ZEA on spermatogenesis in the offspring after prenatal exposure and the underlying mechanisms. And it demonstrated that prenatal low-dose ZEA exposure disrupted the meiosis process to inhibit the spermatogenesis in offspring and even to diminish the semen quality by the decrease in spermatozoa motility and concentration. The DNA methylation marker 5hmC was decreased, the histone methylation markers H3K9 and H3K27 were elevated, and oestrogen receptor alpha was reduced in the offspring testis after prenatal low-dose ZEA exposure. The data suggest that the disruption in spermatogenesis by prenatal low-dose ZEA exposure may be through the modifications on epigenetic pathways (DNA methylation and histone methylation) and the interactions with oestrogen receptor signalling pathway. Moreover, in the current study, the male offspring were indirectly exposed to low-dose ZEA through placenta and the spermatogenesis in offspring was disrupted which suggested that the toxicity of ZEA on reproductive systems was very severe. Therefore, we strongly recommend that greater attention should be paid to this mycotoxin to minimize its adverse impact on human spermatogenesis.

Low dose chlorothalonil impairs mouse spermatogenesis through the intertwining of Estrogen Receptor Pathways with histone and DNA methylation.

Recently, environment contaminants including pesticides, fungicides, mycotoxin and others chemicals have been suggested to be responsible for the decline in the human spermatozoa quality especially motility and the increase in infertility rate. Chlorothalonil is used widely for protection of vegetables and crops because it is a broad spectrum fungicide. It has been reported that chronic occupational exposure to fungicides was associated with poor spermatozoa morphology in young men. The pubertal period is very important for the male reproductive system development due to spermatogonial cell proliferation, the expansion of meiotic and haploid germ cells. Although some investigations have studied the male reproductive toxicity of chlorothalonil, almost no studies focused on spermatogenesis. The aim of our current investigation was to explore the impacts of chlorothalonil on spermatogenesis and the underlying mechanisms. It demonstrates: i) chlorothalonil decreased boar spermatozoa motility in vitro and increased the cell apoptosis; ii) chlorothalonil inhibited mouse spermatogenesis in vivo; iii) chlorothalonil disturbed spermatogenesis through the disruption of estrogen receptor signalling; iv) chlorothalonil disrupted histone methylation and DNA methylation which might be through estrogen signalling pathways. Due to the over use or incorrect use, chlorothalonil might cause serious problems to human health, especially spermatogenesis. Therefore we strongly recommend that greater attention should be paid to this fungicide to minimise its impact on human health especially spermatogenesis.

Chlorothalonil inhibits mouse ovarian development through endocrine disruption.

Although many studies have investigated the toxic effects and even the reproductive toxicity of chlorothalonil, almost no studies have focused on the ovary, the organ of oocyte development. Puberty is a critical window for development of the female reproductive system. Therefore, this investigation aimed to explore the effects and underlying mechanisms of chlorothalonil at low doses on peripubertal mouse ovarian development. Chlorothalonil is frequently used in horticulture with short intervals between applications, therefore, vegetables and fruits may be potential sources of chlorothalonil contamination. For the first time, this study demonstrated that chlorothalonil inhibited ovarian development during puberty in mice, and at levels currently assumed to have no adverse health consequences for humans. Chlorothalonil exposure inhibited mouse ovarian development by increasing the number of primary follicles and decreasing the number of mature follicles. It acted by decreasing the levels of hormone production proteins, such as FSH receptor and estrogen receptor alpha, while increasing the levels of DNA repairing marker RAD51 and cell apoptosis. These results suggest that chlorothalonil may disrupt endocrine function and inhibit murine ovarian development. Therefore it may pose a potential health risk to female reproductive systems in other species, especially to the ovary.

Zinc Oxide Nanoparticle Caused Plasma Metabolomic Perturbations Correlate with Hepatic Steatosis.

Zinc oxide nanoparticles (ZnO NPs), known for their chemical stability and strong adsorption, are used in everyday items such as cosmetics, sunscreens, and prophylactic drugs. However, they have also been found to adversely affect organisms; previously we found that ZnO NPs disrupt pubertal ovarian development, inhibit embryonic development by upsetting γ-H2AX and NF-κB pathways, and even disturb skin stem cells. Non-targeted metabolomic analysis of biological organisms has been suggested as an unbiased tool for the investigation of perturbations in response to NPs and their underlying mechanisms. Although metabolomics has been used in nanotoxicological studies, very few reports have used it to investigate the effects of ZnO NPs exposure. In the current investigation, through a metabolomics-based approach, we discovered that ZnO NPs caused changes in plasma metabolites involved in anti-oxidative mechanisms, energy metabolism, and lipid metabolism in hen livers. These results are in line with earlier findings that ZnO NPs perturb the tricarboxylic acid cycle and in turn result in the use of alternative energy sources. We also found that ZnO NPs disturbed lipid metabolism in the liver and consequently impacted blood lipid balance. Changes in plasma metabolomes were correlated with hepatic steatosis.

MicroRNA-221 may be involved in lipid metabolism in mammary epithelial cells.

Milk lipids, important for infant growth and development, are produced and secreted by mammary gland under the regulation of steroid hormones, growth factors, and microRNAs (miRNAs). miR-221 has been identified in milk and adipocytes and it plays important roles in regulating normal mammary epithelial hierarchy and breast cancer stem cells; however, its roles in lipid metabolism in mammary epithelial cells (MECs), the cells of lipid synthesis and secretion, are as yet unknown. Through overexpression or inhibition of miR-221 expression, we found that it regulated lipid metabolism in MECs and was expressed differentially at various stages during murine mammary gland development. Inhibition of miR-221 expression increased lipid content in MECs through elevation of the lipid synthesis enzyme FASN, while overexpression of miR-221 reduced MEC lipid content. Moreover, the steroid hormones estradiol and progesterone decreased miR-221 expression with a subsequent increase in lipid formation in MECs. The expression of miR-221 was lower during lactation, which suggests that it may be involved in milk production. Therefore, miR-221 might be a useful target for influencing milk lipid production.