Exosomes-mediated retinoic acid disruption: A link between gut microbiota depletion and impaired spermatogenesis.

Gut microbiota symbiosis faces enormous challenge with increasing exposure to drugs such as environmental poisons and antibiotics. The gut microbiota is an important component of the host microbiota and has been proven to be involved in regulating spermatogenesis, but the molecular mechanism is still unclear. A male mouse model with gut microbiota depletion/dysbiosis was constructed by adding combined antibiotics to free drinking water, and reproductive parameters such as epididymal sperm count, testicular weight and paraffin sections were measured. Testicular transcriptomic and serum metabolomic analyses were performed to reveal the molecular mechanism of reproductive dysfunction induced by gut microbiota dysbiosis in male mice.This study confirms that antibiotic induced depletion of gut microbiota reduces sperm count in the epididymis and reduces germ cells in the seminiferous tubules in male mice. Further study showed that exosomes isolated from microbiota-depleted mice led to abnormally high levels of retinoic acid and decrease in the number of germ cells in the seminiferous tubules and sperm in the epididymis. Finally, abnormally high levels of retinoic acid was confirmed to disrupted meiotic processes, resulting in spermatogenesis disorders. This study proposed the concept of the gut microbiota-exosome-retinoic acid-testicular axis and demonstrated that depletion of the gut microbiota caused changes in the function of exosomes, which led to abnormal retinoic acid metabolism in the testis, thereby impairing meiosis and spermatogenesis processes.

METTL3 deficiency leads to ovarian insufficiency due to IL-1ß overexpression in theca cells.

Premature ovarian insufficiency (POI) is a clinical syndrome characterised by a decline in ovarian function in women before 40 years of age and is associated with oestradiol deficiency and a complex pathogenesis. However, the aetiology of POI is still unclear and effective preventative and treatment strategies are still lacking. Methyltransferase like 3 (METTL3) is an RNA methyltransferase that is involved in spermatogenesis, oocyte development and maturation, early embryonic development, and embryonic stem cell differentiation and formation, but its role in POI is unknown. In the present study, METTL3 deficiency in follicular theca cells was found to lead to reduced fertility in female mice, with a POI-like phenotype, and METTL3 knockout promoted ovarian inflammation. Further, a reduction in METTL3 in follicular theca cells led to a decrease in the m6A modification of pri-miR-21, which further reduced pri-miR-21 recognition and binding by DGCR8 proteins, leading to a decrease in the synthesis of mature miR-21-5p. Decrease of miR-21-5p promoted the secretion of interleukin-1ß (IL-1ß) from follicular theca cells. Acting in a paracrine manner, IL-1ß inhibited the cAMP-PKA pathway and activated the NF-κB pathway in follicular granulosa cells. This activation increased the levels of reactive oxygen species in granulosa cells, causing disturbances in the intracellular Ca2+ balance and mitochondrial damage. These cellular events ultimately led to granulosa cell apoptosis and a decrease in oestradiol synthesis, resulting in POI development. Collectively, these findings reveal how METTL3 deficiency promotes the expression and secretion of IL-1ß in theca cells, which regulates ovarian functions, and proposes a new theory for the development of POI disease.

METTL14 mediates nerve growth factor-stimulated testosterone synthesis in porcine theca cells†.

Ovarian theca cells produce testosterone, which acts as a vital precursor substance for synthesizing estrogens during follicular development. Nerve growth factor (NGF) has been shown to participate in reproductive physiology, specifically to follicular development and ovulation. There is currently no available data on the impact of NGF on testosterone synthesis in porcine theca cells. Furthermore, m6A modification is the most common internal modification in eukaryotic mRNAs that are closely associated with female gametogenesis, follicle development, ovulation, and other related processes. It is also uncertain whether the three main enzymes associated with m6A, such as Writers, Erasers, and Readers, play a role in this process. The present study, with an in vitro culture model, investigated the effect of NGF on testosterone synthesis in porcine theca cells and the role of Writers-METTL14 in this process. It was found that NGF activates the PI3K/AKT signaling pathway through METTL14, which regulates testosterone synthesis in porcine theca cells. This study will help to further elucidate the mechanisms by which NGF regulates follicular development and provide new therapeutic targets for ovary-related diseases in female animals. Summary Sentence  The present study investigated the effect of NGF on testosterone synthesis in porcine theca cells. It was found that NGF activates the PI3K/AKT signaling pathway through METTL14, which regulates testosterone synthesis in porcine theca cells.

Heat stress-induced dysbiosis of the gut microbiota impairs spermatogenesis by regulating secondary bile acid metabolism in the gut.

Heat stress (HS) poses a substantial challenge to livestock. Studies have demonstrated that HS reduces fertility and leads to gut microbiota dysbiosis in bulls. However, the impact of the gut microbiota on fertility in bulls during HS is still unclear. Our research revealed that HS exposure decreased semen quality in bulls, and fecal microbiota transplantation (FMT) from heat-stressed bulls to recipient mice resulted in a significant decrease in number of testicular germ cells and epididymal sperm. Untargeted metabolomics methodology and 16S rDNA sequencing conjoint analysis revealed that Akkermansia muciniphila (A. muciniphila) seemed to be a key bacterial regulator of spermatogenesis after HS exposure. Moreover, the research indicated that A. muciniphila regulated secondary bile acid metabolism by promoting the colonization of bile salt hydrolase (BSH)-metabolizing bacteria, leading to increase of retinol absorption in the host gut and subsequently elevation of testicular retinoic acid level, thereby improving spermatogenesis. This study sheds light on the relationship between HS-induced microbiota dysbiosis and spermatogenesis, offering a potential therapeutic approach for addressing bull spermatogenic dysfunction triggered by HS exposure.

Lithium Chloride Promotes Endogenous Synthesis of CLA in Bovine Mammary Epithelial Cells.

Although conjugated linoleic acid (CLA) can promote human health, its content in milk is insufficient to have a significant impact. The majority of the CLA in milk is produced endogenously by the mammary gland. However, research on improving its content through nutrient-induced endogenous synthesis is relatively scarce. Previous research found that the key enzyme, stearoyl-CoA desaturase (SCD) for the synthesis of CLA, can be expressed more actively in bovine mammary epithelial cells (MAC-T) when lithium chloride (LiCl) is present. This study investigated whether LiCl can encourage CLA synthesis in MAC-T cells. The results showed that LiCl effectively increased SCD and proteasome α5 subunit (PSMA5) protein expression in MAC-T cells as well as the content of CLA and its endogenous synthesis index. LiCl enhanced the expression of proliferator-activated receptor-γ (PPARγ), sterol regulatory element-binding protein 1 (SREBP1), and its downstream enzymes acetyl CoA carboxylase (ACC), fatty acid synthase (FASN), lipoprotein lipase (LPL), and Perilipin 2 (PLIN2). The addition of LiCl significantly enhanced p-GSK-3ß, ß-catenin, p-ß-catenin protein expression, hypoxia-inducible factor-1α (HIF-1α), and downregulation factor genes for mRNA expression (P < 0.05). These findings highlight that LiCl can increase the expression of SCD and PSMA5 by activating the transcription of HIF-1α, Wnt/ß-catenin, and the SREBP1 signaling pathways to promote the conversion of trans-vaccenic acid (TVA) to the endogenous synthesis of CLA. This data suggests that the exogenous addition of nutrients can increase CLA content in milk through pertinent signaling pathways.

Lithium Chloride Promotes Milk Protein and Fat Synthesis in Bovine Mammary Epithelial Cells via HIF-1α and ß-Catenin Signaling Pathways.

Lithium is one of the trace elements with many physiological properties, such as being anti-cancer, anti-viral, and anti-inflammatory. However, little is known about its effect on milk synthesis during lactation. Therefore, we selected different concentrations (5 mM, 10 mM, and 20 mM) of lithium chloride (LiCl) and assessed the effect of LiCl on bovine mammary epithelial (MAC-T) cells that underwent 4 days of differentiation induction. Moreover, we analyzed the effect of LiCl on the expression of genes related to milk fat and milk protein synthesis. Herein, LiCl (5-20 mM) significantly increased the expression of ß-casein, promoted mRNA expression and phosphorylated protein expression of the signal transduction molecule and activator of transcription 5ß (STAT5-ß), and inhibited mRNA and protein expression of suppressor of cytokine signaling 2 (SOCS2). In contrast, 5 and 10 mM LiCl significantly inhibited expression of SOCS3. LiCl at concentration of 5-20 mM enhanced phosphorylation level of mTOR protein; at 10 mM and 20 mM, LiCl significantly promoted expression and phosphorylation of downstream ribosomal protein S6 kinase beta-1 (S6K1) protein. Considering milk fat synthesis, mRNA expression of acetyl CoA carboxylase (ACC) and lipoprotein lipase (LPL) genes was considerably increased in the presence of LiCl (5-20 mM). Additionally, increased protein expression levels of stearoyl-CoA desaturase (SCD), peroxisome proliferator-activated receptor-γ (PPARγ), and sterol regulatory element-binding protein 1 (SREBP1) were observed at all LiCl concentrations tested. Subsequently, LiCl (5-20 mM) significantly promoted protein expression and phosphorylation of ß-catenin, while 10 mM and 20 mM of LiCl significantly promoted protein expression of hypoxia-inducible factor-1α (HIF-1α). Collectively, it has been shown that 10 mM LiCl can effectively activate HIF-1α, ß-catenin, and ß-catenin downstream signaling pathways. Conversely, at 10 mM, LiCl inhibited SOCS2 and SOCS3 protein expression through JAK2/STAT5, mTOR, and SREBP1 signaling pathways, improving synthesis of milk protein and fat. Therefore, LiCl can be used as a potential nutrient to regulate milk synthesis in dairy cows.

Beta-Sitosterol Promotes Milk Protein and Fat Syntheses-Related Genes in Bovine Mammary Epithelial Cells.

ß-sitosterol, a phytosterol with multiple biological activities, has been used in the pharmaceutical industry. However, there are only a few reports on the use of ß-sitosterol in improving milk synthesis in dairy cows. This study aimed to investigate the effects of ß-sitosterol on milk fat and protein syntheses in bovine mammary epithelial cells (MAC-T) and its regulatory mechanism. MAC-T cells were treated with different concentrations (0.01, 0.1, 1, 5, 10, 20, 30, or 40 µM) of ß-sitosterol, and the expression levels of milk protein and fat synthesis-related genes and proteins were analyzed. ß-sitosterol at 0.1, 1, and 10 µM concentrations promoted the mRNA and protein expression of ß-casein. ß-sitosterol (0.1, 1, 10 µM) increased the mRNA and protein expression levels of signal transducer activator of transcription 5 (STAT5), mammalian target of rapamycin (mTOR), and ribosomal protein S6 kinase beta-1 (S6K1) of the JAK2/STAT5 and mTOR signaling pathways. It also stimulated the milk fat synthesis-related factors, including sterol regulatory element-binding protein 1 (SREBP1), peroxisome proliferator-activated receptor-gamma (PPARγ), acetyl-CoA carboxylase (ACC), lipoprotein lipase (LPL), and stearyl CoA desaturase (SCD). ß-sitosterol (0.1, 1, 10 µM) also significantly increased the expression of growth hormone/insulin-like growth factor-1 (GH/IGF-1) axis and hypoxia-inducible factor-1α (HIF-1α)-related genes. Notably, the compound inhibited the expression of the negative regulator, the suppressor of cytokine signaling 2 (SOCS2) at the two lower concentrations (0.1, 1 µM), but significantly promoted the expression at the highest concentration (30 µM). These results highlight the role of ß-sitosterol at concentrations ranging from 0.1 to 10 µM in improving milk protein and fat syntheses, regulating milk quality. Therefore, ß-sitosterol can be used as a potential feed additive to improve milk quality in dairy cows.