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The CRISPR/Cas9 system is widely used for genome editing in livestock production, although off-target effects can occur. It is the main method to produce genome-edited goats by somatic cell nuclear transfer (SCNT) of CRISPR/Cas9-mediated genome-edited primary goat fetal fibroblast cells (GFFs). Improving the double-strand break (DSB) efficiency of Cas9 in primary cells would improve the homologous repair (HR) efficiency. The low efficiency of HR remains a major hurdle in CRISPR/Cas9-mediated precise genome editing, increasing the work required to screen the genome-edited primary cell clones. In this study, we modified several essential parameters that affect the efficiency of the CRISPR/Cas9-mediated knock-in GFF cloning system, including establishing a high-efficiency transfection system for primary cells via nucleofection and optimizing homology arm (HA) length during HR. Here, we specifically inserted a recombinant human butyrylcholinesterase gene (rhBChE) into the goat fibroblast growth factor (FGF)-5 locus through the CRISPR/Cas9 system, thereby achieving simultaneous rhBChE insertion and FGF5 knock-out. First, this study introduced the Cas9, FGF5 knock-out small guide RNA, and rhBChE knock-in donors into GFFs by electroporation and obtained positive cell clones without off-target effects. Then, we demonstrated the expression of rhBChE in GFF clones and verified its function. Finally, we obtained a CRISPR/Cas9-mediated rhBChE-overexpression goat.

Oligozoospermia or low sperm count is a leading cause of male infertility worldwide. Despite decades of work on non-coding RNAs (ncRNAs) as regulators of spermatogenesis, fertilization, and male fertility, the literature on the function of long non-coding RNAs (lncRNAs) in human oligozoospermia is scarce. We integrated lncRNA and mRNA sequencing data from 12 human normozoospermic and oligozoospermic samples and comprehensively analyzed the function of differentially expressed lncRNAs (DE lncRNAs) and mRNAs (DE mRNAs) in male infertility. The target genes of DE lncRNAs were identified using a Gaussian graphical model. Gene ontology terms and Kyoto Encyclopedia of Genes and Genomes pathways were primarily enriched in protein transport and localization to the endoplasmic reticulum (ER). The lncRNA–mRNA co-expression network revealed cis- and trans-regulated target genes of lncRNAs. The transcriptome data implicated DE lncRNAs and DE mRNAs and their target genes in the accumulation of unfolded proteins in sperm ER, PERK-EIF2 pathway-induced ER stress, oxidative stress, and sperm cell apoptosis in individuals with oligozoospermia. These findings suggest that the identified lncRNAs and pathways could serve as effective therapeutic targets for male infertility.

Melatonin is a ubiquitous indoleamine hormone synthesized primarily by the pineal gland. Diverse biological actions of melatonin involve quite complex mechanisms via its membrane receptors. More recently, studies have focused on the role of melatonin in male fertility preservation and male reproductive system. The protective effects of melatonin on immature testicular tissue freshness and activity maintenance and the preservation of sperm and spermatogonial stem cells (SSCs) have attracted considerable attention in recent years. Furthermore, since melatonin has strong antioxidant and anti-apoptotic properties, researchers have examined its potential role in male reproductive system. In this article, recent progress regarding melatonin's effects on male fertility preservation and its potential role is reviewed.

Fibroblast growth factor 5 (FGF5) is a famous dominant inhibitor of anagen phase of hair cycle. Mutations of FGF5 gene result in a longer wool in mice, donkeys, dogs, cats, and even in human eyelashes. Sheep is an important source of wool production. How to improve the production of wool quickly and effectively is an urgent problem to be solved. In this study, we generated five FGF5-knockout Dorper sheep by the CRISPR/Cas9 system. The expression level of FGF5 mRNA in knockout (KO) sheep decreased significantly, and all FGF5 proteins were dysfunctional. The KO sheep displayed a significant increase in fine-wool and active hair-follicle density. The crosstalk between androgen and Wnt/β-catenin signaling downstream of FGF5 gene plays a key role. We established downstream signaling cascades for the first time, including FGF5, FGFR1, androgen, AR, Wnt/β-catenin, Shh/Gli2, c-MYC, and KRTs. These findings further improved the function of FGF5 gene, and provided therapeutic ideas for androgen alopecia.

The physiological role of estrogen in the female endometrium is well established. On the basis of responses to steroid hormones (progesterone, androgen, and estrogen), the endometrium is considered to have proliferative and secretory phases. Estrogen can act in the endometrium by interacting with estrogen receptors (ERs) to induce mucosal proliferation during the proliferative phase and progesterone receptor (PR) synthesis, which prepare the endometrium for the secretory phase. Mouse knockout studies have shown that ER expression, including ERα, ERβ, and G-protein-coupled estrogen receptor (GPER) in the endometrium is critical for normal menstrual cycles and subsequent pregnancy. Incorrect expression of ERs can produce many diseases that can cause endometriosis, endometrial hyperplasia (EH), and endometrial cancer (EC), which affect numerous women of reproductive age. ERα promotes uterine cell proliferation and is strongly associated with an increased risk of EC, while ERβ has the opposite effects on ERα function. GPER is highly expressed in abnormal EH, but its expression in EC patients is paradoxical. Effective treatments for endometrium-related diseases depend on understanding the physiological function of ERs; however, much less is known about the signaling pathways through which ERs functions in the normal endometrium or in endometrial diseases. Given the important roles of ERs in the endometrium, we reviewed the published literature to elaborate the regulatory role of estrogen and its nuclear and membrane-associated receptors in maintaining the function of endometrium and to provide references for protecting female reproduction. Additionally, the role of drugs such as tamoxifen, raloxifene, fulvestrant and G-15 in the endometrium are also described. Future studies should focus on evaluating new therapeutic strategies that precisely target specific ERs and their related growth factor signaling pathways.

Skeletal muscle fibers contain a large number of mitochondria, which produce ATP through oxidative phosphorylation (OXPHOS) and provide energy for muscle contraction. In this process, mitochondria also produce several types of “reactive species” as side product, such as reactive oxygen species and reactive nitrogen species which have attracted interest. Mitochondria have been proven to have an essential role in the production of skeletal muscle reactive oxygen/nitrogen species (RONS). Traditionally, the elevation in RONS production is related to oxidative stress, leading to impaired skeletal muscle contractility and muscle atrophy. However, recent studies have shown that the optimal RONS level under the action of antioxidants is a critical physiological signal in skeletal muscle. Here, we will review the origin and physiological functions of RONS, mitochondrial structure and function, mitochondrial dynamics, and the coupling between RONS and mitochondrial oxidative stress. The crosstalk mechanism between mitochondrial function and RONS in skeletal muscle and its regulation of muscle stem cell fate and myogenesis will also be discussed. In all, this review aims to describe a comprehensive and systematic network for the interaction between skeletal muscle mitochondrial function and RONS.

Ovarian cancer is a heterogeneous disease and is also the major cause of death among women from gynecologic malignancies. A combination of surgery and chemotherapy is the major therapy for ovarian cancer. Unfortunately, despite good response rates to initial surgery and chemotherapy, most patients relapse and have a generally poor survival rate. The present research sheds light on the therapeutic effects of multiple natural products in patients with ovarian cancer. Notably, these natural ingredients do not have adverse effects on healthy cells and tissues, indicating that natural products can serve as a safe alternative therapy for ovarian cancer. Trans-3,4,5′-Trihydroxystibene (resveratrol) is a natural product that is commonly found in the human diet and that has been shown to have anticancer effects on various human cancer cells. This review summarizes current knowledge regarding the progress of resveratrol against tumor cell proliferation, metastasis, apoptosis induction, autophagy, sensitization, and antioxidation as well as anti-inflammation. It also provides information regarding the role of resveratrol analogues in ovarian cancer. A better understanding of the role of resveratrol in ovarian cancer may provide a new array for the prevention and therapy of ovarian cancer.

In mammalian, the periodic growth and development of ovarian follicles constitutes the physiological basis of female estrus and ovulation. Concomitantly, follicular angiogenesis exerts a pivotal role in the growth of ovarian follicles. Melatonin (N-acetyl-5-methoxytryptamine, Mel), exists in follicle fluid, was suggested to affect the development of follicles and angiogenesis. This research was conducted to investigate the effects and mechanisms of Mel on the development of ovarian follicles and its angiogenesis. In total, 40 ICR mice at age of 3 weeks were allocated into four groups at liberty: control, Mel, FSH and FSH + Mel for a 12-day trial. Ovaries were collected at 8:00 a.m. on Day 13 for detecting the development of ovarian follicles and angiogenesis. Results indicated that Mel promoted the development of ovarian follicles of 50–250 μm (secondary follicles) and periphery angiogenesis, while FSH remarkably increased the number of antral follicles and periphery angiogenesis. Mechanically, Mel and FSH may regulate the expression of VEGF and antioxidant enzymes in different follicular stages. In conclusion, Mel primarily acted on the secondary follicles, while FSH mainly promoted the development of antral follicles. They both conduced to related periphery angiogenesis by increasing the expression of VEGF. These findings may provide new targets for the regulating of follicular development.

Melatonin is a pleiotropic molecule with a variety of biological functions, which include its immunoregulatory action in mammals. Brucellosis is a worldwide endemic zoonotic disease caused by the Brucella, which not only causes huge economic losses for the livestock industry but also impacts human health. To target this problem, in current study, two marker-free transgenic sheep overexpressing melatonin synthetic enzyme ASMT (acetylserotonin O-methyltransferase) gene were generated and these melatonin enrich transgenic sheep were challenged by Brucella infection. The results showed that the serum melatonin concentration was significantly higher in transgenic sheep than that of wild type (726.92 ± 70.6074 vs 263.10 ± 34.60 pg/mL, P < .05). Brucella challenge test showed that two thirds (4/6) of the wild-type sheep had brucellosis, while none of the transgenic sheep were infected. Whole-blood RNA-seq results showed that differential expression genes (DEGs) were significantly enriched in natural killer cell-mediated cytotoxicity, phagosome, antigen processing, and presentation signaling pathways in overexpression sheep. The DEGs of toll-like receptors (TLRs) and NOD-like receptors (NLRs) families were verified by qPCR and it showed that TLR1, TLR2, TLR7, CD14, NAIP, and CXCL8 expression levels in overexpression sheep were significantly higher and NLRP1, NLRP3, and TNF expression levels were significantly lower than those of wild type. The rectal feces were subjected to 16S rDNA amplicon sequencing, and the microbial functional analysis showed that the transgenic sheep had significantly lower abundance of microbial genes related to infectious diseases compared to the wild type, indicating overexpression animals are likely more resistant to infectious diseases than wild type. Furthermore, exogenous melatonin treatment relieved brucellosis inflammation by upregulating anti-inflammatory cytokines IL-4 and downregulating pro-inflammatory IL-2, IL-6, and IFN-γ. Our preliminary results provide an informative reference for the study of the relationship between melatonin and brucellosis.

Spermatogonial stem cells (SSCs) self-renew and contribute genetic information to the next generation. Pig is wildly used as a model animal for understanding reproduction mechanisms of human being. Inducing directional differentiation of porcine SSCs may be an important strategy in exploring the mechanisms of spermatogenesis and developing better treatment methods for male infertility. Here, we established an in-vitro culture model for porcine small seminiferous tubule segments, to induce SSCs to differentiate into single-tail haploid spermatozoa. The culture model subsequently enabled spermatozoa to express the sperm-specific protein acrosin and oocytes to develop to blastocyst stage after round spermatid injection. The addition of retinoic acid (RA) to the differentiation media promoted the efficiency of haploid differentiation. RT-PCR analysis indicated that RA stimulated the expression of Stra8 but reduced the expression of NANOS2 in spermatogonia. Genes involved in post-meiotic development, transition protein 1 (Tnp1) and protamine 1 (Prm1) were upregulated in the presence of RA. The addition of an RA receptor (RAR) inhibitor, BMS439, showed that RA enhanced the expression of cAMP responsive-element binding protein through RAR and promoted the formation of round spermatids. We established an efficient culture system for in-vitro differentiation of pig SSCs. Our study represents a model for human testis disease and toxicology screening. Molecular regulators of SSC differentiation revealed in this study might provide a therapeutic strategy for male infertility.

Aims: In addition to pineal gland, many cells, tissues, and organs also synthesize melatonin (N-acetyl-5-methoxytryptamine). Embryos are a group of special cells and whether they can synthesize melatonin is still an open question. However, melatonin application promoted embryo development in many species in in vitro condition. The purpose of this study was to investigate whether embryos can synthesize melatonin; if it is so, what are the impacts of the endogenously produced melatonin on embryo development and the associated molecular mechanisms. These have never been reported previously. Results: Melatonin synthesis was observed at different stages of embryonic development. Aanat (aralkylamine N-acetyltransferase), a rate-limiting enzyme for melatonin production, was found to mostly localize in the mitochondria. Aanat knockdown significantly impeded embryonic development, and melatonin supplementation rescued it. The potential mechanisms might be that melatonin preserved mitochondrial intact and its function, thus providing sufficient adenosine 5'-triphosphate for the embryo development. In addition, melatonin scavenged intracellular reactive oxygen species (ROS) and reduced the DNA mutation induced by oxidative stress. In the molecular level, Aanat knockdown reduced tet methylcytosine dioxygenase 2 (Tet2) expression and DNA demethylation in blastocyst and melatonin supplementation rescued these processes. Innovation: This is the first report to show that embryos synthesize melatonin, and its synthetic enzyme Aanat was located in the mitochondria of embryos. An effect of melatonin is to maintain Tet2 expression and normal methylation status, and thereby promote embryonic development. Conclusion: Embryos can produce melatonin that reduces ROS production, preserves mitochondrial function, and maintains Tet2 expression and the normal DNA methylation.

Mammalian fertilization that culminates by fusion of the male and female gametes is intricately regulated within the female reproductive tract. To become competent to fertilize an egg, the mammalian spermatozoa that enter the female reproductive tract must undergo a series of physiological changes, including hyperactivation, and capacitation. For reaching full competency, the acrosome, a specialized membrane-bound organelle that covers the anterior part of the sperm head, must undergo an acrosome reaction. For becoming competent to bind an ovum, and to penetrate the zona pellucida and cumulus, many sperm proteins are released in the course of the acrosome reaction. Ultimately, the acrosome binds to the oolemma and fusion of sperm and egg occurs. In this review, we outline current understanding of the roles and effects of some essential sperm proteins and their functions during fertilization in the female reproductive tract.

Background Gram-negative bacterial infections have a major economic impact on both the livestock industry and public health. Toll-like receptor 4 (TLR4) plays a crucial role in host defence against Gram-negative bacteria. Exploring the defence mechanism regulated by TLR4 may provide new targets for treatment of inflammation and control of bacterial infections. In a previous study, we generated transgenic sheep overexpressing TLR4 by microinjection to improve disease resistance. The defence mechanism through which TLR4 overexpression protected these sheep against pathogens is still not fully understood. Results In the present study, we used Escherichia coli to infect monocytes isolated from peripheral blood of the animal model. The overexpression of TLR4 strongly enhanced the percentage of endocytosis and capacity of elimination in monocytes during the early stages of infection. This phenomenon was mainly due to overexpression of TLR4 promoting caveolae-mediated endocytosis. Pretreatment of the transgenic sheep monocytes with inhibitors of TLR4, Src signalling, or the caveolae-mediated endocytosis pathway reduced the internalization of bacteria, weakened the ability of the monocytes to eliminate the bacteria, and increased the pH of the endosomes. Conclusion Together, our results reveal the effects of TLR4 on the control of E. coli infection in the innate immunity of sheep and provide crucial evidence of the caveolae-mediated endocytosis pathway required for host resistance to invading bacteria in a large animal model, providing theoretical support for breeding disease resistance in the future. Furthermore, Src and caveolin 1 (CAV1) could be potentially valuable targets for the control of infectious diseases.

Female infertility is mainly caused by ovulation disorders, which affect female reproduction and pregnancy worldwide, with polycystic ovary syndrome (PCOS) being the most prevalent of these. PCOS is a frequent endocrine disease that is associated with abnormal function of the female sex hormone estrogen and estrogen receptors (ERs). Estrogens mediate genomic effects through ERα and ERβ in target tissues. The G-protein-coupled estrogen receptor (GPER) has recently been described as mediating the non-genomic signaling of estrogen. Changes in estrogen receptor signaling pathways affect cellular activities, such as ovulation; cell cycle phase; and cell proliferation, migration, and invasion. Over the years, some selective estrogen receptor modulators (SERMs) have made substantial strides in clinical applications for subfertility with PCOS, such as tamoxifen and clomiphene, however the role of ER in PCOS still needs to be understood. This article focuses on the recent progress in PCOS caused by the abnormal expression of estrogen and ERs in the ovaries and uterus, and the clinical application of related targeted small-molecule drugs.

Gram-negative bacterial infections of the testis can lead to infectious orchitis, which negatively influences steroid hormone synthesis and spermatogenesis. Lipopolysaccharide (LPS), a major component of the Gram-negative bacterial cell wall, acts via toll like receptors 4 (TLR4) to trigger innate immune responses and activate nuclear factor kappa B signaling. The protective mechanisms of melatonin on LPS-induced infectious orchitis have not been reported. Herein, we developed an LPS-induced sheep infectious orchitis model. In this model, the phagocytic activity of testicular macrophages (TM) was enhanced after melatonin treatment. Moreover, we found that melatonin suppressed secretion of TM pro-inflammatory factors by suppressing the p38MAPK pathway and promoting Leydig cell testosterone secretion. Expressions of GTP cyclohydrolase-I and NADPH oxidase-2 were reduced by melatonin while heme oxygenase-1 expression was up-regulated. Thus, melatonin reduced the severity of LPS-induced orchitis by stimulating antioxidant activity. The results of this study provide a reference for the treatment of acute infectious orchitis.

Hyperthecosis syndrome is a common endocrine system metabolic disorder in women of childbearing age. The main symptoms are elevated androgen levels, abnormal ovulation, and excessive oxidative stress. Currently, there is no effective treatment for hyperthecosis syndrome. α(1)-adrenergic receptor (ADRA1) is involved in the metabolic pathway of ovarian steroid hormone. This study studied the mechanism of the ADRA1 inhibitor terazosin in the LH-induced bovine theca cells in vitro. We found that terazosin regulates the expression of steroidogenic factor 1 (SF1) and downstream genes through the ERK1/2 pathway, reducing androgen content. Terazosin promotes the expression of HSP90 and reduces the activity of iNOS. In addition, Terazosin up-regulates the expression of nuclear factor erythroid 2-related factor 2 (Nrf2) and its downstream gene γ-GCS, which improves the ability of theca cells to resist oxidative stress. This study provides a reference for the treatment of human hyperthecosis syndrome.

In the field of assisted reproductive technology, female fertility preservation, particularly ovarian tissue cryopreservation in adolescent cancer patients, has attracted much attention. Melatonin (MLT) is well known for its antioxidative and anti-apoptotic properties; however, whether it can ameliorate the cryoinjury and inhibit the generation of reactive oxygen species (ROS) in cryopreserved ovarian tissues (OTs) has not yet been reported. Here, we demonstrated that MLT could protect follicular integrity; prevent cell apoptosis; decrease ROS, malondialdehyde (MDA), and nitric oxide (NO) levels; and increase activities of glutathione peroxidases (GSH-Px), glutathione (GSH), catalase (CAT), and superoxide dismutase (SOD) in cryopreserved OTs. Furthermore, these effects may be related with the activation of the nuclear factor erythroid 2-related factor 2 (Nrf2) signaling pathway, as evidenced by increased mRNA levels of Nrf2 downstream genes, including heme oxygenase-1 (HO-1), glutathione S-transferase M1 (GSTM1), SOD, and CAT. In summary, MLT can not only directly scavenge ROS but also significantly induce the activation of antioxidative enzymes via the Nrf2 signaling pathway, which is a new mechanism underlying the protection effects of MLT on cryopreserved OTs.

Uterine transplantation (UTx) is the only effective treatment for uterine infertility patients to become genetic mothers. After decades of research, the surgical methods of UTx are very developed. There are numerous factors that affect the results of UTx, such as selection of the donor uterus before transplantation, immunosuppressive therapy post-transplantation, rejection monitoring, and immune tolerance. Studies have shown that immune rejection is a crucial factor affecting the survival rate after organ transplantation. Unlike liver or kidney transplantation, the aim of UTx is to obtain a functional uterus that is able to support successful pregnancy and birth of a healthy fetus. Because of the unique purpose of UTx, its immunosuppressive program is relatively specialized. Some immunosuppressive agents can cause perinatal complications, and inducing immune tolerance is necessary to resolve these side effects. Further understanding of the immune mechanism of UTx and the continuous development of new immunosuppressive agents, combined with the application of assisted reproductive technology, will be more conducive to the realization of UTx to breed offspring.

Background Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR associated protein 9 (CRISPR/Cas9) has been wildly used to generate gene knockout models through inducing indels causing frame-shift. However, there are few studies concerning the post-transcript effects caused by CRISPR-mediated genome editing. Results In the present study, we showed that gene knockdown model also could be generated using CRISPR-mediated gene editing by disrupting the boundary of exon and intron in mice (C57BL/6 J). CRISPR induced indel at the boundary of exon and intron (5′ splice site) caused alternative splicing and produced multiple different mRNAs, most of these mRNAs introduced premature termination codon causing down expression of the gene. Conclusions These results showed that alternative splicing mutants were able to generate through CRISPR-mediated genome editing by deleting the boundary of exon and intron causing disruption of 5′ splice site. Although alternative splicing was an unexpected outcome, this finding could be developed as a technology to generate gene knockdown models or to investigate pre-mRNA splicing. Electronic supplementary material The online version of this article (10.1186/s12896-018-0472-8) contains supplementary material, which is available to authorized users.

Cyclin B1 and its interaction with CDK1 are thought to be critical for meiosis I progression in oocytes. However, using oocyte-specific conditional knockouts, Li et al. show that Cyclin B2 activity can compensate for Cyclin B1 to trigger meiosis resumption., Mammalian oocytes are arrested at the prophase of the first meiotic division for months and even years, depending on species. Meiotic resumption of fully grown oocytes requires activation of M-phase–promoting factor (MPF), which is composed of Cyclin B1 and cyclin-dependent kinase 1 (CDK1). It has long been believed that Cyclin B1 synthesis/accumulation and its interaction with CDK1 is a prerequisite for MPF activation in oocytes. In this study, we revealed that oocyte meiotic resumption occurred in the absence of Cyclin B1. Ccnb1-null oocytes resumed meiosis and extruded the first polar body. Without Cyclin B1, CDK1 could be activated by up-regulated Cyclin B2. Ccnb1 and Ccnb2 double knockout permanently arrested the oocytes at the prophase of the first meiotic division. Oocyte-specific Ccnb1-null female mice were infertile due to failed MPF activity elevation and thus premature interphase-like stage entry in the second meiotic division. These results have revealed a hidden compensatory mechanism between Cyclin B1 and Cyclin B2 in regulating MPF and oocyte meiotic resumption.

To explore the anti-inflammatory activity of endogenous produced melatonin, a melatonin-enriched animal model (goat) with AANAT transfer was successfully generated with somatic cell nuclear transfer (SCNT) technology. Basically, a pIRES2-EGFP-AANAT expression vector was constructed and was transferred into the female fetal fibroblast cells (FFCs) via electrotransfection and then the nuclear of the transgenic FFC was transferred to the eggs of the donor goats. The peripheral blood mononuclear cells (PBMCs) of the transgenic offspring expressed significantly higher levels of AANAT and melatonin synthetic function than those PBMCs from the wild-type (WT) animals. After challenge with lipopolysaccharide (LPS), the transgenic PBMCs had increased autophagosomes and LC3B expression while they exhibited suppressed production of the proinflammatory cytokines, IL1B and IL12 (IL12A-IL12B/p70), compared to their WT. The mechanistic analysis indicated that the anti-inflammatory activity of endogenous melatonin was mediated by MTNR1B (melatonin receptor 1B). MTNR1B stimulation activated the MAPK14 signaling pathway to promote cellular macroautophagy/autophagy, thus, suppressing the excessive inflammatory response of cellular. However, when the intact animals challenged with LPS, the serum proinflammatory cytokines were significantly higher in the transgenic goats than that in the WT. The results indicated that endogenous melatonin inhibited the MAPK1/3 signaling pathway and ROS production, subsequently downregulated gene expression of BECN1, ATG5 in PMBCs and then suppressed the autophagy activity of PBMCs and finally elevated levels of serum proinflammatory cytokines in transgenic animals, Herein we provided a novel melatonin-enriched animal model to study the potential effects of endogenously produced melatonin on inflammatory responses and autophagy activity.

Background/Aims: Phagocytosis of bacteria by monocytes/macrophages can trigger the immune response and the clearance of bacteria. This innate immune response involves Toll-like receptor 4 (TLR4). However, much remains unknown about the mechanism of TLR4-regulated phagocytosis of Salmonella enterica serovar Typhimurium (S. typhimurium) within sheep monocytes/macrophages. Here, we aimed to address these knowledge gaps by infecting transgenic sheep overexpressing TLR4 with S. typhimurium and examining the phagocytic mechanisms involved. Methods: Transgenic sheep were generated by microinjection of the constructed plasmids into fertilized eggs. Monocytes/macrophages isolated from sheep blood were stimulated with LPS and S. typhimurium. Phagocytosis-related factor expression, phagocytic ability, and adhesion were then determined. TLR4/phosphatidylinositide 3-kinase (PI3K) signaling was inhibited to investigate if this pathway is involved in changes in bacterial internalization in sheep. Results: We found that TLR4 overexpression effectively activated the PI3K signaling pathway and upregulated the expression of scavenger receptors. Additionally, actin polymerization and adhesive capacity were both enhanced in TLR4-overexpressing sheep monocytes/macrophages. TLR4 inhibition decreased S. typhimurium phagocytosis by reducing the actin polymerization and adhesive capacity of cells. Furthermore, inhibition of PI3K markedly impaired TLR4-dependent phagocytosis by restraining actin polymerization and scavenger receptor expression and reduced the adhesive capacity of the monocytes/macrophages. Conclusion: Our findings indicate that overexpression of TLR4 enhances phagocytosis through PI3K signaling and the subsequent activation of actin polymerization and scavenger receptors in sheep monocytes/macrophages infected with S. typhimurium.

Escherichia coli is one of the most common causal pathogens of mastitis in milk-producing mammals. Toll-like receptor 4 (TLR4) is important for host recognition of this bacteria. Increased activation of TLR4 can markedly enhance the internalization of E. coli. In this study, the relationship between TLR4 and mitogen-activated protein kinase (MAPK) signaling pathways in mediating E. coli internalization was evaluated in sheep monocytes. Using a TLR4-overexpressing transgenic (Tg) sheep model, we explored the bacterial internalization mechanism in sheep. We found that monocytes of Tg sheep could phagocytize more bacteria and exhibited higher adhesive capacity. The specific inhibition of p38 MAPK or c-Jun N-terminal kinase (JNK) or extracellular signal-regulated kinases (ERKs) reduced TLR4-dependent internalization of bacteria into sheep monocytes. Furthermore, the inhibition of MAPK signaling down-regulated the adhesive capacity of monocytes and the expression of scavenger receptors and adhesion molecules. Taken together, the overexpression of TLR4 in transgenic sheep enhanced the internalization of E. coli via MAPK signaling.

Leydig cells synthesize and secrete testosterone, and are regulated by Sertoli cells. These two cell types may work together to regulate testicular androgen production. Studies have shown that Leydig cell androgen synthesis can be dramatically enhanced by Sertoli cells in the presence of melatonin, which can regulate the secretory function of Leydig and Sertoli cells. However, the molecular mechanism of melatonin-regulated Leydig cell androgen production via Sertoli cells remains unclear. Here, we found that 10-7 M melatonin increased testosterone production in co-cultured Leydig and Sertoli cells isolated from sheep. Melatonin increased the expression of stem cell factor and insulin-like growth factor-1 and decreased estrogen synthesis in Sertoli cells. Melatonin promoted insulin-like growth factor-1 and decreased estrogen content via the membrane melatonin receptor 1. It also enhanced stem cell factor expression via the retinoic acid receptor-related orphan receptor alpha. Addition of PD98059, a MEK inhibitor, to Sertoli cell culture demonstrated that the melatonin upregulation of insulin-like growth factor-1 and downregulation of estrogen may be through the MEK/extracellular signal-regulated kinase pathway. Together, these results suggest that melatonin may function through modulating melatonin receptor 1-regulated insulin-like growth factor-1 expression, as well as melatonin receptor 1-induced suppression of estrogen synthesis to increase androgen production in co-cultured Leydig and Sertoli cells.