Extracellular HSPs: The Potential Target for Human Disease Therapy.

Heat shock proteins (HSPs) are highly conserved stress proteins known as molecular chaperones, which are considered to be cytoplasmic proteins with functions restricted to the intracellular compartment, such as the cytoplasm or cellular organelles. However, an increasing number of observations have shown that HSPs can also be released into the extracellular matrix and can play important roles in the modulation of inflammation and immune responses. Recent studies have demonstrated that extracellular HSPs (eHSPs) were involved in many human diseases, such as cancers, neurodegenerative diseases, and kidney diseases, which are all diseases that are closely linked to inflammation and immunity. In this review, we describe the types of eHSPs, discuss the mechanisms of eHSPs secretion, and then highlight their functions in the modulation of inflammation and immune responses. Finally, we take cancer as an example and discuss the possibility of targeting eHSPs for human disease therapy. A broader understanding of the function of eHSPs in development and progression of human disease is essential for developing new strategies to treat many human diseases that are critically related to inflammation and immunity.

Protective effects of melatonin on male fertility preservation and reproductive system.

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.

Oestrogenic Endocrine Disruptors in the Placenta and the Fetus.

Endocrine disrupting chemicals (EDCs) are exogenous substances that interfere with the stability and regulation of the endocrine system of the body or its offspring. These substances are generally stable in chemical properties, not easy to be biodegraded, and can be enriched in organisms. In the past half century, EDCs have gradually entered the food chain, and these substances have been frequently found in maternal blood. Perinatal maternal hormone levels are unstable and vulnerable to EDCs. Some EDCs can affect embryonic development through the blood-fetal barrier and cause damage to the neuroendocrine system, liver function, and genital development. Some also effect cross-generational inheritance through epigenetic mechanisms. This article mainly elaborates the mechanism and detection methods of estrogenic endocrine disruptors, such as bisphenol A (BPA), organochlorine pesticides (OCPs), diethylstilbestrol (DES) and phthalates (PAEs), and their effects on placenta and fetal health in order to raise concerns about the proper use of products containing EDCs during pregnancy and provide a reference for human health.

In-vitro differentiation of early pig spermatogenic cells to haploid germ cells.

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.

Regulation of blood-testis barrier assembly in vivo by germ cells.

The assembly of the blood-testis barrier (BTB) during postnatal development is crucial to support meiosis. However, the role of germ cells in BTB assembly remains unclear. Herein, KitW/KitWV mice were used as a study model. These mice were infertile, failing to establish a functional BTB to support meiosis due to c-Kit mutation. Transplantation of undifferentiated spermatogonia derived from normal mice into the testis of KitW/KitWV mice triggered functional BTB assembly, displaying cyclic remodeling during the epithelial cycle. Also, transplanted germ cells were capable of inducing Leydig cell testosterone production, which could enhance the expression of integral membrane protein claudin 3 in Sertoli cells. Early spermatocytes were shown to play a vital role in directing BTB assembly by expressing claudin 3, which likely created a transient adhesion structure to mediate BTB and cytoskeleton assembly in adjacent Sertoli cells. In summary, the positive modulation of germ cells on somatic cell function provides useful information regarding somatic-germ cell interactions.-Li, X.-Y., Zhang, Y., Wang, X.-X., Jin, C., Wang, Y.-Q., Sun, T.-C., Li, J., Tang, J.-X., Batool, A., Deng, S.-L., Chen, S.-R., Cheng, C. Y., Liu, Y.-X. Regulation of blood-testis barrier assembly in vivo by germ cells.

CRISPR/Cas9-mediated genome editing induces gene knockdown by altering the pre-mRNA splicing in mice.

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.

Melatonin Regulates the Synthesis of Steroid Hormones on Male Reproduction: A Review.

Melatonin is a ubiquitous molecule and exhibits different effects in long-day and short-day breeding animals. Testosterone, the main resource of androgens in the testis, is produced by Leydig cells but regulated mainly by cytokine secreted by Sertoli cells. Melatonin acts as a local modulator of the endocrine activity in Leydig cells. In Sertoli cells, melatonin influences cellular proliferation and energy metabolism and, consequently, can regulate steroidogenesis. These suggest melatonin as a key player in the regulation of steroidogenesis. However, the melatonin-induced regulation of steroid hormones may differ among species, and the literature data indicate that melatonin has important effects on steroidogenesis and male reproduction.

EZH2 deletion promotes spermatogonial differentiation and apoptosis.

Spermatogenesis is crucial for male fertility and is therefore tightly controlled by a variety of epigenetic regulators. However, the function of enhancer of zeste homolog 2 (EZH2) in spermatogenesis and the molecular mechanisms underlying its activity remain poorly defined. Here, we demonstrate that deleting EZH2 promoted spermatogonial differentiation and apoptosis. EZH2 is expressed in spermatogonia, spermatocytes and round and elongated spermatids from stage 9 to 11 but not in leptotene and zygotene spermatocytes. Knocking down Ezh2 in vitro using a lentivirus impaired self-renewal in spermatogonial stem cells (SSCs), and the conditional knockout of Ezh2 in spermatogonial progenitors promoted precocious spermatogonial differentiation. EZH2 functions to balance self-renewal and differentiation in spermatogonia by suppressing NEUROG3 and KIT via a direct interaction that is independent of its histone methyltransferase activity. Moreover, deleting Ezh2 enhanced the activation of CASP3 in spermatids, resulting in reduced spermatozoa production. Collectively, these data demonstrate that EZH2 plays a nonclassical role in the regulation of spermatogonial differentiation and apoptosis in murine spermatogenesis.

Melatonin promotes development of haploid germ cells from early developing spermatogenic cells of Suffolk sheep under in vitro condition.

Promotion of spermatogonial stem cell (SSC) differentiation into functional sperms under in vitro conditions is a great challenge for reproductive physiologists. In this study, we observed that melatonin (10(-7) M) supplementation significantly enhanced the cultured SSCs differentiation into haploid germ cells. This was confirmed by the expression of sperm special protein, acrosin. The rate of SSCs differentiation into sperm with melatonin supplementation was 11.85 ± 0.93% which was twofold higher than that in the control. The level of testosterone, the transcriptions of luteinizing hormone receptor (LHR), and the steroidogenic acute regulatory protein (StAR) were upregulated with melatonin treatment. At the early stage of SSCs culture, melatonin suppressed the level of cAMP, while at the later stage, it promoted cAMP production. The similar pattern was observed in testosterone content. Expressions for marker genes of meiosis anaphase, Dnmt3a, and Bcl-2 were upregulated by melatonin. In contrast, Bax expression was downregulated. Importantly, the in vitro-generated sperms were functional and they were capable to fertilize oocytes. These fertilized oocytes have successfully developed to the blastula stage.

The HPV viral regulatory mechanism of TLRs and the related treatments for HPV-associated cancers.

Infection with human papillomavirus (HPV) typically leads to cervical cancer, skin related cancers and many other tumors. HPV is mainly responsible for evading immune tumor monitoring in HPV related cancers. Toll like receptors (TLRs) are particular pattern recognition molecules. When the body is facing immune danger, it can lead to innate and direct adaptive immunity. TLR plays an important role in initiating antiviral immune responses. HPV can affect the expression level of TLR and interfere with TLR related signaling pathways, resulting in sustained viral infection and even carcinogenesis. This paper introduces the HPV virus and HPV related cancers. We discussed the present comprehension of TLR, its expression and signaling, as well as its role in HPV infection. We also provided a detailed introduction to immunotherapy methods for HPV related diseases based on TLR agonists. This will provide insights into methods that support the therapeutic method of HPV related conditions with TLR agonists.

Increasing GSH-Px Activity and Activating Wnt Pathway Promote Fine Wool Growth in FGF5-Edited Sheep.

Fibroblast growth factor 5 (FGF5) plays key roles in promoting the transition from the anagen to catagen during the hair follicle cycle. The sheep serves as an excellent model for studying hair growth and is frequently utilized in various research processes related to human skin diseases. We used the CRISPR/Cas9 system to generate four FGF5-edited Dorper sheep and only low levels of FGF5 were detected in the edited sheep. The density of fine wool in GE sheep was markedly increased, and the proportion of fine wool with a diameter of 14.4-20.0 µm was significantly higher. The proliferation signal in the skin of gene-edited (GE) sheep was stronger than in wild-type (WT) sheep. FGF5 editing decreased cortisol concentration in the skin, further activated the activity of antioxidant enzymes such as Glutathione peroxidase (GSH-Px), and regulated the expression of Wnt signaling pathways containing Wnt agonists (Rspondins, Rspos) and antagonists (Notum) in hair regeneration. We suggest that FGF5 not only mediates the activation of antioxidant pathways by cortisol, which constitutes a highly coordinated microenvironment in hair follicle cells, but also influences key signals of the Wnt pathway to regulate secondary hair follicle (SHF) development. Overall, our findings here demonstrate that FGF5 plays a significant role in regulating SHF growth in sheep and potentially serves as a molecular marker of fine wool growth in sheep breeding.

A MSTNDel73C mutation with FGF5 knockout sheep by CRISPR/Cas9 promotes skeletal muscle myofiber hyperplasia.

Mutations in the well-known Myostatin (MSTN) produce a 'double-muscle' phenotype, which makes it commercially invaluable for improving livestock meat production and providing high-quality protein for humans. However, mutations at different loci of the MSTN often produce a variety of different phenotypes. In the current study, we increased the delivery ratio of Cas9 mRNA to sgRNA from the traditional 1:2 to 1:10, which improves the efficiency of the homozygous mutation of biallelic gene. Here, a MSTNDel73C mutation with FGF5 knockout sheep, in which the MSTN and FGF5 dual-gene biallelic homozygous mutations were produced via the deletion of 3-base pairs of AGC in the third exon of MSTN, resulting in cysteine-depleted at amino acid position 73, and the FGF5 double allele mutation led to inactivation of FGF5 gene. The MSTNDel73C mutation with FGF5 knockout sheep highlights a dominant 'double-muscle' phenotype, which can be stably inherited. Both F0 and F1 generation mutants highlight the excellent trait of high-yield meat with a smaller cross-sectional area and higher number of muscle fibers per unit area. Mechanistically, the MSTNDel73C mutation with FGF5 knockout mediated the activation of FOSL1 via the MEK-ERK-FOSL1 axis. The activated FOSL1 promotes skeletal muscle satellite cell proliferation and inhibits myogenic differentiation by inhibiting the expression of MyoD1, and resulting in smaller myotubes. In addition, activated ERK1/2 may inhibit the secondary fusion of myotubes by Ca2+-dependent CaMKII activation pathway, leading to myoblasts fusion to form smaller myotubes.

Improving the Efficiency of Precise Genome Editing with CRISPR/Cas9 to Generate Goats Overexpressing Human Butyrylcholinesterase.

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.

Toll-like receptor (TLRs) agonists and antagonists for COVID-19 treatments.

Severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) rapidly infects humans and animals which make coronavirus disease 2019 (COVID-19) a grievous epidemic worldwide which broke out in 2020. According to data analysis of the other coronavirus family, for instance severe acute respiratory syndrome SARS coronavirus (SARS-CoV), can provide experience for the mutation of SARS-CoV-2 and the prevention and treatment of COVID-19. Toll-like receptors (TLRs) as a pattern recognition receptor (PRRs), have an indispensable function in identifying the invader even activate the innate immune system. It is possible for organism to activate different TLR pathways which leads to secretion of proinflammatory cytokines such as Interleukin 1 (IL-1), Interleukin 6 (IL-6), Tumor necrosis factor α (TNFα) and type Ⅰ interferon. As a component of non-specific immunity, TLRs pathway may participate in the SARS-CoV-2 pathogenic processes, due to previous works have proved that TLRs are involved in the invasion and infection of SARS-CoV and MERS to varying degrees. Different TLR, such as TLR2, TLR4, TLR7, TLR8 and TLR9 probably have a double-sided in COVID-19 infection. Therefore, it is of great significance for a correctly acknowledging how TLR take part in the SARS-CoV-2 pathogenic processes, which will be the development of treatment and prevention strategies.

Relationship between Tumor Infiltrating Immune Cells and Tumor Metastasis and Its Prognostic Value in Cancer.

Tumor metastasis is an important reason for the difficulty of tumor treatment. Besides the tumor cells themselves, the tumor microenvironment plays an important role in the process of tumor metastasis. Tumor infiltrating immune cells (TIICs) are one of the main components of TME and plays an important role in every link of tumor metastasis. This article mainly reviews the role of tumor-infiltrating immune cells in epithelial mesenchymal transformation, extracellular matrix remodeling, tumor angiogenesis and formation of pre-metastatic niche. The value of TIICs in the prognosis of cervical cancer, lung cancer and breast cancer was also discussed. We believe that accurate prognosis of cancer treatment outcomes is conducive to further improving treatment regimens, determining personalized treatment strategies, and ultimately achieving successful cancer treatment. This paper elucidates the relationship between tumor and TIICs in order to explore the function of immune cells in different diseases and provide new ideas for the treatment of cancer.

Estrogen Receptor Function: Impact on the Human Endometrium.

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, ERß, 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 ERß 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.

Estrogen Biosynthesis and Signal Transduction in Ovarian Disease.

Estrogen mainly binds to estrogen receptors (ERs) to regulate menstrual cycles and reproduction. The expression of ERalpha (ERα), ERbeta (ERß), and G-protein-coupled estrogen receptor (GPER) mRNA could be detected in ovary, suggesting that they play an important role in estrogen signal transduction in ovary. And many studies have revealed that abnormal expression of estrogen and its receptors is closely related to ovarian disease or malignant tumors. With the continuous development and research of animal models, tissue-specific roles of both ERα and ERß have been demonstrated in animals, which enable people to have a deeper understanding of the potential role of ER in regulating female reproductive diseases. Nevertheless, our current understanding of ERs expression and function in ovarian disease is, however, incomplete. To elucidate the biological mechanism behind ERs in the ovary, this review will focus on the role of ERα and ERß in polycystic ovary syndrome (PCOS), ovarian cancer and premature ovarian failure (POF) and discuss the major challenges of existing therapies to provide a reference for the treatment of estrogen target tissue ovarian diseases.

Mitochondrial Function and Reactive Oxygen/Nitrogen Species in Skeletal Muscle.

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.

Melatonin prevents cyclophosphamide-induced primordial follicle loss by inhibiting ovarian granulosa cell apoptosis and maintaining AMH expression.

Cyclophosphaty -45mide (Cyc) chemotherapy in young female cancer patients is associated with an increased risk of premature ovarian insufficiency (POI). This study was designed to investigate the protective role of melatonin (Mel) as an adjuvant against Cyc-induced POI. Female mice received a single intraperitoneal (i.p.) dose of Cyc (75 mg/kg). Mel protection was achieved in mice after i.p. injection of melatonin (50 mg/kg) every 24 h for four consecutive days prior to chemotherapy initiation and for 14 additional days. Ovarian reserve testing, hormonal assays for follicle-stimulating hormone, luteinizing hormone, and anti-Müllerian hormone (AMH), assessment of the oxidative stress status, and measurement of the relative expression of genes in PTEN/AKT/FOXO3a and mitochondrial apoptosis pathways were performed. The results showed that treatment with 50 mg/kg Mel significantly prevented Cyc-induced over-activation of primordial follicles by maintaining the plasma level of AMH and subsequently preventing litter size reduction in mice treated with Cyc chemotherapy. Importantly, Mel treatment significantly prevented ovarian granulosa cell loss by inhibiting the mitochondrial apoptotic pathway. Identifying the protective actions of Mel against Cyc-induced primordial follicle loss has important implications for fertility maintenance in young cancer patients undergoing chemotherapy.

Advances in Innate Immunity to Overcome Immune Rejection during Xenotransplantation.

Transplantation is an effective approach for treating end-stage organ failure. There has been a long-standing interest in xenotransplantation as a means of increasing the number of available organs. In the past decade, there has been tremendous progress in xenotransplantation accelerated by the development of rapid gene-editing tools and immunosuppressive therapy. Recently, the heart and kidney from pigs were transplanted into the recipients, which suggests that xenotransplantation has entered a new era. The genetic discrepancy and molecular incompatibility between pigs and primates results in barriers to xenotransplantation. An increasing body of evidence suggests that innate immune responses play an important role in all aspects of the xenogeneic rejection. Simultaneously, the role of important cellular components like macrophages, natural killer (NK) cells, and neutrophils, suggests that the innate immune response in the xenogeneic rejection should not be underestimated. Here, we summarize the current knowledge about the innate immune system in xenotransplantation and highlight the key issues for future investigations. A better understanding of the innate immune responses in xenotransplantation may help to control the xenograft rejection and design optimal combination therapies.