Multifunctional Hydrogel of Recombinant Humanized Collagen Loaded with MSCs and MnO2 Accelerates Chronic Diabetic Wound Healing.

Chronic wound repair is a clinical treatment challenge. The development of multifunctional hydrogels is of great significance in the key aspects of treating chronic wounds, including reducing oxidative stress, promoting angiogenesis, and improving the natural remodeling of extracellular matrix and immune regulation. In this study, we prepared a composite hydrogel, sodium alginate (SA)@MnO2/recombinant humanized collagen III (RHC)/mesenchymal stem cells (MSCs), composed of SA, MnO2 nanoparticles, RHC, and MSCs. The hydrogel has high mechanical properties and good biocompatibility. In vitro, SA@MnO2/RHC/MSCs hydrogel effectively enhanced the formation of intricate tubular structures and angiogenesis and showed synergistic effects on cell proliferation and migration. In vivo, the SA@MnO2/RHC/MSCs hydrogel enhanced diabetes wound healing, rapid re-epithelization, favorable collagen deposition, and abundant wound angiogenesis. These findings demonstrated that the combined effects of SA, MnO2, RHC, and MSCs synergistically accelerate healing, resulting in a reduced healing time. These observed healing effects demonstrated the potential of this multifunctional hydrogel to transform chronic wound care and improve patient outcomes.

An endometrial biomimetic extracellular matrix (ECM) for enhanced endometrial regeneration using hyaluronic acid hydrogel containing recombinant human type III collagen.

Endometrial injury poses a significant challenge in tissue regeneration, with type III collagen (COL III) playing a pivotal role in maintaining endometrial integrity and facilitating repair. Our study explored the utility of recombinant human type III collagen (RHC) as an intervention for endometrial damage. To address the challenges associated with the inherent instability and rapid degradation of COL III in vivo, we developed an RHC-HA hydrogel by conjugating RHC with hyaluronic acid (HA), thus ensuring a more stable and sustained delivery. Our findings suggested that the RHC-HA hydrogel significantly promoted endometrial regeneration and restored fertility. The hydrogel facilitated prolonged retention of RHC in the uterus, leading to a substantial improvement in the repair process. The synergistic interaction between RHC and HA greatly enhances cell proliferation and adhesion, surpassing the efficacy of HA or RHC alone. Additionally, the RHC-HA hydrogel demonstrated notable anti-fibrotic effects, which are crucial for preventing abnormalities during endometrial healing. These findings suggested that the RHC-HA hydrogel presented a therapeutic strategy in the treatment of uterine endometrial injuries, which may improve female reproductive health.

Inhibition of miR-143-3p Restores Blood-Testis Barrier Function and Ameliorates Sertoli Cell Senescence.

Due to the increasing trend of delayed childbirth, the age-related decline in male reproductive function has become a widely recognized issue. Sertoli cells (SCs) play a vital role in creating the necessary microenvironment for spermatogenesis in the testis. However, the mechanism underlying Sertoli cell aging is still unclear. In this study, senescent Sertoli cells showed a substantial upregulation of miR-143-3p expression. miR-143-3p was found to limit Sertoli cell proliferation, promote cellular senescence, and cause blood-testis barrier (BTB) dysfunction by targeting ubiquitin-conjugating enzyme E2 E3 (UBE2E3). Additionally, the TGF-ß receptor inhibitor SB431542 showed potential in alleviating age-related BTB dysfunction, rescuing testicular atrophy, and reversing the reduction in germ cell numbers by negatively regulating miR-143-3p. These findings clarified the regulatory pathways underlying Sertoli cell senescence and suggested a promising therapeutic approach to restore BTB function, alleviate Sertoli cell senescence, and improve reproductive outcomes for individuals facing fertility challenges.

miR-143-3p Promotes Ovarian Granulosa Cell Senescence and Inhibits Estradiol Synthesis by Targeting UBE2E3 and LHCGR.

The ovary is a highly susceptible organ to senescence, and granulosa cells (GCs) have a crucial role in oocyte development promotion and overall ovarian function maintenance. As age advances, GCs apoptosis and dysfunction escalate, leading to ovarian aging. However, the molecular mechanisms underpinning ovarian aging remain poorly understood. In this study, we observed a correlation between the age-related decline of fertility and elevated expression levels of miR-143-3p in female mice. Moreover, miR-143-3p was highly expressed in senescent ovarian GCs. The overexpression of miR-143-3p in GCs not only hindered their proliferation and induced senescence-associated secretory phenotype (SASP) but also impeded steroid hormone synthesis by targeting ubiquitin-conjugating enzyme E2 E3 (Ube2e3) and luteinizing hormone and human chorionic gonadotropin receptor (Lhcgr). These findings suggest that miR-143-3p plays a substantial role in senescence and steroid hormone synthesis in GCs, indicating its potential as a therapeutic target for interventions in the ovarian aging process.

In vitroreconstitution of the hormone-responsive testicular organoids from murine primary testicular cells.

Increasing rates of male infertility require more experimental models to understand the mechanisms underlying male infertility.In vitroorganoids hold unprecedented promise for this purpose; however, the development of organoids with tissue architecture similar to that of the testisin vivoremains a challenge. Here, we generated testicular organoids derived from testicular cells by combining a hanging drop culture and a rotation culture system. Our results indicated that testicular cells could self-assemble into spheroid organoids with tubule-like structures in hanging drop culture. The organoids can subsequently be cultured and maintained in a rotation culture system. These established organoids have gene expression profiles similar to those of adult testis tissue, produce testosterone with preserved gonadotropin responsiveness, and exhibit sensitivity to reproductive toxicants. More importantly, each testicular organoid can be generated from only 2000 cells, and they maintain their proliferative ability after freezing and thawing. These features make it possible to obtain fresh primary testis cells from testicular biopsies taken from patients or endangered wild species, and to build individual-specific biobanks. These findings will help enable the exploration of self-organization process of testicular cells and provide an experimental model for reproductive biology research, pharmacotoxicology testing, and regenerative medicine.

Small-Molecule-Driven Direct Reprogramming of Fibroblasts into Functional Sertoli-Like Cells as a Model for Male Reproductive Toxicology.

Sertoli cells (SCs) are vital to providing morphological and nutritional support for spermatogenesis. Defects in SCs often lead to infertility. SCs transplantation is a promising potential strategy to compensate for SC dysfunction. However, isolation of SCs from testes is impractical due to obvious and ethical limitations. Here, a molecular cocktail is identified comprising of pan-BET family inhibitor (I-BET151), retinoic acid, and riluzole that enables the efficient conversion of fibroblasts into functional Sertoli-like cells (CiSCs). The gene expression profiles of CiSCs resemble those of mature SCs and exhibit functional properties such as the formation of testicular seminiferous tubules, engulfment of apoptotic sperms, supporting the survival of germ cells, and suppressing proliferation of primary lymphocytes in vitro. Moreover, CiSCs are sensitive to toxic substances, making them an alternative model to study the deleterious effects of toxicants on SCs. The study provides an efficient approach to reprogram fibroblasts into functional SCs by using pure chemical compounds.

Cervix chip mimicking cervical microenvironment for quantifying sperm locomotion.

As the gate for sperm swimming into the female reproductive tract, cervix is full of cervical mucus, which plays an important role in sperm locomotion. The fact that sperm cannot pass through the cervical mucus-cervix microenvironment will cause the male infertility. However, how the sperm swim across the cervix microenvironment remains elusive. We used hyaluronic acid (HA), a substitute of cervical mucus to mimic cervix microenvironment and designed a cervix chip to study sperm selection and behavior. An accumulation of sperm in HA confirmed that HA served as a reservoir for sperm, similar to cervical mucus. We found that sperm escaping from HA exhibited higher motility than the sperm accessing into HA, suggesting that HA functions as a filter to select sperm with high activity. Our findings construct a practical platform to explore the sophisticated interaction of sperm with cervix microenvironment, with elaborate swimming indicators thus provide a promising cervix chip for sperm selection with kinematic features on-demand. What's more, the cervix chip allows the convenient use in clinical infertility diagnosis, owing to the advantage of simple, fast and high efficiency.

Isolation of Primary Leydig Cells from Murine Testis.

In males, Leydig cells are the primary source of testosterone, which is necessary for testis development, masculinization, and spermatogenesis. Leydig cells are a valuable cellular model for basic research; thus, it is important to develop an improved method for isolation and purification of Leydig cells from testes. The available methods for Leydig cell isolation have some drawbacks, including the need for sophisticated instruments, high cost, tediousness, and time consumption. Here, we describe an improved protocol for isolation of primary Leydig cells from testicular tissue by digestion with collagenase IV.

Sertoli cell-derived exosome-mediated transfer of miR-145-5p inhibits Leydig cell steroidogenesis by targeting steroidogenic factor 1.

In the mammalian testis, two distinct populations of Sertoli cells (SCs), the immature SCs (ISCs) and adult SCs (ASCs), play significant roles in regulating the development and function of Leydig cells. However, the effect of different SC types on the function of Leydig cells is poorly understood. Here, our study showed that miR-145-5p expression was significantly different in SCs at different stages, with the highest expression observed in ISCs. Exosomes mediate the transfer of miR-145-5p from ISCs to Leydig cells. Overexpression of miR-145-5p in Leydig cells significantly downregulated steroidogenic gene expression and inhibited testosterone synthesis. Additionally, miR-145-5p functioned by directly targeted steroidogenic factor-1 (Sf-1) and downregulated the expression of SF-1, which further downregulated the expression of steroidogenic genes, induced accumulation of lipid droplets, and eventually suppressed testosterone production. These findings demonstrate that SC-derived miR-145-5p plays a significant role in regulating the functions of Leydig cells and may therefore serve as a diagnostic biomarker for male hypogonadism developmental abnormalities during puberty.

Sertoli cell-derived exosomal MicroRNA-486-5p regulates differentiation of spermatogonial stem cell through PTEN in mice.

Self-renewal and differentiation of spermatogonial stem cell (SSC) are critical for male fertility and reproduction, both of which are highly regulated by testicular microenvironment. Exosomal miRNAs have emerged as new components in intercellular communication. However, their roles in the differentiation of SSC remain unclear. Here, we observed miR-486-5p enriched in Sertoli cell and Sertoli cell-derived exosomes. The exosomes mediate the transfer of miR-486-5p from Sertoli cells to SSCs. Exosomes release miR-486-5p, thus up-regulate expression of Stra8 (stimulated by retinoic acid 8) and promote differentiation of SSC. And PTEN was identified as a target of miR-486-5p. Overexpression of miR-486-5p in SSCs down-regulates PTEN expression, which up-regulates the expression of STRA8 and SYCP3, promotes SSCs differentiation. In addition, blocking the exosome-mediated transfer of miR-486-5p inhibits differentiation of SSC. Our findings demonstrate that miR-486-5p acts as a communication molecule between Sertoli cells and SSCs in modulating differentiation of SSCs. This provides a new insight on molecular mechanisms that regulates SSC differentiation and a basis for the diagnosis, treatment, and prevention of male infertility.