Piezo1 and Piezo2 collectively regulate jawbone development.

Piezo1 and Piezo2 are recently reported mechanosensory ion channels that transduce mechanical stimuli from the environment into intracellular biochemical signals in various tissues and organ systems. Here, we show that Piezo1 and Piezo2 display a robust expression during jawbone development. Deletion of Piezo1 in neural crest cells causes jawbone malformations in a small but significant number of mice. We further demonstrate that disruption of Piezo1 and Piezo2 in neural crest cells causes more striking defects in jawbone development than any single knockout, suggesting essential but partially redundant roles of Piezo1 and Piezo2. In addition, we observe defects in other neural crest derivatives such as malformation of the vascular smooth muscle in double knockout mice. Moreover, TUNEL examinations reveal excessive cell death in osteogenic cells of the maxillary and mandibular arches of the double knockout mice, suggesting that Piezo1 and Piezo2 together regulate cell survival during jawbone development. We further demonstrate that Yoda1, a Piezo1 agonist, promotes mineralization in the mandibular arches. Altogether, these data firmly establish that Piezo channels play important roles in regulating jawbone formation and maintenance.

High Anti-Müllerian Hormone Strongly Correlates with Reproductive Outcomes in Women Undergoing Assisted Reproduction.

BACKGROUND: In a retrospective cohort of 881 women with gynecologic and unexplained infertility, we aimed to study the relationship between serum AMH levels and ART outcomes. This retrospective cohort includes 881 infertile women aged 20 - 45 who underwent their first fresh autologous non-preimplantation genetic diagnosis ART cycles between 2012 and 2020. METHODS: We assessed the correlation between AMH levels and reproductive outcomes among infertile women with different causes of infertility (including endometriosis, polycystic ovary syndrome (PCOS), and unexplained infertility). RESULTS: We found a strong correlation between high AMH levels and reproductive outcomes independent of age and the cause of infertility in women undergoing ART. In all patients with gynecologic and unexplained infertility, higher AMH correlated with the improved number of oocytes (p < 0.001), MII oocytes (p < 0.001), good-quality embryos (p < 0.001), chemical pregnancy rate (p < 0.001 in women < 37; and p = 0.002 in women over 37), clinical pregnancy rate (p < 0.05), and live birth rate (p = 0.05). CONCLUSIONS: Serum AMH concentrations can be invaluable for predicting ovarian reserve and reproductive outcomes in young and advanced-age infertile patients undergoing ART. However, it should not be used as the sole predictive marker for disqualifying infertile women from ART treatment. Further large cohort studies are warranted to determine an exact cutoff point for AMH to provide an accurate ART success prediction.

Melatonin Promotes Differentiation of Human Spermatogonial Stem Cells Cultured on Three-Dimensional Decellularized Human Testis Matrix.

PURPOSE: The use of 3D (3-Dimensional) culture systems supported cell-to-cell and cell-to-extracellular matrix (ECM) interactions, proliferation, and differentiation of SSCs (Spermatogonial stem cells). The potential advantages of ECM-based scaffolds for in vitro spermatogenesis have been indicated in human and animal experiments. Furthermore, the strong antioxidant and anti-inflammatory activities of melatonin have improved in vitro manipulation of human SSCs in culture conditions. MATERIALS AND METHODS: SSCs were isolated from the testis of three dead-brain patients and then propagated for four weeks. The characterization of SSC colonies was done using real-time PCR (Polymerase chain reaction), ICC (Immunocytochemistry), and xenotransplantation to mice model. Decellularization of the human testis was performed using 0.3% sodium dodecyl sulfate (SDS) solution and 1% Triton X-100. Also, various characterizations of DTM (Decellularized testicular matrix ) were carried out using histological staining and DNA content analysis. The optimum dose of melatonin was selected by MTT (Methyl thiazol tetrazolium). SSCs were cultured in 4 groups: control, melatonin, ECM, and ECM-melatonin in a differentiation medium for four weeks. The expression of differentiation genes was evaluated by real-time polymerase chain reaction. In addition, the viability of cultured cells was assessed by MTT assay. RESULTS: The results of ICC and real-time PCR showed the expression of undifferentiated SSC markers (PLZF and GRFA1) in SSC colonies following the 2D culture of isolated SSCs. The presence of testicular ECM components after different staining methods; and the reduction of DNA content confirmed the proper decellularization process. Germ cell apoptosis significantly decreased in melatonin and ECM groups, and the higher viability of SSCs was seen in the ECM-melatonin group. The relative expression of GFRA1 and PRM2 decreased and increased in ECM and ECM-melatonin groups, respectively. CONCLUSION: Our study showed that the addition of melatonin to the human naturally-derived ECM scaffold could provide a suitable platform for inducing the differentiation and preserving the viability of SSCs.

Differentiation of human primary testicular cells in the presence of SCF using the organoid culture system.

PURPOSE: Development of organoids using human primary testicular cells has remained a challenge due to the complexity of the mammalian testicular cytoarchitecture and culture methods. In this study, we generated testicular organoids derived from human primary testicular cells. Then, we evaluated the effect of stem cell factor (SCF) on cell differentiation and apoptosis in the testicular organoid model. METHODS: The testicular cells were harvested from the three brain-dead donors. Human spermatogonial stem cells (SSCs) were characterized using immunocytochemistry (ICC), RT-PCR and flow cytometry. Testicular organoids were generated from primary testicular cells by hanging drop culture method and were cultured in three groups: control group, experimental group 1 (treated FSH and retinoic acid (RA)), and experimental group 2 (treated FSH, RA and SCF), for five weeks. We assessed the expression of SCP3 (Synaptonemal Complex Protein 3) as a meiotic gene, PRM2 (Protamine 2) as a post-meiotic marker and apoptotic genes of Bax (BCL2-Associated X Protein) and Bcl-2 (B-cell lymphoma 2), respectively by using RT-qPCR. In addition, we identified the expression of PRM2 by immunohistochemistry (IHC). RESULTS: Relative expression of SCP3, PRM2 and Bcl-2 were highest in group 2 after five weeks of culture. In contrast, BAX expression level was lower in experimental group 2 in comparison with other groups. IHC analyses indicated the highest expression of PRM2 as a postmeiotic marker in group 2 in comparison to 2D culture and control groups but not find significant differences between experimental group 1 and experimental group 2 groups. Morphological evaluations revealed that organoids are compact spherical structures and in the peripheral region composed of uncharacterized elongated fibroblast-like cells. CONCLUSION: Our findings revealed that the testicular organoid culture system promote the spermatogonial stem cell (SSC) differentiation, especially in presence of SCF. Developed organoids are capable of recapitulating many important properties of a stem cell niche.

Advances of three-dimensional (3D) culture systems for in vitro spermatogenesis.

The loss of germ cells and spermatogenic failure in non-obstructive azoospermia are believed to be the main causes of male infertility. Laboratory studies have used in vitro testicular models and different 3-dimensional (3D) culture systems for preservation, proliferation and differentiation of spermatogonial stem cells (SSCs) in recent decades. The establishment of testis-like structures would facilitate the study of drug and toxicity screening, pathological mechanisms and in vitro differentiation of SSCs which resulted in possible treatment of male infertility. The different culture systems using cellular aggregation with self-assembling capability, the use of different natural and synthetic biomaterials and various methods for scaffold fabrication provided a suitable 3D niche for testicular cells development. Recently, 3D culture models have noticeably used in research for their architectural and functional similarities to native microenvironment. In this review article, we briefly investigated the recent 3D culture systems that provided a suitable platform for male fertility preservation through organ culture of testis fragments, proliferation and differentiation of SSCs.

Substance P aggravates ligature-induced periodontitis in mice.

Periodontitis is one of the most common oral diseases in humans, affecting over 40% of adult Americans. Pain-sensing nerves, or nociceptors, sense local environmental changes and often contain neuropeptides. Recent studies have suggested that nociceptors magnify host response and regulate bone loss in the periodontium. A subset of nociceptors projected to periodontium contains neuropeptides, such as calcitonin gene-related peptide (CGRP) or substance P (SP). However, the specific roles of neuropeptides from nociceptive neural terminals in periodontitis remain to be determined. In this study, we investigated the roles of neuropeptides on host responses and bone loss in ligature-induced periodontitis. Deletion of tachykinin precursor 1 (Tac1), a gene that encodes SP, or treatment of gingiva with SP antagonist significantly reduced bone loss in ligature-induced periodontitis, whereas deletion of calcitonin related polypeptide alpha (Calca), a gene that encodes CGRP, showed a marginal role on bone loss. Ligature-induced recruitment of leukocytes, including neutrophils, and increase in cytokines leading to bone loss in periodontium was significantly less in Tac1 knockout mice. Furthermore, intra-gingival injection of SP, but not neurokinin A, induced a vigorous inflammatory response and osteoclast activation in alveolar bone and facilitated bone loss in ligature-induced periodontitis. Altogether, our data suggest that SP plays significant roles in regulating host responses and bone resorption in ligature-induced periodontitis.

Protective effects of human amniotic membrane derived mesenchymal stem cells (hAMSCs) secreted factors on mouse spermatogenesis and sperm chromatin condensation following unilateral testicular torsion.

Testicular torsion is considered a urological disorder that requires immediate detorsion surgery. Ischemia/reperfusion (I/R) injury after testicular torsion detorsion causes of drastic impairment of spermatogenesis and infertility. Cell-free-based approaches seem to be a promising strategy to prevent I/R injury, they have more stable biological properties, and they contain paracrine factors of mesenchymal stem cells. The purpose of this study was to evaluate the protective effects of human amniotic membrane derived mesenchymal stem cells (hAMSCs) secreted factors on mouse sperm chromatin condensation and spermatogenesis improvement after I/R injury. hAMSCs were isolated and characterized by RT- PCR and flow cytometry, preparation of hAMSCs secreted factors was performed. Forty male mice were randomly divided into 4 groups: sham-operated, torsion detorsion, torsion detorsion+ intratesticular injection of DMEM/F-12, and torsion detorsion+ intratesticular injection of hAMSCs secreted factors. After one cycle of spermatogenesis, the mean number of germ cells, Sertoli, Leydig, myoid as well as tubular parameters, Johnson score, and spermatogenesis indexes were evaluated by H& E and PAS stainings. Sperm chromatin condensation and relative expression of c-kit and prm 1 genes were assessed by aniline blue staining and real-time PCR, respectively. The mean number of spermatogenic cells, Leydig, myoid, Sertoli, spermatogenesis parameters, Johnson score, as well as germinal epithelial height and diameters of seminiferous tubules decreased significantly after I/R injury. The thickness of basement membrane and percentage of sperm with excessive histone significantly increased, while the relative expression of c-kit and prm 1 significantly decreased in torsion detorsion group (p 0.001). hAMSCs secreted factors remarkably restored normal sperm chromatin condensation, spermatogenesis parameters and histomorphometric organization of seminiferous tubules via intratesticular injection (p 0.001). Thus, hAMSCs secreted factors may potentially salvage torsion-detorsion-induced infertility.

Differentiation of human spermatogonial stem cells using a human decellularized testicular scaffold supplemented by platelet-rich plasma.

BACKGROUND: Effective culture systems for attachment, migration, proliferation, and differentiation of spermatogonial stem cells (SSCs) can be a promising therapeutic modality for preserving male fertility. Decellularized extracellular matrix (ECM) from native testis tissue creates a local microenvironment for testicular cell culture. Furthermore, platelet-rich plasma (PRP) contains various growth factors for the proliferation and differentiation of SSCs. METHODS: In this study, human testicular cells were isolated and cultured for 4 weeks, and SSCs were characterized using immunocytochemistry (ICC) and flow cytometry. Human testicular tissue was decellularized (0.3% SDS, 1% Triton), and the efficiency of the decellularization process was confirmed by histological staining and DNA content analysis. SSCs were cultured on the human decellularized testicular matrix (DTM) for 4 weeks. The viability and the expression of differentiation genes were evaluated by MTT and real-time polymerase chain reaction (PCR), respectively. RESULTS: Histological evaluation and DNA content analysis showed that the components of ECM were preserved during decellularization. Our results showed that after 4 weeks of culture, the expression levels of BAX, BCL-2, PLZF, and SCP3 were unchanged, while the expression of PRM2 significantly increased in the cells cultured on DTM supplemented with PRP (ECM-PRP). In addition, the expression of GFRA1 was significantly decreased in the ECM group compared to the control and PRP groups. Furthermore, the MTT test indicated that viability was significantly enhanced in cells plated on DTM supplemented with PRP. CONCLUSION: Our study demonstrated that DTM supplemented with PRP can provide an effective culture system for the differentiation and viability of SSCs.

Testicular Tissue Vitrification: a Promising Strategy for Male Fertility Preservation.

Destruction of spermatogonial stem cells in juvenile men survivors of pediatric cancers leads to infertility as a side effect of gonadotoxic therapies. Sperm freezing before cancer treatment is commonly used in the clinic for fertility preservation, but this method is not applicable for prepubertal boys due to the lack of mature sperm. In these cases, cryopreservation of testicular tissues is the only option for fertility preservation. Although controlled slow freezing (CSF) is the most common procedure for testicular tissue cryopreservation, vitrification can be used as an alternative method. Controlled vitrification has prevented cell damage and formation of ice crystals. Procedures were done easily and quickly with a brief exposure time to high concentration of cryoprotectants without expensive equipment. Different studies used vitrification of testicular tissues and they assessed the morphology of seminiferous tubules, apoptosis, and viability of spermatogonial cells. Transplantation of vitrified testicular tissue into infertile recipient mice as well as in vitro culture of vitrified tissues was done in previous studies and their findings showed complete spermatogenesis and production of mature sperm. Review articles usually have compared controlled slow freezing with vitrification. In this review, we focused only on the vitrification method and its results. Despite promising results, many studies have been done for finding an optimal cryopreservation protocol in order to successfully preserve fertility in prepubertal boys.

Evaluation of Apoptosis-related Genes and Hormone Secretion Profiles Using Three Dimensional Culture System of Human Testicular Organoids.

BACKGROUND: In reproductive biology, testicular organoids can be used to treat infertility and to study testicular development and spermatogonial stem cells (SSCs) differentiation. Generating organoid from primary cells is challenging. In this study, testicular organoids were created using human primary testicular cells and evaluated the apoptotic gene expression and hormone secretion profiles of the organoids. MATERIALS AND METHODS: Primary human testicular cells were isolated using 2-step enzymatic digestion from three brain-dead donors. Immunocytochemistry and flow cytometry analyses were performed to confirm human SSCs. Isolated cells were cultured in three experimental groups: control group (2 dimensional (2D)), group 1 (organoid culture after 2D culture), and group 2 (organoid culture immediately after enzymatic digestion). Testicular organoids were cultured in DMEM/F-12 media supplemented with follicle-stimulating hormone (FSH) and fetal bovine serum (FBS) for four weeks. After 24 hours and four weeks of culture, reverse transcription quantitative real-time PCR (RT-qPCR) was used to investigate the relative expression of apoptotic genes (caspase 3, 9, Bax, and Bcl-2). At 24 hours, two weeks, and four weeks after culture, enzyme-linked immunoassay (ELISA) was used to determine the testosterone and inhibin B concentrations. Light microscopy and toluidine blue staining were also used for morphological analysis. RESULTS: RT-qPCR results revealed that pro-apoptotic (caspase 3, 9, Bax) gene expression levels were highest in group 2 after 24 h and four weeks of culture. In contrast, the expression level of Bcl-2 (anti-apoptotic) was lower in group 2 compared to other groups. The hormone secretion levels decreased in a time-dependent manner during the cultivation. According to morphological evaluations, testicular organoids are compact, spherical structures with two to three elongated cells organized along their border. CONCLUSION: Our findings revealed that the testicular organoid culture system maintained hormonal secretory abilities, demonstrating the function of Sertoli and Leydig cells in the absence of testis-specific environments.

Chitosan Scaffold Containing Periostin Enhances Sternum Bone Healing and Decreases Serum Level of TNF-α and IL-6 after Sternotomy in Rat.

BACKGROUND: In the aftermath of bone injuries, such as cranium and sternum, bone wax (BW) is used to control bleeding from the bone surfaces during surgery. Made up of artificial substances, however, it is associated with many complications such as inflammation, increased risk for infection, and bone repair delay. We, therefore, in this study set out to design and evaluate a novel BW without the above-mentioned side-effects reported for other therapies. METHODS: The pastes (new BW(s)) were prepared in the laboratory and examined by MTT, MIC, MBC, and degradability tests. Then, 60 adult male Wistar rats, divided into six equal groups including chitosan (CT), CT-octacalcium phosphate (OCP), CT-periostin (Post), CT-OCP-Post, Control (Ctrl), and BW, underwent sternotomy surgery. Once the surgeries were completed, the bone repair was assessed radiologically and thereafter clinically in vivo and in vitro using CT-scan, H&E, ELISA, and qRT-PCR. RESULTS: All pastes displayed antibacterial properties and the CT-Post group had the highest cell viability compared to the control group. In contrast to the BW, CT-Post group demonstrated weight changes in the degradability test. In the CT-Post group, more number of osteocyte cells, high trabeculae percentage, and the least fibrous connective tissue were observed compared to other groups. Additionally, in comparison to the CT and Ctrl groups, higher alkaline phosphatase activity, as well as decreased level of serum tumor necrosis factor-α, interleukin-6, and OCN in the CT-Post group was evident. Finally, Runx2, OPG, and RANKL genes' expression was significantly higher in the CT-Post group than in other groups. CONCLUSION: Our results provide insights into the desirability of pastes in terms of cellular viability, degradability, antibacterial properties, and surgical site restoration compared to the BW group. Besides, Periostin could enhance the osteogenic properties of bone tissue defect site.

Fe3O4 magnetic nanoparticles improve the vitrification of mouse immature oocytes and modulate the pluripotent genes expression in derived pronuclear-stage embryos.

The vitrification of Germinal Vesicle (immature) oocytes is beneficial for preservation of fertility in cases involving reproductive problems. The use of nanoparticles (NP(s)) as vitrification aid is a novel approach towards improving vitrification efficiency. The efficacy of use of iron oxide (Fe3O4) nanoparticles as vitrification aid is reported in this paper. Immature oocytes from NMRI mice were collected and divided into non-vitrified (nVit), Vitrified (Vit) and Vitrified + NP (Vit+NP) groups. In the Vit+NP group, solutions containing Fe3O4 nanoparticles at three different concentrations (0.004%, 0.008% and 0.016% w/v) were separately added to the vitrification solution and their effects on the vitrification of the oocytes were compared. The concentration that was found to be best performing (0.004% w/v) was used in vitrification studies in subsequent experiments. Mitochondrial function, apoptosis incidence, ultrastructure alteration, nuclear maturity, embryo formation and genes expression (Nanog, Oct4, Cdx2, and Sox2) were evaluated in response to the addition of the nanoparticle solution during vitrification. Nuclear maturity of oocyte and embryo formation increased significantly (P ≤ 0.05) in the vitrified + NP group. Expression of Sox2 also increased significantly in both vitrified and vitrified + NP groups. While there was a significant increase in Oct4 expression in the vitrified group as compared to control, there was no significant difference between vitrified and Vit+NP groups. The expression of Cdx2 decreased significantly (P ≤ 0.05) in the Vit+NP group. From these observations, Fe3O4 nanoparticles could protect immature oocytes from cryodamages, positively affect vitrification and modulate the pluripotency of derived pronuclear-stage embryos.