Morteza Koruji

Correction for ‘Artificial testis: a testicular tissue extracellular matrix as a potential bio-ink for 3D printing’ by Zahra Bashiri et al. , Biomater. Sci. , 2021, 9 , 3465–3484, https://doi.org/10.1039/D0BM02209H.

Background Cryopreservation can lead to sperm damage, which is why incorporating extracellular vesicles derived from normal semen can help protect against the damage caused by freezing. Materials and methods Extracellular vesicles were obtained from normozoospermic semen samples (n = 15), and their effects on the cryopreservation process were studied in three groups: the control group (A, n = 6), the extracellular vesicles treated before freezing group (B, n = 6), and the extracellular vesicles treated after freezing group (C, n = 6). Characteristics of sperm after thawing, including morphology, viability, motility, total antioxidant capacity (TAC), the rate of apoptosis, mitochondrial membrane potential (MMP), and the DNA fragmentation index (DFI), as well as the expression levels of the BAX, BCL₂, CRISP₃, and KRAS genes, were examined. Results Group B displayed a significant increase in total motility, progressive motility, normal morphology, and MMP compared to groups A, and DNA fragmentation (DFI) showed a significant decrease compared to groups A and C. Also, vitality and TAC were significantly increased in group B compared to groups A and C. Decreased level of apoptosis was evident in group B compared to group A. Group A exhibited a significant increase in the expression of BAX/BCL₂ ratio compared to groups B and C. Significantly higher expression of KRAS and CRISP₃ was evident in group B compared to groups A and C. Conclusion Extracellular vesicles obtained from normozoospermic seminal plasma could protect against the adverse cryopreservation effects through increased MMP and TAC, as well as decreased expression of BAX/BCL₂ ratio and increased KRAS and CRISP₃.

Advancements in biomaterial engineering have enabled the creation of sophisticated in vitro culture platforms that closely mimic natural cellular microenvironments. This study focuses on designing an innovative bioprinted scaffold using a bioink composed of gelatin methacrylate (GelMA) and human testicular extracellular matrix (htECM) for constructing a bioartificial testis. Human testis was decellularized and confirmed using hematoxylin and eosin (H&E) staining and DNA quantification, while maintaining htECM components, as shown by Masson's trichrome, Alcian blue, and Orcein staining. This ECM was combined with GelMA to form a bioink for scaffold fabrication. Human spermatogonial stem cells (hSSCs) isolated using enzymatic digestion and confirmed by RT‐PCR. Then, we evaluated the mechanical and biological properties of the scaffolds, focusing on surface morphology, degradation rate, swelling behavior, MTT assay, and biocompatibility. The decellularization protocol for testicular tissue was effective in eliminating cellular debris while conserving the ECM compounds. GelMA and GelMA/htECM scaffolds revealed a porous structure with suitable pore size and porosity, while the GelMA/htECM scaffold had more porosity (p < 0.05). The swelling ratio of the GelMA scaffold was enhanced by adding htECM, whereas its degradation rate decreased. The MTT assay showed that both scaffolds have no cytotoxic properties. Moreover, the GelMA/htECM scaffold displayed more cell attachment and better biocompatibility properties compared to the GelMA scaffold. These results highlight the potential of the GelMA/htECM scaffold as an advanced platform for in vitro spermatogenesis models, facilitating sperm production from hSSCs. This approach offers a promising tool for studying SSC behavior and developing personalized treatments for male infertility.

Developing a bioactive testicular scaffold has been proposed as a potential option to preserve male fertility. Using the gas foaming method, this study aimed to fabricate an effective and highly porous scaffold derived from a gelatin-testicular extracellular matrix. Male ram testis decellularization was performed using a combination of NaCl buffer and Triton. Then, evaluations were done for 4′,6-diamidino-2-phenylindole, hematoxylin-eosin staining, and quantitative DNA content. Masson’s trichrome, Alcian blue, and orcein staining were conducted to ensure the maintenance of ECM components post-decellularization. Porous scaffolds were fabricated using gelatin incorporation with various concentrations of extracted ECM via the gas foaming method. The mechanical and structural characteristics of the scaffolds, along with evaluations of cell spreading and penetration depth, were performed. Furthermore, scaffolds were used to culture mouse spermatogonial stem cells to investigate the morphology, viability, and adhesion of the cells on the scaffolds. Our results showed successful decellularization of testicular tissue, resulting in significant removal of DNA content while preserving ECM major components. The hybrid scaffolds exhibited uniform porous microstructures with a pore size average ranging from 298-330 μm. There were no significant differences in biodegradation and swelling ratios between the scaffolds. The cell penetration index significantly increased in gel-ECM by 5% compared to other groups. Also, ECM5% led to increased cell attachment and viability, proper compressive strength, and a decrease in Young’s module. Our study suggests that using a combination of testicular ECM and gelatin shows promise in constructing bioartificial testes through the gas-foaming method.

Male infertility can be caused by genetic anomalies, endocrine disorders, inflammation, and exposure to toxic chemicals or gonadotoxic treatments. Therefore, several recent studies have concentrated on the preservation and restoration of fertility to enhance the quality of life for affected individuals. It is currently recommended to biobank the tissue extracted from testicular biopsies to provide a later source of spermatogonial stem cells (SSCs). Another successful approach has been the in vitro production of haploid male germ cells. The capacity of SSCs to transform into sperm, as in testicular tissue transplantation, SSC therapy, and in vitro or ex vivo spermatogenesis, makes them ideal candidates for in vivo fertility restoration. The transplantation of SSCs or testicular tissue to regenerate spermatogenesis and create embryos has been achieved in nonhuman mammal species. Although the outcomes of human trials have yet to be released, this method may soon be approved for clinical use in humans. Furthermore, regenerative medicine techniques that develop tissue or cells on organic or synthetic scaffolds enriched with bioactive molecules have also gained traction. All of these methods are now in different stages of experimentation and clinical trials. However, thanks to rigorous studies on the safety and effectiveness of SSC-based reproductive treatments, some of these techniques may be clinically available in upcoming decades.

Utilizing natural scaffold production derived from extracellular matrix components presents a promising strategy for advancing in vitro spermatogenesis. In this study, we employed decellularized human placental tissue as a scaffold, upon which neonatal mouse spermatogonial cells (SCs) were cultured three‐dimensional (3D) configuration. To assess cellular proliferation, we examined the expression of key markers (Id4 and Gfrα1) at both 1 and 14 days into the culture. Our quantitative reverse transcription‐polymerase chain reaction (qRT‐PCR) analysis revealed a notable increase in Gfrα1 gene expression, with the 3D culture group exhibiting the highest levels. Furthermore, the relative frequency of Gfrα1‐positive cells significantly rose from 38.1% in isolated SCs to 46.13% and 76.93% in the two‐dimensional (2D) and 3D culture systems, respectively. Moving forward to days 14 and 35 of the culture period, we evaluated the expression of differentiating markers (Sycp3, acrosin, and Protamine 1). Sycp3 and Prm1 gene expression levels were upregulated in both 2D and 3D cultures, with the 3D group displaying the highest expression. Additionally, acrosin gene expression increased notably within the 3D culture. Notably, at the 35‐day mark, the percentage of Prm1‐positive cells in the 3D group (36.4%) significantly surpassed that in the 2D group (10.96%). This study suggests that the utilization of placental scaffolds holds significant promise as a bio‐scaffold for enhancing mouse in vitro spermatogenesis.

Background: Spermatogenesis is a fundamental process crucial for male reproductive health and fertility. Exosomes, small membranous vesicles released by various cell types, have recently garnered attention for their role in intercellular communication.Objective: This review aims to comprehensively explore the role of exosomes in regulating spermatogenesis, focusing on their involvement in testicular development and cell‐to‐cell communication.Methods: A systematic examination of literature was conducted to gather relevant studies elucidating the biogenesis, composition, and functions of exosomes in the context of spermatogenesis.Results: Exosomes play a pivotal role in orchestrating the complex signaling networks required for proper spermatogenesis. They facilitate the transfer of key regulatory molecules between different cell populations within the testes, including Sertoli cells, Leydig cells, and germ cells.Conclusion: The emerging understanding of exosome‐mediated communication sheds light on novel mechanisms underlying spermatogenesis regulation. Further research in this area holds promise for insights into male reproductive health and potential therapeutic interventions.

Introduction: Methamphetamine (METH) is an addictive psychostimulant that facilitates dopamine transmission to the nucleus accumbens (NAc), resulting in alterations in the mesocorticolimbic brain regions. Cannabidiol (CBD) is considered the second most abundant component of cannabis and is believed to decrease the METH effects. Reversing psychostimulant-induced abnormalities in the mesolimbic dopamine system is the main mechanism for this effect. Various other mechanisms have been proposed: increasing endocannabinoid system activity and modulating gamma-aminobutyric acid (GABA) and glutamate neurons in NAc. However, the exact CBD action mechanisms in reducing drug addiction and relapse vulnerability remain unclear. Methods and Results: The present study aimed to investigate the effects of intracerebroventricular (ICV) administrating 5, 10, and 50 μg/5 μL CBD solutions on the extinction period and reinstatement phase of a METH-induced conditioned place preference. This research also aimed to examine the NAc D1-like dopamine receptor (D1R) and D2-like dopamine receptor (D2R) roles in the effects of CBD on these phases, as mentioned earlier, using SCH23390 and sulpiride microinjections as an antagonist of D1R and D2R. The obtained results showed that microinjection of CBD (10 and 50 μg/5 μL, ICV) suppressed the METH-induced reinstatement and significantly decreased mean extinction latency in treated groups compared to both vehicles and/or untreated control groups. In addition, the results demonstrated that administrating intra-accumbal SCH23390 (1 and 4 μg/0.5 μL saline) reversed the inhibitory effects of CBD on extinction and reinstatement phases while different doses of sulpiride (0.25, 1, and 4 μg/0.5 μL; dimethyl sulfoxide 12%) could not alter the CBD effects. Conclusions: In summary, this study showed that CBD made shorter extinction latencies and suppressed the METH reinstatement, in part, by interacting with D1R but not D2R in the NAc.