Review of Sertoli cell dysfunction caused by COVID-19 that could affect male fertility.

Spermatogenesis is a complex and elaborate differentiation process and is vital for male fertility. Sertoli cells play a major role in fertility and induce spermatogenesis by protecting, nourishing, and supporting germ cells. It has been speculated that severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) could directly affect the male reproductive system, and therefore heredity and fertility. The similarity of SARS-CoV-2 to SARS-CoV could confirm this hypothesis because both viruses use angiotensin-converting enzyme (ACE2) as the receptor to enter human cells. ACE2 is expressed by Sertoli cells and other testicular cells, therefore COVID-19 has the potential to impair fertility by destroying Sertoli cells. This hypothesis should be evaluated and confirmed by monitoring fertility in patients with COVID-19.

Successful transplantation of spermatogonial stem cells into the seminiferous tubules of busulfan-treated mice.

BACKGROUND: Spermatogonial stem cells (SSCs) in the testis are crucial for transferring genetic information to the next generation. Successful transplantation of SSCs to infertile men is an advanced therapeutic application in reproductive biology research. METHODS: In this experimental research, both in vitro and in vivo characterization of undifferentiated and differentiated SSCs were performed by morphology-immunocytochemistry (ICC), immunohistochemistry (IMH), Fluidigm Real-Time polymerase chain reaction (RT-PCR) and flow cytometry analysis. The isolated SSCs were finally microinjected into the rete testis of busulfan-treated mice. The compact undifferentiated and more loosely connected round differentiated SSCs were isolated during testicular cell expansion from their specific feeder layer. RESULTS: ICC analysis indicated high and low expression levels of Zbtb16 in undifferentiated and differentiated germ cells. Also, IMH analysis showed different expression levels of Zbtb16 in the two different germ stem cell populations of the testicular tissue. While Fluidigm RT-PCR analysis indicated overexpression of the TAF4B germ cell gene, the expression of DAZL, VASA, and Zbtb16 were down-regulated during the differentiation of SSCs (P < 0.05). Also, flow cytometry analysis confirmed the significant downregulation of Itgb1 and Itga4 during differentiation. By transplantation of SSCs into busulfan-treated NOD/SCID mice, GFP-labeled sperm cells developed. CONCLUSIONS: In the current study, we performed a transplantation technique that could be useful for the future microinjection of SSCs during infertility treatment and for studying in vivo differentiation of SSCs into sperm.

Spermatogonia (SSCs) in the testis transmit genetic information to the next generation. Successful SSC transplantation into infertile men is an advanced therapeutic application in reproductive biology research. In this experimental research, both in vitro and in vivo characterization of undifferentiated and differentiated SSCs were performed by morphology­immunocytochemistry (ICC), immunohistochemistry (IMH), Fluidigm Real-Time polymerase chain reaction (RT-PCR) and flow cytometry analysis. The isolated SSCs were finally microinjected into the rete testis of busulfan-treated mice. ICC analysis indicated high and low expression levels of Zbtb16 in undifferentiated and differentiated germ cells. IMH analysis showed different expression levels of Zbtb16 in both populations. Fluidigm RT-PCR analysis indicated overexpression of the TAF4B germ cell gene and the down-regulated expression of DAZL, VASA, and Zbtb16 during SSCs differentiation of (P < 0.05). Flow cytometry analysis confirmed the significant downregulation of Itgb1 and Itga4 during differentiation. By transplantation of SSCs into busulfan-treated NOD/SCID mice, GFP-labeled sperm cells developed. We performed a transplantation technique that could be useful for the future microinjection of SSCs during infertility treatment and for studying in vivo differentiation of SSCs into sperm. Data analysis confirmed that zbtb16 is expressed in the undifferentiated germ cells located on the basal membrane of seminiferous tubules and SSCs in vitro. Also, spermatogenesis was resumed, and fertility improved after transplantation of undifferentiated cells into busulfan-treated mice; thus, improvements in vitro SSCs transplantation, isolation and culture would be helpful in future clinical treatments to solve the reproductive problems of families influenced by infertility.

Testicular Localization and Potential Function of Vimentin Positive Cells during Spermatogonial Differentiation Stages.

Vimentin is a type of intermediate filament (IF) and one of the first filaments expressed in spermatogenesis. Vimentin plays numerous roles, consisting of the determination of cell shape, differentiation, cell motility, the maintenance of cell junctions, intracellular trafficking, and assisting in keeping normal differentiating germ cell morphology. This study investigated the vimentin expression in two populations of undifferentiated and differentiated spermatogonia. We examined vimentin expression in vivo and in vitro by immunocytochemistry (ICC), immunohistochemistry (IMH), and Fluidigm real-time polymerase chain reaction. IMH data showed that the high vimentin expression was localized in the middle of seminiferous tubules, and low expression was in the basal membrane. ICC analysis of the colonies by isolated differentiated spermatogonia indicated the positive expression for the vimentin antibody, but vimentin's expression level in the undifferentiated population was negative under in vitro conditions. Fluidigm real-time PCR analysis showed significant vimentin expression in differentiated spermatogonia compared to undifferentiated spermatogonia (p < 0.05). Our results showed that vimentin is upregulated in the differentiation stages of spermatogenesis, proving that vimentin is an intermediate filament with crucial roles in the differentiation stages of testicular germ cells. These results support the advanced investigations of the spermatogenic process, both in vitro and in vivo.

In Vitro and In Vivo Determinations of The Anti-GDNF Family Receptor Alpha 1 Antibody in Mice by Immunochemistry and RT-PCR.

BACKGROUND: The glial cell-derived neurotrophic factor (GDNF) family plays essential roles in the maintenance, growth, regulatory and signalling pathways of spermatogonial stem cells (SSCs). In this study, we analysed the expression of anti-GDNF family receptor alpha 1 antibody (GFRa1) by immunohistochemistry (IHC), immunocytochemistry (ICC), Fluidigm real-time polymerase chain reaction (RT-PCR) and flow cytometry analyses. MATERIALS AND METHODS: In this experiment study, ICC, IHC, Fluidigm RT-PCR and flow cytometry were used to analyse the expression of the germ cell marker GFRa1 in testis tissue and SSC culture. RESULTS: IHC analysis showed that there were two groups of GFRa1 positive cells in the seminiferous tubules based on their location and expression shape - a small round punctuated shape on the basal compartment donut shape and a C-shaped expression located between the basal and the luminal compartments of the seminiferous tubules. OCT4 and PLZF positive cells may have similar patterns of expression as the first group. Assessment of the seminiferous tubule sections demonstrated that about 27% of the SSCs were positive for GFRa1. Fluidigm RT-PCR confirmed the significant expression (P<0.001) of GFRa1 in the SSCs compared to testicular stromal cells (TSCs). Flow cytometry analysis demonstrated that about 75% of the isolated SSCs colonies were positive for GFRa1. CONCLUSION: The results indicated that GFRa1 had a specific expression pattern both in vivo and in vitro. This finding could be helpful for understanding the proliferation, maintenance and signalling pathways of SSCs, and differentiation of meiotic and haploid germ cells.