The Effects of Pifithrin-µ on Spermatogonial Stem Cell Viability and Pluripotency

Spermatogonial stem cells (SSCs) as the most primitive spermatogonia in the testis contribute to supporting vastly productive spermatogenesis through self-renewal and prolonged establishment of daughter spermatogonia. The daughter spermatogonia, in turn, differentiate into spermatozoa, thereby conveying genetic information to the following generation [Kubota et al., 2003; Oatley and Brinster, 2006]. SSC biology provides significant capabilities for animal reproduction and defeating human disorders in the regenerative medicine [Tan and Wilkinson, 2020]. SSCs transmit genetic information to the subsequent generation and thereby can be used for gene targeting. In contrast to embryonic stem (ES) cells, SSCs can be exploited without any ethical concerns as they are adult cells. Accumulating evidence suggests that SSC biotechnologies can circumvent fertility-associated problems, such as fertility restoration in oncological patients, infertility therapy, as well as the reproduction of endangered species [David and Orwig, 2020]. Moreover, these stem cells bring about eminent progress in the cryopreservation of germ cells and the developing approaches of spermatogenesis in vitro.

The small molecular 2-phenyl ethyne sulfonamide, also known as pifithrin-μ (PFT-μ) is a well-known inhibitor of P53 (also known as TP53 or tumor protein)-elicited apoptosis that suppresses interrelations between P53 and B-cell lymphoma-extra large Bcl2l1 (BCL2-like 1) as well as Bcl2 (B cell leukemia/lymphoma 2) at the mitochondrial surface without modifying P53 transactivational functions [Dong et al., 2012; Maj et al., 2017]. The P53 protein forms a homotetrameric transcription factor that regulates the expression of ∼500 target genes, thus controlling a diversity of cellular processes, such as cell cycle arrest, cell senescence, DNA repair, metabolic adaptation, and cell death [Fridman and Lowe, 2003; He et al., 2020]. PFT-µ can alleviate inflammatory oxidative stress, autophagy, and mitophagy mechanisms, leading to improved survival of target cells by binding to P53 [Yang et al., 2020]. It also inhibits DNA damage-induced apoptosis in the human ESCs [Zhu et al., 2020]. Also, PFT-µ enhances cell recovery for dissociated human ESCs after cryopreservation, as reported by Xu et al. [Xu et al., 2010]. It also augments the survival of grafted stem cells through the inhibition of P53 translocation into the nucleus in vivo [Lei et al., 2013]. Thanks to the pivotal role of P53 in suppressing the pluripotency of ESCs after DNA damage and blocking the reprogramming of somatic cells into induced pluripotent stem cells, it has been suggested that downregulation of P53 by PFT-µ could support pluripotency in such stem cells and also improve their survival [García et al., 2014]. There is some evidence indicating that PFT-µ could potentiate stem cell proliferation and conversely inhibit their apoptosis by downregulation of the ROS-SIRT1-p53-p53 upregulated modulator of apoptosis pathway [He et al., 2017; Jung et al., 2021]. In the present study, we investigated the effects of P53 inhibitor PFT-µ on the expression of pluripotency genes as well as the survival rate of SSCs in vitro.

This study was carried out according to the recommendations of the Helsinki Declaration. The protocol was approved by the Ethical Committee of the National Institute of Genetic Engineering and Biotechnology (NIGEB-2019-178). Adult NMRI mice having a weight of 20–30 g were obtained from the Pasteur Institute and were placed in plastic cages and were housed under standard laboratory conditions of temperature (21–23°C), humidity (55%), and light-dark cycle (12-h light/dark). All animals were permitted to have access to water and food. The testes of adult NMRI mice pups (6–8 weeks old), were collected in phosphate-buffered saline (PBS) (Thermo Scientific PBS, Cat. No. 18912014). Following decapsulation, the testes were pounded into small fragments in DMEM (Thermo Scientific DMEM, Cat. No. 11966025). Then, testicular pieces were digested in a collagenase type IV solution (1 mg/mL) (Thermo Scientific collagenase type IV, Cat. No. 17104019) and incubated at 37°C for 15 min, homogenized by pipetting for 5 min. The suspension upon the first digestion stage was centrifuged at 161 RCF for 1 min. Single cells were isolated by second enzymatic digestion with trypsin 0.25% (Thermo Scientific trypsin 0.25%, Cat. No. 25200056) under the same circumstance followed by pipetting for 1 min. Then, the effect of trypsin was neutralized by adding a DMEM/F12 (Thermo Scientific DMEM/F12, powder, Cat. No. 32500043) medium containing 10% FBS (Thermo Scientific FBS, Cat. No. A4766801). The isolated SSCs were kept at 32°C with 5% CO₂ for 7 days. The culture medium was changed every 2 days.

The cultivated SSC colonies were first fixed in 4% paraformaldehyde (paraformaldehyde solution, 4% in PBS, Thermo Scientific Cat. No. J19943.K2) in PBS (pH 7.4) for 20 min at 25°C using coverslips. Afterward, SSCs were washed twice with 0.2% Tween 20 (Sigma-Aldrich Cat. No. P9416-100 ML) in PBS for 2 min at 25°C before blocking in 10% normal goat serum (Thermo Scientific normal goat serum, Cat. No. PCN5000) in PBS for 10 min. Then, SSCs were incubated with primary antibody solution: 1 μg/mL mouse polyclonal anti-c-Kit (Thermo Scientific polyclonal anti-c-Kit, Cat. No. BS-0672R), and 1 μg/mL mouse polyclonal anti-PLZF (Thermo Scientific polyclonal PLZF, Cat. No. PA5-81949) overnight at 4°C. The SSCs were washed twice with 0.2% Tween 20 (Sigma-Aldrich Cat. No. P9416-100 ML) in PBS for 2 min and then incubated with the appropriate secondary antibody (rabbit anti-mouse labeled with fluorescein isothiocyanate [FITC]; 1:200; Sigma-Aldrich) for 1 h and then cells were washed with 0.2% Tween 20 in PBS for 2 min. Nuclei were counterstained with 4,6-diamidino-2-phenylindole (DAPI) (Roche DAPI, Cat. No. 10236276001) in PBS. Finally, labeled cells were assessed using a fluorescent microscope (Nikon inverted fluorescence microscope).

Upon trypsinization of SSC colonies, the viability of SSCs following exposure to PFT-µ was assessed using the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay (Sigma-Aldrich, Cat. No. M5655-5X1G) based on manufacturer’s instructions. A total of 5 × 10³ SSCs in 100 μL of culture medium were seeded into each well of a 96-well plate. Cells were treated with 0–5µM PFT-µ (Sigma-Aldrich, Cat. No. P0122-10MG) for 24 and 48 h. Upon 24 and 48 h of exposure, 20 μL of 5 mg MTT/mL medium was added to each SSC-containing well. SSCs were kept at 37°C for 4 h, and finally, the optical density of wells was measured at 570 nm wavelengths using an ELISA reader (Biocompare ELISA Plate Reader).

The soft agar colony formation assay utilizing a 6-well plate was used to further investigate the effect of PFT-µ on self-renewal and colony formation of SSCs. The base agar layer was primed from a 0.6% soft agar solution (Sigma-Aldrich, Cat. No. A1296-500G) containing DMEM and 10% FBS. Then, 2 × 10⁵ cells were suspended in DMEM containing 10% FBS and 0.3% agar solution and plated onto the base layer. Plates were incubated at 37°C with 5% CO₂ for 7 and 14 days. Then, colony formation was evaluated using a microscope.

The apoptosis percentages were estimated utilizing combined staining with FITC-conjugated annexin V (Thermo Scientific Annexin V Conjugates for Apoptosis Detection, Cat. No. A13199) and propidium iodide (Sigma-Aldrich, Cat. No. P4864) following SSC exposure to PFT-µ. Initially, the colonies were trypsinized into single cells. Then, 1 × 10⁶ cells were washed once with Ca²⁺-binding buffer (HEPES, NaCl, and CaCl₂) (Sigma-Aldrich, Cat. No. P4864) and resuspended in 100 μL of the same buffer containing 5 μL FITC-conjugated annexin V, and SSCs were kept at 4°C for 20 min in dark. Cells were diluted by 400 μL of binding buffer and then 5 μL propidium iodide was added before flow cytometric analysis (FACS). Finally, cells were evaluated using the FACSCalibur flow cytometer (Becton-Dickinson, Mountain View, CA, USA) and FlowJo software v9.9.5.

The RNAiso Plus reagent (TaKaRa RNAiso Plus, Cat. No. 9108/9109) was used to extract total RNA from the cells. The quality and concentrations of total RNA were assessed by using a NanoDrop instrument (Thermo Scientific, MA, USA), and cDNA was synthesized employing Transcriptor First Strand cDNA Synthesis Kit (Norgen Biotek TruScript First Strand cDNA Synthesis Kit, Cat. No. 54420). Real-time quantitative polymerase chain reaction was conducted using the SYBR Green reagent (Kiagene Fanavar 2X SYBR Green Master Mix, Cat. No. FPLF009.1000) to quantify the signaled by retinoic acid 8 genes (Stra8) and Octamer-binding transcription factor 4 (Oct4) expression in SSC following exposure to PFT-µ. Relative gene expression was calculated utilizing the Pfaffl method [Harshitha and Arunraj, 2021]. The primers used in this study and target sequence data have been summarized in Table 1. After designing the primers, they were evaluated in terms of specificity, efficiency, reproducibility, and cross-reactivity [Tanabe et al., 2019]. Gapdh (as a housekeeping gene) was exploited as an internal control, and triplicate analysis was done for all samples. The 2^−ΔΔ cycle threshold (Ct) approach was used to define the relative mRNA expression.

Descriptive and statistical analyses were performed using GraphPad Prism 8.0 (GraphPad Software, CA, USA). Each experiment was replicated three times, and all values are shown as the mean ± SD. The t test was carried out to determine the statistical difference between groups. Finally, a p value <0.05 was considered statistically significant.

PLZF [Buaas et al., 2004; Costoya et al., 2004] and c-Kit markers [Schrans-Stassen et al., 1999] were used to characterize undifferentiated SSCs (Fig. 1). Immunocytochemistry results showed that PLZF, a specific marker for SSCs, was highly expressed in our cell colonies, while c-Kit was expressed at very low levels, thus confirming the SSC identity of these cells (Fig. 1).

MTT assay results showed that 0.6 and 1.2 µm PFT-µ concentrations promoted the viability of SSCs within 24 and 48 h of exposure in vitro (p < 0.05) (Fig. 2). This improvement was more obvious at 48 h of treatment than at 24 h of treatment. The 2.5 µm PFT-µ treatment increased slightly SSC viability within 24 h of treatment, whereas this reduction was not significant (p < 0.05) (Fig. 2). Besides, 2.5 µm PFT-µ attenuated SSC viability significantly within 48 h of exposure. Finally, 5 µm PFT-µ had remarkable cytotoxicity against SSCs at 24 and 48 h of treatment (p < 0.05) (Fig. 2).

To investigate colony formation, SSCs were treated with 0.6 and 1.2 µm PFT-µ concentrations. The number and diameter of the colonies were evaluated after 2 weeks (Fig. 3). The area of the colonies was checked and calculated with the help of ImageJ software [Schneider et al., 2012]. The results and statistical tests were performed using the t test method, considering 50 samples for each group. This comparison shows that the samples treated with 0.6 μm PFT-µ have more changes in size compared to the control group as shown in Table 2 and the samples treated with 1.2 μm PFT-µ and the control group show fewer changes in Table 3. T stat <-t critical two-tail or T stat> t critical two-tail is true for both tables, so we reject the null hypothesis and p value ≤0.05 to show that the data have normal statistical dispersion and significant differences.

FACS analysis revealed that 0.6 and 1.2 µm PFT-µ concentrations reduced SSCs apoptosis percentage compared with control cells at 48 h of treatment (p < 0.05) (Fig. 4). This reduction was more prominent in cells treated with 0.6 μm PFT-µ than 1.2 μm. Accordingly, the percentage of apoptotic cells in SSC treated with 0.6 μm PFT-µ, 1.2 μm PFT-µ, and also control cells (untreated SSCs) were 21.65 ± 2.19, 45.84 ± 3.08, and 54.89 ± 2.03 (Fig. 4).

Stra8 and c-Kit expression levels were evaluated after 3, 7, and 14 days of exposure to PFT-µ by real-time PCR. Results showed that 0.6 μm PFT-µ and 1.2 μm increased c-Kit expression levels within 7 and 14, but not 3 days of exposure compared with control groups (p < 0.05) (Fig. 5a). This increase was higher after 14 than after 7 days of treatment. In contrast, 0.6 PFT-µ and 1.2 μm reduced Stra8 expression levels at 7 and 14 days of treatment (p < 0.05) (Fig. 5b).

SSCs are the most undifferentiated spermatogonia in the testis and have an essential role to maintain highly productive spermatogenesis through self-renewal and continuous generation of daughter spermatogonia that differentiate into spermatozoa, transmitting genetic information to the next generation [Subash et al., 2021]. In 1994, a spermatogonial transplantation method was reported that established a quantitative functional assay to identify SSCs by evaluating their ability to both self-renew and differentiate into spermatozoa [Brinster and Zimmermann, 1994; Russell and Griswold, 2000; Takashima and Shinohara, 2018]. The system was originally developed using mice and subsequently extended to non-rodents, including domestic animals and humans [Fayomi and Orwig, 2018]. The availability of the functional assay for SSCs has made it possible to develop culture systems for their in vitro expansion, which has dramatically advanced germ cell biology and allowed medical applications [de Michele et al., 2017]. SSCs convey genetic information to the next generation, a property that can be exploited for gene targeting [Zheng et al., 2014; Xie et al., 2020]. Additionally, SSCs can be induced to become ES cell-like pluripotent cells in vitro. Previous studies have reported the role of P53 in suppressing the pluripotency of ES cells after DNA damage and blocking the reprogramming of somatic cells into the induced pluripotent stem cells [Abdelalim and Tooyama, 2012]. This report highlights the central role of P53 in affecting stem cell biological procedures like proliferation.

PFT-μ was initially identified as a small-molecule inhibitor of the binding of P53 to the mitochondria [Yang et al., 2020]. Thereafter, this molecule was found to selectively interact with HSP70 and inhibit its functions [Yang et al., 2021]. Thereby, it has been suggested that it can promote stem cell proliferation, reduce apoptosis, and also maintain their stemness. Based on previous reports, PFT-µ improves the ESCs cell recovery after cryopreservation and reduces their loss [Xu et al., 2010]. It also increases the survival of implanted stem cells by suppressing P53 translocation into the nucleus in animal models [Lei et al., 2013]. Accumulating evidence also exhibited that PFT-µ can promote stem cell proliferation and their viability through suppression of ROS/SIRT1/p53/p53 upregulated modulator of apoptosis pathway [He et al., 2017; Jung et al., 2021]. This pathway is typically induced by oxidative stress, and in turn, leads to cell apoptosis. In the present study, we showed that the optimum dose of PFT-µ (0.6 and 1.2 µm) induced the viability of SSC, while a higher dose of PFT-µ did not improve SSC viability in vitro. Moreover, PFT-µ (0.6 and 1.2 µm) enhanced SSC colony formation, conferring the positive effects of PFT-µ on SSC viability and growth. Importantly, PFT-µ also reduced SSC apoptosis compared with untreated cells. It seems that the observed effect might be attributable to reducing TP53 activation and downregulation of oxidative stress-induced damages. In addition, other studies have shown that downregulation of P53 by PFT-µ could support pluripotency in pluripotent stem cells and also improve their survival [García et al., 2014]. We showed that PFT-µ, more evidently at 0.6 µm concentrations, improved expression of pluripotency-related gene Oct4, and conversely decreased differentiation-related marker gene Stra8. Thus, it appeared that PFT-µ could maintain SSC pluripotency and constrain their differentiation.

According to the achieved results, low dose of PFT-µ, particularly the 0.6 µm concentration, not only improves SSC viability but also attenuates their apoptosis. As well, PFT-µ can also support the pluripotency and self-renewal capabilities of SSC by increasing Oct4 and declining Stra8 expression. Thus, it seems that the treatment of SSC with an optimized concentration of PFT-µ may benefit their biological process, making it a favored source to apply in regenerative medicine.

We would like to thank the Kiagene Fanavar Aria company for their assistance with the collection of our data.

This study was carried out according to the recommendations of the Helsinki Declaration. The protocol was approved by the Ethical Committee of the National Institute of Genetic Engineering and Biotechnology (NIGEB-2019-178).

The authors have no relevant financial or non-financial interests to disclose.

The authors declare that no funds, grants, or other support were received during the preparation of this manuscript.

All authors contributed to the conception and the main idea of the work. Sara Moghadasi, Ehsan Razeghian, Mahdi Shamsara, and Farid Heidari drafted the main text, figures, and tables. Farid Heidari supervised the work and provided comments and additional scientific information. Ehsan Razeghian and Mahdi Shamsara also reviewed and revised the text. All authors read and approved the final version of the work to be published.