Cryoprotective Effect of Pentoxifylline on Spermatogonial Stem Cell During Transplantation into Azoospermic Torsion Mouse Model

Preserving the spermatogonial stem cells (SSCs) in long periods of time during the treatment of male infertility using stem cell banking systems and transplantation is an important issue. Therefore, this study was conducted to develop an optimal cryopreservation protocol for SSCs using 10 mM pentoxifylline (PTX) as an antioxidant in basal freezing medium. Testicular torsion—a mouse model for long-term infertility—was used to transplant fresh SSCs (n = 6), fresh SSCs treated with PTX (n = 6), cryopreserved SSCs with basal freezing medium (n = 6), and cryopreserved SSCs treated with PTX (n = 6). Eight weeks after germ cell transplantation, samples were assessed for proliferation, through evaluation of Ddx4 and Id4 markers, and differentiation via evaluation of C‐Kit and Sycp3, Tnp1, Tnp2, and Prm1 markers. According to morphological and flow cytometry results, SSCs are able to form colonies and express Gfra1, Id4, α6‐integrin, and β1‐integrin markers. We found positive influence from PTX on proliferative and differentiative markers in SSCs transplanted to azoospermic mice. In the recipient testis, donor SSCs formed spermatogenic colonies and sperm. Respecting these data, adding pentoxifylline is a practical way to precisely cryopreserve germ cells enriched for SSCs in cryopreservation, and this procedure could become an efficient method to restore fertility in a clinical setup. However, more studies are needed to ensure its safety in the long term.


Introduction
A considerable number of childhood cancer survivors are at risk of infertility due to the loss of spermatogenic cells after treatment [1]. For adult male cancer patients, fertility can be preserved by cryopreservation of semen [2]. Since spermatogenesis has not commenced in pre-pubertal boys, cryopreservation of ejaculated or surgically retrieved spermatozoa is not feasible. Therefore there is a need for a clinical application to preserve and restore fertility in these boys [3,4]. Spermatogonial stem cell autotransplantation is an experimental technique that is still in a pre-clinical phase [5,6]. Spermatogonial stem cell transplantation (SSCT) has proved to restore spermatogenesis in various animal models, including non-human primates [7]. The procedure of treatment requires a testicular biopsy before cancer therapy to preserve the fine spermatogonial stem cells (SSCs). These cells will be transplanted after successful cancer treatment and proven sterility [8]. Currently, in many centers across the globe, cryopreservation of testicular biopsies is offered to children with cancer [9]. Due to lack of active spermatogenesis at the time of cancer diagnosis, SSC freezing cannot be helpful by its own; thus, long-term preservation techniques, such as cell culture and cryopreservation in combination with SSCT, may be the best strategy for these patients as a possible fertility preservation [9].
The two most commonly used cryopreservation procedures are slow (controlled or uncontrolled) freezing and vitrification. Development of cryopreservation techniques for SSCs has utilized uncontrolled slow-freezing methods coupled with rapid thawing. These methods have proven to be convenient methods for the long-term preservation of SSCs capable of restoring fertility after thawing and transplantation into infertile recipient mice [7,10]. Osmotic stress leads to development of cryoinjury as a common consequence of cryopreservation using uncontrolled slow freezing. However, the osmotic stress can be overcome by including permeable cryoprotectant agents (PCAs), which reduce intracellular ice formation, in cryopreservation media. Furthermore, the addition of additive cryoprotectant agents (ACAs) can further improve cellular viability after thawing [11]. The process of cryopreservation results in unfavorable cryoinjuries, which disrupts the normal biological function of cells. Also, previous reports of SSC cryopreservation have indicated that the functional capacity of thawed SSCs is less than ideal. These disruptions include DNA fragmentation, mitochondrial dysfunction, osmotic stress, oxidative stress [12], induction of apoptosis [13], and increased generation of reactive oxygen species (ROS) [14,15].
Studies have demonstrated that the addition of exogenous antioxidant in the freezing extender can improve sperm quality and function and increase the quality and viability of SSCs after thawing, providing indirect evidence that oxidative stress during cryopreservation can be a potential harm to these cells [16,17]. Pentoxifylline (PTX), a methylxanthine derivate, is known as an inhibitor of cyclic adenosine monophosphate [18]. There are studies indicating that PTX can increase partial pressure of oxygen and pose anti-inflammatory activity, eliminate free radicals, block the expression of NF-κB and the macrophagic nitric oxide synthesis induced by TNF-α messenger ribonucleic acid (mRNA), and reduce cellular apoptosis [19]. Indeed, PTX possesses both antioxidative and ROS scavenging (ROS identification and inhibition) which demonstrates the antioxidative activity of PTX properties [20]. The results suggest that adding PTX to the basic freezing medium can effectively protect the SSCs by increasing the viability and reducing the ROS production after cryopreservation and can improve the transplantation efficiency of thawed SSCs. The methods developed here will serve as foundations for the long-term preservation of SSCs, but more studies should be performed on this subject.
We hypothesized that transplantation of SSCs treated with PTX may help to improve fertility after testicular torsion (TT). To investigate the effect of PTX, we optimized an infertility model representing an impaired spermatic cord by TT. Next, we transplanted cryopreserved SSCs treated with PTX to pursue its effect on markers of pre-meiotic and post-meiotic SSCs for the first time ( Supplementary Fig. 1).

Animals and Study Design
A total number of 10 male NMRI mice (3-6 days old) underwent testicular surgery and were used as donners for SSC.
Six-to eight-week-old male NMRI mice (n = 66) were used as recipients of SSC transplantation. All NMRI mice were obtained from the Pharmacy Faculty of Tehran University of Medical Sciences. Animals were preserved under standard conditions of 12-h/12-h light/dark. All surgical procedures were done under xylazine/ketamine (10/90 mg/kg, i.p.) anesthesia using sterile conditions. All procedures in this study were conducted in accordance with the Guide for the Care and Use of Laboratory Animals (1996, published by National Academy Press, 2101 Constitution Ave. NW, Washington, DC 20,055, USA), and also this study was approved by the Ethical Committee of Tehran University of Medical Sciences (IR.TUMS.MEDICINE.REC.1397.210). Forty-eight mice in four testicular torsion transplantation groups (12 mice in each group), 9 mice in the control group, and 9 mice in the torsion group were considered as experimental groups. After testicular torsion model was confirmed, transplantation groups were divided as follows: the group that received SSCs (TT + fresh), the group receiving SSCs after freezing (TT + cryo), the group which received SSCs treated with PTX after freezing (TT + cryo + PTX), and the group receiving SSCs with PTX (TT + fresh + PTX). Also, the control group received Dulbecco's modified Eagle's medium (DMEM). The TT group had testicular torsion operation but did not undergo transplantation. Two weeks after testicular torsion, 3 mice from each of 6 groups were sacrificed to confirm the torsion by cervical dislocation. Two weeks after transplantation, 3 mice in each transplantation group were sacrificed to confirm the transplantation. Recipient mice were euthanized, and testes were collected and decapsulated 8 weeks after transplantation. Finally, 36 mice in all groups (6 groups of mice, 6 mice in each group) included four testicular torsion transplantation groups, and control and torsion groups (as a sham-transplanted testes) were used to evaluate the testes at the end of the study which was 8 weeks after transplantation ( Supplementary Fig. 2).

Testicular Surgery, SSC Isolation, and Enrichment
After anesthesia, testes were dissected and transferred into the phosphate-buffered saline (PBS; Sigma-Aldrich). Then, SSCs were isolated by a two-step enzymatic digestion according to the protocol of Kanatsu-Shinohara et al. [21]. First, the testes were suspended in 3 ml DMEM containing 5 µg/ml DNase (Sigma-Aldrich, Germany), 1 mg/ml collagenase type IV (Gibco, CA, USA), and 1 mg/ml hyaluronidase (Sigma-Aldrich). Then, they were transferred to an incubator for 20 min at 37 °C until forming a cellular suspension [22]. Interstitial cells were digested after pipetting and centrifuging at 1500 g for 5 min. In the second step, cellular pellets were enriched by the same medium for 15 min. Cell detachment from seminiferous tubules occurred in this step. The suspension was centrifuged at 1500 g for 5 min, and the cell pellets were transferred to the gelatinized dishes for further pelleting with a duration of 5 h. For SSC enrichment, the cellular suspension was transferred into gelatin-coated (Sigma-Aldrich) culture dishes for 2 h at 37 °C. The nonadherent SSCs were cultured in minimal essential medium (Gibco-Invitrogen, USA) containing 2% fetal bovine serum (FBS) (Life Technologies), 1000 U/ml leukemia inhibitory factor (LIF; Sigma, Haverhill, MA, USA), 10 ng/ml basic fibroblast growth factor (PeproTech, Rocky Hill, NJ, USA), 0.1 mM β-mercaptoethanol (Sigma-Aldrich), 1% non-essential amino acids (Gibco, Invitrogen, UK), 10 µg/ml glial cell line-derived neurotrophic factor (GDNF; Sigma-Aldrich, USA), 100 U/ml penicillin (Sigma-Aldrich, Darmstadt), and 100 µg/ml streptomycin (Sigma, Germany). Afterward, the cells were transferred into an incubator (5% CO 2 , 37 °C) for 2 weeks. The medium was changed every 2 days [23].

Alkaline Phosphatase Staining
Fast Red TR/Naphthol AS-MX tablets (Sigma-Aldrich) were used for assessment of alkaline phosphatase activity. For this purpose, 0.1 M Tris buffer was added to the 0.2 mg/ ml Fast Red TR/Naphthol AS-MX tablet to form an alkaline dye. After fixing the cells with 4% formaldehyde and washing twice with PBS, alkaline dye was added to the cells that were incubated for 30 min at 37 °C and was observed under an inverted microscope (IX71, Olympus, Japan) at room temperature (at a magnification of × 20).

SSC Cryopreservation
Single cells were suspended in 2.5 × 10 5 cells/ml of freezing medium containing alpha-modified Eagle's minimum essential medium (α-MEM) (Invitrogen) with 10% dimethyl sulfoxide (DMSO; 1.4 M, Sigma, Germany), MEM-α (Sigma, Germany), and 10% FBS (Sigma, Germany) without additional supplements (basal freezing medium) [24] or basal freezing medium with the addition of 10 mM PTX (pH = 7.4, osmotic pressure 300 mOsm/kg, purity (TLC) ≥ 98%, cat. no. P1784, Sigma-Aldrich), and were placed in 1.8-ml cryovials (Corning). Cryovials were frozen in a Nalgene freezing container (cat. no. Z359017, Sigma) at a rate of − 1 °C per min to − 80 °C and stored overnight at − 80 °C. After overnight storage, cryovials were placed in liquid nitrogen for at least a week of storage. After removal from liquid nitrogen, samples were maintained at room temperature for 30 s and then in a water bath at 37 °C for 2.5 min. The cryovial contents were transferred to a tube with pre-warmed medium and diluted 1:10 with α-MEM containing 10% FBS in a drop-wise manner. The cells were washed two times with medium and centrifuged at 1200 × g for 5 min [24].

Cell Viability Assay
After thawing, cell viability was determined using methylthiazolyldiphenyl-tetrazolium bromide (MTT; Sigma-Aldrich) assay in control and treatment groups at doses of 5 mM, 10 mM, and 15 mM of PTX. One hundred microliters of MTT reagent (5 mg/ml in PBS, pH 7.6) was added to each well of 96-well plates, and plates were incubated in 5% CO 2 for 2 h at 37 °C. After that, an equal volume of DMSO was added. The absorbance was measured at 570 nm with background subtraction at 630 nm.

Measurement of Cellular Reactive Oxygen Species
Cellular ROS was detected using specific ROS probes by flow cytometry. Ten thousand SSC cells were loading with 50 µm 2'-7'dichlorofluorescin diacetate (DCFH-DA) (ROS Assay Kit; Beyotime, Haimen, Jiangsu, China) according to the manufacturer's instructions. DCFH-DA fluorescence was measured using logarithmic amplification in the flow cytometer (Becton Dickinson, USA). Data were reported as peak fluorescence intensity between 500 and 530 nm. Experiments were performed in triplicate and repeated three times. ROS production was calculated as the intensity in the fluorescence compared with the control group. The data was analyzed with FlowJo software (version 7.6.1).

Optimization of the Mouse Model for Infertility
For this purpose, the scrotum was excised through a midline incision. The tunica vaginalis was opened, and the left testis was exposed to the surgical field. The left testis was rotated 720° in a clockwise direction and maintained in this torsion position by fixing the testicle to the scrotum with a 6-0 silk suture [25]. Animals underwent 2 h of unilateral testicular ischemia. Then, the suture was removed, the ischemic testis was untwisted and replaced in the scrotum, and the incision area was closed [26]. For confirmation of the model, 3 mice in each group (control, torsion, and transplantation groups) were sacrificed and excluded from the study in order to examine the seminiferous tubules and confirm the torsion by cervical dislocation. After 2 weeks, TT and the testes of mice were removed, fixed in a Bouin's solution for 48 h, embedded in paraffin, sectioned at 5 µm thickness, and finally stained with hematoxylin-eosin (H&E) and the testis tissue sections were examined using an optical microscope (Nikon, Japan).

Germ Cell Transplantation
To detect the transplanted cells and purify them from testicular endogenous cells, 2 × 10 5 cells/ml were exposed to 2 µg di-alkyl indocarbocyanine (DiI; Eugene, OR, USA) before transplantation for 5 min. One milliliter of PBS preservative solution was placed at room temperature and then placed in a dark place for 20 min at 4 °C. After ensuring that the cells were stained under a fluorescent microscope (LX71, Olympus, Japan), the cell surface was washed with PBS and then isolated from petri dish by trypsin enzyme (25%) in 0.1% ethylenediaminetetraacetic acid (EDTA) (Sigma, USA). After washing 3 times in the medium, they were ready to be transplanted into the host testis.
Two weeks after testicular torsion, the recipient mice in four transplantation groups, as described above (n = 36), received transplantation. By clipping the abdominal hair and disinfection of the area with cedium chlorhexidini alcoholicus 0.5% (BE351513; Laboratoires Gifrer Barbezat, Décines-Charpieu, France), the surgical area was ready for use. The testes were exteriorized by incision of the abdomen. Afterward, the fatty tissue around the efferent ducts was gently removed, and 12 µl of the cell suspension containing 2 × 10 5 cells/ml was injected into the efferent ducts using a microinjection needle under a stereomicroscope. If the tracking dye (trypan blue at the tip of the pipette) along with the injected solution entered the seminiferous tubules, the transplantation was considered as "successful."

Immunofluorescent Staining of Testis Cross Sections and Selected Germ Cells
The immunofluorescent analysis was used to explore the expression of Ddx4 and C-Kit in the testes of mice. After dissection of testes from the mice, testes were fixed in 4% paraformaldehyde for 12 h at 4 °C, embedded in paraffin, and sectioned. Sodium citrate buffer (10 mM sodium citrate [pH 6.0]) was used for antigen retrieval by boiling the sections for 15 min. Endogenous peroxidase activity was blocked using blocking solution for 10 min at room temperature. Sections were then blocked with 10% normal goat serum, followed by incubation with respective primary antibodies of Ddx4 (1:500, ab13840, Abcam, UK) and C-Kit (1:500, ab5506, Abcam, UK) diluted in PBS containing 0.5% bovine serum albumin (BSA) at 4 °C overnight, followed by incubation with the appropriate Alexa Fluor dyeconjugated secondary antibodies FITC goat anti-rabbit IgG H&L (ab6717, Abcam, UK). The sections were washed and incubated with 4′,6-diamidino-2-phenylindole dihydrochloride hydrate (DAPI) (Vector Laboratories, CA, USA) in PBS to label the cell nuclei, followed by a fluorescence microscope with a magnification of × 40. All image analysis was done by ImageJ 1.5 software.

Quantitative Real-Time PCR
Expression rates of Tnp1, Tnp2, and Prm1 genes were assessed by quantitative real-time PCR. Extraction of total RNA from tissue samples was performed using a TRIzol reagent (Roche, Germany). Reverse transcription of 500 ng of the extracted RNA into complementary DNA (cDNA) was carried out by a cDNA synthesis kit (PrimeScript™ RT Reagent Kit Fast, RR037A, TaKaRa, Japan). PCR assay was performed using a thermocycler (Bio-Rad Laboratories) and a SYBR Green master mix (SYBR Premix Ex Taq II [Tli Plus], TaKaRa, RR820L). Samples were undergoing an initial melting stage for 5 min at 95 °C followed by melting stage (40 cycles) for 5 s at 95 °C and synthesis for 30 s at 60 °C (n = 3). The gene expression cycle threshold (ΔΔCt) values were calculated after normalizing with Hprt internal control. Sequences of primers are listed in Table 1.

Statistical Analysis
GraphPad Prism 8.3.0 (GraphPad Software, Inc., La Jolla, CA, USA) was used for statistical analysis. Values are expressed as the mean ± standard deviation (SD). Statistical comparisons were made between the experimental and control groups using one-way analysis of variance (ANOVA) followed by Tukey's test. Statistical significance was set as a P value less than 0.05.

High Expression of Gfra1, Id4, α6-integrin, and β1-integrin in SSCs
SSCs depicted distinguish unicellular features having the ability to attach to the bottom of the culture plate. Progression into forming small clusters of cells and then colonies is apparent after extending the time of culture. These colonies are easily seen in red colors after staining with the alkaline phosphatase (Fig. 1). Also, expression rates of markers of SSC, Gfra1, Id4, α6-integrin, and β1-integrin were high in the SSCs. The highest rate was for β1-integrin at 94.20 ± 2.60%. Expression rates for the three markers were 93.70 ± 2.28% (α6-integrin), 90.9 ± 9.13% (Gfra1), and 73.70 ± 8.03% (Id4) (Fig. 1).

Cell Viability
Statistically significant changes in the cell viability after administration of 10 mM PTX were seen, while there was a decrease in cellular viability after administration of this factor at doses of 5 mM and 15 mM. Therefore, we used the 10 mM [27] dose of this factor for the subsequent experiments (data not shown).

Optimization of the Mouse Model for Infertility
Our aim was to create a mouse model that can represent the clinical condition for infertility by damaging SSC niche through testicular torsion. The histological findings in the sections are illustrated in Fig. 3 (control and torsion groups). Histological examinations of the control testes revealed seminiferous tubules (Fig. 3a). The epithelium of all seminiferous tubules in the ischemic testis was severely disrupted. Most tubules were depleted to the extent that in some of them, only the basement membrane was detectable (Fig. 3b).
SSC-labeled cells were transplanted to confirm the presence of SSCs in cell suspension as well as to assess SSC colonization in the testis. Eight weeks after transplantation, fluorescent-labeled cells were considered transplanted cells. The labeled cells were localized in the seminiferous tubules of the recipient testes. Eight weeks after transplantation, colonization and proliferation of transplanted cells were observed. Fifteen days after transplantation, SSCs were labeled with DiI, showing the donor-derived origin of germ cells. Red light indicated that the DiI-positive cells are localized in the base of seminiferous tubules, and they showed homing of the transplanted cells (Fig. 4).

Immunofluorescent Findings Showed that PTX Increased Expression of Proliferation and Differentiation Markers After Transplantation
As expected, expression rates of Ddx4 and C-Kit were significantly decreased in the torsion (4.11 ± 2.73) vs. control (48.20 ± 1.99) group (P ≤ 0.001), and importantly, data from double immunofluorescence staining assays revealed that all of the transplantation groups showed a significant increase in the expression of both markers compared to the torsion group (Fig. 5A-D). Similar to immunofluorescence images, the results of quantitative analysis showed that the percentage of Ddx4 protein expression was 4.11 ± 2.73 (P < 0.0001 vs. control), 21.54 ± 2.26 (P < 0.0001 vs. control), 16.12 ± 1.85 (P < 0.0001 vs. HPRT 5′-GCA GCG TTT CTG AGC CAT TG-3′  3′-TCA TCG CTA ATC ACG ACG CT-5′  172  PRM1 5′-ATG GCC AGA TAC CGA TGC TG-3′  3′-GCA GCA TCT TCG CCT CCT C-5′  114  TP1  5′-GAG GAG AGG CAA GAA CCG AG-3′  3′-CGG TAA TTG CGA CTT GCA TCA-5′ 120  TP2  5′-AGC TCA GGG CGA AGA TAC AAGT-3′ 3′-TCC TGT GAC ATC ATC CCA ACA-5′ 107 control), 43.31 ± 1.00 (P < 0.0001 vs. control), and 35.92 ± 1.28 (P < 0.0001 vs. control) in the TT, TT + fresh, TT + cryo, TT + fresh + PTX, and TT + cryo + PTX groups, respectively. The expression of Ddx4 protein was significantly higher in the TT + cryo + PTX in comparison with the TT + cryo group (P ≤ 0.001) (Fig. 5A, C). These data showed that cryopreservation leads to the reduction of  (scale bar = 50 µm). B Gfra1, Id4, α6-integrin, and β1-integrin were assessed by the flow cytometry to determine whether the isolated cells had SSC identity. As it is evident, these markers were expressed at high percentages in the cells P < 0.0001). C-Kit protein expression displayed a significant increase in the TT + cryo + PTX group compared to the TT + cryo group (P < 0.0001) (Fig. 5B, D). The expression of C-Kit was also higher in the PTX-treated donor cells compared with the nontreated groups (P < 0.0001) (Fig. 5D). The significant expression of Ddx4 and C-Kit proteins in the cryopreservation group with PTX indicates the protective effect of PTX on cryopreservation of the SSCs before transplantation as well as its effect on proliferation (Ddx4) and differentiation (C-Kit) markers after transplantation.
Tnp2 showed a significant increase in the rate of expression for the TT + cryo + PTX group (0.81 ± 0.03), compared to the TT + cryo group (0.39 ± 0.04) (P < 0.001). The rate of expression for this maker in the TT + fresh (0.77 ± 0.14) and TT + fresh + PTX (0.92 ± 0.10) groups was almost the same (P > 0.05). An increase in the rate of expression for Tnp2 in the TT + cryo + PTX group was also noticeable as compared to the torsion (0.03 ± 0.02) (P < 0.0001) (Fig. 7B).
However, there was a significant increase in the expression of these genes in the cryopreservation group with PTX compared to the cryopreservation group (P < 0.0001).

Discussion
Azoospermia following cancer therapy, immunosuppressive drugs, genetic factors, environmental toxins, testicular injuries, and torsion is considered to be a major factor of quality of life. Given the rising proportion of patients surviving cancer due to improved therapeutic protocols, it is an issue of growing importance. Hence, the efforts to preserve fertility have motivated researchers to develop options for the Fig. 6 Western blot assay for evaluation of protein expression levels for proliferative (Id4) (A) and differentiation (Sycp3) (B) markers in spermatogonial stem cells (SSCs) assessed at 8 weeks after transplantation. GAPDH was used as an internal control. C, D Graphs are indicative of the ratio for normalization of the density of the markers to the GAPDH. (n = 6; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001) pediatric population facing fertility-threatening cancer therapies. In pre-pubertal boys who do not yet produce sperm, cryobanking of testicular tissue containing spermatogonial stem cells (SSCs) is the only viable option for future fertility preservation. Transplanting the SSCs, or testicular tissue containing SSCs, back to the cured patient appears the most promising strategy [28].
To improve cell survival during freezing and thawing, cryoinjury caused by intracellular ice crystal formation has to be avoided. The addition of cryoprotective agents (CPAs) and controlling freezing and thawing rates are intended to diminish the width of the possible damage window during cryopreservation. Here, we demonstrate the effectiveness of 10 mM PTX as an antioxidant and apoptosis inhibitor on the recovery, proliferation, and differentiation capacity of thawed germ cells enriched for SSCs. In recent years, several studies have evaluated the effect of PTX as a cryoprotectant on the sperm freezing medium [29][30][31]. Similar to our study, Baek et al. [31] found that adding 10 mM PTX to the freezing medium increased sperm viability after thawing. Also, Yovich [32] suggests that a higher concentration of PTX, up to a 10 mM solution, is required to complete ROS release suppression. However, Esteves et al. [33] demonstrated that 5 mM PTX, as proposed, may improve the fertilizing ability of cryopreserved spermatozoa.
We found that the cells were able to form colonies by expanding the culture time. It means that the cells have selfrenewal potential. By assessing Gfra1, Id4, α6-integrin, and β1-integrin, the cells were also assayed for their stem cell identity and the high rates of expression rates for these markers in the cells indicate that the cells are in their early stages of development in the spermatogenic lineage and do not initiate differentiation processes.
Although the previous studies demonstrate an effective way for cryopreservation, the current techniques can cause cryoinjuries due to the high rate of ROS production. In the present study, our aim was to use PTX as an antioxidant to favorably inhibit ROS production after cryopreservation and testicular torsion. However, some tissues require ROS for self-renewal, and in many self-renewing tissues, ROS is harmful to stem cells. It is necessary to be mentioned that the positive role of ROS in self-renewal division has only recently begun to be analyzed. Morimoto et al. [34] showed that in SSCs, ROS amplification plays a critical role in driving self-renewal division. Moreover, excessive ROS production in the cryptorchid condition induces DNA damage in spermatogenic cells, and spermatogonia in SOD1 KO mice showed poor resistance to heat stress [35]. Since ROS can be toxic to germ cells and cause the spermatogenic defect, the success of spermatogenesis seems to be dependent on the delicate control of ROS levels. The excessive ROS leads to the peroxidation of membrane lipids, DNA damage, and protein oxidation and has an effect on the production of mitochondrial ATP.
The data of this study confirmed a significant improvement in the reduction of ROS after the treatment of the cryopreserved SSCs with PTX for a week. In addition to PTX, the inclusion of DMSO in cryopreservation media has been proven to be beneficial for post-thaw survival and function of various stem cells [36]. To the best of our knowledge, literature has not discussed specifically about the effect of PTX on SSC culture, and the therapeutic efficacy of PTX on spermatozoa has been indicated in some previous studies. In Results were normalized at first to the HPRT and then to the control (n = 4, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001) a study conducted by Esteves et al. [33], the acrosome reaction to ionophore challenge in cryopreserved spermatozoa was improved with PTX treatment before freezing. It has also been shown that treatment of the poor-quality human sperm with PTX may enhance post-thaw sperm fertilizing ability [29].
Therefore, we believe that PTX protects spermatogonia cells as a cryoprotective agent. Although its exact mechanism is not fully understood, two reasons are suggested: (1) PTX may cause changes in the physical state of the freezing medium or minimize cell dehydration by forming a sheath around the cells, and (2) PTX can attenuate oxidative metabolism and preserve antioxidant enzyme activities.
Intracellular cAMP concentration, produced by PTX, is effective on in vitro survival, proliferation, and differentiation of human germ cells cultured in system retaining [37]. So, PTX treatment and stem cell transplantation are a hope for significant efficiency of stem cell therapy in some diseases.
After thawing, the cells were transplanted to recipient mice, and the mice were sacrificed after 8 weeks for evaluation. Our results showed that transplanted SSCs started to express proliferation or pre-meiotic markers (i.e., Id4 and Ddx4) and differentiation or meiotic markers (i.e., Sycp3 and C-Kit). Interestingly, greater expression of Id4 and Ddx4 was observed in the presence of 10 mM PTX. As noted in the results, the expression of Id4 and Ddx4 proliferation proteins was markedly reduced in ischemic testes and increased after transplantation. In the transplantation groups, the increase in the expression of these markers was significant in the cryopreservation group with PTX compared to the cryopreservation in the basic medium. The increased expression of these markers indicates an increase in the number and proliferation of SSCs in ischemic testicles. These results confirmed other findings of the present study indicating that the freezing medium containing PTX was able to protect SSCs from cryoinjuries during the freezing-thawing process.
C-Kit is known as an early marker of spermatogonial differentiation [38]. As a marker of spermatogonial (Spg) differentiation, C-Kit functions as an anti-apoptotic factor in primordial germ cells (PGCs), promoting cell replication in PGCs and Spg and initiating the entry of Spg into meiosis. Activation of early meiotic markers such as Dmc1 and Sycp3 is an outcome of C-Kit activity [39]. Here, we noticed a noticeable increase in protein expression for C-Kit and Sycp3 proteins in the transplanted group with cryopreserved SSCs under the exposure to PTX in comparison with the group with the basal freezing medium.
To have more outlook toward possible influences of PTX on the differentiation of SSCs in transplanted mice, we then tried to assess post-meiotic-related factors (i.e., Tnp1, Tnp2, and Prm1). We noticed increased mRNA expression levels for Tnp1, Tnp2, and Prm1 in transplanted mice with cryopreserved SSCs after exposure to PTX. Increased expression levels for Tnp1 and Tnp2 in the SSCs have also been reported by others after incubation of the cells with both retinoic acid and stem cell factor [40]. Prm1 incorporated into chromatin is required to be modified in both transcriptional and post-transcriptional levels [41]. Therefore, it would not be out of expectation to see different functions for PTX on Prm1 in transcriptional and post-transcriptional levels. This demands ongoing studies to have more accurate interpretations of the possible effects of PTX on SSC Prm1 expression traits.
The cAMP also appears to play important regulatory roles within germ cells, since post-meiotic germ cell differentiation is disrupted in mice carrying the mutation of the cAMP-responsive element modulator gene. Intracellular cAMP concentration, produced by inhibition of phosphodiesterase with PTX, mimics the effect of FSH on in vitro survival and differentiation of human germ cells [37].
Therefore, our study showed successful spermatogenesis due to the use of PTX for long-term storage of SSCs, which can increase the probability of banking SSCs for infertile men in future recovery of spermatogenesis.

Conclusion
The only option for improvement of fertility in pre-pubertal boys who suffered from cancer or testicular torsion is preservation of SSCs. The autologous SSC transplantation is a technique with high promise toward a clinical application. Taken together, the data have demonstrated that the presence of using 10 mM PTX in SSC basal freezing media containing DMSO can significantly improve the post-thaw proliferation capacity of germ cells enriched for SSCs. The results suggest that adding PTX to the basic freezing medium can effectively protect the SSCs by increasing the viability and reducing the ROS production after cryopreservation and can improve the transplantation efficiency of thawed SSCs. The methods developed here will serve as foundations for the long-term preservation of SSCs, but more studies should be performed on this subject.
Sadeghiani analyzed the data, Somayeh Solhjoo and Heidar Toolee interpreted the data and prepared the manuscript for publication, Nasrin Takzaree and Nasrin Khanmohammadi supervised the data collection and analyzed the data, and Maryam Shabani reviewed the draft of the manuscript. All authors have read and approved the manuscript.
Funding This study was supported in part by a grant received from the Tehran University of Medical Sciences and Health Services, Tehran, Iran (grant number: 37741).

Data Availability
The data that support the findings of this study are available from the corresponding author upon reasonable request.

Declarations
Ethics Approval and Consent to Participate This article does not contain any studies with human participants performed by any of the authors. All experiments involving the use of animals were approved by the Institutional Animal Care and Use Committee (IACUC) of the Tehran University of Medical Science. All applicable international, national, and institutional guidelines for the care and use of animals were followed. All participants signed informed consent forms approved by the Ethical Committee of Tehran University of Medical Sciences (IR.TUMS.MEDICINE.REC.1397.210).

Consent for Publication Not applicable.
Competing Interests The authors declare no competing interests.