Sertoli Cell-Conditioned Medium Induces Differentiation of Bone Marrow-Derived Mesenchymal Stem Cells to Male Germ-Like Cells in Busulfan-Induced Azoospermic Mouse Model.

Non-obstructive azoospermia is a severe form of male infertility, with limited effective treatments. Bone marrow mesenchymal stem cells (BMSCs) can differentiate to different cell lines; therefore, transplantation of these cells is used for treatment of several diseases. Since these cells require induction factors to differentiate into germ cells, we co-transplanted bone marrow stem cells (BMSCs) with Sertoli cell-conditioned medium (SCCM) into the testis of azoospermic mice. This study was carried out in two sections, in vitro and in vivo. For in vitro study, differentiating factors (c-kit and ID4) were examined after 15 days of co-culture of bone marrow cells with Sertoli cell-conditioned medium, while for in vivo study, the azoospermia model was first created by intraperitoneal administration of a single-dose busulfan (40 mg/kg) followed by single-dose CdCl2 (2 mg/kg) after 4 weeks. Mice were divided into 4 groups including control (azoospermia), BMSC, SCCM, and BMSC + SCCM. Eight weeks after transplantation, samples were assessed for proliferation and differentiation via the expression level of MVH, ID4, SCP3, Tp1, Tp2, and Prm1 differentiation markers. The results showed that BMSC co-cultured with SCCM in vitro differentiated BMSC to germ-like cells. Similarly, in vivo studies revealed a higher level of BMSC differentiation into germ-like cells with significant higher expression of differentiation markers in transplanted groups compared to the control. This study confirmed the role of SCCM as an inductive factor for BMSC differentiation to germ cells both in vivo and in vitro conditions.

Pentoxifylline promotes spermatogenesis via upregulation of the Nrf2-ARE signalling pathway in a mouse model of germ-cell apoptosis induced by testicular torsion-detorsion.

CONTEXT: Testicular torsion-detorsion results in loss of germ cells and infertility. Pentoxifylline has been shown to prevent tissue damage. AIMS: To determine the effect of pentoxifylline on germ cell survival in torsion-detorsion induced apoptosis Methods: Twenty male mice were divided into four groups of five animals each: Control, T1 (Torsion-detorsion+single dose 100mg/kg Pentoxifylline/IP), T2 (Torsion-detorsion+daily 20mg/kg pentoxifylline/IP for 2weeks, and T/D (Torsion-detorsion only). 35thday after torsion-detorsion, the left testes of all the animals were harvested for histological and biochemical analysis. KEY RESULTS: Histomorpholoical analysis showed significant increase (P <0.05) in seminiferous tubule diameter, Johnsen's score and germ cells of Control and T1 compared to T2 and T/D, with no significant difference (P >0.05) in testis weight, sertoli, leydig and myoid cells. Tunnel assay showed significant increase (P <0.05) in apoptotic cells of T/D and T2 animals compared to Control and T1. RT-PCR analysis showed significant high (P <0.01) mRNA expression of Bax gene in T/D compared to T1 and T2 and significant increase (P <0.05) of Bcl2 in Control, T1, T2 compared to T/D. Nrf2-ARE transcripts revealed significant increase (P <0.05) in Control and T1 compared to T2 and T/D. Western blot showed significantly increased (P <0.05) caspase-3 in T/D compared to Control, T1 and T2. CONCLUSION: Pentoxifylline promotes spermatogenesis and suppressed apoptosis induced by testicular torsion-detorsion. IMPLICATION: Pentoxifylline could serve as adjunct therapy to surgery in the treatment of torsion-detorsion induced germ cell apoptosis.

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 ß1-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.

Pentoxifylline improves the survival of spermatogenic cells via oxidative stress suppression and upregulation of PI3K/AKT pathway in mouse model of testicular torsion-detorsion.

Testicular torsion-detorsion results in enhanced formation of free radicals which contribute to the pathophysiology of testicular tissue damage. Recent reports have identified protective role of pentoxifylline (PTX) against free radicals. Thus, we determined the protective effect of pentoxifylline against testicular damage in mouse model of testicular torsion-detorsion. Twenty (6 weeks old) male mice were divided into 4 groups of 5 animals each namely: Control (sham operated group), T1 (Torsion-detosion + single dose 100 mg/kg PTX, T2 (torsion-detorsion + 20 mg/kg PTX for 2 weeks and T/D (torsion-detorsion only). Animals in T1, T2 and T/D groups underwent 2 h of testicular torsion with the left testes rotated 720° (clockwisely) followed by 30 min of detorsion. After detorsion, drug administration was done intraperitoneally. The left testes of all the animals were excised on the 35th day after torsion-detortion for histopathological and biochemical assay. Histomorphological analysis of the seminiferous tubules showed that there were significant increase (P < 0.01 or 0.05) in the mean seminiferous tubule diameter, Johnson score and germ cells of animals in Control and T1 compared to T2 and T/D with no significant difference (P > 0.05) in testes weight, sertoli, leydig and myoid cells in all groups. IHC results showed significant increase (P < 0.01 or 0.05) in id4 and scp3 protein markers in Control, T1 and T2 compared to T/D. Oxidative stress analysis revealed that Pentoxifylline significantly increased (P < 0.01 or 0.05) the level of SOD, catalase, mRNA expression of akt and pi3k genes but significantly suppress (P < 0.01 or 0.05) MDA and Caspase-3 level in Control, T1 and T2 compared to T/D. Pentoxifylline could be used as an adjunct therapy to surgery in the treatment of torsion-detorsion related testicular injury, However, Further studies are needed to evaluate the effects of pentoxifylline on testicular torsion.

Effects of Co-Administration of Bone Marrow Stromal Cells and L-Carnitine on The Recovery of Damaged Ovaries by Performing Chemotherapy Model in Rat.

BACKGROUND: L-carnitine (Lc) as a type of flavonoid antioxidants and bone marrow stromal cells (BMSCs) as a type of mesenchymal stem cells may recover damaged ovaries. It seems that Lc has favorable effects on differentiation, increasing lifespan and decreasing apoptosis in BMSCs. The aim of this study was to investigate effects of co-administration of BMSC+Lc on damaged ovaries after creating a chemotherapy model with cyclophosphamide in rats. MATERIALS AND METHODS: In this experimental study, cyclophosphamide was intraperitoneally (IP) injected to forty female wistar rats for 14 days, in terms of chemotherapy-induced ovarian destruction. The rats were then randomly divided into four groups: control, Lc, BMSCs and co-administration of BMSC+Lc. Injection of BMSCs into bilateral ovaries and intraperitoneal injection of Lc were performed individually and together. Four weeks later, levels of serum estradiol (E2) and follicle-stimulating hormone (FSH) using enzyme-linked immunosorbent assay (ELISA) kit, number of ovarian follicles at different stages using hematoxylin and eosin (H and E) staining and expression of ovarian Bcl-2 and Bax proteins using western blot were assessed. RESULTS: Co-administration of BMSC+Lc increased E2 and decreased FSH levels compared to the control group (P<0.001). The number of follicles was higher in the co-administrated group compared to the control group (P<0.001). Co-administration of BMSC+Lc increased Bcl-2 protein level, decreased Bax protein level and increased Bcl-2/Bax ratio (P<0.001). CONCLUSION: The effect of co-administration of BMSC+Lc is probably more effective than the effect of their separate administration on the recovery of damaged ovaries by chemotherapy.

Bone Marrow Stromal Cells with the Granulocyte Colony-Stimulating Factor in the Management of Chemotherapy-Induced Ovarian Failure in a Rat Model.

BACKGROUND: Bone marrow stromal cells (BMSCs), as a type of mesenchymal stem cells, and the granulocyte colony-stimulating factor (G-CSF), as a type of growth factor, may recover damaged ovaries. The aim of the present study was to investigate the effects of the coadministration of BMSCs and the G-CSF on damaged ovaries after creating a chemotherapy model with cyclophosphamide (CTX) in rats. METHODS: The present study was performed in Semnan, Iran, in the late 2016 and the early 2017. BMSCs were cultured and were confirmed using the CD markers of stromal cells. Forty female Wistar rats were randomly divided into 4 groups. The rats were injected intraperitoneally with CTX for 14 days to induce chemotherapy and ovarian destruction. Then, the BMSCs were injected into bilateral ovaries and the G-CSF was injected intraperitoneally, individually and together. Four weeks later, the number of ovarian follicles using H&E staining, the number of apoptotic granulosa cells using the TUNEL assay, the number of produced oocytes from the ovaries, and the levels of serum E2 and FSH using an ELISA reader were assessed. Statistical analysis was done using one-way ANOVA with SPSS, version 16.0. RESULTS: The results showed that the effects of the coadministration of 2×106 BMSCs and 70 µg/kg of the G-CSF were significantly more favorable than those in the control group (P<0.001), the BMSC group (P=0.016), and the G-CSF group (P<0.001) on the recovery of damaged ovaries. CONCLUSION: The efficacy of the coadministration of BMSCs and the G-CSF in the recovery of ovaries damaged by chemotherapy was high by comparison with the administration of either of them separately.

Effect of quercetin on the number of blastomeres, zona pellucida thickness, and hatching rate of mouse embryos exposed to actinomycin D: An experimental study.

BACKGROUND: Quercetin is a flavonoid with the ability to improve the growth of embryos in vitro, and actinomycin D is an inducer of apoptosis in embryonic cells. OBJECTIVE: The aim was to evaluate the effect of quercetin on the number of viable and apoptotic cells, the zona pellucida (ZP) thickness and the hatching rate of preimplantation embryos exposed to actinomycin D in mice. MATERIALS AND METHODS: Two-cell embryos were randomly divided into four groups (Control, Quercetin, actinomycin D, and Quercetin + actinomycin D group). Blastocysts percentage, hatched blastocysts, and ZP thickness of blastocysts was measured. The number of blastomeres was counted by Hoechst and propidium iodide staining and the apoptotic cells number was counted by TUNEL assay. RESULTS: The results showed that the use of quercetin significantly improved the growth of embryos compared to the control group (p=0.037). Moreover, quercetin reduced the destructive effects of actinomycin D on the growth of embryos significantly (p=0.026). CONCLUSION: quercetin may protect the embryos against actinomycin D so that increases the number of viable cells and decreases the number of apoptotic cells, which can help the expansion of the blastocysts, thinning of the ZP thickness and increasing the hatching rate in mouse embryos.

Effect of Transplantation of Bone Marrow Stromal Cell- Conditioned Medium on Ovarian Function, Morphology and Cell Death in Cyclophosphamide-Treated Rats.

OBJECTIVES: Although stem cell transplantation has beneficial effects on tissue regeneration, but there are still problems such as high cost and safety issues. Since stem cell therapy is largely dependent on paracrine activity, in this study, utilization of transplantation of bone marrow stromal cells (BMSCs)-secretome instead of the cells, into damaged ovaries was evaluated to overcome the limitations of stem cell transplantation. MATERIALS AND METHODS: In this experimental study, BMSCs were cultured and 25-fold concentrated conditioned medium (CM) from BMSCs was prepared. Female rats were injected intraperitoneally with cyclophosphamide (CTX) for 14 days. Then, BMSCs and CM were individually transplanted into bilateral ovaries, and the ovaries were excised after four weeks of treatment. The follicle count was performed using hematoxylin and eosin (H and E) staining and the apoptotic cells were counted using TUNEL assay. Ovarian function was evaluated by monitoring the ability of ovulation and the levels of serum estradiol (E2) and follicle-stimulating hormone (FSH). RESULTS: Evaluation of the ovarian function and structure showed that results of secretome transplantation were almost similar to those of BMSCs transplantation and there was no significant differences between them. CONCLUSIONS: BMSCs-secretome is likely responsible for the therapeutic paracrine effect of BMSCs. Stem cellsecretome is expected to overcome the limitations of stem cell transplantation and become the basis of a novel therapy for ovarian damage.

Effect of L-carnitine on in vitro developmental rate, the zona pellucida and hatching of blastocysts and their cell numbers in mouse embryos.

L-carnitine (LC) is an antioxidant with the ability to promote the growth in vitro embryo. OBJECTIVE: The goal was to evaluate the effect of LC on some indicators of embryo development and blastocyst quality including zona pellucid (ZP) thickness, the hatching of blastocysts and their cell numbers. MATERIALS AND METHODS: Mouse embryos were randomly divided into five groups and incubated with different concentrations of LC (I; 0, II; 0.5, III; 1, IV; 2 and V; 4 mg/ml) from 2-cell to hatched blastocyst. The percentage of blastocysts and hatched blastocysts was calculated. Blastocysts ZP thickness was measured and the number of blastocyst cells was counted using Hoechst and propidium iodide (PI) staining. RESULTS: The results showed concentration of 0.5 mg/ml of LC had an antioxidant effect as in this group, the percentage of blastocysts and hatched blactocysts (p=0.01), the ZP thickness (p=0.00) and the number of blastocyst inner cell mass were significantly more favorable than the control group (p=0.03); and concentration of 4 mg/ml of LC had a toxic effect on embryo development and blastocyst quality (p=0.00). CONCLUSION: The results suggest that LC may increase the number of blastocyst cells, which probably helps to expand the blastocyst and thinning of the ZP thickness and, therefore, creating a successful hatching for implantation.