Stem cell transplantation has become a new therapeutic strategy for restoring organ or tissue structure and function [1
]. In the treatment of male infertility, spermatogonial stem cell transplantation (SSCT) was first reported by Brinster in 1994 [3
] and has since been established as a technological breakthrough in stem cell research and the study of Sertoli cell-germ cell interactions. Autologous, homologous, and exogenous SSCT has been carried out in various species including rodents, bovines, monkeys and even humans [3
]. Since spermatogonial stem cells (SSCs) transmit genetic information to their offspring, homologous and exogenous SSCT will inevitably be challenged in terms of reproductive ethics [7
Genetically-modified somatic cells or stem cells have been intensively investigated [8
]. Although the induced stem cells express functional genes and act as sperm cells, the risk and safety of using these cells have been of concern. Non-genetically manipulated stem cell therapy might provide a safe, effective solution to male infertility, especially following anticancer chemotherapy [16
]. Mesenchymal stem cells (MSCs) from bone marrow or adipose tissues have great potential [17
] for tissue repair. MSCs can differentiate into bone, fat, cartilage, muscle, neurons, hepatocytes, insulin-producing cells and skin in the appropriate conditions in vivo
]. Furthermore, bone marrow derived MSCs (BMSCs), which are easy to isolate, have high proliferation rates and have a high potential for differentiation. Based on these characteristics, they may be valuable for use in autologous transplantation. Nayernia et al.
] demonstrated that murine BMSCs are able to differentiate into early germ cells in vitro
and in vivo
. Cakici et al.
] recently demonstrated that GFP-traced adipose-tissue-derived mesenchymal stem cells (ASCs) can give rise to sperm-like cells, leading to recovery of fertility in the busulfan-treated azoospermatic rat model. However, in the busulfan-induced azoospermatism model, self-repair of spermatogenesis might not be excluded due to endogenous stem cells. In this pilot study, we tested the role of BMSC in recovery of fertility in azoospermia. We examined the spermatogenic differentiation of BSMC in vitro
to evaluate the survival and basic biological characteristics of transplanted BMSCs in an azoospermia rat model. We are also investigating sperm cell development in vivo
in our ongoing study.
Approximately 1% of all men in the general population suffer from azoospermia, obstructive or non-obstructive, and azoospermic men constitute approximately 10%~15% of all infertile men [21
]. Among this population, men with non-obstructive azoospermia (NOA) are the most difficult to treat. Various conditions can cause NOA, including genetic or congenital abnormalities, infectious issues, exposure to gonadotoxins, medications such as chemotherapy reagents, varicocele, trauma, endocrine disorders, and idiopathic causes. Current medical therapy, including hormone or surgical methods, shows little benefit to NOA [22
Recent regenerative medical research has shed light on this problem. Human somatic stem cells could be induced to differentiate into multi-lineage cells that help to replace and rebuild damaged or mis-functioning tissues and organs [16
]. Among the stem cells used in regenerative medicine, BMSCs are easily collected, have a high proliferation and differentiation potential, and have low immunological suppression and rejection, therefore BMSCs are suitable for autologous stem cell therapy [17
The particular microenvironments in which BMSCs are implanted plays a vital role in the course of their differentiation [2
]. Seminiferous tubules physically provide dynamic and cyclic regulation of spermatogenesis, and testicular Sertoli cells form a microenvironment that is conducive to sperm cell differentiation and proliferation. In this study, rat BMSCs which were co-cultured with Sertoli cells in a transwell system and in conditioned media in vitro
, were transplanted into the seminiferous tubules of busulfan-treated infertile rats in vivo
. This provided an appropriate spermatogenic microenvironment (niche) to investigate whether they could transdifferentiate into sperm cells. Because cells in normal seminiferous tubules are hierarchically organized with regard to spermatogenesis [26
], endogenous germ cells must be removed so that spermatogonial stem cell niches are opened and rendered accessible to the transplanted donor cells [27
]. Busulfan-treated rats are excellent and well-established recipients for evaluating stem cell activity in rat testis cell populations [27
]. They were used as recipients in this study. Having vacant stem cell niches, favorable growth factors/hormonal milieu, and no Sertoli cell tight junctions provide a suitable physical microenvironment for spermatogenesis.
In this study, rat BMSCs were co-cultured with Sertoli cells in conditioned media in vitro
, which mimics the spermatogenic microenvironment. The BMSCs became round and allied, and exhibited specific morphological characteristics of spermatogonia. After induction, BMSCs expressed integrin-β. Both the post-induced and pre-induced BMSCs expressed c-kit and GCNF, although the former exhibited these markers at higher levels. This suggests that because BMSCs and sperm cells share some markers, BMSCs have the potential to transform into sperm cells. Although Lassalle et al.
] reported that mouse BMSCs could not be transformed into sperm cells in vivo
, we found that rat BMSCs pre-induced with vitamin A in vitro
can survive in recipient rat testes in vivo
, migrate, and become implanted in the basement membranes of seminiferous tubules, considered the unique biological characteristics of spermatogenic stem cells [26
]. Furthermore, the molecular markers of spermatogonial stem cells and spermatogonia—Vasa, Stella, SMAD1, Dazl, GCNF, HSP90α, integrinβ1 and c-kit—were expressed in the recipient testicular tissue after transplantation, indicating that BMSCs transplanted into seminiferous tube probably transdifferentiate into spermatogenic cells. Only a few of the numerous donor BMSCs survived in the recipient and became implanted in the basement membranes of the seminiferous tubules, which indicates that only a small portion of BMSCs have the potential to differentiate into germ cells. Up to 8 weeks after transplantation, no meiosis was found. The reason for this arrest of differentiation is still unknown; therefore, long-term observation must be continued.
Another feature of BMSCs is that they are not only hypo-immunogenic but also produce immunosuppression or immunosurveillance upon transplantation, so they are suitable for allogeneic transplantation [24
]. Also, Sertoli cells are immune tolerant cells [32
]. This ultimately benefits the survival of the donor BMSCs in the recipient seminiferous tubules, so it is not surprising that no immune or inflammatory reaction occurred post transplantation. Although no tumor mass was found 8 weeks post-injection in the recipient rats, the safety and tumorigenic potential of BMSC transplantation still requires long-term observation.
Several previous studies demonstrated that mesenchymal stem cell transplantation recovered fertility in busulfan-treated azoospermatic rats [19
]. Although the results were encouraging, the mechanism is unclear. There are three possibilities for MSCs to recover cell or tissue function during the tissue regeneration process: (1) MSCs differentiated into the target function cells via appropriate induction conditions [1
]; (2) Stem cells secreted trophic factors to stimulate the endogenous stem cells or restore the injured host cell function [2
]; or (3) MSCs merge with the resident cells to recover the injured cell function [35
]. In this study, we demonstrated that spermatogenically-differentiated BMSCs expressed integrin-β, c-kit, and GCNF in vitro
and located at the basement of seminiferous tubule after implanted. However, the role of induced BMSCs in vivo
is still unclear. Further experiments are needed to determine the effect of trans-differentiation, trophic effect or cell fusion of BMSCs on spermatogenesis. Bhartiya et al.
] recently reported that there is a small population of pluripotent stem cells, described as “very small embryonic-like stem cells (VSELs)”, which exists in various adult body tissues, including bone marrow. In our experiment, BMSCs may have been contaminated with VSELs; This explains the following: (1) only a small amount of BMSCs exhibited the morphology of spermatogenic stem cells when co-cultured with Sertoli cells in vitro
; (2) only a small amount of BMSCs survived and located in the basement of the seminiferous tubule in the azoospermatic rat model after transplantation; (3) the possibility of MSCs, the mesoderm originated cells can give rise to sperm cells, the endoderm lineage cells, without genetic modifications.
There are some limitations to this study. Long term observation is difficult without a long term traceable labeling method, and without a negative control group (injection with only medium and trypan blue without cells), the possibility of endogenous recovery of spermatogenesis in the busulfan-induced azoospermic model cannot be excluded. Further experiments on genetic traceable GFP+ cell labeling, appropriate control groups, and long-term follow up will help better understand the spermatogenesis support of BMSCs.
4. Materials and Methods
4.1. Experimental Animals
Male Sprague-Dawley (SD) rats of 4–6 weeks old were obtained from the Experimental Animal Center, Chongqing Medical University, Chongqing, China. The Animal Ethical Committees of the Institute of Zoology approved the use of animals for the study.
4.2. BMSCs Collection, Culture and Differentiation Potential Test
BMSCs were isolated from the rats following the method described by Gnecchi et al.
] and Pereira et al.
]. Cells were cultured in Dulbecco’s Modified Eagle’s Medium/nutrient mixture F12 Ham medium (DMEM/F12 1:1, Gibco, New York, NY, USA) with 10% FBS, 37 °C, 5% CO2
, and passaged when they reached 80%–90% confluence. To test the differentiation ability, cells were subcultured on poly-l
-lysine coated slides in 24-well plates, 10 nmol/L hexadecadrol, 10 μmol/L 5-azacytidine, 100 g/L Salvia miltiorrhiza, and were incubated for 1 week, 1 week, and 1 day respectively. The osteoblastic marker cAKP was detected by the modified Kaplow method. Myocardial cell marker cTnT and nerve cell marker nestin were detected by immunocytochemistry according to the instructions.
4.4. Preparation of Donor MSCs for Transplantation in Vivo
The BMSCs of passage 4 were incubated in DMEM/F12 medium with 10% FBS and 20 μmol/L retinoic acid (Sigma, St. Louis, MI, USA) for 3 days before transplantation. They were then incubated in 10 mL medium containing 10 μg/mL Hoechst 33342 (Promega, Madison, WI, USA) for 15 min, washed at least three times with PBS and trypsinized. The final cell suspension (106 cells/mL) in FBS-free DMEM/F12 with 10% (v/v) trypan blue was ready for transplantation.
4.5. Preparation of Recipient Rats: Busulfan-Induced Azoospermatism Model
The busulfan-treated infertile rat model was prepared as described by Brinster et al.
]. SD rats were used as recipients 4 weeks after single dose intraperitoneal injection with busulfan (40 mg/kg, Sigma, St. Louis, MI, USA) at 4 weeks of age. Hematoxylin-Eosin stain of testicular cross section was performed to evaluate the recipient model 4 weeks after busulfan injection.
4.6. BMSCs Transplantation and Testicular Tissue Collection
The donor BMSCs suspended in serum-free DMEM/F12 were injected into the seminiferous tubules of the recipient rats, with introduction into the rete (is this supposed to be rat?) testes, as described by Brinster and Ogawa [3
]. Approximate 100 μL (105
cells) of BMSCs suspension was introduced into the tubules in the recipient testis, which filled more than half the surface seminiferous tubules. The recipient rats were anesthetized with chloral hydrate injection (30 mg/kg, i.p.) for transplantation. The testes of the recipient rats were collected and fixed in 10% neutral buffered formalin or kept in −80 °C for paraffin sections and spermatogenic markers were detected immediately after the injection at 1, 2, 3, 4 and 8 weeks later.
4.7. RNA Extraction and Quantitative Real-Time RT-PCR
Total RNA was extracted from the testis tissue using an RNApure total RNA isolation kit (Bioteke, Beijing, China) according to the manufacturer’s instructions. First-strand cDNA was obtained from 5 μL of the total RNA using an AMV First Strand cDNA Synthesis Kit (QiaGEN, Shanghai, China). Amplification reactions were performed in a total volume of 25 μL of PCR mixture from the SYBR Green I Real Time PCR KIT (Takara, Beijing, China) containing 5 μL 5× PrimeSTAR™ buffer (Mg2+
plus), 2 μL dNTPs (2.5 mM each), 0.25 μL PrimeSTAR™ HS DNA polymerase (2.5 U/μL), 0.5 μL first-strand cDNA, 0.5 μL (20 pmol) each of the specific primers for Vasa
, and β-actin
(as reference) (for primer sequences see Table 2
), and 16.25 μL RNase-free water. The samples were denatured at 94 °C for 3 min, followed by 40 amplification cycles of 94 °C for 30 s, 50, 51, 56, 58, 55, 58 and 56 °C (for Vasa
, and β-actin
respectively) for 15 s, and 72 °C for 30 s, in a thermal cycler (Roche, Basel, Switzerland); fluorescence signal intensity was measured at 72 °C during each cycle. The PCR products were identified by melting curves: 95 °C for 2 min, 72 °C for 1 min, 95 °C for 30 s with steps of 0.5 °C/s, 30 °C for 1 min. Each product represented a single peak.
4.8. SDS-PAGE and Western Blotting
Proteins were extracted from the cells or testis tissue using Radioimmunoprecipitation assay (RIPA) buffer (Sangon, Shanghai, China) containing 1 mM phenylmethylsulfonylfluoride (PMSF). SSCs and testicular tissue from normal 6-week-old male rats were used as a positive control. Each protein sample (20 μL) was separated on a 12% SDS-polyacrylamide gel (Invitrogen, Carlsbad, NM, USA) and blotted on to a polyvinylidene fluoride (PVDF) membrane (Bio-Rad, Shanghai, China). Protein binding sites were blocked for 1~2 h with blocking buffer: TBST (Tris-buffered saline with Tween-20: 10 mM Tris–HCl, pH 7.5, 150 mM NaCl, 0.05% Tween-20) containing 5% nonfat dry milk at room temperature (RT). Primary antibodies were diluted in blocking buffer and incubated overnight at 4 °C: GCNF (1:600), HSP90α, integrinβ1, c-kit (1:300), β-actin (1:3000). The membranes were washed three times with TBST for 10 min. After incubation with secondary antibodies: goat-anti-rabbit-HRP or rabbit-anti-mouse-HRP (1:5000, Santa Cruz, Dallas, TX, USA) in blocking buffer for 1 h at RT, the membranes were again washed three times in TBST for 10 min. Protein bands were detected by the enhanced chemiluminescence method (ECL, QiaGEN, Shanghai, China). The area and density of each protein band were measured and the relative level of target protein was evaluated by the ratio of its area density to that of β-actin.
Primer sets used for quantitative real-time PCR.
Primer sets used for quantitative real-time PCR.
|Target Gene||Locus No.||Sequence of Primers||Product Size|
|Vasa||S75275||F: 5'-GCGAGACTACATCTACAAC-3'||135 bp|
|SMAD1||AF067727||F: 5'-CTCATGTCATTTATTGCCG-3'||138 bp|
|Stella||BK001414||F: 5'-CTATCATCGTCGTCAAAGG-3'||177 bp|
|Dazl||NM_001025742||F: 5'-CGACGAAATCGGGAAGCTC-3'||94 bp|
|GCNF||AJ783965||F: 5'-CAACTGAACAAGCGGTATT-3'||114 bp|
|c-kit||NM_022264||F: 5'-TGCCCGAAACAAGTCATCTCC-3'||112 bp|
|β-actin||NM_031144||F: 5'-GCTCGTCGTCGACAACGGCTC-3'||353 bp|
4.9. Statistical Analysis
Experiments were repeated at least three times. qPCR results were expressed by Ct values and calculated as follows: ΔCt = Ct(target gene) − Ct(β-actin); ΔΔCt = ΔCt(transplantation group) − ΔCt(control group). The relative levels of target gene expression were evaluated as 2−ΔΔCt. Relative protein levels were expressed as means ± SEM and analyzed by the Student-Newman-Keuls test using SPSS 18.0. A value of p < 0.05 was chosen as an indication of statistical significance.