1. Restoration of Fertility in experimental Azoospermic rat by transplantation of Mesenchymal stem cells
Samir A.M. Zahkook1; Ahmed A M Atwa2;Shahat,M.M2;Ahmed M Mansour3 and Sayed Bakry1
1- Zoology Department , faculty of Science , Al Azhar University, Nasr City, Cairo, Egypt
2- Zoology Department , faculty of Science , Al Azhar University , Assuit, Egypt
3- Department of pharmacology & Toxicology, Faculty of Pharmacy -Al-Azhar University-Cairo-Egypt
4- Surgical process and MSCs transplantation:
ABSTRACT
MSCs cells were transplanted into the recipient rat through the efferent ducts into the seminiferous tubules (Ogawa et al., 1999). A total of ten animals received the cell suspension in one testis (Right) while the other(left) was used as internal control, receiving 100 µL of PBS. Cell transplantation was performed few hours after cell isolation (cells were kept in room temperature meanwhile). During surgery, recipient rats were kept under anesthesia by intraperitoneal administration of Xylazine (20 mg/kg), Ketamine (5 mg/kg), and Athropine (0.04 mg/kg). The testes were surgically exteriorized through a midline abdominal incision, and stem cells suspension were injected using a micropipette via efferent ducts under a stereomicroscope (Carl Zeiss Stemi 2000). Cell concentration for transplantation was 5–10X106cells/ml and approximately 100 µL of the cell suspension was transplanted. Histological examination to evaluate restoring of spermatogenesis will performed after 12 weeks of MSCs transplantation in transplanted testis .
Defective spermatogenesis due to the failure in germ cell proliferation and differentiation is the major sign of male infertility pathogenesis; and male factor is involved in up to half of all infertile couples. The pathogenesis of male infertility can be reflected by defective spermatogenesis due to failure in germ cell proliferation and differentiation. Here, we use the capacity of mesenchymal stem cells (MSCs), as multipotent adult stem cells to revive spermatogenesis in an induced azoospermic rat. Successful transplant`ation of mesenchymal stem cells (MSCs) into testes requires depletion of the host germ cells to allow efficient colonization of the donor MSC in seminiferous tubules of recipient. To induce complete depletion of endogenous spermatogenesis, we used different doses of Busulfan (Myleran); anticancer drug is commonly used for preparing a recipient rat before transplantation (chemo sterilization). For preparation of the transplanted cells, MSCs were collected by flushing the femurs of 4–6 week old male rats and cultured. The injection of MSCs into efferent duct of testis was conducted as previously described by Ogawa et al (1997). Currently different physiological parameters of testes are being measured and analyzed (testis weight, sperm count and histological assessment of spermatogenic recovery). Moreover, Histopathological examination of rat testicular tissue will be conducted to refer spermatogenesis and their changes in control, busulfan treated and MSCs transplanted testis. Taken together, this study is a trial to evaluate the capacity of mesenchymal stem cells in azoospermic rat testis to estimate the capacity of stem cells in spermatogenesis revival. This approach may provide a powerful tool to investigate the basic biology of stem cells for infertility treatment.
RESULTS
1-Histological changes of testes
In the control group at week , most of the seminiferous tubules showed a thick wall with a very limited lumen (Figure 1 A&B). The wall consists of several layers of seminiferous epithelial cells, with the spermatogonia in the outer layer, the spermatocytes in the middle and the spermatozoa and spermatids protruding toward the lumen, which we defined in the present study as the ‘fully spermatogenic’ seminiferous tubule. After administration of double injections of 10→15 mg/kg−1 busulfan, the walls of the majority of the seminiferous tubules became thinner at the end of week 8 because of the depletion of the spermatids and the spermatozoa (Figure 1 C&D).
INTRODUCTION
Infertility is defined as the failure to achieve a pregnancy after one year of regular (at least twice weekly) unprotected intercourse with the same partner. Infertility affects 15% of couples, with male factors responsible for 40%–60% of cases of infertility. Moreover, of these cases, approximately 50% result from defective spermatogenesis due to complete blockage of spermatogenesis, low sperm counts, or abnormal sperm motility, morphology, or function. Adult spermatogenesis is a complex and tightly regulated process in which a small pool of germ-line stem cells develops to ultimately form spermatozoa(Kobayashi et al.,2013) . Some report has suggested that adult bone marrow derived stem cells (BMSCs) are able to regenerate various non-hematopoietic cell lineages in several organs, and that tissue-specific and pluripotent stem cells are present in the bone marrow. Mesenchymal stem cells possess the capacity to trans-differentiate into epithelial cells and lineages derived from the neuro- ectoderm, and in addition, these cells can migrate to the sites of injury, inflammation, and to tumors. These properties of mesenchymal stem cells make them promising candidates for use in regenerative medicine
This study aims to investigate the regenerative potency of PKH26 Red Fluorescent Cell Linker Kit labeled bone marrow derived-mesenchymal stem cells (BM-MSCs), implanted into the azoospermic rat testis. This trial experiment and long time follow up studies can help to introduce novel therapeutic approaches in patients suffering non obstructive Azoospermia.
Figure 1. Representative micrographs of testes of mal rat after the injection of busulfan.(A): Testis of control animals. (B): An amplified local region of A, showing a typical ‘fully spermatogenic’ seminiferous tubule with spermatogonia (white arrow), spermatocytes (Black arrow), spermatids (Δ) and spermatozoa (▲). (C): Testis of the animals 6 weeks after double injections of mg kg−1 busulfan; (D) An amplified local region of E showing a typical‘non-spermatogenic’ seminiferous tubule, sertoli cells are present.
2-Derivation of bone marrow MSCs:
MATERIAL AND METHODS
During primary cell culture, the attached cells expanded normally with a spindle like appearance, as expected. These adherent cells were readily grown in vitro by successive cycles of trypsinization, seeding and culture, every 3 days, for 4 passages without visible morphological changes. As it is known, during trypsinization and in consequence passages, fibroblast cells would be eliminated .Thus, we designated these fibroblasts- like cell populations as bone marrow derived mesenchymal stem cells (Fig.2 the lower small box).⇩
30 Male Sprague-Dawley rats 8 weeks old, weighing 150–180 g, were obtained from VACSERA .Cairo. Egypt and maintained in the (Experimental Animal Unit) of the Center of Genetic Engineering. faculty of science . Al Azhar University. The recipient animals received a standard laboratory chow and water ad libitum.
Recipient animals were treated with a system of divided injections was used in which 10 then 15 mg/kg Busulfan (GlaxoSmithKline Inc., Mississauga, Ontario, Canada) was administered by intraperitoneal injection with a 14 day interval . MSCs cell transplantation was performed 4 or more weeks after the initial Busulfan injection, to induce testicular degeneration(experimental Azoospermia).
Briefly, MSCs were collected by flushing the femurs of 4–6 week old male rats. Cells were cultured in 25 cm2 flasks in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco, Scotland) supplemented with 15% fetal bovine serum (FBS) (Gibco, Scotland), 100 ll/ml penicillin, and 100 mg/ml streptomycin (Gibco, Scotland). every 3 days. According to the fact that fibroblast cells will be eliminated during trypsinization in consequent passages, we considered these fibroblasts-like cells as MSCs.
MSCs cells were labeled with PKH26; PKH26 is a red fluorochrome. It has excitation (551 nm) and emission (567 nm) characteristics compatible with rhodamine or phycoerythrin detection systems. In the current work, MSCs cells were labeled with PKH26 according to the manufacturer’s recommendations (Sigma).
1- Recipient animals
2- Isolation and expansion of bone marrow derived mesenchymal stem cells
CONCLOUSION
In conclusion, these primary current results showed the protocol tested in this study can prepare recipient Azoospermic malt rat model suitable for the autologous transplantation of bone marrow stem cells, thereby providing a model with which to explore treatments for testicular infertility.
Stem cell research has shown remarkable progress over the past 5 years. However, improvements are still needed before stem cells can be used clinically. For the treatment of male infertility, future advances may enable spermatids to be differentiated from( Induced Pluripotent Stem Cells) iPS cells.
3- Labeling of bone marrow-derived mesenchymal stem cells: