| Part One:Enhancement of Mouse Germ Cell-Associated Genes Expression by Injection of HUCMSCs into the Testis of Chemical-Induced Azoospermic MicePurposeTo explore how to use human umbilical cord-derived mesenchymal stem cells （HUCMSCs） treat azoospermia, Transplant UCMSCs into the model of azoospermia mouse, and then detect the expression of germ cell specific genes. All the expression of the genes is increased. We have demonstrated HUCMSCs could enhance mouse germ cell-specific gene expression.This study provides further evidence for preclinical therapeutic effects of HUCMSCs, and explores a new approach to the treatment of azoospermia.Methods1. Animal model of Azoospermia. A total of80male BALB/c mice aged8weeks （22-25g） were weighed and given a single injection. All the mice were given a single dose of35mg kg-1busulfan as previously described. At five weeks after the single injection, the mice survived were prepared for transplantation. The testes of five mice were selected for histology examination by Hematoxylin and Eosin staining. HE staining was performed as previously described.2. Parent’s written consent for tissue donation was required prior to the collection of umbilical cords from full-term birth babies. The umbilical cords from three different individuals were separately processed within6hours of vaginal delivery. HUCMSCs were isolated from the Wharton’s Jelly （WJ） without enzyme digestion or dissection. After washing in phosphate buffered saline （PBS） to remove blood on the surface, the vessels were removed and the cord was cut into small pieces （0.5-lcm）. The cells were cultured in Dulbecco’s modified Eagle’s medium （DMEM） supplemented with antibiotics （penicillin100mg mL-1, streptomycin10mg mL-1,） and10%fetal bovine serum in5%CO2in a37℃incubator. HEK293cells were cultured in DMEM medium containing10%fetal bovine serum under standard condition of5%CO2at37℃. HUCMSCs from Wharton’s jelly after harvesting at the third passage were immediately detected Fluorescein isothiocynate （FITC）-conjugated or phycoerythryne （PE）-conjugated Anti-CD31, anti-CD105, anti-CD73antibodies （Biolegend, San Diego, USA） were used. Cells were analyzed using a FACScan flow cytometer. The karyotypes of HUCMSCs were performed by G banding staining. To induce adipogenic differentiation, cells were stained with Oil Red O. To induce osteogenic differentiation, cells were stained by von Kossa staining after21days.3. The third passage of HUCMSCs was prepared for transplantation. All40busulfan-treated mice were randomly divided into four groups （each group included10mice）: Group one was model mice without injection as a blank control. Group two was injected with physiological saline as negative control in the left side testis. Group three was injected with HEK293cells also in the left side testis. Group four was injected with HUCMSCs in the left side testis.10μl of the cell suspension （approximately1×105cells） was directly injected under the tunica albuginea.4. Detecting the germ cell specific gene expression. Three weeks after injection, five mice of each group were selected and total RNA was extracted from testis. The genes including vasa, Scp3, Cyclin A1, Dazl, Stra8, miwi, Tnp2, Pgk2, Akap3, Tex18were detected. Three weeks after injection, five mice of each group were selected and western blots were performed. Data are from the experiment presented as a mean±standard deviation （SD）. All p values<0.05were considered statistically significant.Results1. Azoospermia model. After busulfan administration （35mg kg-1）, the testes of five mice were selected for the HE staining. Testicular sections show most of the seminiferous tubules walls became thinner at the end of week5, the wall consisted of seminiferous epithelial cells and the spermatogonium is in the outer layer. Spermatogenic cells, including spermatocytes, spermatids and spermatozoa were severely depleted. Spermatogonia and sertoli cells remained. 2. Cell culture and characterizing. The umbilical cord tissues were cut into small pieces, after two weeks, the cells migrate out of the tissue and reach confluence. Most of the cells were spindle-shaped and fibroblast-like. Immunostaining of HUCMSCs showed strong positive signals against CD73and CD105. Cells were not immunostained by CD31which is a marker of endothelial cells. After21days of osteogenic differentiation induction, mineralized deposits were observed. The cells contained a large amount of small lipid vacuoles stained using Oil Red O solution at25days.3. Transplant HUCMSCs into azoospermia model with the third passage of HUCMSCs. Then under normal condition, keep for3weeks. The mice’s conditions were well and no mouse died.4. Three weeks later, after azoospermia model were prepare, five mice were chosen randomly and were evaluated separately. The expression of meiosis associated genes in injected testis was higher than the other side which as a control. Meanwhile, when the saline water and HEK293cells were separately injected into the testis as a negative group, the expression of all the genes we detected were unchanged compared to control side testis. The testes of five mice were selected and the expression of miwi, vasa, scp3were detected at a higher level in injected testis than control side on week3after injecting HUCMSCs.However, no difference can be detected between the testes which treated with saline and without treatment. The expression level of these three genes in saline-treated group was low. Meanwhile, when injecting HEK293cells into the testes, as negative control group, no difference can be detected between two sides, and the level of expression was also very low.Conclusion1. Our study shows using optimized dose of busulfan to establish azoospermia model considering the survival rate. In busulfan-treated mice, we transplant HUCMSCs into the testis of these mice.2. Using tissue method to culture HUCMSCs, to decrease the effects of digestive enzymes. Using lower passage of HUCMSCs for transplantation, the stem cells possess potent immunosuppressive function. The detection of HUCMSCs property is effective.3. When transplanting HUCMSCs into the testis of azoospermia model mouse, enhanced the expression of germ cell specific gene compared to the HEK293cells and saline water injection. It proved this kind of enhancement is not indeced by injection and Heterologous cells.4. Our study provides experimental evidence in preclinical models of infertility suggesting the possible clinical benefits of HUCMSCs. It is a promising candidate, at least partly, for promoting spermatogenesis. Part Two:Elucidation of RNA Binding Regions ofGRTH protein to Transcripts of a Chromatin Remodeling Protein TP2Essential for SpermatogenesisPurposeGonadotropin regulated testicular RNA helicase （GRTH/DDX25）, is a testis-specific protein, which is a member of the DEAD-box protein family. It is highly expressed in pachytene and metaphase spermatocytes and round spermatids. GRTH is regulated developmentally, and control the spermatogenesis progress posttranscriptionally.To better understand the mechanism of GRTH regulating TP2posttranscriptionally, this study focus on elucidating the interaction region of GRTH protein binding with TP2mRNA which is a germ cell specificgene.Identification of RNA binding motifs of GRTH to relevant germ cells transcripts and the determination of the cis-elements in3’UTR is very important.To determine3’ UTR interacting regions and further understand the control mechanisms of binding with TP2during germ cell development. This study is very valuable for understand function of male reproduction.Methods1. DNA Constructs:The cDNA of the GRTH gene, encoding the mouse Gonadotropin-regulated testicular RNA helicase （NM013932.4）, and3’untranslated region （3’-UTR） of transition protein2（TP2, NM013694, nt408-535）3’-UTR were amplified by PCR using cDNA template prepared from mouse testis tissue. To construct GRTH truncated deletions plasmids, GRTH full length cDNA was inserted into the Hind3and Xbal site of a pcDNA3.1（+） plasmid vector. GRTH DNA fragments were digested with Hind3and Xbal and ligated into pcDNA3.1vectors to generate pcDNA3.1-G423（a.a1-423）, pcDNA3.1-G115-483（a.a116-483）, pcDNA3.1-G138（a.a1-138）, pcDNA3.1-G166（a.a1-166）, pcDNA3.1-G244（a.a1-244）, pcDNA3.1-G325（a.a1-325）, pcDNA3.1-G388（a.a1-388）. DNA fragment from TP2-3’UTR were digested with Hind3and Eco Rl and ligated into pST18vector to generate pST18-TP2-3’UTR plasmid.2. Mutagenesis:The mutation of G483（GRTH full length） motif V from ARGID to AAAID was introduced into the G483construct with QuickChange mutagenesis kit. It was verified by sequencing （G483Vx）. Mutation of motif Ia from PTYELA to PTSALA was followed using mutated G483Vx as the template to produce G483IaxVx and verify by sequencing3. In vitro transcription and translation of GRTH deletions cDNA. One microgram each of the GRTH full length and deleted constructs above were transcribed and translated in vitro at30℃for90min with the T7TNT coupled reticulocyte lysate system according to the manufacturer’s instructions Western Blot Analysis:The expression of GRTH full length and truncated deletions proteins from in vitro system was detected by western blot4. Generation of3’Biotin labeled RNA of Tp2:The plasmid of pST18-TP2-3’ UTR was linearized and used for in vitro transcription with T7RNA polymerase. RNA probe from full length3’UTR of Tp2was generated by in vitro T7polymerase transcription and purified on denaturing polyacrylamide gels.50pmol of RNAs were labeled at the3’end with biotinylated cytidine bisphosphate using T4RNA. Oligo synthesis of three consecutive10bp overlapped3’UTR of TP2RNA （50bp each）, T1（381/430）, T2（421/470）, T3（461/510）, either biotylanted or unlabeled, was synthesized by IDTDNA.5. RNA electrophoretic mobility shift assay （REMSA）. REMSAs were performed by using the LightShift Chemiluminescent EMSA kit.20fmol of biotinylated RNA （1nM） and4pmol of proteins （0.2μM） were incubated. First, detect the GRTH full length（G483） to bind with TP23’UTR RNA. Following, G483、G115/483、G423、 G388、G325、G244、G166、G138binded with T1（381/430）, T2（421/470）, T3（461/510） probe. In supershift assay, anti-V5antibody was added to detect G483、 G115/483、G423、G388、G325、G244binding with T1（381/430）、T3（461/510）. Then G483Iax、G483Vx、G483Iax/Vxwere used to bind with T1（381/430） probe. All binding assay were performed by using Chemiluminescent Nucleic Acid Detection Module Kit.Results1.RNA-EMSA analysis of GRTH with127bp3’UTR of Tp2transcript.To define specific binding motif of GRTH protein to3’UTR of Tp2transcript, deletion of GRTH sequence was designed based on the conserved motifs of RNA helicase protein known for its individual function as the guide line. Each deletion construct was established in frame at C-terminus with V5as the marker. Western blot showed expected protein size from the in vitroTnt translated individual deletion construct respectively. To first evaluate if GRTH protein interacts with Tp23’UTR transcript,127bp of3’UTR of mouse Tp2was cloned and in vitro transcribed as the probe. Apparent retarded Tp23’UTR RNA with full length GRTH protein （#） was observed in RNA EMSA analysis.2. RNA-EMSA analysis of sequential3’deletion of GRTH protein with Tp23’UTR region. To further define the cis-element in3’UTR region of Tp2interacting with GRTH,3consecutive overlapped mRNA oligomers were designed and end-labeled with biotin. For systemically dissecting GRTH binding motif with Tp2transcript, GRTH protein was further visualized as2domains followed the homology of GRTH protein to the crystal structure of Ddx19. Full length GRTH protein （G483） formed specific complexes with probe T1,48nt immediately downstream of TGA termination codon and one specific retarded complex with probe T3,78-127nt3’to TGA codon. Similar retarded complexes were observed when probe T1but not probe T3when interacted with G423with deletion of conserved RNA helicase motif Ⅵ None of those retarded complexes were present in GRTH388with further sequences deletion including motif Ⅴ known as RNA binding motif in the family of RNA helicase. Loss a specific retarded band in the G388with deleted Motif V compared to G423implicated the importance of sequence containing conserved RNA binding motif ARGID in GRTH associated with Tp2transcript. There is no complex formation when probe T2located between nt43-92overlapped with3’end of T1and 5’ end of T3（Fig.3）. This suggests sequences between T1and T3do not functional cis element（s） to interact with GRTH.3. When domain II with conserved motifs IV, V and VI was absent in G325, two retarded complexes with probe T1of Tp2were observed. Only one retarded complex was detected in G244when conserved motifs Ⅱ and Ⅲ were also deleted. No complex was detected when used either probe T2or T3. Presence of specific retarded bands with T1probe in the complete deleted domain Ⅱ of GRTH protein in the case of G325or G244suggests an additional RNA binding site located in the domain of GRTH protein. Loss of retarded bands in G166and G138in the absence of conserved motifs Ia/Ib also known as the RNA binding site revealed those region might also important for GRTH binding to the RNA transcript.4. When deletion of N terminus114aa in GRTH （G115/483）, similar retarded bands noted in the full length GRTH （G483） by interacting with probe T1or T3of Tp2were observed. This is consistent with the finding of absence of any retarded complexes in the G138containing the114aa of5’N terminus sequence only. This also reflected5’114aa is not essential for RNA binding. However loss of114aa might enhance T3association with deleted G115/483GRTH with the presence of additional band.5. Antibody Supershift analysis of GRTH-Tp2RNA complexes. Most retarded complexes identified in G483, G115/483, G423, G325when interacting with probe T1of Tp2were supershifted by V5antibody. Similar supershifted complexes was observed when probe T3probe interacting with G483and G115/483.6. RNA-EMSA analysis of GRTH mutation protein binding with TP23’UTR. G483Iax, G483Vx, G483Iax/Vx bind with T1（381/430） probe separately. The results showed when mutating only single RNA binding site, no matter which one, the binding between GRTH and TP2still existed. However, when mutating both RNA binding site, the binding disappeared. This result demonstrated that two RNA binding site（Ia domain and V domain） play role in the binding with RNA cooperately. Conclusion1. We have demonstrated the RNA binding motifs of GRTH protein with RNA derived from Tp23’UTR. These correspond to the RNA binding motifs （Ia） and （V） proposed for the RNA helicase family.2. The3’sequence immediately downstream of termination codon of Tp2（50bp） appears to be important for the interaction with GRTH.3. Demonstration of a sequence-specific RNA binding complex between Tp2and GRTH/DDX25is consistent with GRTH as mRNP associated with Tp2RNA message.4. We provide insights for the regulation of Tp2expression via binding to the conserved RNA binding motifs of GRTH protein, previously demonstrated an association of the complex at the polysomal sites.5. The homology of GRTH/DDX25protein to the crystal structure of its closely related DDX19shows RNA binding domains （I/Ia and V） accessible for interaction with RNA for functional regulation. These studies will facilitate further understanding on the manner that GRTH contributes in the regulation of the expression of genes which are essential for germ cell elongation and completion of spermatogenesis. |
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