- Email: [email protected]
Expression Profiling of microRNA in Cryptorchid Testes: miR-135a Contributes to the Maintenance of Spermatogonial Stem Cells by Regulating FoxO1
Expression Profiling of microRNA in Cryptorchid Testes: miR-135a Contributes to the Maintenance of Spermatogonial Stem Cells by Regulating FoxO1 Yoshinobu Moritoki, Yutaro Hayashi,* Kentaro Mizuno, Hideyuki Kamisawa, Hidenori Nishio, Satoshi Kurokawa, Shinya Ugawa, Yoshiyuki Kojima and Kenjiro Kohri From the Departments of Nephro-Urology (YM, YH, KM, HK, HN, SK, KK) and Neurobiology and Anatomy (SU), Graduate School of Medical Sciences, Nagoya City University, Nagoya and Department of Urology, Fukushima Medical University (YK), Fukushima, Japan
Abbreviations and Acronyms DT ¼ descended testis FoxO1 ¼ Forehead box protein O1 ISH ¼ in situ hybridization n-SSC ¼ nucleus positive SCC PCR ¼ polymerase chain reaction qRT-PCR ¼ quantitative RT-PCR RT-PCR ¼ reverse transcriptasePCR SSC ¼ spermatogonial stem cell UDT ¼ undescended testis UTR ¼ untranslated region WT ¼ wild type Accepted for publication October 28, 2013. Study received institutional animal care and use committee approval. * Correspondence: Department of NephroUrology, Graduate School of Medical Sciences, Nagoya City University, 1, Kawasumi, Mizuho-cho, Mizuho-ku, Nagoya 467-8601, Japan (telephone: þ81-52-853-8266; FAX: þ81-52-852-3179; e-mail: [email protected]).
Purpose: We elucidated the mechanism of spermatogonial stem cell disturbance of cryptorchidism and investigated the expression of miRNAs and their target genes in undescended testes. Materials and Methods: Using microarray analysis we compared total miRNA expression in unilateral undescended testes with that in contralateral descended and normal testes in a rat model of cryptorchidism. The model was derived by administering flutamide to pregnant Sprague DawleyÒ rats. We identified mRNA targets of miRNAs by bioinformatic analysis, followed by in situ hybridization and immunohistochemistry to localize candidate miRNAs and mRNAs, respectively. We also investigated whether miRNAs could inhibit target protein expression in vitro. Results: Microarray analysis and subsequent quantitative reverse transcriptasepolymerase chain reaction showed that only miR-135a was expressed at a lower level in undescended testes. We identified its target as FoxO1, which is essential for stem cell maintenance. miR-135a and FoxO1 localized to spermatogonial stem cells. FoxO1 localized to the spermatogonial stem cell nucleus less frequently in undescended testes, indicating that the activity of FoxO1, which acts as a transcription factor, is altered in undescended testes. Finally, miR-135a transfection into spermatogonia in vitro resulted in down-regulation of FoxO1 expression. Conclusions: In cryptorchid testes there is a decreased number of spermatogonial stem cells in which FoxO1 is activated, indicating that failure of spermatogonial stem cell maintenance results in spermatogenesis alteration. We also noted interaction between miR-135a and FoxO1, and propose that miR-135a contributes to spermatogonial stem cell maintenance through modulation of FoxO1 activity. Key Words: testis; cryptorchidism; spermatogenesis; Foxo1 protein, rat; gene expression
CRYPTORCHIDISM is one of the most common congenital abnormalities1 leading to male infertility. Testicular biopsy of UDTs in boys younger than
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2 years revealed low levels of type A dark spermatogonia or SSCs, which are strongly associated with future oligospermia.2 Thus, this is considered
0022-5347/14/1914-1174/0 THE JOURNAL OF UROLOGY® © 2014 by AMERICAN UROLOGICAL ASSOCIATION EDUCATION AND RESEARCH, INC.
http://dx.doi.org/10.1016/j.juro.2013.10.137 Vol. 191, 1174-1180, April 2014 Printed in U.S.A.
EXPRESSION PROFILING OF microRNA IN CRYPTORCHID TESTES
a putative cause of clinically proven infertility. Our previous studies of rat UDTs showed that infertility is caused in part by attenuation of SSC activity in early spermatogenesis around postnatal day 9, when gonocytes are transitioning into spermatogonia.3,4 miRNAs are RNA molecules about 23 nucleotides long that target the 30 -UTR of mRNAs to regulate mRNA turnover or translational efficiency.5 miRNAs can inhibit protein synthesis up to 50%6 and yet total loss of miRNA function represses spermatogenesis.7 Recent reports demonstrated the contribution of miRNAs to SSC and spermatid differentiation.8,9 Therefore, we hypothesized that some miRNAs must be associated with maintenance of the SSC phenotype during early spermatogenesis. In this study we predicted candidate miRNAs associated with SSCs in a UDT animal model. We then identified target mRNAs and analyzed their interactions in vitro.
MATERIALS AND METHODS Animal Protocol We used a cryptorchid rat testes model4,10 in which early stage spermatogonia express stem cell functions and are analogous to human adult dark spermatogonia.11 Sprague Dawley rats were housed under controlled environmental conditions and fed with complete pellet chow with free access to tap water. The rats were sacrificed under inhalation anesthesia with isoflurane. Rats were handled in accordance with Nagoya City University institutional animal care policies. Cryptorchidism was induced by daily injections of 7.5 mg flutamide in the abdomen of pregnant Sprague Dawley rats on gestation days 14 to 20. We defined a unilateral UDT as a testis located in a high position with an obviously thin gubernaculum compared to the contralateral testis.12 We divided the rats into 3 groups, including group 1dcontrol testis (bilateral DTs induced by intra-abdominal saline injection), group 2dDT (a contralateral UDT) and group 3dUDT (a contralateral DT). In our previous study we noted possible attenuation of SSC activity in the UDT with no attenuation in the contralateral DT on postnatal day 9.3 Therefore, we used rats from postnatal day 9 in these experiments.
Microarray Analysis We performed microarray analysis of prepubertal rat testes on postnatal day 9. To detect differential miRNA expression in UDTs we used Rat miRNA Microarray, Release 10.1 (Agilent Technologies, Santa Clara, California) according to the manufacturer protocol. The array included all miRNA transcripts in the Sanger Institute miRBase (http://www.mirbase.org/) available at the time of analysis (release 10.0). GeneSpring GX 10.0 (Agilent Technologies) was used to analyze raw data. We harvested 3 testes from each of the control, DT and UDT groups. Total RNA was isolated and quantified by determining the RNA integrity number,13 which was greater than 10, showing that samples were intact. UDT and DT sample
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results were directly compared. The DT, UDT and control groups were then compared to normalize for flutamide injection.
Quantitative RT-PCR To quantify miRNA levels qRT-PCR was performed using the TaqManÒ MicroRNA Assay according to the manufacturer protocol.14 Total RNA was extracted using a mirVanaÔ miRNA Isolation Kit and converted to cDNA using the TaqMan MicroRNA Reverse Transcription Kit. The expression level of each miRNA was normalized to that of an endogenous sno202 control and relative expression values were determined using the 2eDDCt method.15 To quantify mRNA levels RNA was converted to cDNA using the SuperScriptÒ First-Strand Synthesis System. The PCR reaction was performed using Power SYBRÒ Green PCR Master Mix and the 7500 Fast Real-Time PCR System (Life TechnologiesÔ). FoxO1 primer sequences were rat (forward) 50 -aaccagtccaactcgaccac-30 and (reverse) 50 -tgctc ataaagtcggtgctg-30 , and mouse (forward) 50 -GCTGGGTGT CAGGCTAAGAG-30 and (reverse) 50 -GGACTGCTCCTCA GTTCCTG-30 . Gapdh primer sequences were rat (forward) 50 -GTGGTGCCAAAAGGGTCAT-30 and (reverse) 50 -ATTT CTCGTGGTTCACACCA-30 , and mouse (forward) 50 -agaac atcatccctgcatcc-30 and (reverse) 50 -cacattgggggtaggaacac-30 . All experiments were done in triplicate.
In Situ Hybridization To localize miRNA in tissues we performed ISH using a miRCURY LNAÔ ISH Optimization Kit according to the manufacturer protocol. Testes were flash frozen in liquid nitrogen and stored at e80C. Frozen sections (10 mm) were mounted on 3-aminopropyltriethoxysilane coated slides. Digoxigenin labeled LNA probes for each miRNA were obtained from the manufacturer. The hybridization temperature of each probe was 21C below the melting temperature (calculated melting temperature of the miRCURY detection probe). After freeze drying tissue sections were washed and fixed in 4% paraformaldehyde solution, as previously described,12 followed by incubation overnight in hybridization buffer (Life Technologies). The next day tissue sections were incubated in hybridization buffer containing 2 mM probe in a humidified chamber overnight at 50C. After washes and incubation in blocking buffer (Life Technologies) tissue sections were incubated in blocking buffer containing Anti-Digoxigenin-AP antibody (Roche, Basel, Switzerland). The experiment was performed 3 times independently and reproducibility was confirmed.
Target mRNA Bioinformatics Prediction We screened target mRNAs for binding to each candidate miRNA using TargetScan, version 6 (http://www. targetscan.org/). The TargetScan algorithm searches the UTR of an mRNA for segments of perfect Watson-Crick complementarity to bases 2 to 8 of the miRNA, numbered from the 50 end and referred to as the miRNA seed. It extends each seed match with additional complementary base pairs as far as possible in each direction. This algorithm allows for guanine-uracil pairs, prevents mismatches and assigns a free energy of folding to each miRNA-target site interaction.
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Reporter Vectors and DNA Construct We generated a direct match miRNA target site and cloned the insert into the multiple cloning sites in the luciferase reporter vector of the Dual-LuciferaseÒ Reporter Assay System. The miR-135a binding site was predicted to be in the 30 -UTR of the rat and mouse according to miRBase, as described. Inserts containing the FoxO1 30 -UTR were amplified from mouse genomic DNA by PCR amplification using KOD-Plus-NEO (Toyobo, Osaka, Japan) according to the manufacturer protocol. Primers and product size were Luc vector WT, including the putative miR-135a binding site, forward primer 50 - TTTC AGCTGGGGAGTGATTG-30 and reverse primer 50 - GCAAT CTCTGTGAATGGGTCA-30 with a product size of 512 bp. The product was gel purified using a Gel Extraction Kit (QiagenÒ). It was then subcloned into a pGEMÒ-T Easy vector and sequenced using the T7 primer 50 -TAATA CGACTCACTATAGGG-30 and the Sp6 primer 50 -CAAGCT ATTTAGGTGACACTATAG-30 . We then repeated PCR using the same polymerase described with forward primer (WT-F1) 50 -AAAAAAGAGC TCTTTCAGCTGGGGAGTGATTG-30 and reverse primer (WT-R1) 50 -AAAAAAGCTAGCGCAATCTCTGTGAATGG GTCA-30 , in which the SacI and NheI sites were added to the forward and reverse primers, respectively. The second amplified product was gel purified, cut by the restriction enzymes SacI and NheI, and cloned into the multicloning site of the destination vector. Luc vector MUT, a part of the seed region of the miR-135a binding site in the WT vector, was produced using an overlapping strategy.16 Two fragments (Mut-1 and Mut-2) were created by PCR amplification using the mutagenesis primers WT-F1 and 50 -CACTTAAGATGATTTATGTACATTgcgttaggTAT ACAACGCACAGTAAGGATTG-30 for Mut-1 generation and WT-R1 and 50 -CAATCCTTACTGTGCGTTGTATA cctaacgcAATGTACATAAATCATCTTAAGTGGCTTG-30 for Mut-2 generation. The underlined sequences indicate a mutated region of the seed sequence. The 2 fragments shared an overlapping region that was generated by mutagenesis primers. In a second PCR the 2 fragments were mixed equally and amplified using the primer pair WT-F1 and WT-R1, which have a restriction enzyme site. The second PCR products were cloned into the destination vector after gel purification and enzymatic treatment. A Luc vector positive control plasmid was engineered using a pair of primers complementary to each other and containing the perfect match sequence to the targeting miRNA plus the SacI and NheI restriction sites at the 50 and 30 ends, respectively. The 2 primers were annealed to each other and inserted in the multicloning site of the destination vector. The primers used in this procedure were 50 -CTCA CATAGGAATAAAAAGCCATAG-30 and 50 -CTAGCTATG GCTTTTTATTCCTATGTGAGAGCT-30 .
Dual Luciferase Assay NIH3T3 cells were seeded 24 hours before transfection at 6 103 per well in 96-well plates. miR-135a mimic miRNAs or scrambled RNAs (each 2 pmol, Exiqon) were co-transfected with 60 ng WT plasmid, mutant plasmid and empty vector using LipofectamineÒ 2000. At 24 hours after transfection cells were washed with phosphate
buffered saline and lysed using the Dual-GloÒ Luciferase Reporter Assay System. Firefly and Renilla luciferase activity was measured consecutively with the DualLuciferaseÒ Reporter Assay System using the Mithras LB940 luminometer (Berthold Technologies, Bad Wildbad, Germany). All luciferase assays were performed in 4 biological replicates with 3 technical replicates each.
Immunohistochemistry Testes fixed with 4% paraformaldehyde were sectioned in 7 mm slices and stained with hematoxylin and eosin. To detect FoxO1 in rat testes we used rabbit polyclonal antibody (Cell Signaling Technology, Inc.). Three urologists who were experts in testicular histopathology and blinded to the groups were randomly assigned slides from each group according to respective hematoxylin and eosin staining as well as FoxO1 immunohistochemistry. The experts randomly selected 100 round seminiferous tubules and scored SSCs as FoxO1 nucleus positive or cytoplasm positive. At the same time we performed fertility index analysis, as previously reported,17 to determine the difference in germ cell numbers in each group.
Western Blot Western blot was performed as described previously.18 We extracted proteins from GC-1 cells using Cell Culture Lysis Reagent (PromegaÒ) according to the manufacturer instructions. Samples were separated by electrophoresis using sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels. Proteins were subsequently transferred to ImmobilonÒ-P polyvinylidene difluoride membranes. Protein bands were visualized using the ECL Western Blot Analysis Kit (Thermo Scientific, Rockford, Illinois) according to manufacturer instructions.
Cell Culture and Transfection The GC-1 spermatogonia cell line was cultured at 37C in Dulbecco modified Eagle medium (Life Technologies) supplemented with 10% fetal calf serum, 2 mM l-glutamine, penicillin and streptomycin in a 5% CO2 atmosphere. GC-1 cells were transfected using HiPerFectÒ Transfection Reagent according to manufacturer instructions. Briefly, 2.5 105 cells were plated the day before transfection in a 6-well tissue culture dish, followed by transfection using 0.5 mg DNA and U6 snRNA (Exiqon) as a control.
Statistical Analysis The Student t-test was used for normally distributed models and the Mann-Whitney test was used for nonparametric comparisons. Statistical significance was considered at p <0.05. On microarray analysis the statistical significance (p <0.05) of a differences in values of 2.0 and less than 0.5 was determined by the Student t-test.
RESULTS miR-135a Expression Significantly attenuated in UDTs. The table shows
up-regulated and down-regulated miRNAs in UDTs compared to DTs. Levels of miR-22-5p, 376b-3p, 7a-1-3p and 742 were up-regulated more than
EXPRESSION PROFILING OF microRNA IN CRYPTORCHID TESTES
Table Gene
Fold Change
p Value (t-test)
miR-22-5p miR-376b-3p miR-71-1-3p miR-742 miR-135a
2.53 2.54 2.40 2.14 0.29
<0.05 0.01 <0.01 <0.01 <0.05
twofold and only miR-135a was downregulated less than 0.5-fold. For further experimentation we focused on miR-135a because qRT-PCR revealed that differential levels of miR-135a expression were reproducible (fig. 1), in contrast to those of the other miRNAs. Testis specific and located in SSCs. Extensive analysis
of miR-135a organ specific expression on qRT-PCR showed significantly higher expression in the testis than in other organs (p <0.01, fig. 2). ISH revealed miR-135a in SSCs but it was not detected in Leydig cells or other interstitial cells (fig. 3). FoxO1 as miR-135a Putative Target Using TargetScan, version 6.0 we identified putative mRNA targets of miR-135a (supplementary table, http://jurology.com/). Of the targets we chose FoxO1 mRNA for further analysis based on its considerably high prediction score and complementary sequence. Computer analysis indicated that its sequence is a thermodynamic complement of miR135a. Figure 4, A shows a predicted miRNA binding site in the FoxO1 30 -UTR and miRNA sequences. Because the seed match region of oligonucleotides
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was 8 bp long, FoxO1 mRNA was predicted to undergo strong base pairing with miR-135a. On luciferase assay only the WT sequence cotransfected with miR-135a mimic showed significant repression relative to scRNA (fig. 4, B). FoxO1 Localized in SSCs and Less Often Activated in UDTs When localized to the nucleus, FoxO1 proteins are activated, allowing them to regulate transcription, while they are inactivated in cytoplasm.19 Our results showed that SSCs were FoxO1 positive (fig. 5, A to C ). High magnification imaging revealed 2 staining patterns, including cytoplasm positive and nucleus positive (fig. 5, D). Figure 6, A and B shows the number of n-SSCs per tubule and the ratio of n-SSCs divided by cytoplasm positive SSCs. In the UDT group there were significantly fewer n-SSCs than in the other 2 groups. Figure 6, C shows that there was no fertility index difference among the 3 groups. Figure 6, D demonstrates that the FoxO1 mRNA level was significantly higher in UDTs than in controls or DTs. miR-135a In Vitro Over Expression Suppressed FoxO1 Expression GC-1 cells transfected with miR-135a expressed lower levels of FoxO1 mRNA and protein than untransfected cells (fig. 7).
DISCUSSION Early stage spermatogonia (SSCs) must function properly to ensure fertility. This stem cell compartment
Figure 1. Relative miR-135a, miR-22-5p, miR-376b-3p, miR-7a-1-3p and miR-742 levels were determined by qRT-PCR in normal rat testes on postnatal day 9. Cont., control.
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Figure 2. Relative tissue miR-135a levels in normal rat at postnatal day 9.
ensures a constant supply of spermatocytes, which drive spermatogenesis. To perform this function they must precisely control gene expression posttranscriptionally as well as transcriptionally.20 miRNAs are representative of posttranscriptional regulators. They promote target mRNA degradation or inhibit its translation. Emerging evidence shows that miRNAs are involved in germ cell development9 and their differential expression changes through early stages of spermatogenesis.21 In clinical and animal models cryptorchidism leads to SSC disturbance2 and it is reported to be a partial cause of infertility. Taken together we hypothesized that examining differential expression of miRNAs and their target mRNAs between UDTs and DTs might reveal miRNAs that function to maintain SSCs. Using microarray analysis and qRT-PCR we initially identified miR-135a, which was differentially expressed in UDTs and DTs. miR-135a is widely conserved among vertebrates (miRBase,
Figure 3. ISH of normal rat testes at postnatal day 9 using miR-135a control sense and miR-135a antisense probes. Arrowheads indicate SSCs characterized by more cytoplasm showing miR-135a expression. Reduced from 1,000.
Figure 4. A, predicted miRNA binding site in FoxO1 30 UTR (wild ) and miRNA. Solid lines indicate Watson-Crick base pairing. Dotted lines indicate guanine-uracil wobbles. Gray areas indicate seed match areas. FoxO1 mut sequence shows luciferase assay changed seed sequences. B, normalized Renilla/Firefly luciferase activity in NIH3T3 cells cotransfected with miR-135a mimic or scRNA. Y axis represents relative expression of each vector cotransfected with miR-135a with scRNA. empty, empty dual-luciferase vector or luc vector. WT, WT sequence inserted in luc vector. MUT, mutated sequence inserted in luc vector. POS, miR-135a reverse complementary sequence inserted in luc vector.
version 37) and it is specifically expressed during the zygotic stage as well as by certain carcinomas.22,23 However, to our knowledge no study is available of miR-135a expression during neonatal development.
Figure 5. FoxO1 immunohistochemistry in control, DT and UDT rats. A to C, spermatogonia were stained by FoxO1 antibody. Reduced from 400. A, normal rat testis. B, DT. C, UDT. D, normal rat testis at postnatal day 9 shows 2 types of staining by FoxO1 antibody. Arrowhead indicates stained nucleus. Arrows indicate spermatogonia cytoplasm. Reduced from 1,000. Inset, example of UDT seminiferous tubules. Reduced from 1,000.
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Figure 7. Effects of miR-135a over expression in GC-1 cells. A, RT-PCR revealed lower FoxO1 mRNA expression in cells transfected with miRNA. Cont., control. B, Western blot shows lower FoxO1 levels in miRNA transfected cells. Figure 6. A, number of n-SSCs per seminiferous tubule stained by FoxO1 antibody. B, number of n-SSCs divided by number of cytoplasm positive SSCs. C, number of SSCs scored as fertility index, that is number of germ cells per tubule. N.S., not significant. D, FoxO1 relative expression in vivo by qRT-PCR. Cont., control.
In our experiments miR-135a expression was localized to the testes in the normal rat. We then focused on FoxO1 as a target candidate because it ranked within the top 15 candidates of 531 genes on our bioinformatic analysis (supplementary table, http://jurology.com/). To confirm that FoxO1 was a target of miR-135a we performed luciferase assay, which revealed that miR-135a affects FoxO1 expression via a target sequence in the 30 -UTR of this transcript (fig. 4, B). miRNAs are generally thought to predominantly cause translational blockades in animals while in plants they mainly function in direct degradation of target mRNAs.24 Transfection of miR-135a in GC-1 cells showed down-regulation of FoxO1 (fig. 7). In vivo RT-PCR showed that the FoxO1 expression level was inversely related to that of miR-135a (fig. 6, D). These data support the view that miR-135a is biologically relevant in mRNA degradation. FoxO1 functions to maintain cellular homeostasis, as indicated by certain evidence. 1) FoxO1 over expression induced cell death in the presence of inappropriately high levels of proliferative stress signaling.25 2) FoxO1 is essential for stem cell multipotency, including mouse SSCs.26,27 Taken together these findings indicate that a specific level of FoxO1 expression might be essential to maintain stem cell function. Because FoxO1 functions as a transcription factor in nuclei, the current data suggest that a decrease in the number of n-SSCs in UDTs may attenuate SSC activity. Infertility associated with cryptorchid testes is a major problem faced by urologists. The current
consensus recommends early UDT fixation in the scrotum (orchiopexy) based on histological examination.17 However, in some cases orchiopexy does not always guarantee subsequent fertility and paternity.28 Figure 6, C shows that there was no significant decrease in the fertility index, indicating that hematoxylin and eosin staining demonstrated no histological degradation that would lead to infertility in day 9 rats. However, considering the described clinical fact, evaluation of SSC activity according to FoxO1 at surgery could be used to predict future fertility. Moreover, the observation that FoxO1 translocation is induced by exogenous stimulation29 suggests that the higher temperature of the UDT in vivo might affect SSC function, although to our knowledge this supposition remains to be determined. Therefore, we propose that miR-135a maintains SSC homeostasis through FoxO1 modulation. Based on our findings in vitro miR-135a over expression suppressed FoxO1 expression and the expression of miR-135a was down-regulated in UDTs, which have fewer n-SSCs than controls. However, there are several limitations of our study and some questions remain. We could not identify the mechanism that regulates miR-135a expression. Also, whether our results can be extrapolated to humans requires more investigation.
CONCLUSIONS In cryptorchid testes there was a decreased number of SSCs in which FoxO1 was activated, indicating that a failure of SSC maintenance altered spermatogenesis. We also noted interaction between miR-135a and FoxO1. Correspondingly, we propose that miR-135a contributes to SSC maintenance through the modulation of FoxO1 activity.
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REFERENCES 1. Huff DS, Fenig DM, Canning DA et al: Abnormal germ cell development in cryptorchidism. Horm Res 2001; 55: 11. 2. Hadziselimovic F and Herzog B: The importance of both an early orchidopexy and germ cell maturation for fertility. Lancet 2001; 358: 1156. 3. Kamisawa H, Kojima Y, Mizuno K et al: Attenuation of spermatogonial stem cell activity in cryptorchid testes. J Urol 2012; 187: 1047.
11. Lee PA, Coughlin MT and Bellinger MF: Inhibin B: comparison with indexes of fertility among formerly cryptorchid and control men. J Clin Endocrinol Metab 2001; 86: 2576. 12. Mizuno K, Hayashi Y, Kojima Y et al: Early orchiopexy improves subsequent testicular development and spermatogenesis in the experimental cryptorchid rat model. J Urol 2008; 179: 1195.
4. Mizuno K, Hayashi Y, Kojima Y et al: Influence for testicular development and histological peculiarity in the testes of flutamide-induced cryptorchid rat model. Int J Urol 2007; 14: 67.
13. Schroeder A, Mueller O, Stocker S et al: The RIN: an RNA integrity number for assigning integrity values to RNA measurements. BMC Mol Biol 2006; 7: 3.
5. Bartel DP: MicroRNAs: genomics, biogenesis, mechanism, and function. Cell 2004; 116: 281.
14. Melton C, Judson RL and Blelloch R: Opposing microRNA families regulate self-renewal in mouse embryonic stem cells. Nature 2010; 463: 621.
6. Mendell JT and Olson EN: MicroRNAs in stress signaling and human disease. Cell 2012; 148: 1172. 7. Maatouk DM, Loveland KL, McManus MT et al: Dicer1 is required for differentiation of the mouse male germline. Biol Reprod 2008; 79: 696. 8. Tong M-H, Mitchell DA, McGowan SD et al: Two miRNA clusters, Mir-17-92 (Mirc1) and Mir-106b25(Mirc3), are involved in the regulation of spermatogonial differentiation in mice. Biol Reprod 2012; 86: 72.
15. Schmittgen TD and Livak KJ: Analyzing real-time PCR data by the comparative C(T) method. Nat Protoc 2008; 3: 1101. 16. Nicolas FE: Experimental validation of microRNA targets using a luciferase reporter system. In: Methods in Molecular Biology. Edited by JM Walker. New York: Springer 2011; chapt 11, p 139. 17. McAleer IM, Packer MG, Kaplan GW et al: Fertility Index analysis in cryptorchidism. J Urol 1995; 153: 1255.
9. Dai L, Tsai-Morris CH, Sato H et al: Testis-specific miRNA-469 up-regulated in gonadotropinregulated testicular RNA helicase (GRTH/ DDX25)-null mice silences transition protein 2 and protamine 2 messages at sites within coding region. Implications of its role in germ cell development. J Biol Chem 2011; 286: 52.
18. Mizuno K, Kojima Y, Kurokawa S et al: Identification of differentially expressed genes in human cryptorchid testes using suppression subtractive hybridization. J Urol 2009; 181: 1330.
10. Kamisawa H, Kojima Y, Mizuno K et al: Spermatogenesis after 1-stage Fowler-Stephens orchiopexy in experimental cryptorchid rat model. J Urol 2010; 183: 2380.
19. Haftmann C, Stittrich AB, Sgouroudis E et al: Lymphocyte signaling: regulation of FoxO transcription factors by microRNAs. Ann NY Acad Sci 2012; 1247: 46.
20. Papaioannou MD and Nef S: microRNAs in the testis: building up male fertility. J Androl 2010; 31: 26. 21. Mclver SC, Stanger SJ, Santarelli DM et al: A unique combination of male germ cell miRNAs coordinates gonocyte differentiation. PLoS One 2012; 7: e35553. 22. Pang RT, Liu WM, Leung CO et al: miR-135A regulates preimplantation embryo development through down-regulation of E3 Ubiquitin Ligase Seven in Absentia Homolog 1A (SIAH1A) expression. PLoS One 2011; 6: e27878. 23. Liu S, Guo W, Shi J et al: MicroRNA-135a contributes to the development of portal vein tumor thrombus by promoting metastasis in hepatocellular carcinoma. J Hepatol 2012; 56: 389. 24. Chen X: A microRNA as a translational repressor of APETALA2 in Arabidopsis flower development. Science 2004; 303: 2022. 25. Shen M, Lin F, Zhang J et al: Involvement of the up-regulated FoxO1 expression in follicular granulosa cell apoptosis induced by oxidative stress. J Biol Chem 2012; 287: 25727. 26. Zhang X, Yalcin S, Lee DF et al: FOXO1 is an essential regulator of pluripotency in human embryonic stem cells. Nat Cell Biol 2011; 13: 1092. 27. Goertz MJ, Wu Z, Gallardo TD et al: Foxo1 is required in mouse spermatogonial stem cells for their maintenance and the initiation of spermatogenesis. J Clin Invest 2011; 121: 3456. 28. Gracia J, Sanchez Zalabardo J, Sanchez Garcia J et al: Clinical, physical, sperm and hormonal data in 251 adults operated on for cryptorchidism in childhood. BJU Int 2000; 85: 1100. 29. Yan L, Lavin VA, Moser LR et al: PP2A regulates the pro-apoptotic activity of FOXO1. J Biol Chem 2008; 283: 7411.
Abbreviations and Acronyms DT ¼ descended testis FoxO1 ¼ Forehead box protein O1 ISH ¼ in situ hybridization n-SSC ¼ nucleus positive SCC PCR ¼ polymerase chain reaction qRT-PCR ¼ quantitative RT-PCR RT-PCR ¼ reverse transcriptasePCR SSC ¼ spermatogonial stem cell UDT ¼ undescended testis UTR ¼ untranslated region WT ¼ wild type Accepted for publication October 28, 2013. Study received institutional animal care and use committee approval. * Correspondence: Department of NephroUrology, Graduate School of Medical Sciences, Nagoya City University, 1, Kawasumi, Mizuho-cho, Mizuho-ku, Nagoya 467-8601, Japan (telephone: þ81-52-853-8266; FAX: þ81-52-852-3179; e-mail: [email protected]).
Purpose: We elucidated the mechanism of spermatogonial stem cell disturbance of cryptorchidism and investigated the expression of miRNAs and their target genes in undescended testes. Materials and Methods: Using microarray analysis we compared total miRNA expression in unilateral undescended testes with that in contralateral descended and normal testes in a rat model of cryptorchidism. The model was derived by administering flutamide to pregnant Sprague DawleyÒ rats. We identified mRNA targets of miRNAs by bioinformatic analysis, followed by in situ hybridization and immunohistochemistry to localize candidate miRNAs and mRNAs, respectively. We also investigated whether miRNAs could inhibit target protein expression in vitro. Results: Microarray analysis and subsequent quantitative reverse transcriptasepolymerase chain reaction showed that only miR-135a was expressed at a lower level in undescended testes. We identified its target as FoxO1, which is essential for stem cell maintenance. miR-135a and FoxO1 localized to spermatogonial stem cells. FoxO1 localized to the spermatogonial stem cell nucleus less frequently in undescended testes, indicating that the activity of FoxO1, which acts as a transcription factor, is altered in undescended testes. Finally, miR-135a transfection into spermatogonia in vitro resulted in down-regulation of FoxO1 expression. Conclusions: In cryptorchid testes there is a decreased number of spermatogonial stem cells in which FoxO1 is activated, indicating that failure of spermatogonial stem cell maintenance results in spermatogenesis alteration. We also noted interaction between miR-135a and FoxO1, and propose that miR-135a contributes to spermatogonial stem cell maintenance through modulation of FoxO1 activity. Key Words: testis; cryptorchidism; spermatogenesis; Foxo1 protein, rat; gene expression
CRYPTORCHIDISM is one of the most common congenital abnormalities1 leading to male infertility. Testicular biopsy of UDTs in boys younger than
1174
j
www.jurology.com
2 years revealed low levels of type A dark spermatogonia or SSCs, which are strongly associated with future oligospermia.2 Thus, this is considered
0022-5347/14/1914-1174/0 THE JOURNAL OF UROLOGY® © 2014 by AMERICAN UROLOGICAL ASSOCIATION EDUCATION AND RESEARCH, INC.
http://dx.doi.org/10.1016/j.juro.2013.10.137 Vol. 191, 1174-1180, April 2014 Printed in U.S.A.
EXPRESSION PROFILING OF microRNA IN CRYPTORCHID TESTES
a putative cause of clinically proven infertility. Our previous studies of rat UDTs showed that infertility is caused in part by attenuation of SSC activity in early spermatogenesis around postnatal day 9, when gonocytes are transitioning into spermatogonia.3,4 miRNAs are RNA molecules about 23 nucleotides long that target the 30 -UTR of mRNAs to regulate mRNA turnover or translational efficiency.5 miRNAs can inhibit protein synthesis up to 50%6 and yet total loss of miRNA function represses spermatogenesis.7 Recent reports demonstrated the contribution of miRNAs to SSC and spermatid differentiation.8,9 Therefore, we hypothesized that some miRNAs must be associated with maintenance of the SSC phenotype during early spermatogenesis. In this study we predicted candidate miRNAs associated with SSCs in a UDT animal model. We then identified target mRNAs and analyzed their interactions in vitro.
MATERIALS AND METHODS Animal Protocol We used a cryptorchid rat testes model4,10 in which early stage spermatogonia express stem cell functions and are analogous to human adult dark spermatogonia.11 Sprague Dawley rats were housed under controlled environmental conditions and fed with complete pellet chow with free access to tap water. The rats were sacrificed under inhalation anesthesia with isoflurane. Rats were handled in accordance with Nagoya City University institutional animal care policies. Cryptorchidism was induced by daily injections of 7.5 mg flutamide in the abdomen of pregnant Sprague Dawley rats on gestation days 14 to 20. We defined a unilateral UDT as a testis located in a high position with an obviously thin gubernaculum compared to the contralateral testis.12 We divided the rats into 3 groups, including group 1dcontrol testis (bilateral DTs induced by intra-abdominal saline injection), group 2dDT (a contralateral UDT) and group 3dUDT (a contralateral DT). In our previous study we noted possible attenuation of SSC activity in the UDT with no attenuation in the contralateral DT on postnatal day 9.3 Therefore, we used rats from postnatal day 9 in these experiments.
Microarray Analysis We performed microarray analysis of prepubertal rat testes on postnatal day 9. To detect differential miRNA expression in UDTs we used Rat miRNA Microarray, Release 10.1 (Agilent Technologies, Santa Clara, California) according to the manufacturer protocol. The array included all miRNA transcripts in the Sanger Institute miRBase (http://www.mirbase.org/) available at the time of analysis (release 10.0). GeneSpring GX 10.0 (Agilent Technologies) was used to analyze raw data. We harvested 3 testes from each of the control, DT and UDT groups. Total RNA was isolated and quantified by determining the RNA integrity number,13 which was greater than 10, showing that samples were intact. UDT and DT sample
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results were directly compared. The DT, UDT and control groups were then compared to normalize for flutamide injection.
Quantitative RT-PCR To quantify miRNA levels qRT-PCR was performed using the TaqManÒ MicroRNA Assay according to the manufacturer protocol.14 Total RNA was extracted using a mirVanaÔ miRNA Isolation Kit and converted to cDNA using the TaqMan MicroRNA Reverse Transcription Kit. The expression level of each miRNA was normalized to that of an endogenous sno202 control and relative expression values were determined using the 2eDDCt method.15 To quantify mRNA levels RNA was converted to cDNA using the SuperScriptÒ First-Strand Synthesis System. The PCR reaction was performed using Power SYBRÒ Green PCR Master Mix and the 7500 Fast Real-Time PCR System (Life TechnologiesÔ). FoxO1 primer sequences were rat (forward) 50 -aaccagtccaactcgaccac-30 and (reverse) 50 -tgctc ataaagtcggtgctg-30 , and mouse (forward) 50 -GCTGGGTGT CAGGCTAAGAG-30 and (reverse) 50 -GGACTGCTCCTCA GTTCCTG-30 . Gapdh primer sequences were rat (forward) 50 -GTGGTGCCAAAAGGGTCAT-30 and (reverse) 50 -ATTT CTCGTGGTTCACACCA-30 , and mouse (forward) 50 -agaac atcatccctgcatcc-30 and (reverse) 50 -cacattgggggtaggaacac-30 . All experiments were done in triplicate.
In Situ Hybridization To localize miRNA in tissues we performed ISH using a miRCURY LNAÔ ISH Optimization Kit according to the manufacturer protocol. Testes were flash frozen in liquid nitrogen and stored at e80C. Frozen sections (10 mm) were mounted on 3-aminopropyltriethoxysilane coated slides. Digoxigenin labeled LNA probes for each miRNA were obtained from the manufacturer. The hybridization temperature of each probe was 21C below the melting temperature (calculated melting temperature of the miRCURY detection probe). After freeze drying tissue sections were washed and fixed in 4% paraformaldehyde solution, as previously described,12 followed by incubation overnight in hybridization buffer (Life Technologies). The next day tissue sections were incubated in hybridization buffer containing 2 mM probe in a humidified chamber overnight at 50C. After washes and incubation in blocking buffer (Life Technologies) tissue sections were incubated in blocking buffer containing Anti-Digoxigenin-AP antibody (Roche, Basel, Switzerland). The experiment was performed 3 times independently and reproducibility was confirmed.
Target mRNA Bioinformatics Prediction We screened target mRNAs for binding to each candidate miRNA using TargetScan, version 6 (http://www. targetscan.org/). The TargetScan algorithm searches the UTR of an mRNA for segments of perfect Watson-Crick complementarity to bases 2 to 8 of the miRNA, numbered from the 50 end and referred to as the miRNA seed. It extends each seed match with additional complementary base pairs as far as possible in each direction. This algorithm allows for guanine-uracil pairs, prevents mismatches and assigns a free energy of folding to each miRNA-target site interaction.
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Reporter Vectors and DNA Construct We generated a direct match miRNA target site and cloned the insert into the multiple cloning sites in the luciferase reporter vector of the Dual-LuciferaseÒ Reporter Assay System. The miR-135a binding site was predicted to be in the 30 -UTR of the rat and mouse according to miRBase, as described. Inserts containing the FoxO1 30 -UTR were amplified from mouse genomic DNA by PCR amplification using KOD-Plus-NEO (Toyobo, Osaka, Japan) according to the manufacturer protocol. Primers and product size were Luc vector WT, including the putative miR-135a binding site, forward primer 50 - TTTC AGCTGGGGAGTGATTG-30 and reverse primer 50 - GCAAT CTCTGTGAATGGGTCA-30 with a product size of 512 bp. The product was gel purified using a Gel Extraction Kit (QiagenÒ). It was then subcloned into a pGEMÒ-T Easy vector and sequenced using the T7 primer 50 -TAATA CGACTCACTATAGGG-30 and the Sp6 primer 50 -CAAGCT ATTTAGGTGACACTATAG-30 . We then repeated PCR using the same polymerase described with forward primer (WT-F1) 50 -AAAAAAGAGC TCTTTCAGCTGGGGAGTGATTG-30 and reverse primer (WT-R1) 50 -AAAAAAGCTAGCGCAATCTCTGTGAATGG GTCA-30 , in which the SacI and NheI sites were added to the forward and reverse primers, respectively. The second amplified product was gel purified, cut by the restriction enzymes SacI and NheI, and cloned into the multicloning site of the destination vector. Luc vector MUT, a part of the seed region of the miR-135a binding site in the WT vector, was produced using an overlapping strategy.16 Two fragments (Mut-1 and Mut-2) were created by PCR amplification using the mutagenesis primers WT-F1 and 50 -CACTTAAGATGATTTATGTACATTgcgttaggTAT ACAACGCACAGTAAGGATTG-30 for Mut-1 generation and WT-R1 and 50 -CAATCCTTACTGTGCGTTGTATA cctaacgcAATGTACATAAATCATCTTAAGTGGCTTG-30 for Mut-2 generation. The underlined sequences indicate a mutated region of the seed sequence. The 2 fragments shared an overlapping region that was generated by mutagenesis primers. In a second PCR the 2 fragments were mixed equally and amplified using the primer pair WT-F1 and WT-R1, which have a restriction enzyme site. The second PCR products were cloned into the destination vector after gel purification and enzymatic treatment. A Luc vector positive control plasmid was engineered using a pair of primers complementary to each other and containing the perfect match sequence to the targeting miRNA plus the SacI and NheI restriction sites at the 50 and 30 ends, respectively. The 2 primers were annealed to each other and inserted in the multicloning site of the destination vector. The primers used in this procedure were 50 -CTCA CATAGGAATAAAAAGCCATAG-30 and 50 -CTAGCTATG GCTTTTTATTCCTATGTGAGAGCT-30 .
Dual Luciferase Assay NIH3T3 cells were seeded 24 hours before transfection at 6 103 per well in 96-well plates. miR-135a mimic miRNAs or scrambled RNAs (each 2 pmol, Exiqon) were co-transfected with 60 ng WT plasmid, mutant plasmid and empty vector using LipofectamineÒ 2000. At 24 hours after transfection cells were washed with phosphate
buffered saline and lysed using the Dual-GloÒ Luciferase Reporter Assay System. Firefly and Renilla luciferase activity was measured consecutively with the DualLuciferaseÒ Reporter Assay System using the Mithras LB940 luminometer (Berthold Technologies, Bad Wildbad, Germany). All luciferase assays were performed in 4 biological replicates with 3 technical replicates each.
Immunohistochemistry Testes fixed with 4% paraformaldehyde were sectioned in 7 mm slices and stained with hematoxylin and eosin. To detect FoxO1 in rat testes we used rabbit polyclonal antibody (Cell Signaling Technology, Inc.). Three urologists who were experts in testicular histopathology and blinded to the groups were randomly assigned slides from each group according to respective hematoxylin and eosin staining as well as FoxO1 immunohistochemistry. The experts randomly selected 100 round seminiferous tubules and scored SSCs as FoxO1 nucleus positive or cytoplasm positive. At the same time we performed fertility index analysis, as previously reported,17 to determine the difference in germ cell numbers in each group.
Western Blot Western blot was performed as described previously.18 We extracted proteins from GC-1 cells using Cell Culture Lysis Reagent (PromegaÒ) according to the manufacturer instructions. Samples were separated by electrophoresis using sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels. Proteins were subsequently transferred to ImmobilonÒ-P polyvinylidene difluoride membranes. Protein bands were visualized using the ECL Western Blot Analysis Kit (Thermo Scientific, Rockford, Illinois) according to manufacturer instructions.
Cell Culture and Transfection The GC-1 spermatogonia cell line was cultured at 37C in Dulbecco modified Eagle medium (Life Technologies) supplemented with 10% fetal calf serum, 2 mM l-glutamine, penicillin and streptomycin in a 5% CO2 atmosphere. GC-1 cells were transfected using HiPerFectÒ Transfection Reagent according to manufacturer instructions. Briefly, 2.5 105 cells were plated the day before transfection in a 6-well tissue culture dish, followed by transfection using 0.5 mg DNA and U6 snRNA (Exiqon) as a control.
Statistical Analysis The Student t-test was used for normally distributed models and the Mann-Whitney test was used for nonparametric comparisons. Statistical significance was considered at p <0.05. On microarray analysis the statistical significance (p <0.05) of a differences in values of 2.0 and less than 0.5 was determined by the Student t-test.
RESULTS miR-135a Expression Significantly attenuated in UDTs. The table shows
up-regulated and down-regulated miRNAs in UDTs compared to DTs. Levels of miR-22-5p, 376b-3p, 7a-1-3p and 742 were up-regulated more than
EXPRESSION PROFILING OF microRNA IN CRYPTORCHID TESTES
Table Gene
Fold Change
p Value (t-test)
miR-22-5p miR-376b-3p miR-71-1-3p miR-742 miR-135a
2.53 2.54 2.40 2.14 0.29
<0.05 0.01 <0.01 <0.01 <0.05
twofold and only miR-135a was downregulated less than 0.5-fold. For further experimentation we focused on miR-135a because qRT-PCR revealed that differential levels of miR-135a expression were reproducible (fig. 1), in contrast to those of the other miRNAs. Testis specific and located in SSCs. Extensive analysis
of miR-135a organ specific expression on qRT-PCR showed significantly higher expression in the testis than in other organs (p <0.01, fig. 2). ISH revealed miR-135a in SSCs but it was not detected in Leydig cells or other interstitial cells (fig. 3). FoxO1 as miR-135a Putative Target Using TargetScan, version 6.0 we identified putative mRNA targets of miR-135a (supplementary table, http://jurology.com/). Of the targets we chose FoxO1 mRNA for further analysis based on its considerably high prediction score and complementary sequence. Computer analysis indicated that its sequence is a thermodynamic complement of miR135a. Figure 4, A shows a predicted miRNA binding site in the FoxO1 30 -UTR and miRNA sequences. Because the seed match region of oligonucleotides
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was 8 bp long, FoxO1 mRNA was predicted to undergo strong base pairing with miR-135a. On luciferase assay only the WT sequence cotransfected with miR-135a mimic showed significant repression relative to scRNA (fig. 4, B). FoxO1 Localized in SSCs and Less Often Activated in UDTs When localized to the nucleus, FoxO1 proteins are activated, allowing them to regulate transcription, while they are inactivated in cytoplasm.19 Our results showed that SSCs were FoxO1 positive (fig. 5, A to C ). High magnification imaging revealed 2 staining patterns, including cytoplasm positive and nucleus positive (fig. 5, D). Figure 6, A and B shows the number of n-SSCs per tubule and the ratio of n-SSCs divided by cytoplasm positive SSCs. In the UDT group there were significantly fewer n-SSCs than in the other 2 groups. Figure 6, C shows that there was no fertility index difference among the 3 groups. Figure 6, D demonstrates that the FoxO1 mRNA level was significantly higher in UDTs than in controls or DTs. miR-135a In Vitro Over Expression Suppressed FoxO1 Expression GC-1 cells transfected with miR-135a expressed lower levels of FoxO1 mRNA and protein than untransfected cells (fig. 7).
DISCUSSION Early stage spermatogonia (SSCs) must function properly to ensure fertility. This stem cell compartment
Figure 1. Relative miR-135a, miR-22-5p, miR-376b-3p, miR-7a-1-3p and miR-742 levels were determined by qRT-PCR in normal rat testes on postnatal day 9. Cont., control.
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Figure 2. Relative tissue miR-135a levels in normal rat at postnatal day 9.
ensures a constant supply of spermatocytes, which drive spermatogenesis. To perform this function they must precisely control gene expression posttranscriptionally as well as transcriptionally.20 miRNAs are representative of posttranscriptional regulators. They promote target mRNA degradation or inhibit its translation. Emerging evidence shows that miRNAs are involved in germ cell development9 and their differential expression changes through early stages of spermatogenesis.21 In clinical and animal models cryptorchidism leads to SSC disturbance2 and it is reported to be a partial cause of infertility. Taken together we hypothesized that examining differential expression of miRNAs and their target mRNAs between UDTs and DTs might reveal miRNAs that function to maintain SSCs. Using microarray analysis and qRT-PCR we initially identified miR-135a, which was differentially expressed in UDTs and DTs. miR-135a is widely conserved among vertebrates (miRBase,
Figure 3. ISH of normal rat testes at postnatal day 9 using miR-135a control sense and miR-135a antisense probes. Arrowheads indicate SSCs characterized by more cytoplasm showing miR-135a expression. Reduced from 1,000.
Figure 4. A, predicted miRNA binding site in FoxO1 30 UTR (wild ) and miRNA. Solid lines indicate Watson-Crick base pairing. Dotted lines indicate guanine-uracil wobbles. Gray areas indicate seed match areas. FoxO1 mut sequence shows luciferase assay changed seed sequences. B, normalized Renilla/Firefly luciferase activity in NIH3T3 cells cotransfected with miR-135a mimic or scRNA. Y axis represents relative expression of each vector cotransfected with miR-135a with scRNA. empty, empty dual-luciferase vector or luc vector. WT, WT sequence inserted in luc vector. MUT, mutated sequence inserted in luc vector. POS, miR-135a reverse complementary sequence inserted in luc vector.
version 37) and it is specifically expressed during the zygotic stage as well as by certain carcinomas.22,23 However, to our knowledge no study is available of miR-135a expression during neonatal development.
Figure 5. FoxO1 immunohistochemistry in control, DT and UDT rats. A to C, spermatogonia were stained by FoxO1 antibody. Reduced from 400. A, normal rat testis. B, DT. C, UDT. D, normal rat testis at postnatal day 9 shows 2 types of staining by FoxO1 antibody. Arrowhead indicates stained nucleus. Arrows indicate spermatogonia cytoplasm. Reduced from 1,000. Inset, example of UDT seminiferous tubules. Reduced from 1,000.
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Figure 7. Effects of miR-135a over expression in GC-1 cells. A, RT-PCR revealed lower FoxO1 mRNA expression in cells transfected with miRNA. Cont., control. B, Western blot shows lower FoxO1 levels in miRNA transfected cells. Figure 6. A, number of n-SSCs per seminiferous tubule stained by FoxO1 antibody. B, number of n-SSCs divided by number of cytoplasm positive SSCs. C, number of SSCs scored as fertility index, that is number of germ cells per tubule. N.S., not significant. D, FoxO1 relative expression in vivo by qRT-PCR. Cont., control.
In our experiments miR-135a expression was localized to the testes in the normal rat. We then focused on FoxO1 as a target candidate because it ranked within the top 15 candidates of 531 genes on our bioinformatic analysis (supplementary table, http://jurology.com/). To confirm that FoxO1 was a target of miR-135a we performed luciferase assay, which revealed that miR-135a affects FoxO1 expression via a target sequence in the 30 -UTR of this transcript (fig. 4, B). miRNAs are generally thought to predominantly cause translational blockades in animals while in plants they mainly function in direct degradation of target mRNAs.24 Transfection of miR-135a in GC-1 cells showed down-regulation of FoxO1 (fig. 7). In vivo RT-PCR showed that the FoxO1 expression level was inversely related to that of miR-135a (fig. 6, D). These data support the view that miR-135a is biologically relevant in mRNA degradation. FoxO1 functions to maintain cellular homeostasis, as indicated by certain evidence. 1) FoxO1 over expression induced cell death in the presence of inappropriately high levels of proliferative stress signaling.25 2) FoxO1 is essential for stem cell multipotency, including mouse SSCs.26,27 Taken together these findings indicate that a specific level of FoxO1 expression might be essential to maintain stem cell function. Because FoxO1 functions as a transcription factor in nuclei, the current data suggest that a decrease in the number of n-SSCs in UDTs may attenuate SSC activity. Infertility associated with cryptorchid testes is a major problem faced by urologists. The current
consensus recommends early UDT fixation in the scrotum (orchiopexy) based on histological examination.17 However, in some cases orchiopexy does not always guarantee subsequent fertility and paternity.28 Figure 6, C shows that there was no significant decrease in the fertility index, indicating that hematoxylin and eosin staining demonstrated no histological degradation that would lead to infertility in day 9 rats. However, considering the described clinical fact, evaluation of SSC activity according to FoxO1 at surgery could be used to predict future fertility. Moreover, the observation that FoxO1 translocation is induced by exogenous stimulation29 suggests that the higher temperature of the UDT in vivo might affect SSC function, although to our knowledge this supposition remains to be determined. Therefore, we propose that miR-135a maintains SSC homeostasis through FoxO1 modulation. Based on our findings in vitro miR-135a over expression suppressed FoxO1 expression and the expression of miR-135a was down-regulated in UDTs, which have fewer n-SSCs than controls. However, there are several limitations of our study and some questions remain. We could not identify the mechanism that regulates miR-135a expression. Also, whether our results can be extrapolated to humans requires more investigation.
CONCLUSIONS In cryptorchid testes there was a decreased number of SSCs in which FoxO1 was activated, indicating that a failure of SSC maintenance altered spermatogenesis. We also noted interaction between miR-135a and FoxO1. Correspondingly, we propose that miR-135a contributes to SSC maintenance through the modulation of FoxO1 activity.
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