world - wide , infertility affects 1015% of couples who are trying to conceive , and about 15% of these cases are caused by male factors , which affect 1 out of 20 men in the general population .
most cases of male infertility are idiopathic , apart from several etiologies , such as obstruction of deferent duct , varicocele , sexual dysfunction , and cryptorchidism .
although assisted reproductive technology ( art ) has helped many sterile couples to conceive , non - obstructive azoospermia ( noa ) , which accounts for a considerable proportion of male infertility , has a dramatically lower rate of sperm retrieval and clinical pregnancy .
the etiological mechanism of noa is unknown , but factors such as oxidative stress were considered to have effects on spermatogenesis , and some antioxidants have been effective in protecting spermatogenesis .
therefore , it is helpful to explore the underlying pathogenesis of noa in these patients . micrornas are a class of small rnas that do not code amino acid sequences , but they play fundamental roles in regulating gene expression after transcription .
lian et al . found 154 down - regulated mirnas and 19 up - regulated mirnas in testes of noa patients compared to fertile males , by using microarray technologies .
furthermore , some of the mirnas have been shown to affect the proliferation , apoptosis , and dna damage in germ cells [ 911 ] .
mir-210 is one of the 19 up - regulated mirnas in testes of noa patients , located within the genomic loci of transcript ak123483 .
it can be induced by hypoxia , and plays an essential role in cell adaptation to hypoxia .
mir-210 also affects regulation of diverse physiological processes , such as angiogenesis , cell survival , proliferation , cell cycle arrest , protein modification , and dna damage repair .
although mir-210 has been shown to be involved in regulation of physiological processes in various diseases and to be an up - regulated mirna in testes of noa patients , it remains unknown how mir-210 affects spermatogenesis .
hence , the aim of this study was to investigate the underlying mechanisms by which mir-210 is involved in the pathogenesis of spermatogenesis .
we enrolled 25 patients ( aged 1841 years ) with azoospermia ( proven by 3 semen analyses from testicular biopsies from the first affiliated hospital of anhui medical university ) .
pathological examinations were performed on each testicular specimen . combined with clinical features , 4 patients were diagnosed as having sertoli - cell - only syndrome ( scos ) , 7 patients were diagnosed as having maturation arrest ( ma ) , 8 patients were diagnosed as having hypospermatogenesis , and the other 6 patients were diagnosed as having obstructive azoospermia ( oa ) . all patients provided informed consent before their participation in this study .
our local medical ethics committee approved this study before it began . to examine the location of insulin - like growth factor ii
( igf2 ) in human testicular tissues , we performed immunohistochemistry staining to detect the igf2 expression .
tissues were cut into sections for immunoperoxidase staining after being treated with 4% pfa and paraffin wax .
after the specific treatment with standard - procedure immunohistochemistry staining as described as lian et al . , sections were incubated with igf2 antibody ( abcam ) overnight at 4c and biotinylated secondary antibody ( abcam ) for 2 h at room temperature . to detect expression of mir-210 ,
rnas were extracted from nt-2 cells or tissues and subjected to real - time pcr as described as lian et al . .
briefly , rna extraction was performed following a standard trizol protocol , real - time pcr was carried out with the abi step one system ( applied biosystems ) , and the sybr premix ex taq ii kit ( takara bio , inc . ) was used .
primers for q - rt pcr were as follows : forward primer : 5-caataactgtgcgtgtgacagc-3 reverse primer : 5-tatggttttgacgactgtgtgat-3 forward primer : 5-cagcacatatactaaaattggaacg-3 reverse primer : 5-acgaatttgcgtgtcatcc-3 western blot analysis was carried out to detect protein expression of igf2 in the human testicular tissues in the 3 groups and in nt2 cells .
anti - igf2 ( abcam ) was used for western blot analysis , and we used -actin as a loading control to detect expression of igf2 .
we supplemented the medium with 10% fetal bovine serum ( life technology inc . ) , 1% antibiotics ( 100 units / ml penicillin , and 100 ug / ml streptomycin , life technology inc . ) .
cells were incubated at 37c in a humidified incubator with 5% co2 . to transfect oligonucleotides and plasmids into nt-2 cells , lipofectamine rnaimax ( invitrogen ) and fugene hd ( roche )
all processes were performed in accordance with the protocols supplied by manufacturers . in this study , all the experiments were performed independently at least 3 times .
we enrolled 25 patients ( aged 1841 years ) with azoospermia ( proven by 3 semen analyses from testicular biopsies from the first affiliated hospital of anhui medical university ) .
pathological examinations were performed on each testicular specimen . combined with clinical features , 4 patients were diagnosed as having sertoli - cell - only syndrome ( scos ) , 7 patients were diagnosed as having maturation arrest ( ma ) , 8 patients were diagnosed as having hypospermatogenesis , and the other 6 patients were diagnosed as having obstructive azoospermia ( oa ) . all patients provided informed consent before their participation in this study .
to examine the location of insulin - like growth factor ii ( igf2 ) in human testicular tissues , we performed immunohistochemistry staining to detect the igf2 expression .
tissues were cut into sections for immunoperoxidase staining after being treated with 4% pfa and paraffin wax .
after the specific treatment with standard - procedure immunohistochemistry staining as described as lian et al .
, sections were incubated with igf2 antibody ( abcam ) overnight at 4c and biotinylated secondary antibody ( abcam ) for 2 h at room temperature .
rnas were extracted from nt-2 cells or tissues and subjected to real - time pcr as described as lian et al . .
briefly , rna extraction was performed following a standard trizol protocol , real - time pcr was carried out with the abi step one system ( applied biosystems ) , and the sybr premix ex taq ii kit ( takara bio , inc . ) was used .
primers for q - rt pcr were as follows : forward primer : 5-caataactgtgcgtgtgacagc-3 reverse primer : 5-tatggttttgacgactgtgtgat-3 forward primer : 5-cagcacatatactaaaattggaacg-3 reverse primer : 5-acgaatttgcgtgtcatcc-3
western blot analysis was carried out to detect protein expression of igf2 in the human testicular tissues in the 3 groups and in nt2 cells .
anti - igf2 ( abcam ) was used for western blot analysis , and we used -actin as a loading control to detect expression of igf2 .
we supplemented the medium with 10% fetal bovine serum ( life technology inc . ) , 1% antibiotics ( 100 units / ml penicillin , and 100 ug / ml streptomycin , life technology inc . ) .
cells were incubated at 37c in a humidified incubator with 5% co2 . to transfect oligonucleotides and plasmids into nt-2 cells , lipofectamine rnaimax ( invitrogen ) and fugene hd ( roche )
the igf2 gene is part of a cluster of imprinted genes expressing the single polypeptide as igf2 , which is only produced from the paternal allele .
the maternal allele is transcriptionally silent . to clarify the location of igf2 in human testicular tissues ,
we found igf2 located in spermatocytes in the testes of patients with oa ( figure 1 ) . because igf2 is located in spermatocytes of the testis , we detected the expression of igf2 in cases with ma , hypospermatogenesis , and oa , but not in the scos patients .
we found that igf2 was down - regulated in patients with ma and hypospermatogenesis compared to oa patients , which was considered as the control group with normal spermatogenesis , although without a significant difference ( figures 2 , 3 ) , possibly because there were fewer samples and longer preservation times of some samples .
quantitative real - time pcr was performed to examined mir-210 expression in the testis of patients with ma , hypospermatogenesis , and oa . we found that mir-210 was significantly up - regulated in the testis of ma and hypospermatogenesis patients compared to oa patients ( figure 4 ) . however , due to errors in the rna extraction in the preliminary experiment , 3 testis samples ( 1 each ) from ma , hypospermatogenesis , and oa patients were damaged and were not tested . in the targetscan database , because the 3utr of the igf2-mrna has a putative mir-210-binding site , igf2 was predicted to be a potential target of mir-210 . to identify whether the igf2 gene was targeted by mir-210 directly , renilla luciferase reporters , which include the wild - type full - length 3utr forms of mir-210 seeding sites ,
figure 5 shows that there was a 60% decrease in luciferase activity after cotransfection of the mir-210 mimic and the renilla luciferase reporters into nt2 cells , and inhibiting mir-210 expression increased activity of the reporter renilla luciferase .
expression of igf2 protein was also significantly lower in the nt2 cells transfected with mir-210 mimics than in control cells , and knockdown of mir-210 with mir-210 inhibitor increased protein expression of igf2 ( figures 6 ) .
the igf2 gene is part of a cluster of imprinted genes expressing the single polypeptide as igf2 , which is only produced from the paternal allele .
the maternal allele is transcriptionally silent . to clarify the location of igf2 in human testicular tissues ,
we found igf2 located in spermatocytes in the testes of patients with oa ( figure 1 ) .
because igf2 is located in spermatocytes of the testis , we detected the expression of igf2 in cases with ma , hypospermatogenesis , and oa , but not in the scos patients .
we found that igf2 was down - regulated in patients with ma and hypospermatogenesis compared to oa patients , which was considered as the control group with normal spermatogenesis , although without a significant difference ( figures 2 , 3 ) , possibly because there were fewer samples and longer preservation times of some samples .
quantitative real - time pcr was performed to examined mir-210 expression in the testis of patients with ma , hypospermatogenesis , and oa . we found that mir-210 was significantly up - regulated in the testis of ma and hypospermatogenesis patients compared to oa patients ( figure 4 ) . however , due to errors in the rna extraction in the preliminary experiment , 3 testis samples ( 1 each ) from ma , hypospermatogenesis , and oa patients were damaged and were not tested .
in the targetscan database , because the 3utr of the igf2-mrna has a putative mir-210-binding site , igf2 was predicted to be a potential target of mir-210 . to identify whether the igf2 gene was targeted by mir-210 directly , renilla luciferase reporters , which include the wild - type full - length 3utr forms of mir-210 seeding sites , were used .
figure 5 shows that there was a 60% decrease in luciferase activity after cotransfection of the mir-210 mimic and the renilla luciferase reporters into nt2 cells , and inhibiting mir-210 expression increased activity of the reporter renilla luciferase .
expression of igf2 protein was also significantly lower in the nt2 cells transfected with mir-210 mimics than in control cells , and knockdown of mir-210 with mir-210 inhibitor increased protein expression of igf2 ( figures 6 ) .
during recent decades several studies have focused on the effects of mirnas on spermatogenesis in male infertility [ 911,17 ] .
however , it was not understood how mir-210 , which is one of the up - regulated mirnas in testes of patients with noa , was involved in spermatogenesis in male infertility .
the transformation of diploid spermatogonia into mature haploid cells in spermatogenesis is a complex biological process in the testes of males .
the insulin / igf system takes part in the processes of cell proliferation , cell growth , differentiation , and survival , which affects nearly every organ in the body .
also , insulin / igf plays an important role in the proper function of the testis in males .
igf2 binds to igf1r and insr - a with a high affinity and binds to insr - a / igf1r , insr - b / igf1r , but with lower affinity .
found that in inactivated insr and igf1r , there was a 79% reduction in daily sperm production in adult mouse testes by a conditional ko approach . taken together , the aforementioned data suggest that igf2 might be involved in the process of spermatogenesis . to examine the specific mechanism by which mir-210 is associated with the process of spermatogenesis , quantitative real - time pcr was performed to detect mir-210 expression .
we found that mir-210 was significantly up - regulated in the testes of subjects with ma and hypospermatogenesis patients compared to oa .
these results agree with findings of lian et al . using microarray technologies performed in noa and normal controls .
several studies have suggested that this mirna could be mediated by hypoxia and participate in various types of regulation of angiogenesis , cell survival , proliferation , cell cycle arrest , and protein modification [ 1214 ] .
furthermore , some researchers even found that mir-210 might be considered as one of the indicated markers in some diseases , such as clear cell renal cell carcinoma and acute myeloid leukemia . in the present study we found that igf2 was targeted by mir-210 directly in the in vitro experiment in nt2 cells , and mir-210 might be associated with spermatogenesis by targeting igf2 in male infertility .
firstly , as some errors occurred in the rna extraction in the preliminary experiment , mir-210 of 3 testes samples were damaged and not detected in the subsequent quantitative real - time pcr experiment , which might have affected our results .
secondly , we did not investigate the functions of mir-210 and igf2 in vitro or in vivo , and we plan to do this in future research .
we demonstrated that mir-210 might be associated with spermatogenesis by targeting igf2 in male infertility .
future mechanistic studies on the role of mir-210/igf2 in the process of spermatogenesis in male infertility will provide new insights into the diagnosis and management of male infertility .
 backgroundmicrornas ( mirnas ) play pivotal roles in spermatogenesis . 
 microrna-210 ( mir-210 ) expression was up - regulated in the testes of sterile men with non - obstructive azoospermia ( noa ) . 
 however , the underlying mechanisms of mir-210 involved in the spermatogenesis in patients with noa are unknown.material/methodsexpression of mir-210 and insulin - like growth factor ii ( igf2 ) in the testes of noa cases ( only including maturation arrest and hypospermatogenesis ) were detected in this study . 
 we carried out in vitro experiments to determine if igf2 was directly targeted by mir-210 in nt2 cells.resultscompared with obstructive azoospermia ( oa ) as normal control , our results suggest that mir-210 was significantly up - regulated in testis of patients with noa ( p<0.05 ) , and igf2 was down - regulated , but without a significant difference . 
 the results also indicated that igf2 was directly targeted by mir-210 in nt2 cells.conclusionsthe results showed that mir-210 was involved in spermatogenesis by targeting igf2 in male infertility . 
Background
Material and Methods
Human testicular samples
Immunohistochemistry Staining
RNA extraction and real-time PCR
Western blotting
Cell lines/cell culture and transfection
Statistical analysis
Results
IGF2 located in spermatocytes in the testis of patients with OA
Down-regulation of IGF2 in testes of MA and hypospermatogenesis patients
Up-regulation of miR-210 in testis of MA and hypospermatogenesis patients
IGF2 was targeted directly by miR-210
Discussion
Conclusions