{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,5,11]],"date-time":"2026-05-11T15:36:55Z","timestamp":1778513815636,"version":"3.51.4"},"reference-count":51,"publisher":"MDPI AG","issue":"1","license":[{"start":{"date-parts":[[2019,1,3]],"date-time":"2019-01-03T00:00:00Z","timestamp":1546473600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100012166","name":"National Key Research and Development Program of China","doi-asserted-by":"publisher","award":["2016YFD0501205, 2016YFD0500503"],"award-info":[{"award-number":["2016YFD0501205, 2016YFD0500503"]}],"id":[{"id":"10.13039\/501100012166","id-type":"DOI","asserted-by":"publisher"}]},{"name":"Natural Science Foundation of China program","award":["31872435 and 31472163"],"award-info":[{"award-number":["31872435 and 31472163"]}]},{"name":"The Key Project of Guangdong Provincial Nature Science Foundation","award":["2018B030311015, 2016A030313413"],"award-info":[{"award-number":["2018B030311015, 2016A030313413"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>It has been reported that the miR-125 family plays an important role in regulating embryo development. However, the function of miR-125b-2 in spermatogenesis remains unknown. In this study, we used a model of miR-125b knockout (KO) mice to study the relationship between miR-125b-2 and spermatogenesis. Among the KO mice, the progeny test showed that the litter size decreased significantly (p = 0.0002) and the rate of non-parous females increased significantly from 10% to 38%. At the same time, the testosterone concentration increased significantly (p = 0.007), with a remarkable decrease for estradiol (p = 0.02). Moreover, the sperm count decreased obviously (p = 0.011) and the percentage of abnormal sperm increased significantly (p = 0.0002). The testicular transcriptome sequencing revealed that there were 173 up-regulated genes, including Papolb (PAP), and 151 down-regulated genes in KO mice compared with wild type (WT). The Kyoto Encyclopedia of Genes and Genomes (KEGG) and gene ontology (GO) analysis showed that many of these genes were involved in sperm mitochondrial metabolism and other cellular biological processes. Meanwhile, the sperm mitochondria DNA (mtDNA) copy number increased significantly in the KO mice, but there were no changes observed in the mtDNA integrity and mutations of mt-Cytb, as well as the mt-ATP6 between the WT mice and KO mice. In the top 10 up-regulated genes, PAP, as a testis specific expressing gene, affect the process of spermatogenesis. Western blotting and the Luciferase assay validated that PAP was the target of miR-125b-5p. Intriguingly, we also found that both miR-125b and PAP were only highly expressed in the germ cells (GC) instead of in the Leydig cells (LC) and Sertoli cells (SC). Additionally, miR-125b-5p down regulated the secretion of testosterone in the TM3 cell by targeting PAP (p = 0.021). Our study firstly demonstrated that miR-125b-2 regulated testosterone secretion by directly targeting PAP, and increased the sperm mtDNA copy number to affect semen quality. The study indicated that miR-125b-2 had a positive influence on the reproductive performance of animals by regulating the expression of the PAP gene, and could be a potential drugs and diagnostic target for male infertility.<\/jats:p>","DOI":"10.3390\/ijms20010148","type":"journal-article","created":{"date-parts":[[2019,1,3]],"date-time":"2019-01-03T03:36:30Z","timestamp":1546486590000},"page":"148","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":9,"title":["MiR-125b-2 Knockout in Testis Is Associated with Targeting to the PAP Gene, Mitochondrial Copy Number, and Impaired Sperm Quality"],"prefix":"10.3390","volume":"20","author":[{"given":"Longlong","family":"Li","sequence":"first","affiliation":[{"name":"Guangdong Provincial Key Laboratory of Animal Nutrition Control, College of Animal Science, South China Agricultural University, Guangzhou 510642, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Yanling","family":"Zhu","sequence":"additional","affiliation":[{"name":"Guangdong Provincial Key Laboratory of Animal Nutrition Control, College of Animal Science, South China Agricultural University, Guangzhou 510642, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Ting","family":"Chen","sequence":"additional","affiliation":[{"name":"Guangdong Provincial Key Laboratory of Animal Nutrition Control, College of Animal Science, South China Agricultural University, Guangzhou 510642, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Jiajie","family":"Sun","sequence":"additional","affiliation":[{"name":"Guangdong Provincial Key Laboratory of Animal Nutrition Control, College of Animal Science, South China Agricultural University, Guangzhou 510642, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Junyi","family":"Luo","sequence":"additional","affiliation":[{"name":"Guangdong Provincial Key Laboratory of Animal Nutrition Control, College of Animal Science, South China Agricultural University, Guangzhou 510642, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Gang","family":"Shu","sequence":"additional","affiliation":[{"name":"Guangdong Provincial Key Laboratory of Animal Nutrition Control, College of Animal Science, South China Agricultural University, Guangzhou 510642, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Songbo","family":"Wang","sequence":"additional","affiliation":[{"name":"Guangdong Provincial Key Laboratory of Animal Nutrition Control, College of Animal Science, South China Agricultural University, Guangzhou 510642, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Xiaotong","family":"Zhu","sequence":"additional","affiliation":[{"name":"Guangdong Provincial Key Laboratory of Animal Nutrition Control, College of Animal Science, South China Agricultural University, Guangzhou 510642, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Qingyan","family":"Jiang","sequence":"additional","affiliation":[{"name":"Guangdong Provincial Key Laboratory of Animal Nutrition Control, College of Animal Science, South China Agricultural University, Guangzhou 510642, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Yongliang","family":"Zhang","sequence":"additional","affiliation":[{"name":"Guangdong Provincial Key Laboratory of Animal Nutrition Control, College of Animal Science, South China Agricultural University, Guangzhou 510642, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Qianyun","family":"Xi","sequence":"additional","affiliation":[{"name":"Guangdong Provincial Key Laboratory of Animal Nutrition Control, College of Animal Science, South China Agricultural University, Guangzhou 510642, China"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2019,1,3]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"1506","DOI":"10.1093\/humrep\/dem046","article-title":"International estimates of infertility prevalence and treatment-seeking: Potential need and demand for infertility medical care","volume":"22","author":"Boivin","year":"2007","journal-title":"Hum. Reprod."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"581","DOI":"10.1002\/mrd.22197","article-title":"A novel m.6307A>G mutation in the mitochondrial COXI gene in asthenozoospermic infertile men","volume":"80","author":"Bakloutigargouri","year":"2013","journal-title":"Mol. Reprod. Dev."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"719","DOI":"10.1093\/molehr\/8.8.719","article-title":"Can mitochondrial DNA mutations cause sperm dysfunction?","volume":"8","author":"Spiropoulos","year":"2002","journal-title":"Mol. Hum. Reprod."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"1815","DOI":"10.1095\/biolreprod.103.019323","article-title":"Proliferation and Differentiation of Spermatogonial Stem Cells in the W\/Wv Mutant Mouse Testis1","volume":"69","author":"Ohta","year":"2003","journal-title":"Biol. Reprod."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"83","DOI":"10.1111\/j.1365-2605.1993.tb01156.x","article-title":"Histological and Histopathological Evaluation of the Testis","volume":"16","author":"Russell","year":"2010","journal-title":"Int. J. Androl."},{"key":"ref_6","first-page":"229","article-title":"Chromosome Y microdeletions in the pathogenesis of male infertility","volume":"13","author":"Slezak","year":"2002","journal-title":"Polski Merkuriusz Lekarski Organ Polskiego Towarzystwa Lekarskiego"},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"802","DOI":"10.1111\/j.1365-2605.2012.01284.x","article-title":"The role of male chromosomal polymorphism played in spermatogenesis and the outcome of IVF\/ICSI-ET treatment","volume":"35","author":"Guo","year":"2012","journal-title":"Int. J. Androl."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"901","DOI":"10.1530\/REP-08-0494","article-title":"Small RNA molecules in the regulation of spermatogenesis","volume":"137","author":"He","year":"2009","journal-title":"Reproduction"},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"351","DOI":"10.1146\/annurev-biochem-060308-103103","article-title":"Regulation of mRNA translation and stability by microRNAs","volume":"79","author":"Fabian","year":"2010","journal-title":"Annu. Rev. Biochem."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"2699","DOI":"10.1074\/jbc.M114.621565","article-title":"Gonadotrope-Specific Deletion of Dicer Results in Severely Suppressed Gonadotropins and Fertility Defects","volume":"290","author":"Wang","year":"2015","journal-title":"J. Biol. Chem."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"124","DOI":"10.1016\/j.mce.2015.10.006","article-title":"Fshb-iCre mice are efficient and specific Cre deleters for the gonadotrope lineage","volume":"419","author":"Wang","year":"2016","journal-title":"Mol. Cell. Endocrinol."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"501","DOI":"10.1159\/000445642","article-title":"MicroRNA-125b Suppresses Ovarian Cancer Progression via Suppression of the Epithelial-Mesenchymal Transition Pathway by Targeting the SET Protein","volume":"39","author":"Ying","year":"2016","journal-title":"Cell. Physiol. Biochem."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"2434","DOI":"10.1016\/j.yexcr.2010.06.006","article-title":"Regulation of p53 in embryonic stem cells","volume":"316","author":"Solozobova","year":"2010","journal-title":"Exp. Cell Res."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"65","DOI":"10.2527\/jas.2015-9456","article-title":"Critical role of miR-125b in lipogenesis by targeting stearoyl-CoA desaturase-1 (SCD-1)","volume":"94","author":"Cheng","year":"2016","journal-title":"J. Anim. Sci."},{"key":"ref_15","doi-asserted-by":"crossref","unstructured":"Kim, K.H., Seo, Y.M., Kim, E.Y., Lee, S.Y., Kwon, J., Ko, J.J., and Lee, K.A. (2016). The miR-125 family is an important regulator of the expression and maintenance of maternal effect genes during preimplantational embryo development. Open Biol., 6.","DOI":"10.1098\/rsob.160181"},{"key":"ref_16","first-page":"1549","article-title":"Effects of testosterone on PPAR\u03b3 and P450arom expression in polycystic ovary syndrome patients and related mechanisms","volume":"22","author":"He","year":"2018","journal-title":"Eur. Rev. Med. Pharmacol. Sci."},{"key":"ref_17","unstructured":"Frazee, A.C., Pertea, G., Jaffe, A.E., Langmead, B., Salzberg, S.L., and Leek, J.T. (2014). Flexible isoform-level differential expression analysis with Ballgown. bioRxiv."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"289","DOI":"10.1111\/j.2517-6161.1995.tb02031.x","article-title":"Controlling The False Discovery Rate\u2014A Practical And Powerful Approach To Multiple Testing","volume":"57","author":"Benjamini","year":"1995","journal-title":"J. R. Stat. Soc."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"935","DOI":"10.1093\/hmg\/ddh109","article-title":"Mitochondrial transcription factor A regulates mtDNA copy number in mammals","volume":"13","author":"Ekstrand","year":"2004","journal-title":"Hum. Mol. Genet."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"295","DOI":"10.1016\/j.bbrc.2017.08.129","article-title":"Effect of sodium fluoride on the sperm mitochondrial DNA in mice","volume":"492","author":"Sun","year":"2017","journal-title":"Biochem. Biophys. Res. Commun."},{"key":"ref_21","first-page":"81","article-title":"Significance of microRNA targeted estrogen receptor in male fertility","volume":"17","author":"Abhari","year":"2014","journal-title":"Iran. J. Basic Med. Sci."},{"key":"ref_22","doi-asserted-by":"crossref","unstructured":"Zhang, Q., Wang, Q., Zhang, Y., Cheng, S., Hu, J., Ma, Y., and Zhao, X. (2018). Comprehensive Analysis of MicroRNA(-)Messenger RNA from White Yak Testis Reveals the Differentially Expressed Molecules Involved in Development and Reproduction. Int. J. Mol. Sci., 19.","DOI":"10.3390\/ijms19103083"},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"498","DOI":"10.1016\/j.mce.2013.04.015","article-title":"Small RNAs in spermatogenesis","volume":"382","author":"Noora","year":"2014","journal-title":"Mol. Cell. Endocrinol."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"871","DOI":"10.1016\/j.fertnstert.2004.02.132","article-title":"Human sperm superoxide anion generation and correlation with semen quality in patients with male infertility","volume":"82","author":"Said","year":"2004","journal-title":"Fertil. Steril."},{"key":"ref_25","first-page":"345","article-title":"Mitochondria in Mammalian Sperm Physiology and Pathology: A Review","volume":"44","author":"Tapia","year":"2010","journal-title":"Reprod. Domest. Anim."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"94","DOI":"10.1016\/j.theriogenology.2017.02.015","article-title":"The relationship between mitochondrial DNA copy number and stallion sperm function","volume":"94","author":"Darr","year":"2017","journal-title":"Theriogenology"},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"231","DOI":"10.1038\/ng0398-231","article-title":"Mitochondrial transcription factor A is necessary for mtDNA maintenance and embryogenesis in mice","volume":"18","author":"Larsson","year":"1998","journal-title":"Nat. Genet."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"843","DOI":"10.1093\/molehr\/gah243","article-title":"Mitochondrial DNA content and 4977 bp deletion in unfertilized oocytes","volume":"11","author":"Chan","year":"2006","journal-title":"Mol. Hum. Reprod."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"e68","DOI":"10.1093\/nar\/gnf067","article-title":"Detection and quantification of mitochondrial DNA deletions in individual cells by real-time PCR","volume":"30","author":"He","year":"2002","journal-title":"Nucleic Acids Res."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"1999","DOI":"10.1126\/science.1074632","article-title":"Regulation of spermatogenesis by testis-specific, cytoplasmic poly(A) polymerase TPAP","volume":"298","author":"Kashiwabara","year":"2002","journal-title":"Science"},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"207","DOI":"10.1262\/jrd.50.207","article-title":"Transgenic expression of testis-specific poly (A) polymerase TPAP in wild-type and TPAP-deficient mice","volume":"50","author":"Zhuang","year":"2004","journal-title":"J. Reprod. Dev."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"607","DOI":"10.1262\/jrd.2016-116","article-title":"Adenylation by testis-specific cytoplasmic poly(A) polymerase, PAPOLB\/TPAP, is essential for spermatogenesis","volume":"62","author":"Kashiwabara","year":"2016","journal-title":"J. Reprod. Dev."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"106","DOI":"10.1006\/dbio.2000.9894","article-title":"Identification of a novel isoform of poly (A) polymerase, TPAP, specifically present in the cytoplasm of spermatogenic cells","volume":"228","author":"Kashiwabara","year":"2000","journal-title":"Dev. Biol."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"5010","DOI":"10.1128\/MCB.18.9.5010","article-title":"Deregulation of poly(A) polymerase interferes with cell growth","volume":"18","author":"Zhao","year":"1998","journal-title":"Mol. Cell. Biol."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"6603","DOI":"10.1093\/emboj\/cdf633","article-title":"Control of poly(A) polymerase level is essential to cytoplasmic polyadenylation and early development in Drosophila","volume":"21","author":"Juge","year":"2014","journal-title":"EMBO J."},{"key":"ref_36","first-page":"73","article-title":"Levels of FSH, LH and testosterone, and sperm DNA fragmentation","volume":"35","author":"Wdowiak","year":"2014","journal-title":"Neuro Endocrinol. Lett."},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"81","DOI":"10.6061\/clinics\/2013(Sup01)09","article-title":"Hypogonadotropic Hypogonadism Revisited","volume":"68","author":"Renato","year":"2013","journal-title":"Clinics"},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"2536","DOI":"10.1210\/jc.2005-2847","article-title":"Testosterone therapy in adult men with androgen deficiency syndromes: An endocrine society clinical practice guideline","volume":"91","author":"Bhasin","year":"2006","journal-title":"J. Clin. Endocrinol. Metab."},{"key":"ref_39","first-page":"1988","article-title":"Pathophysiology of small testes in beef bulls: Relationship between scrotal circumference, histopathologic features of testes and epididymides, seminal characteristics, and endocrine profiles","volume":"47","author":"Veeramachaneni","year":"1986","journal-title":"Am. J. Vet. Res."},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"45","DOI":"10.1007\/BF02872388","article-title":"Evaluation of FSH, LH and testosterone levels in different subgroups of infertile males","volume":"19","author":"Babu","year":"2004","journal-title":"Indian J. Clin. Biochem."},{"key":"ref_41","unstructured":"Ferlin, A., and Foresta, C. (2009). Infertility, Male, Springer."},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"63","DOI":"10.1186\/1477-7819-9-63","article-title":"Primary testicular necrotizing vasculitis clinically presented as neoplasm of the testicle: A case report","volume":"9","year":"2011","journal-title":"World J. Surg. Oncol."},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"182","DOI":"10.4103\/0971-6580.97220","article-title":"To Study the Effect of Vitamin D and E on Sodium-Fluoride-induced Toxicity in Reproductive Functions of Male Rabbits","volume":"19","author":"Kumar","year":"2012","journal-title":"Toxicol. Int."},{"key":"ref_44","doi-asserted-by":"crossref","first-page":"787","DOI":"10.1016\/S0092-8674(03)01018-3","article-title":"Prediction of Mammalian MicroRNA Targets","volume":"115","author":"Lewis","year":"2003","journal-title":"Cell"},{"key":"ref_45","doi-asserted-by":"crossref","first-page":"44","DOI":"10.1038\/nprot.2008.211","article-title":"Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources","volume":"4","author":"Huang","year":"2009","journal-title":"Nat. Protoc."},{"key":"ref_46","doi-asserted-by":"crossref","first-page":"2238","DOI":"10.1016\/j.fertnstert.2007.10.059","article-title":"Mitochondrial DNA integrity and copy number in sperm from infertile men","volume":"90","author":"Song","year":"2008","journal-title":"Fertil. Steril."},{"key":"ref_47","doi-asserted-by":"crossref","first-page":"20","DOI":"10.3109\/19401736.2013.840612","article-title":"Polymorphisms in the MT-ATP6 and MT-CYB genes in in vitro fertilization failure","volume":"26","author":"Mao","year":"2015","journal-title":"Mitochondrial DNA"},{"key":"ref_48","doi-asserted-by":"crossref","first-page":"2827","DOI":"10.1210\/en.2011-0031","article-title":"Toll-like receptor-initiated testicular innate immune responses in mouse Leydig cells","volume":"152","author":"Shang","year":"2011","journal-title":"Endocrinology"},{"key":"ref_49","doi-asserted-by":"crossref","first-page":"2709","DOI":"10.1172\/JCI59901","article-title":"Estrogen promotes Leydig cell engulfment by macrophages in male infertility","volume":"124","author":"Yu","year":"2014","journal-title":"J. Clin. Investig."},{"key":"ref_50","doi-asserted-by":"crossref","first-page":"867","DOI":"10.1387\/ijdb.092874gk","article-title":"Dicer is required for Sertoli cell function and survival","volume":"54","author":"Kim","year":"2015","journal-title":"Int. J. Dev. Biol."},{"key":"ref_51","doi-asserted-by":"crossref","first-page":"341","DOI":"10.2144\/000113764","article-title":"Isolation of Sertoli, Leydig, and spermatogenic cells from the mouse testis","volume":"51","author":"Chang","year":"2011","journal-title":"Biotechniques"}],"container-title":["International Journal of Molecular Sciences"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1422-0067\/20\/1\/148\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,11]],"date-time":"2025-10-11T12:23:17Z","timestamp":1760185397000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1422-0067\/20\/1\/148"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2019,1,3]]},"references-count":51,"journal-issue":{"issue":"1","published-online":{"date-parts":[[2019,1]]}},"alternative-id":["ijms20010148"],"URL":"https:\/\/doi.org\/10.3390\/ijms20010148","relation":{"has-preprint":[{"id-type":"doi","id":"10.20944\/preprints201811.0563.v1","asserted-by":"object"}]},"ISSN":["1422-0067"],"issn-type":[{"value":"1422-0067","type":"electronic"}],"subject":[],"published":{"date-parts":[[2019,1,3]]}}}