Experimental Study On The Effect Of ATP5D On Male Fertility

Objective Infertility presents as a global medical and social problem affecting human reproduction and social development.Little is known about the specific role of ATP5 D implicated in the male reproductive system,and thus it is of great scientific value and practical significance to investigate the role of ATP5 D in male fertility and identify core target molecules involved in male fertility regulation.Methods In this study,the semen of normal and infertile men were collected from the male outpatient department of the Affiliated Hospital of Zunyi Medical University from June to August in 2021,of which the indicators were detected by automatic sperm analyzer.Enzyme-linked-immunosorbent serologic assay(ELISA)was then used to detect the ATP5 D protein level in semen.Following extracting DNA in semen,we employed polymerase chains reaction(PCR)for amplification and agarose electrophoresis for purification.PCR products was further sequenced using the sequencer to detect potential genetic mutation.The correlation of ATP5 D protein expression with semen indicators was preliminarily evaluated by Pearson.The prepared transgenic mice using CRISPR/CAS9 technology underwent genotype identification and construction efficiency verification.Afterwards,ATP5 D knockout(KO)and knockin(KI)male mice aged 8-12 weeks were mated with adult wild type(WT)female mice for more than two months for fertility assessment.The morphological changes of testis and epididymis tissues of mice in each group were observed by Hematoxylin and Eosin(H&E)staining.Sperm from the epididymis were collected for sperm counting,and morphological changes were observed by Diff-Quik staining to further clarify the role of ATP5 D in spermatogenic function.Results The expression level of ATP5 D protein in seminal plasma of male infertility patients was lower than that of normal male participants(P < 0.001),and the morphological staining results showed the normal morphology rate of sperm in infertile patients was also much lower than that of normal males(P < 0.001).The ATP5 D levels were positively correlated with various important indicators in male semen.Singer sequencing results showed that the sequences of ATP5 D consisted with the normal group and no mutation occurred.The outcomes of animal experiments demonstrated no significant difference in the number of births between KO mice and WT mice,but the litter time of KO mice was significantly longer than WT mice(P = 0.02).The testes of KO mice were significantly reduced in size and weight(P = 0.01)compared with WT mice,while the testes of KI mice showed no significant changes in size and weight.Morphologically,HE staining of KO mice exhibited disorder arrangement of testicular cells,larger lumen,invisible interstitial cells,atrophy and the decreased number of spermatocytes at all levels in the germinal tubules.Meanwhile,the testicular structure of KI mice was slightly loosened,and the outer edge was no longer tight and smooth,with the inner edge irregularly arranged and a few immature spermatocytes found in the lumen of the tubules.The number of spermatocytes in the testicular lumen of KO mice was substantially reduced compared with WT mice(P = 0.002),while there was no statistically significant difference in KI mice relative to WT mice.Nevertheless,the number of spermatozoa in the epididymis was significantly reduced in both KO and KI male mice compared to WT group(P < 0.05).Conclusions In this study,ATP5 D was found to be associated with male infertility,indicated by significant decreased protein levels of ATP5 D in the semen of male infertile patients in absence of genetic mutation.ATP5 D dysregulation in mice resulted in longer bearing time and decreased fertility,lesioned testes,and a series of pathological changes such as hypospermatogenesis,oligospermia,and decreased normal morphology rate.These results indicate that ATP5 D is an essential protein for spermatogenesis and fertility in male mice and may be used as a possible biomarker of male fertility.