领域：学术论文 · 自然科学

Spermatogenesis is a precisely regulated and highly ordered process, including mitotic proliferation of spermatogonia, meiosis of spermatocytes, and differentiation of haploid spermatids during spermiogenesis. Any errors in this process will lead to male reproductive disorders. Male infertility, accounting for 50% of infertile couples, is a serious reproductive health problem Nonobstructive azoospermia (NOA) is the most serious form, accounting for 10–15% of male infertility. 
Only one in three cases of azoospermia can be explained by genetic defects, such as abnormalities and deletion of the azoospermia-factor (AZF) region in the Y chromosome, whereas more than 70% of the cases are idiopathic NOA (iNOA). In addition to genetic defects, infectious and inflammatory conditions in the reproductive system contribute to male infertility. Genome-wide association studies of idiopathic male infertility have revealed a series of NOA susceptibility loci, including PEX10, PRMT6, and SIRPA. Several single- gene mutations have been reported in patients with testicular- phenotype NOA, including TEX11, TEX14, TEX15, DMC1, and MEIOB. Recent studies have used transcriptome analysis to investigate the pathogenic mechanism of NOA. Bulk transcriptome analyses have revealed that PILRA and ZNF676 are related to NOA susceptibility. Two-dimensional coculture of multiple stem cell types and three-dimensional testicular organ culture have been used to simulate the environment of testicular sperm production and promote spermatogenesis, with promising results for the treatment of azoospermia. 
Treatments for azoospermia require a better understanding of the classification of azoospermia. Single-cell RNA sequencing (scRNA-seq) enables the study of highly heterogeneous cell populations in the testes at a single-cell resolution. We previously conducted scRNA-seq analysis of adult human testis samples from normal donors and iNOA patients and reconstructed the transcriptional programs inherent to sequential cell fate transition during human spermatogenesis. The results showed that the gene expression patterns of somatic cells in iNOA differed from those in normal spermatogenesis and mainly included DNA damage genes and other stress-responsive genes. A recent study revealed that maturation disorders in Sertoli cells are in- volved in iNOA, and inhibition of the Wnt signaling pathway promoted the maturation of these cells. However, the number of reported study cases is very small, and the explanation of the iNOA mechanism is limited from a somatic perspective. The gene expression characteristics of germ cells and somatic cells, cell–cell interactions between germ cells and somatic cells, as well as the regulatory networks of transcription factors (TFs) in iNOA patients remain largely unknown. 
We performed scRNA-seq analysis of 3696 individual testicular cells from 17 iNOA donors to construct transcriptional land- scapes of human spermatogenesis in the context of NOA. In comparison with healthy subjects with normal spermatogenesis, we assessed the gene expression characteristics of germ cells and somatic cells, cell–cell interactions between germ cells and somatic cells, as well as the regulatory networks of TFs in iNOA patients. In particular, we demonstrated that CD164 played an important role in the apoptosis of spermatogonia. Moreover, we identified a series of genes predicting spermatogenic capacity in different testicular diseases. In general, our study provides new insights into the molecular pathogenesis as well as the clinical diagnosis and therapeutic strategy of iNOA. 
Our results revealed that germ cells, including SSC, from azoospermia patients had enhanced cell cycle activity and weakened energy production, which may provide clues for the selection of factors to be added to the in vitro culture medium for round sperm. As SSC can self-renew to maintain the stem cell population and further produce sperm, sperm can be produced uninterruptedly throughout adulthood. 
To identify candidate iNOA pathogenic genes, we analyzed the key DEGs between the four classes iNOA and the NC group. Among them, HMGB4, a member of the high-mobility group box (HMGB) family, is present in spermatocytes, spermatids, and spermatozoa in human and mice. A significant decline of HMGB4 in all cell types in the three classes of iNOA was observed. Though male fertility was not affected in Hmgb4 knock- out mice, which might be due to species variation. We speculated that HMGB4 might be a key pathogenic gene of iNOA and played an important role in spermatogenesis. More studies should be carried out in the future. Cell–cell interactions between ST and developing SSC have long received research interest. We found that the CD226/NECTIN2, LGR4/RSPO3, and NOTCH1/JAG2 interaction pairs might have an effect on human spermatogenesis. Moreover, the CSF1/SIRPA and CXCL12/CXCR4 interaction pairs were downregulated in all classes of iNOA. CSF1 (colony-stimulating factor 1) exerts a crucial impact on the development of SSC. Polymorphisms in SIRPA, its interaction partner, are strongly associated with iNOA susceptibility. Additionally, CXCL12 encodes a chemokine ex- pressed and secreted by ST and binds to the CXCR4 receptor on SSC to regulate the self-renewal and maintenance of SSC. Therefore, the downregulation of these key interaction signals may provide ideas for targeted therapy of iNOA in the future. 
Apart from intercellular communication, TFs are essential in spermatogenesis. We found that HES7 (Hes family bHLH TF 7), as a transcriptional repressor. was significantly upregulated in iNOA cells. Another key TF, SOX5, is necessary for correct gene expression patterns during spermatogenesis and its expression was significantly downregulated in iNOA cells. Therefore, we speculate that the increase in HES7 transcriptional activity and decrease in SOX5 transcriptional activity may disrupt the key regulatory network during spermatogenesis and cause iNOA. 
None of the iNOA patients in our study had mature sperm, but other indicators, such as genetic factors, were normal in all patients. We classified them into four groups based on gene expression and pathological features, and identified a series of genes that were highly associated with spermatogenic capacity. At present, testicular biopsy is an invasive method to diagnose the spermatogenic ability of patients, and a safe and noninvasive diagnostic strategy is still lacking. The genes identified in this work may be integrated into a panel for the rapid and noninvasive diagnosis of male infertility in the future. 
As demonstrated in this study, the interactions between germ cells and somatic cells were speculated only by expression data, computational software prediction, protein localization, and quantification. Additional experiments are needed to further clarify the cell–cell communications. In addition, we used cell lines for functional validation of the key pathogenic genes we screened, indicating the reliability of our bioinformatics analysis results. Further studies using in vivo animal models are still needed to better characterize the effect of these pathogenic genes on spermatogenesis. Moreover, we used different testicular diseases to find common diagnostic predictor of spermatogenic ability, providing a large amount of data resources for clinical diagnosis. However, a large number of clinical samples still need to be included to verify its accuracy. 
Although there have been enormous advances in iNOA testicular microenvironment, transcriptional network of autophagy- related genes, and hub genes of azoospermia to our knowledge, this was the first study to investigate the occurrence and development of idiopathic NOA from a testicular germ cell perspective, including the largest number of cases to date. The data comprehensively and systematically reveal the transcriptional regulatory network of iNOA. Our findings not only provide valuable knowledge to improve our understanding of the molecular basis and mechanism of iNOA but also provide new insights for clinical diagnosis and treatment. 
Experimental Model and Subject Details: the donors in this study underwent sperm isolation surgery for in vitro fertilization due to male-related infertility. Men with spermatogenic cells, but without mature sperm, abnormal hormone levels, chromosomal abnormalities, Y chromosome microdeletion, and no other medical history were selected. Testis tissues were collected from 17 iNOA patients between 27 and 37 years old to map single-cell transcriptome landscapes. All testis samples for transcriptome analysis were obtained from Peking University Third Hospital with informed consent from the donors. The study was approved by the Ethics Committee of Peking University Third Hospital (2017SZ-048). All experimental procedures and animal care were approved by the Animal Care and Use Committee of the Peking University Health Science Center (LA2021579). Normal Samples data included 2854 testicular cells from 9 donors, have been deposited in NCBI GEO: GSE106487. 
Isolation of iNOA Male Testicular Cells for Transcriptional Profiling: Idiopathic NOA testis tissues were washed three times with Dulbecco’s modified Eagle’s medium (DMEM, C11995500BT, Thermo Scientific) containing 10% fetal bovine serum (FBS, 10437028, Gbico) and minced using sterilized scissors. Then, 500 μL of Accutase Cell Detachment Solution (A6964, Sigma- Aldrich) was added, and the samples were incubated at 37 °C for 15 min. To completely digest the tissues into single cells, the mixture was pipetted 30 times at 7 min and at the end of digestion. The cell suspension was filtered through 40 μm Pre-Separation Filters and centrifuged at 300× g for 8 min, and the cells were resuspended in 500 μL of DMEM (containing 10% FBS). 
ScRNA-seq Library Preparation and Sequencing: After digestion, the cells were diluted in 1 mg mL−1 Ac-Bovine Serum Albumin (A9418, Sigma-Aldrich) and single cells were randomly picked using a mouth pipette and added to lysis buffer prepared in advance. Mouth pipette method can make sure that there’s only one cell in each tube. A previously reported modified STRT- seq protocol was used to construct the scRNA-seq library. In brief, after total RNA was extracted from the single cells, the mRNAs were captured using an oligo (dT) primer and subjected to reverse transcription. The cDNAs were PCR-amplified in 18 cycles to increase yields. After barcoding, the cDNAs from 48 single cells were pooled, and a biotin-modified index sequence was added to the 3′ end in four cycles of PCR. Dynabeads MyOne Streptavidin C1(65 002, Invitrogen) was used to capture the fragmented 3′ cDNAs, which were sheared using Covaris (S2). The libraries were constructed using a Kapa Hyper Prep Kit (KK8505, Kapa Biosystems) and were subjected to 150 bp paired-end sequencing on the Illumina 4000 platform. 


翻译考点：
考点1：“Donors” 应译为“供体”
考点2：“high-mobility group box (HMGB) family” 必须译为“高迁移率族蛋白”，固定译法
考点3：“DMEM”必须译为“杜尔贝科改良鹰培养基”，固定译法
考点4：“PCR”必须译为“聚合酶链式反应”，固定译法