scRNA-seq reveals that origin recognition complex subunit 6 regulates mouse spermatogonial cell proliferation and apoptosis via activation of Wnt/ß-catenin signaling.

ABSTRACT: The regulation of spermatogonial proliferation and apoptosis is of great significance for maintaining spermatogenesis. The single-cell RNA sequencing (scRNA-seq) analysis of the testis was performed to identify genes upregulated in spermatogonia. Using scRNA-seq analysis, we identified the spermatogonia upregulated gene origin recognition complex subunit 6 (Orc6), which is involved in DNA replication and cell cycle regulation; its protein expression in the human and mouse testis was detected by western blot and immunofluorescence. To explore the potential function of Orc6 in spermatogonia, the C18-4 cell line was transfected with control or Orc6 siRNA. Subsequently, 5-ethynyl-2-deoxyuridine (EdU) and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assays, flow cytometry, and western blot were used to evaluate its effects on proliferation and apoptosis. It was revealed that ORC6 could promote proliferation and inhibit apoptosis of C18-4 cells. Bulk RNA sequencing and bioinformatics analysis indicated that Orc6 was involved in the activation of wingless/integrated (Wnt)/ ß-catenin signaling. Western blot revealed that the expression of ß-catenin protein and its phosphorylation (Ser675) were significantly decreased when silencing the expression of ORC6. Our findings indicated that Orc6 was upregulated in spermatogonia, whereby it regulated proliferation and apoptosis by activating Wnt/ß-catenin signaling.

Efficacy of stepwise mini-incision microdissection testicular sperm extraction for nonobstructive azoospermia with varied etiologies.

Stepwise mini-incision microdissection testicular sperm extraction (mTESE) is a procedure that attempts to minimize testicular damage. However, the mini-incision approach may vary in patients with different etiologies. Here, we performed a retrospective analysis of 665 men with nonobstructive azoospermia (NOA) who underwent stepwise mini-incision mTESE (Group 1) and 365 men who underwent standard mTESE (Group 2). The results showed that the operation time (mean ± standard deviation) for patients with successful sperm retrieval in Group 1 (64.0 ± 26.6 min) was significantly shorter than that in Group 2 (80.2 ± 31.3 min), with P <0.001. The total sperm retrieval rate (SRR) was 23.1% in our study, and there was no significant difference between Group 1 and Group 2 ( P >0.05), even when the etiologies of NOA were taken into consideration. The results of consecutive multivariate logistic regression analysis (odds ratio [OR]: 0.57; 95% confidence interval [CI]: 0.38-0.87; P =0.009) and receiver operating characteristic (ROC) analysis (area under the ROC curve [AUC]=0.628) showed that preoperative anti-Müllerian hormone (AMH) level in idiopathic NOA patients was a potential predictor for surgical outcomes after initial three small incisions made in the equatorial region without sperm examined under an operating microscope (Steps 2-4). In conclusion, stepwise mini-incision mTESE is a useful technique for NOA patients, with comparable SRR, less surgical invasiveness, and shorter operation time compared with the standard approach. Low AMH levels may predict successful sperm retrieval in idiopathic patients even after a failed initial mini-incision procedure.

Novel mutation in ODF2 causes multiple morphological abnormalities of the sperm flagella in an infertile male.

Numerous genes have been associated with multiple morphological abnormalities of the sperm flagella (MMAF), which cause severe asthenozoospermia and lead to male infertility, while the causes of approximately 50% of MMAF cases remain unclear. To reveal the genetic causes of MMAF in an infertile patient, whole-exome sequencing was performed to screen for pathogenic genes, and electron microscope was used to reveal the sperm flagellar ultrastructure. A novel heterozygous missense mutation in the outer dense fiber protein 2 (ODF2) gene was detected, which was inherited from the patient's mother and predicted to be potentially damaging. Transmission electron microscopy revealed that the outer dense fibers were defective in the patient's sperm tail, which was similar to that of the reported heterozygous Odf2 mutation mouse. Immunostaining of ODF2 showed severe ODF2 expression defects in the patient's sperm. Therefore, it was concluded that the heterozygous mutation in ODF2 caused MMAF in this case. To evaluate the possibility of assisted reproductive technology (ART) treatment for this patient, intracytoplasmic sperm injection (ICSI) was performed, with the help of a hypo-osmotic swelling test and laser-assisted immotile sperm selection (LAISS) for available sperm screening, and artificial oocyte activation with ionomycin was applied to improve the fertilization rate. Four ICSI cycles were performed, and live birth was achieved in the LAISS-applied cycle, suggesting that LAISS would be valuable in ART treatment for MMAF.

Clinical benefits of a modified Cryopiece system for cryopreservation of rare ejaculated and testicular spermatozoa for ICSI.

Cryopreservation of rare testicular-retrieved spermatozoa for intracytoplasmic sperm injection (ICSI) in patients with severe oligozoospermia and azoospermia remains a major challenge in clinical practice. This study evaluated the Cryopiece system as a potential technique to cryopreserve rare human spermatozoa for ICSI. Small numbers of ejaculated (24 patients) and testicular (13 patients) spermatozoa were cryopreserved using the Cryopiece system. The total number of recovered spermatozoa and motility were assessed after thawing. Thirty-seven couples underwent ICSI using spermatozoa cryopreserved by the Cryopiece system, and ICSI outcomes (rates of fertilization, embryo cleavage, and clinical pregnancy) were evaluated. The average sperm post-thaw retrieval rate was 79.1%, and motility was 29.7%. Ejaculated spermatozoa had a higher post-thaw motility (32.5%) than testicular spermatozoa (21.8%; P = 0.005). ICSI achieved a fertilization rate of 61.9%, embryo cleavage rate of 84.6%, and clinical pregnancy rate of 43.3%. The ICSI outcomes in the ejaculated and testicular frozen-thawed spermatozoa were similar. Assisted oocyte activation (AOA) after ICSI with motile (72.1%) or immotile (71.9%) spermatozoa resulted in a significantly higher fertilization rate than that when using motile spermatozoa without AOA (52.0%; P = 0.005). However, AOA did not enhance the clinical pregnancy rate (55.6% or 40.0% vs 35.3%; P = 0.703). The Cryopiece system is simple and useful for the cryopreservation of small numbers of ejaculated or testicular spermatozoa for ICSI in patients with severe oligozoospermia or nonobstructive azoospermia.

Cryopiece, a novel carrier with faster cooling rate, high recovery rate and retrieval rate, for individual sperm cryopreservation.

BACKGROUND: Cryopreservation of extremely few spermatozoa is still a major challenge for male fertility preservation. This study aims to evaluate the cooling rate, recovery rate, and retrieval rate, along with other parameters of spermatozoa that cryopreserved using Cryopiece, a novel carrier, for individual sperm cryopreservation. METHODS: Semen samples from 60 fertile donors were collected, and each semen sample was screened for motile sperm and mixed with cryoprotective agent (CPA), and then frozen using Cryopiece, micro-straw, and mini-straws. The cooling rate, retrieval rate, and recovery rate, morphology, DNA fragmentation index (DFI) and mitochondrial membrane potential (MMP), were compared among the un-frozen sperm and the sperm cryopreserved using these carriers. RESULTS: Cryopiece possessed the fastest cooling rate. After freeze-thaw, the average retrieval rate of sperm cryopreserved using Cryopiece was 96.25%, and the average recovery rate was 64.40%, which were higher than that of sperm cryopreserved using the other two carriers (71.42% and 54.30% for micro-straw, and 63.54% and 58.04% for mini-straw, respectively). There was no significant impact on DFI after sperm cryopreservation, and no significant difference in morphology between sperm cryopreserved using these carriers was observed. Though MMP of sperm changed significantly after cryopreservation, micro-straw maintained sperm MMP better than Cryopiece and mini-straw did, while no significant difference was observed in MMP between sperm cryopreserved using Cryopiece and mini-straw. CONCLUSIONS: Cryopiece produced satisfying retrieval and recovery rates in sperm cryopreservation and should be an ideal carrier for cryopreservation of small number of sperm.

[Impact of plateau environment on seminal characteristics of native Tibetans and immigrated Tibetan Hans].

OBJECTIVE: To investigate the characteristics of the semen parameters of native Tibetans and immigrated Tibetan Hans in the high-altitude area and analyze the influence of altitude adaptation on male fertility. METHODS: This study included 1 563 infertile male patients, including 698 native Tibetans and 865 immigrated Tibetan Hans, and 56 normal fertile men, including 33 native Tibetans and 23 Tibetan Hans. We obtained semen samples from the subjects for routine semen analysis and sperm DNA fragmentation index (DFI) examination and collected peripheral blood for determination of the reproductive hormone levels. RESULTS: In the infertile patients, the native Tibetans, as compared with the immigrated Hans, showed significantly higher incidence rates of azoospermia (5.87% vs 2.89%, P <0.05), severe oligozoospermia (3.15% vs 1.73%, P <0.05) and abnormal seminal viscosity (43.12% vs 25.89%, P<0.01), but no statistically significant differences in the percentages of normozoospermia (81.08% vs 87.39%, P >0.05), oligozoospermia (5.44% vs 3.93%, P >0.05), severe asthenozoospermia (4.44% vs 4.04%, P >0.05) or severe teratozoospermia (4.58% vs 6.59%, P >0.05). In the normal fertile men, there were no statistically significant differences between the native Tibetans and immigrated Hans in age (ï¼»32.42 ± 4.82ï¼½ vs ï¼»34.57 ± 6.01ï¼½ yr, P >0.05), sperm concentration (ï¼»143.69 ± 85.74ï¼½ vs ï¼»155.11 ± 82.56ï¼½ ×106/ml, P >0.05), straight line velocity (ï¼»25.74 ± 3.94ï¼½ vs ï¼»27.24 ± 3.46ï¼½ µm/s, P >0.05), percentage of morphologically normal sperm (ï¼»8.22 ± 4.35ï¼½ vs ï¼»7.28±2.46ï¼½ %, P >0.05), total testosterone concentration (ï¼»17.97 ± 2.98ï¼½ vs ï¼»15.72 ± 6.38ï¼½ nmol/L, P >0.05), or follicle stimulating hormone level (ï¼»5.51 ± 1.62ï¼½ vs ï¼»4.17 ± 2.08ï¼½ IU/L, P >0.05). However, the immigrated Hans, in comparison with the native Tibetans, exhibited a higher sperm motility (ï¼»79.75 ± 14.67ï¼½ vs ï¼»66.58 ± 17.21ï¼½%, P <0.05), a lower curvilinear velocity (ï¼»60.97 ± 2.71ï¼½ vs ï¼»71.14 ± 82.13ï¼½ µm/s, P <0.05) and a lower level of luteinizing hormone (ï¼»4.28 ± 1.20ï¼½ vs ï¼»5.84 ± 1.15ï¼½ IU/L, P <0.05). CONCLUSIONS: During the acclimatization to the plateau hypoxia environment, the immigrated Tibetan Hans undergo adaptive changes in sperm concentration and motility and have lower incidence rates of azoospermia and severe oligozoospermia than native Tibetan males.

[Qilin Pills promote testicular spermatogenesis in azoospermia mice].

OBJECTIVE: To investigate the effect of Qilin Pills (QP) in facilitating the recovery of spermatogenic function in azoospermia (AS) mice and to explore its mechanism of regulating testicular spermatogenesis. METHODS: Fifteen 4-week-old male mice were equally randomized into an AS model control, a low-dose QP and a high-dose QP group. The AS model was established in the mice by intraperitoneal injection of busulfan at 35 mg/kg. After modeling, the animals in the low- and high-dose QP groups were treated with Qilin Pills intragastrically at 2 000 and 8 000 mg/kg/d respectively while those in the model control group fed on a normal diet, all for 28 days. Then, all the mice were sacrificed for examination of the ultrastructures of the epididymis and testis by HE staining, detection of the specific markers of spermatogenic, Sertoli and Leydig cells by Western blot, and determination of the expressions of these markers in the testis tissue by immunofluorescence assay. RESULTS: The number of spermatogenic cells in the testis tissue was significantly decreased in the AS model controls, with no spermatozoa in most of the seminiferous tubules in the epididymis (Johnsen's score: 5.2 ± 0.5). In the high-dose QP group, spermatogenic cells were tightly arranged with distinct layers in the seminiferous tubules, with a large number of spermatozoa but no non-sperm cells in the lumens of the epididymis (Johnsen's score: 9.4 ± 0.6). The number of spermatogenic cells in the testis was increased in the low-dose QP group with some spermatozoa in the seminiferous tubules as compared with that in the model control, but lower than in the high-dose group (Johnsen's score: 7.6 ± 0.6). The Johnsen's score was significantly lower in the model control than in the high- and low-dose QP groups (P < 0.01), and higher in the high-dose than in the low-dose QP group (P < 0.05). The expressions of the specific markers of Sertoli cells SCF, BMP4, SYCP3, DMC1 and Ki67 were also remarkably lower in the model control than in the high- and low-dose QP groups (P < 0.01), and higher in the high-dose than in the low-dose QP group (P < 0.05 or P < 0.01). No statistically significant differences were observed among the three groups of mice in the markers of spermatogonial stem cells (SSC) and undifferentiated SSCs UCHL1, STRA8, NGN3 and PLZF3 (P > 0.05). The expressions of the spermatocyte markers DMC1 and SYCP3 were markedly lower in the model control than in the high- and low-dose QP groups (P < 0.05 or P < 0.01), and higher in the high-dose than in the low-dose QP group (P < 0.05 or P < 0.01). The Ki67 fluorescence signals were distributed in the spermatogonia, with a higher intensity in the model control than in the high- and low-dose QP groups. The acrosome marker PNA was found mainly in the seminiferous tubules, with abundant fluorescence signals in the high- and low-dose QP groups but no obvious dot signals in the model controls. CONCLUSIONS: Qilin Pills may contribute to the meiosis of spermatogonia and promote spermatogenesis by improving the function of Sertoli cells in the testis.

Association of a TDRD1 variant with spermatogenic failure susceptibility in the Han Chinese.

PURPOSE: Piwi-interacting RNAs (piRNAs) are a broad group of noncoding small RNAs that have important biological functions in germline cells and can maintain genome integrity via silencing of retrotransposons. In this study, we aimed to explore the associations between genetic variants of important genes involved in piRNA biogenesis and male infertility with spermatogenic impairment. METHODS: To this end, five single-nucleotide polymorphisms (SNPs) in the ASZ1, PIWIL1, TDRD1, and TDRD9 genes were genotyped by TaqMan allelic discrimination assays in 342 cases of nonobstructive azoospermia (NOA) and 493 controls. RESULTS: The SNP rs77559927 in TDRD1 was associated with a reduced risk of spermatogenic impairment. The genotypes TC and TC + CC showed odds ratios and 95 % confidence intervals of 0.73 (0.55-0.98, P = 0.034) and 0.73 (0.56-0.97, P = 0.030), respectively, in patients with NOA compared with those in the controls. CONCLUSION: Thus, our results provided the first epidemiological evidence supporting the involvement of TDRD1 genetic polymorphisms in piRNA processing genes in determining the risk of spermatogenic impairment in a Han Chinese population.

Transcriptome research on spermatogenic molecular drive in mammals.

It is known that spermatogenic disorders are associated with genetic deficiency, although the primary mechanism is still unclear. It is difficult to demonstrate the molecular events occurring in testis, which contains germ cells at different developmental stages. However, transcriptomic methods can help us reveal the molecular drive of male gamete generation. Many transcriptomic studies have been performed on rodents by utilizing the timing of the first wave of spermatogenesis, which is not a suitable strategy for research in fertile men. With the development of separation methods for male germ cells, transcriptome research on the molecular drive of spermatogenesis in fertile men has seen great progress, and the results could be ultimately applied to improve the diagnosis and treatment for male infertility.

NODAL secreted by male germ cells regulates the proliferation and function of human Sertoli cells from obstructive azoospermia and nonobstructive azoospermia patients.

This study was designed to explore the regulatory effects of male germ cell secreting factor NODAL on Sertoli cell fate decisions from obstructive azoospermia (OA) and nonobstructive azoospermia (NOA) patients. Human Sertoli cells and male germ cells were isolated using two-step enzymatic digestion and SATPUT from testes of azoospermia patients. Expression of NODAL and its multiple receptors in human Sertoli cells and male germ cells were characterized by reverse transcription-polymerase chain reaction (RT-PCR) and immunochemistry. Human recombinant NODAL and its receptor inhibitor SB431542 were employed to probe their effect on the proliferation of Sertoli cells using the CCK-8 assay. Quantitative PCR and Western blots were utilized to assess the expression of Sertoli cell functional genes and proteins. NODAL was found to be expressed in male germ cells but not in Sertoli cells, whereas its receptors ALK4, ALK7, and ACTR-IIB were detected in Sertoli cells and germ cells, suggesting that NODAL plays a regulatory role in Sertoli cells and germ cells via a paracrine and autocrine pathway, respectively. Human recombinant NODAL could promote the proliferation of human Sertoli cells. The expression of cell cycle regulators, including CYCLIN A, CYCLIN D1 and CYCLIN E, was not remarkably affected by NODAL signaling. NODAL enhanced the expression of essential growth factors, including GDNF, SCF, and BMP4, whereas SB431542 decreased their levels. There was not homogeneity of genes changes by NODAL treatment in Sertoli cells from OA and Sertoli cell-only syndrome (SCO) patients. Collectively, this study demonstrates that NODAL produced by human male germ cells regulates proliferation and numerous gene expression of Sertoli cells.

[Isolation, culture, and identification of human spermatogonial stem cells].

OBJECTIVE: To isolate, identify and culture human spermatogonial stem cells (SSC) and then obtain purified and enriched human SSCs for research and application. METHODS: We detected the expression of CD90 in the human testis using the immunofluorescence technique and isolated human testicular spermatogenic cells by two-step enzymatic digestion, followed by differential plating and magnetic-activated cell sorting (MACS) with CD90 as an SSC marker. Then we identified the isolated CD90-positive spermatogenic cells by RT-PCR and immunocytochemistry, and meanwhile cocultured them with Sertoli cells in SG medium in vitro. RESULTS: The isolated CD90-positive cells showed a relatively homogeneous characteristic in size and morphology and expressed the genes specific for human SSCs, with high expressions (90.5%) of GFRA1, GPR125, and UCHL1. After coculture with Sertoli cells in the SG medium for 2 weeks, the isolated CD90-positive cells maintained a good activity. CONCLUSION: CD90 can be regarded as a speci- fic marker for human SSCs and used to obtain highly enriched human SSCs by differential plating and MACS. Furthermore, the isolated human SSCs can be cultured in SG medium in vitro.

The role of Dby mRNA in early development of male mouse zygotes.

Ejaculated mammalian spermatozoa contain a complex yet specific population of mRNA. However, the possible roles that mRNA has in early zygotic and embryonic development remain unclear. We found that Dby mRNA is selectively retained in capacitated mouse spermatozoa, and is transferred into the oocyte during fertilization by reverse transcription-polymerase chain reaction even though no DBY protein expression is detected. The cellular location of Dby mRNA is seen in the post-acrosome region, and it comprises nearly half of the mouse spermatozoa in in situ hybridization. In contrast, transcripts of the control gene, Smcy, are not detected in capacitated mouse spermatozoa, although the H-Y antigen encoded by Smcy is expressed on the surface of the spermatozoa. In our microinjection experiment, the zygotic development rate of the as-Dby male pronucleus injection group was significantly lower than that of the as-Smcy male pronucleus injection group (35.9% vs. 95%, P = 0.001) and the as-Dby female pronucleus injection group (35.9% vs. 93.8%, P = 0.001). The rate of male-developed zygotes was also lower than that of the as-Smcy male pronucleus injection group (17.4% vs. 57.9%, P = 0.002) and the as-Dby female pronucleus injection group (17.4% vs. 54.1%, P = 0.002). Thus, we conclude that Dby mRNA is selectively retained in capacitated mouse spermatozoa, and it has an important role in the early zygotic development of male mouse zygotes. This might imply that spermatozoa mRNA is involved in early zygotic and embryonic stages of reproduction.