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 ofDby 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 capaci- tated 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.

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

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 scRNAseq 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)/β-catenin signaling.Western blot revealed that the expression ofβ-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/β-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 Group1(64.0±26.6min)was significantly shorter than that in Group2(80.2±31.3min),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[0R]:0.57;95%confidence interval[Cl]:0.38-0.87;P=0.009)and receiver operating characteristic(ROC)analysis(area under the ROC curve[AUC]=O.628)showed that preoperative anti-Mullerian 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.

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-PCRI 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.

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.

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.

A de novo frameshift mutation of the SRY gene leading to a patient with 46,XY complete gonadal dysgenesis

Dear Editor,Sex in mammals is genetically determined and defined at the cellular level by the sex chromosome constitution(XY males and XX females)and at the phenotypic level by the development of genderspecific anatomy,physiology,and behavior.146,XY complete gonadal dysgenesis(46,XY CGD),first reported by Swyer in 1955,^2 is a rare congenital condition with completely or partially disordered gonadal development,leading to discordance between the genetic,gonadal,and phenotypic sex.Affected patients have a 46,XY karyotype,a female phenotype,normal female external genitalia,and CGD("streak gonads")without sperm production and follicular or steroid function.

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.

Sperm DNA Fragmentation may Influence IUI Outcome but could be Treated by ICSI: Evidence from Human Sperm Bank

Objective To determine the correlation between semen parameters, sperm DNA damage, progressive motility （PR）, morphology and intrauterine insemination （IUI）/ intracyto- plasmic sperm injection （ICSI） outcomes. Methods All the donors providing the samples in this study were recruited by Shang- hai Human Sperm Bank. For IUI, 122 donors were divided into group A （n=60） and group B （n=62）. Group A had a higher pregnancy rate while group B had a lower pregnancy rate （3.86 ± 1.50% vs 0.18 ± 0.52%）. For ICSI, 45 donors were divided into group C with a higher pregnancy rate （77. 78 ± 17.21%, n=23）, group D with a lower pregnancy rate （40. 73 ± 19.19%, n=22） and group E with an average preg- nancy rate in the sperm bank （48.96 ± 12.08%, n=23）. Semen analysis, morphology and DNA damage were assessed on samples retained in the sperm bank. Fresh semen samples were also collected and corresponding semen analyses data was included along with the pregnancy rates. Results No significant difference was found in the population characteristics between groups A and B, while there was a significant difference in sperm DNA fragmetation index （DFI） and morphology between the two groups （P〈0.05）, but not in other semen parameters （P〉0.05）. There was no significant difference in population characteristics between groups C, D and E while the DFI of group D was significant higher than groups C and E （P〈0.01）, but there was no significant difference in other semen parameters （P〉0.05）.Conclusion DFI might be a good predictor for IUI outcomes. Infertile couples with a high DFI should choose ICSI treatment instead of IUI. DFI should be a routine screening marker used to screen for sperm donors.

A nonobstructive azoospermic patient with Trichomonas vaginalis infection in testes

Dear Editor, Trichomonas vaginalis （ T. vaginalis）, a flagellated protozoan parasite emerged as one of the most common nonviral sexually transmitted infections worldwide, often inhabits the vagina, urethra, prostate, and epididymis.X It has been estimated that there are more than 170 million new cases of T. vaginalis infections per year worldwide. However, current knowledge of T. vaginalis and trichomoniasis is based mainly on studies in female vaginal infections. The prevalence of trichomoniasis in males is far less well characterized than that in females, probably because the infection seems to be asymptomatic in most men and can be resolved after treatment with one dose of metronidazole.