Ascorbic Acid-Caused Quenching Effect of Protein Clusteroluminescence Probe: The Fast Fluorescent Detection of Ascorbic Acid in Vegetables.

It is interesting and meaningful to explore fluorescent probes for novel rapid detection methods. In this study, we discovered a natural fluorescence probe, bovine serum albumin (BSA), for the assay of ascorbic acid (AA). Due to clusterization-triggered emission (CTE), BSA has the character of clusteroluminescence. AA shows an obvious fluorescence quenching effect on BSA, and the quenching effect increases with increasing concentrations of AA. After optimization, a method for the rapid detection of AA is established by the AA-caused fluorescence quenching effect. The fluorescence quenching effect reaches saturation after 5 min of incubation time and the fluorescence is stable within more than one hour, suggesting a rapid and stable fluorescence response. Moreover, the proposed assay method shows good selectivity and a wide linear range. To further study the mechanisms of AA-caused fluorescence quenching effect, some thermodynamic parameters are calculated. The main intermolecular force between BSA and AA is electrostatic, presumably leading to the inhibiting CTE process of BSA. This method also shows acceptable reliability for the real vegetable sample assay. In summary, this work will not only provide an assay strategy for AA, but also open an avenue for the application expansion of CTE effect of natural biomacromolecules.

Novel mutations of PMFBP1 in a man with acephalic spermatozoa defects.

BACKGROUND: Acephalic spermatozoa (AS) is a serious but rare reproductive genetic disorder that causes infertility in men. To date, only a few genes associated with AS defects have been identified, including the polyamine modulated factor 1 binding protein 1 (PMFBP1) gene. Consistent with this, PMFBP1 localizes to the head-neck connection, which bridges the implantation fossa and basal body. METHODS: A male patient was diagnosed as having an AS defect. Blood samples from all family members and a sample of the patient's semen were collected to determine the genetic causes of his infertility. RESULTS: Compound heterozygote mutation in the PMFBP1 gene, which is associated with AS defects in the present case: two loss-of-function mutations, with one a nonsense mutation c.361C > T p.Gln121Ter, and another a splice donor mutation c.414 + 1G > T. The current study, together with previous studies, suggests that the nonsense mutation is responsible for a truncated PMFBP1 protein during its formation; a splice donor mutation c.414 + 1G > T might lead to new open reading frames, from which the dysfunction of an abnormal PMFBP1 protein might be predicted. Additionally, the expression of outer dense fiber 1 (ODF1) and ODF2 proteins has been experimentally shown to be regulated by the truncated PMFBP1 protein. CONCLUSION: We herein present a case with AS defects associated with heterozygote mutations of PMFBP1, which have been shown to be rare and pathogenic; the association with an AS defect is a monogenic disorder with a recessive inherited pattern in the patient's family.

H3K36me2 methyltransferase NSD2 orchestrates epigenetic reprogramming during spermatogenesis.

Spermatogenesis is precisely controlled by sophisticated gene expression programs and is driven by epigenetic reprogramming, including histone modification alterations and histone-to-protamine transition. Nuclear receptor binding SET domain protein 2 (Nsd2) is the predominant histone methyltransferase catalyzing H3K36me2 and its role in male germ cell development remains elusive. Here, we report that NSD2 protein is abundant in spermatogenic cells. Conditional loss of Nsd2 in postnatal germ cells impaired fertility owing to apoptosis of spermatocytes and aberrant spermiogenesis. Nsd2 deficiency results in dysregulation of thousands of genes and remarkable reduction of both H3K36me2 and H3K36me3 in spermatogenic cells, with H3K36me2 occupancy correlating positively with expression of germline genes. Nsd2 deficiency leads to H4K16ac elevation in spermatogenic cells, probably through interaction between NSD2 and PSMA8, which regulates acetylated histone degradation. We further reveal that Nsd2 deficiency impairs EP300-induced H4K5/8ac, recognized by BRDT to mediate the eviction of histones. Accordingly, histones are largely retained in Nsd2-deficient spermatozoa. In addition, Nsd2 deficiency enhances expression of protamine genes, leading to increased protamine proteins in Nsd2-deficient spermatozoa. Our findings thus reveal a previously unappreciated role of the Nsd2-dependent chromatin remodeling during spermatogenesis and provide clues to the molecular mechanisms in epigenetic abnormalities impacting male reproductive health.

Effects of varicocele and microsurgical varicocelectomy on the metabolites in semen.

The influence of varicocele and microsurgical varicocelectomy on semen quality remains unclear. Few studies have investigated the relationship between semen metabolism and the abnormalities in reproductive function caused by varicocele, however, there is no study on the changes of semen metabolism after microsurgical varicocelectomy. Here, we used the non-targeted and targeted metabolic analysis to investigate the different metabolites in seminal plasma within normal, varicocele, and varicocelectomy groups. We clearly showed that varicocele significantly affects semen metabolism, and microsurgical varicocelectomy can reverse this metabolic abnormality. Moreover, we characterized the landscape of three dipeptides in the seminal plasma of patients with varicocele that have not been identified previously in human tissues or biofluids. Interestingly, the levels of these three dipeptides decreased after microsurgical varicocelectomy coincident with an improvement in semen quality. Western blotting confirmed the downregulation of DPEP3 (dipeptidase 3) in the varicocele group and the upregulation of DPEP3 in the varicocelectomy group. Furthermore, we found that eight metabolites may be helpful to distinguish varicocele patients from normal subjects. Our results may be applied to earlier diagnosis or to predict the outcome of microsurgery for varicocele.

Studying the mechanism of sperm DNA damage caused by folate deficiency.

Sperm DNA injury is one of the common causes of male infertility. Folic acid deficiency would increase the methylation level of the important genes, including those involved in DNA double-strand break (DSB) repair pathway. In the early stages, we analysed the correlation between seminal plasma folic acid concentration and semen parameters in 157 infertility patients and 91 sperm donor volunteers, and found that there was a significant negative correlation between seminal folic acid concentration and sperm DNA Fragmentation Index (DFI; r = -0.495, p < 0.01). Then through reduced representation bisulphite sequencing, global DNA methylation of sperm of patients in the low folic acid group and the high folic acid group was analysed, it was found that the methylation level in Rad54 promoter region increased in the folic acid deficiency group compared with the normal folic acid group. Meanwhile, the results of animal model and spermatocyte line (GC-2) also found that folic acid deficiency can increase the methylation level in Rad54 promoter region, increased sperm DFI in mice, increased the expression of γ-H2AX, that is, DNA injury marker protein, and increased sensitivity of GC-2 to external damage and stimulation. The study indicates that the expression of Rad54 is downregulated by folic acid deficiency via DNA methylation. This may be one of the mechanisms of sperm DNA damage caused by folate deficiency.

Tex13a Optimizes Sperm Motility via Its Potential Roles in mRNA Turnover.

mRNAs have been found to undergo substantial selective degradation during the late stages of spermiogenesis. However, the mechanisms regulating this biological process are unknown. In this report, we have identified Tex13a, a spermatid-specific gene that interacts with the CCR4-NOT complex and is implicated in the targeted degradation of mRNAs encoding particular structural components of sperm. Deletion of Tex13a led to a delayed decay of these mRNAs, lowered the levels of house-keeping genes, and ultimately lowered several key parameters associated with the control of sperm motility, such as the path velocity (VAP, average path velocity), track speed (VCL, velocity curvilinear), and rapid progression.

Bioprinting of a Blue Light-Cross-Linked Biodegradable Hydrogel Encapsulating Amniotic Mesenchymal Stem Cells for Intrauterine Adhesion Prevention.

Intrauterine adhesion (IUA) is a common and prevailing complication after uterine surgery, which can lead to clinical symptoms such as a low menstrual volume, amenorrhea, periodic lower abdominal pain, infertility, and so on. Placing a three-dimensional printing hydrogel between the injured site and the adjacent tissue is considered to be a physical barrier to prevent adhesion, which can isolate the damaged area during the healing process. In this work, a tissue hydrogel with various proportions of a methacrylated gelatin (GelMA) and methacrylated collagen (ColMA) composite hydrogel loaded with amniotic mesenchymal stem cells (AMSCs) was constructed by using three-dimensional biological printing technology. Compared with the single GelMA hydrogel, the composite antiadhesion hydrogel (GelMA/ColMA) showed an appropriate swelling ratio, enhanced mechanical properties, and impressive stability. Meanwhile, the microstructure of the GelMA/ColMA composite hydrogel showed a denser and interconnected microporous structure. In addition, the cytotoxicity study indicated that the GelMA/ColMA hydrogel has a cytocompatibility nature toward AMSCs. Finally, the fabrication of stem cell encapsulation hydrogels was studied, and the cells could be released continuously for more than 7 days with the normal cell function. The results of in vivo experiments indicated that the GelMA/ColMA/hAMSC (human amnion mesenchymal stem cell) hydrogel can prevent cavity adhesion in a rat IUA model. Therefore, bioprinting a biodegradable hydrogel cross-linked by blue light has satisfactory anticavity adhesion effects with excellent physical properties and biocompatibility, which could be used as a preventive barrier for intrauterine adhesion.

OTOGL, a gelforming mucin protein, is nonessential for male germ cell development and spermatogenesis in mice.

Otogelin-like protein (encoded by Otogl) was highly structural similar to the gelforming mucin proteins. Although human OTOG mutations have been linked to deafness, the biological function of OTOGL in male germ cell development remains enigmatic. In screening 336 patients with non-obstructive azoospermia (NOA), OTOGL displays the high mutant ratio (13.99 %). Then, we examined the expression of OTOGL in developing mouse testes. Otogl mRNA and protein are continually expressed in postnatal developing testes from postnatal day 0 (P0) testes to P21 testes exhibiting a decreased trend with the age growth. We thus generated a global Otogl knockout mouse (KO) model using the CRISPR/Cas9 technology; however, Otogl KO mice displayed normal development and fertility. Further histological analysis of Otogl knockout mouse testes revealed that all types of spermatogenic cells are present in Otogl KO seminiferous tubules. Together, our study suggested that OTOGL is nonessential for male germ cell development and spermatogenesis.

Triptonide is a reversible non-hormonal male contraceptive agent in mice and non-human primates.

There are no non-hormonal male contraceptives currently on the market despite decades of efforts toward the development of "male pills". Here, we report that triptonide, a natural compound purified from the Chinese herb Tripterygium Wilfordii Hook F displays reversible male contraceptive effects in both mice and monkeys. Single daily oral doses of triptonide induces deformed sperm with minimal or no forward motility (close to 100% penetrance) and consequently male infertility in 3-4 and 5-6 weeks in mice and cynomolgus monkeys, respectively. Male fertility is regained in ~4-6 weeks after cessation of triptonide intake in both species. Either short- or long-term triptonide treatment causes no discernable systematic toxic side effects based on histological examination of vital organs in mice and hematological and serum biochemical analyses in monkeys. Triptonide appears to target junction plakoglobin and disrupts its interactions with SPEM1 during spermiogenesis. Our data further prove that targeting late spermiogenesis represents an effective strategy for developing non-hormonal male contraceptives.

Reproductive outcomes of intracytoplasmic sperm injection using testicular sperm and ejaculated sperm in patients with AZFc microdeletions: a systematic review and meta-analysis.

Studies have explored the assisted reproductive technology (ART) outcomes of Y-chromosome azoospermia factor c (AZFc) microdeletions, but the effect of sperm source on intracytoplasmic sperm injection (ICSI) remains unknown. To determine the ART results of ICSI using testicular sperm and ejaculated sperm from males with AZFc microdeletions, we searched Embase, Web of Science, and PubMed to conduct a systematic review and meta-analysis. The first meta-analysis results for 106 cycles in five studies showed no significant differences in the live birth rate between the testicular sperm group and the ejaculated sperm group (risk ratio: 0.97, 95% confidence interval [CI]: 0.73-1.28, P = 0.82). The second meta-analysis of 106 cycles in five studies showed no difference in the abortion rate between the testicular sperm group and ejaculated sperm group (risk ratio: 1.06, 95% CI: 0.54-2.06, P = 0.87). The third meta-analysis of 386 cycles in seven studies showed no significant difference in clinical pregnancy rates between the testicular sperm group and the ejaculated sperm group (risk ratio: 1.24, 95% CI: 0.66-2.34, P = 0.50). Inevitable heterogeneity weakened our results. However, our results indicated that testicular sperm and ejaculated sperm yield similar ART outcomes, representing a meaningful result for clinical treatment. More properly designed studies are needed to further confirm our conclusions.

Epigenetic Regulation of Spermatogonial Stem Cell Homeostasis: From DNA Methylation to Histone Modification.

Spermatogonial stem cells(SSCs)are the ultimate germline stem cells with the potential of self-renewal and differentiation, and a dynamic balance of SSCs play an essential role in spermatogenesis. During the gene expression process, genomic DNA and nuclear protein, working together, contribute to SSC homeostasis. Recently, emerging studies have shown that epigenome-related molecules such as chromatin modifiers play an important role in SSC homeostasis through regulating target gene expression. Here, we focus on two types of epigenetic events, including DNA methylation and histone modification, and summarize their function in SSC homeostasis. Understanding the molecular mechanism during SSC homeostasis will promote the recognition of epigenetic biomarkers in male infertility, and bring light into therapies of infertile patients.Graphical Abstract.

High Throughput scRNA-Seq Provides Insights Into Leydig Cell Senescence Induced by Experimental Autoimmune Orchitis: A Prominent Role of Interstitial Fibrosis and Complement Activation.

Leydig cells (Lc), located in the interstitial space of the testis between seminiferous tubules, produce 95% of testosterone in male individuals, which is pivotal for male sexual differentiation, spermatogenesis, and maintenance of the male secondary sex characteristics. Lc are prone to senescence in aging testes, resulting in compromised androgen synthesis capability upon aging. However, little is known about whether Lc undergo senescence in a chronic inflammatory environment. To investigate this question, mouse models of experimental autoimmune orchitis (EAO) were used, and Lc were analyzed by high throughput scRNA-Seq. Data were screened and analyzed by correlating signaling pathways with senescence, apoptosis, androgen synthesis, and cytokine/chemokine signaling pathways. EAO did induce Lc senescence, and Lc senescence in turn antagonized androgen synthesis. Based on the correlation screening of pathways inducing Lc senescence, a plethora of pathways were found to play potential roles in triggering Lc senescence during EAO, among which the Arf6 and angiopoietin receptor pathways were highly correlated with senescence signature. Notably, complement and interstitial fibrosis activated by EAO worsened Lc senescence and strongly antagonized androgen synthesis. Furthermore, most proinflammatory cytokines enhanced both senescence and apoptosis in Lc and spermatogonia (Sg) during EAO, and proinflammatory cytokine antagonism of the glutathione metabolism pathway may be key in inducing cellular senescence during EAO.

Dynamic Profiles and Transcriptional Preferences of Histone Modifications During Spermiogenesis.

During spermiogenesis, extensive histone modifications take place in developing haploid spermatids besides morphological alterations of the genetic material to form compact nuclei. Better understanding on the overall transcriptional dynamics and preferences of histones and enzymes involved in histone modifications may provide valuable information to dissect the epigenetic characteristics and unique chromatin status during spermiogenesis. Using single-cell RNA-Sequencing, the expression dynamics of histone variants, writers, erasers, and readers of histone acetylation and methylation, as well as histone phosphorylation, ubiquitination, and chaperones were assessed through transcriptome profiling during spermiogenesis. This approach provided an unprecedented panoramic perspective of the involving genes in epigenetic modifier/histone variant expression during spermiogenesis. Results reported here revealed the transcriptional ranks of histones, histone modifications, and their readers during spermiogenesis, emphasizing the unique preferences of epigenetic regulation in spermatids. These findings also highlighted the impact of spermatid metabolic preferences on epigenetic modifications. Despite the observed rising trend on transcription levels of all encoding genes and histone variants, the transcriptome profile of genes in histone modifications and their readers displayed a downward expression trend, suggesting that spermatid nuclei condensation is a progressive process that occurred in tandem with a gradual decrease in overall epigenetic activity during spermiogenesis.

Inhibitory effect of Lycium barbarum polysaccharide on sperm damage during cryopreservation.

With the development of cryopreservation technology, marked progress has been made regarding sperm cryopreservation. However, as conventional cryopreservation agents are not effective at freezing weak sperm, improved cryopreservation agents are in demand. In the present study, the addition of Lycium barbarum polysaccharides to glycerol-egg-yolk-citrate sperm cryopreservation agent was determined to improve sperm forward speed, reduce the DNA fragmentation index and improve the mitochondrial membrane potential. Furthermore, during the freezing and thawing of sperm, the improved cryopreservative increased the content of Bcl-2 while reducing the content of Bax, cytochrome C and caspase-3. These results indicated that polysaccharides added as a protective agent preserved the normal function of sperm mitochondria. Transmission electron microscopy also confirmed the protective effect of the polysaccharides on the structure of mitochondria. It was also indicated that improved cryopreservative lowered the levels of reactive oxygen species (ROS) during the freeze-thawing process. Therefore, it is hypothesized that improved cryopreservative agents may be beneficial for maintaining the structure and function of the mitochondria of weak sperm when cryopreserved, which may be facilitated via reducing the production of ROS in the freezing-thawing process, thus avoiding activation of the apoptotic pathway in sperm mitochondria and protecting mitochondrial structure and sperm function.

Risk and mechanism of glucose metabolism disorder in the offspring conceived by female fertility maintenance technology.

Although female fertility maintenance technology (FFMT) provides an effective option for preserving fertility in patients with cancer suffering from fertility loss due to cancer treatment, previous studies have shown that the technique has certain potential risks and requires an assessment of the health status of the offspring since FFMT may lead to glucose metabolism disorder in offspring mice. The present animal study examined the glucose metabolism of adult mice offspring born from ovarian tissue cryopreservation and orthotopic allotransplantation. The mice were divided into three groups: normal, fresh ovary transplantation, and cryopreserved ovary transplantation. We recorded fasting blood glucose, glucose tolerance, and fasting serum insulin level for six months. Liver DNA, RNA, and proteins were extracted to detect the interaction between DNA methylation and Grb10 expression and insulin signaling pathway factors such as P-IGF1R, P-IRS2, P-AKT, and Grb10. Female recipient mice that received FFMT could successfully give birth after mating. The average litter size and total litter size of the cryopreserved and fresh groups showed marked differences compared with the normal group. Compared with the normal group, the fasting blood glucose and fasting serum insulin levels were higher in the cryopreserved and fresh groups. The mRNA and protein expressions of Grb10 were higher in the fresh and cryopreserved groups. Compared with the normal group, the DNA methylation status of four of the 11 sites of the Grb10 promoter was lower in the cryopreserved group. Grb10 overexpression inhibited the downstream phosphorylation protein factor expression (p-IGF-1R, p-IRS2, and p-Akt) of the IGF-1R signaling pathway. Female fertility maintenance technology (FFMT), including ovarian tissue cryopreservation (OTC), and orthotopic allotransplantation techniques might lead to glucose metabolism disorders in offspring mice.

Beyond Acephalic Spermatozoa: The Complexity of Intracytoplasmic Sperm Injection Outcomes.

This review analyses the genetic mechanisms of acephalic spermatozoa (AS) defects, which are associated with primary infertility in men. Several target genes of headless sperms have been identified but intracytoplasmic sperm injection (ICSI) outcomes are complex. Based on electron microscopic observations, broken points of the sperm neck are AS defects that are based on various genes that can be classified into three subtypes: HOOK1, SUN5, and PMFBP1 genes of subtype II; TSGA10 and BRDT genes of subgroup III, while the genetic mechanism(s) and aetiology of AS defects of subtype I have not been described and remain to be explored. Interestingly, all AS sperm of subtype II achieved better ICSI outcomes than other subtypes, resulting in clinical pregnancies and live births. For subtype III, the failure of clinical pregnancy can be explained by the defects of paternal centrioles that arrest embryonic development; for subtype I, this was due to a lack of a distal centriole. Consequently, the embryo quality and potential ICSI results of AS defects can be predicted by the subtypes of AS defects. However, this conclusion with regard to ICSI outcomes based on subtypes still needs further research, while the existence of quality of oocyte and implantation failure in women cannot be ignored.

Histone H3 methylation orchestrates transcriptional program in mouse spermatogenic cell line.

Changes in histone modifications always correlate with altered transcriptional activities of genes. Recent studies have shown that the mutation of certain lysine residues to methionine in the histone variant H3.3 can act as a valuable tool to reduce specific H3 methylation levels. In our study, we used the mouse spermatogenic cell line GC-2 as a model to generate cells stably expressing H3.3 K4, H3.3 K9, H3.3 K27, and H3.3 K36M. The expression of these H3.3 K-to-M mutants influenced the expression of different subsets of genes, and a total of 891 differentially expressed genes were identified through global gene expression profiling. Moreover, the H3.3 K-to-M transgenes, especially H3.3 K36M, impacted the expression of endogenous retrovirus ERVK. This study gives a global view of how different H3 modifications regulate transcriptomes in spermatogenic cell lines, and identifies potential targets of H3 modifications in male germ line.

m6A-dependent biogenesis of circular RNAs in male germ cells.

The majority of circular RNAs (circRNAs) spliced from coding genes contain open reading frames (ORFs) and thus, have protein coding potential. However, it remains unknown what regulates the biogenesis of these ORF-containing circRNAs, whether they are actually translated into proteins and what functions they play in specific physiological contexts. Here, we report that a large number of circRNAs are synthesized with increasing abundance when late pachytene spermatocytes develop into round and then elongating spermatids during murine spermatogenesis. For a subset of circRNAs, the back splicing appears to occur mostly at m6A-enriched sites, which are usually located around the start and stop codons in linear mRNAs. Consequently, approximately a half of these male germ cell circRNAs contain large ORFs with m6A-modified start codons in their junctions, features that have been recently shown to be associated with protein-coding potential. Hundreds of peptides encoded by the junction sequences of these circRNAs were detected using liquid chromatography coupled with mass spectrometry, suggesting that these circRNAs can indeed be translated into proteins in both developing (spermatocytes and spermatids) and mature (spermatozoa) male germ cells. The present study discovered not only a novel role of m6A in the biogenesis of coding circRNAs, but also a potential mechanism to ensure stable and long-lasting protein production in the absence of linear mRNAs, i.e., through production of circRNAs containing large ORFs and m6A-modified start codons in junction sequences.

Ran-binding protein 3 is associated with human spermatogenesis and male infertility.

Ran-binding protein 3 (RanBP3) is a Ran-interacting protein, which participates in the Ran GTPase system in cancer cell biology. However, the expression pattern and physiological role of RanBP3 remain largely unknown. In this study, we found that RanBP3 was expressed in human testes and localised to spermatogonium and spermatocyte of germ cells. In subcellular structure, its localisation is in the nucleus and cytoplasm. Interestingly, compared with normal groups, RanBP3 expression was lower in groups of patients with Maturation Arrest (MA) and Sertoli cell-only syndrome (SCO) when considered by the Johnson Score. RanBP3 expression in the MA group and SCO groups was dramatically lower than that in the normal control group. Studies have shown that RanBP3, which is one of the helper factors of Ran, is mainly participate in the nucleocytoplasmic transport of cells. RanBP3 helps Ran to achieve some functions such as nucleocytoplasmic transport, spindle assembly during mitosis and nuclear assembly after mitosis. Consequent changes in the expression of RanBP3 may associate with human spermatogenesis disorders and male infertility. The identification and characterisation of RanBP3 enhances our understanding of the molecular mechanisms underpinning its function in human spermatogenesis and male infertility.