Detection of Cadmium(II) in Aquatic Products Using a Rolling-Circle Amplification-Coupled Ratio Fluorescent Probe Based on an Aptamer-Peptide Conjugate.

The existing aptamers for cadmium (Cd2+), the common toxic heavy metal contaminant in food, cannot meet the requirements for detecting Cd2+ in rapid detection methods. In previous work, we found that coupling aptamer-peptide conjugates (APCs) with peptides and aptamers can provide a less disruptive method with a significantly improved affinity. Moreover, we found that the spatial conformation of aptamers and peptides is crucial for obtaining proper affinity in APC. Therefore, we describe a simple design strategy to obtain a series of APCs with different affinities by designing peptide orientations (N-terminal, C-terminal). The best affinity was found for APC(C1-N) with a binding constant (Ka) of 2.23 × 106 M-1, indicating that the APC(C1-N) affinity was significantly increased by 829.17% over aptamer. Finally, a rolling-circle amplification (RCA)-coupled ratio fluorescence-based biosensor for Cd2+ detection was established with a detection limit of 0.0036 nM, which has great potential for practical aquatic product detection.

Germ cell-specific deletion of Pex3 reveals essential roles of PEX3-dependent peroxisomes in spermiogenesis.

Peroxisomes are organelles enclosed by a single membrane and are present in various species. The abruption of peroxisomes is correlated with peroxisome biogenesis disorders and single peroxisomal enzyme deficiencies that induce diverse diseases in different organs. However, little is known about the protein compositions and corresponding roles of heterogeneous peroxisomes in various organs. Through transcriptomic and proteomic analyses, we observed heterogenous peroxisomal components among different organs, as well as between testicular somatic cells and different developmental stages of germ cells. As Pex3 is expressed in both germ cells and Sertoli cells, we generated Pex3 germ cell- and Sertoli cell-specific knockout mice. While Pex3 deletion in Sertoli cells did not affect spermatogenesis, the deletion in germ cells resulted in male sterility, manifested as the destruction of intercellular bridges between spermatids and the formation of multinucleated giant cells. Proteomic analysis of the Pex3-deleted spermatids revealed defective expressions of peroxisomal proteins and spermiogenesis-related proteins. These findings provide new insights that PEX3-dependent peroxisomes are essential for germ cells undergoing spermiogenesis, but not for Sertoli cells.

Biallelic mutations in RNA-binding protein ADAD2 cause spermiogenic failure and non-obstructive azoospermia in humans.

STUDY QUESTION: What are some pathogenic mutations for non-obstructive azoospermia (NOA) and their effects on spermatogenesis? SUMMARY ANSWER: Biallelic missense and frameshift mutations in ADAD2 disrupt the differentiation of round spermatids to spermatozoa causing azoospermia in humans and mice. WHAT IS KNOWN ALREADY: NOA is the most severe cause of male infertility characterized by an absence of sperm in the ejaculate due to impairment of spermatogenesis. In mice, the lack of the RNA-binding protein ADAD2 leads to a complete absence of sperm in epididymides due to failure of spemiogenesis, but the spermatogenic effects of ADAD2 mutations in human NOA-associated infertility require functional verification. STUDY DESIGN SIZE DURATION: Six infertile male patients from three unrelated families were diagnosed with NOA at local hospitals in Pakistan based on infertility history, sex hormone levels, two semen analyses and scrotal ultrasound. Testicular biopsies were performed in two of the six patients. Adad2 mutant mice (Adad2Mut/Mut) carrying mutations similar to those found in NOA patients were generated using the CRISPR/Cas9 genome editing tool. Reproductive phenotypes of Adad2Mut/Mut mice were verified at 2 months of age. Round spermatids from the littermates of wild-type (WT) and Adad2Mut/Mut mice were randomly selected and injected into stimulated WT oocytes. This round spermatid injection (ROSI) procedure was conducted with three biological replicates and >400 ROSI-derived zygotes were evaluated. The fertility of the ROSI-derived progeny was evaluated for three months in four Adad2WT/Mut male mice and six Adad2WT/Mut female mice. A total of 120 Adad2Mut/Mut, Adad2WT/Mut, and WT mice were used in this study. The entire study was conducted over 3 years. PARTICIPANTS/MATERIALS SETTING METHODS: Whole-exome sequencing was performed to detect potentially pathogenic mutations in the six NOA-affected patients. The pathogenicity of the identified ADAD2 mutations was assessed and validated in human testicular tissues and in mouse models recapitulating the mutations in the NOA patients using quantitative PCR, western blotting, hematoxylin-eosin staining, Periodic acid-Schiff staining, and immunofluorescence. Round spermatids of WT and Adad2Mut/Mut mice were collected by fluorescence-activated cell sorting and injected into stimulated WT oocytes. The development of ROSI-derived offspring was evaluated in the embryonic and postnatal stages. MAIN RESULTS AND THE ROLE OF CHANCE: Three recessive mutations were identified in ADAD2 (MT1: c.G829T, p.G277C; MT2: c.G1192A, p.D398N; MT3: c.917_918del, p.Q306Rfs*43) in patients from three unrelated Pakistani families. MT1 and MT2 dramatically reduced the testicular expression of ADAD2, likely causing spermiogenesis failure in the NOA patients. Immunofluorescence analysis of the Adad2Mut/Mut male mice with the corresponding MT3 mutation showed instability and premature degradation of the ADAD2 protein, resulting in the spermiogenesis deficiency phenotype. Through ROSI, the Adad2Mut/Mut mice could produce pups with comparable embryonic development (46.7% in Adad2Mut/Mut versus 50% in WT) and birth rates (21.45 ± 10.43% in Adad2Mut/Mut versus 27.5 ± 3.536% in WT, P = 0.5044) to WT mice. The Adad2WT/Mut progeny from ROSI (17 pups in total via three ROSI replicates) did not show overt developmental defects and had normal fertility. LARGE SCALE DATA: N/A. LIMITATIONS REASONS FOR CAUTION: This is a preliminary report suggesting that ROSI can be an effective treatment for infertile Adad2Mut/Mut mice. Further assisted reproductive attempts need to be carefully examined in humans during clinical trials. WIDER IMPLICATIONS OF THE FINDINGS: Our work provides functional evidence that mutations in the ADAD2 gene are deleterious and cause consistent spermiogenic defects in both humans and mice. In addition, preliminary results show that ROSI can help Adad2Mut/Mut to produce biological progeny. These findings provide valuable clues for genetic counselling on the ADAD2 mutants-associated infertility in human males. STUDY FUNDING/COMPETING INTERESTS: This work was supported by the National Natural Science Foundation of China (32000587, U21A20204, and 32061143006), and the National Key Research and Developmental Program of China (2019YFA0802600 and 2021YFC2700202). This work was also supported by Institute of Health and Medicine, Hefei Comprehensive National Science Center, Hefei, China. The authors declare no competing interests.

A prime editor efficiently repaired human induced pluripotent stem cells with AR gene mutation (c.2710G > A; p. V904M).

Prime Editor (PE) is a precise genome manipulation technology based on the CRISPR-Cas9 system, while its application in human induced pluripotent stem cells (iPSCs) remains limited. Here, we established a repaired hiPS cell line (SKLRMi001-A-1) from hiPSCs with androgen receptor (AR) mutation (c.2710G > A; p.V904M). The repaired iPSC line expressed pluripotency markers, retained normal karyotype, showed the capability of differentiating into three germ layers and was absence of mycoplasma infection. The repaired iPSC line will help to elucidate the mechanism of androgen insensitivity syndrome (AIS) and benefit treatment for AIS in the future.

A novel protein encoded by circRsrc1 regulates mitochondrial ribosome assembly and translation during spermatogenesis.

BACKGROUND: Circular RNAs (circRNAs) are a large class of mammalian RNAs. Several protein products translated by circRNAs have been reported to be involved in the development of various tissues and systems; however, their physiological functions in male reproduction have yet not been explored. RESULTS: Here, we report an endogenous circRNA (circRsrc1) that encodes a novel 161-amino-acid protein which we named Rsrc1-161aa through circRNA sequencing coupled with mass spectrometry analysis on mouse testicular tissues. Deletion of Rsrc1-161aa in mice impaired male fertility with a significant decrease in sperm count and motility due to dysfunctions of mitochondrial energy metabolism. A series of in vitro rescue experiments revealed that circRsrc1 regulates mitochondrial functions via its encoded protein Rsrc1-161aa. Mechanistically, Rsrc1-161aa directly interacts with mitochondrial protein C1qbp and enhances its binding activity to mitochondrial mRNAs, thereby regulating the assembly of mitochondrial ribosomes and affecting the translation of oxidative phosphorylation (OXPHOS) proteins and mitochondrial energy metabolism. CONCLUSIONS: Our studies reveal that Rsrc1-161aa protein encoded by circRsrc1 regulates mitochondrial ribosome assembly and translation during spermatogenesis, thereby affecting male fertility.

Derivation of new pluripotent stem cells from human extended pluripotent stem cells with formative features and trophectoderm potential.

Previous studies have demonstrated the existence of intermediate stem cells, which have been successfully obtained from human naive pluripotent stem cells (PSCs) and peri-implantation embryos. However, it is not known whether human extended pluripotent stem cells (hEPSCs) can be directly induced into intermediate stem cells. Moreover, the ability of extra-embryonic lineage differentiation in intermediate stem cells has not been verified. In this issue, we transformed hEPSCs into a kind of novel intermediate pluripotent stem cell resembling embryonic days 8-9 (E8-E9) epiblasts and proved its feature of formative epiblasts. We engineered hEPSCs from primed hPSCs under N2B27-LCDM (N2B27 plus Lif, CHIR, DiH and MiH) conditions. Then, we added Activin A, FGF and XAV939 to modulate signalling pathways related to early humans' embryogenesis. We performed RNA-seq and CUT&Tag analysis to compare with AF9-hPSCs from different pluripotency stages of hPSCs. Trophectoderm (TE), primordial germ cells-like cells (PGCLC) and endoderm, mesoderm, and neural ectoderm induction were conducted by specific small molecules and proteins. AF9-hPSCs transcription resembled that of E8-E9 peri-implantation epiblasts. Signalling pathway responsiveness and histone methylation further revealed their formative pluripotency. Additionally, AF9-hPSCs responded directly to primordial germ cells (PGCs) specification and three germ layer differentiation signals in vitro. Moreover, AF9-hPSCs could differentiate into the TE lineage. Therefore, AF9-hPSCs represented an E8-E9 formative pluripotency state between naïve and primed pluripotency, opening new avenues for studying human pluripotency development during embryogenesis.

Multi-Omics Approaches for Revealing the Epigenetic Regulation of Histone H3.1 during Spermatogonial Stem Cell Differentiation In Vitro.

Epigenetic regulation, particularly post-translational modifications (PTMs) of histones, participates in spermatogonial stem cell (SSCs) differentiation. However, there is a lack of systemic studies of histone PTM regulation during the differentiation of SSCs due to its low number in vivo. Herein, we quantified dynamic changes of 46 different PTMs on histone H3.1 by targeted quantitative proteomics using mass spectrometry during SSCs differentiation in vitro, in combination with our RNA-seq data. We identified seven histone H3.1 modifications to be differentially regulated. In addition, we selected H3K9me2 and H3S10ph for subsequent biotinylated peptide pull-down experiments and identified 38 H3K9me2-binding proteins and 42 H3S10ph-binding proteins, which contain several transcription factors, such as GTF2E2 and SUPT5H, which appear to be crucial for epigenetic regulation of SSC differentiation.

Functional screening of congenital heart disease risk loci identifies 5 genes essential for heart development in zebrafish.

Congenital heart disease (CHD) is the most common birth defect worldwide and a main cause of perinatal and infant mortality. Our previous genome-wide association study identified 53 SNPs that associated with CHD in the Han Chinese population. Here, we performed functional screening of 27 orthologous genes in zebrafish using injection of antisense morpholino oligos. From this screen, 5 genes were identified as essential for heart development, including iqgap2, ptprt, ptpn22, tbck and maml3. Presumptive roles of the novel CHD-related genes include heart chamber formation (iqgap2 and ptprt) and atrioventricular canal formation (ptpn22 and tbck). While deficiency of maml3 led to defective cardiac trabeculation and consequent heart failure in zebrafish embryos. Furthermore, we found that maml3 mutants showed decreased cardiomyocyte proliferation which caused a reduction in cardiac trabeculae due to inhibition of Notch signaling. Together, our study identifies 5 novel CHD-related genes that are essential for heart development in zebrafish and first demonstrates that maml3 is required for Notch signaling in vivo.

A male germ-cell-specific ribosome controls male fertility.

Ribosomes are highly sophisticated translation machines that have been demonstrated to be heterogeneous in the regulation of protein synthesis1,2. Male germ cell development involves complex translational regulation during sperm formation3. However, it remains unclear whether translation during sperm formation is performed by a specific ribosome. Here we report a ribosome with a specialized nascent polypeptide exit tunnel, RibosomeST, that is assembled with the male germ-cell-specific protein RPL39L, the paralogue of core ribosome (RibosomeCore) protein RPL39. Deletion of RibosomeST in mice causes defective sperm formation, resulting in substantially reduced fertility. Our comparison of single-particle cryo-electron microscopy structures of ribosomes from mouse kidneys and testes indicates that RibosomeST features a ribosomal polypeptide exit tunnel of distinct size and charge states compared with RibosomeCore. RibosomeST predominantly cotranslationally regulates the folding of a subset of male germ-cell-specific proteins that are essential for the formation of sperm. Moreover, we found that specialized functions of RibosomeST were not replaceable by RibosomeCore. Taken together, identification of this sperm-specific ribosome should greatly expand our understanding of ribosome function and tissue-specific regulation of protein expression pattern in mammals.

Single-Cell Quantitative Proteomic Analysis of Human Oocyte Maturation Revealed High Heterogeneity in In Vitro-Matured Oocytes.

Oocyte maturation is pertinent to the success of in vitro maturation (IVM), which is used to overcome female infertility, and produced over 5000 live births worldwide. However, the quality of human IVM oocytes has not been investigated at single-cell proteome level. Here, we quantified 2094 proteins in human oocytes during in vitro and in vivo maturation (IVO) by single-cell proteomic analysis and identified 176 differential proteins between IVO and germinal vesicle oocytes and 45 between IVM and IVO oocytes including maternal effect proteins, with potential contribution to the clinically observed decreased fertilization, implantation, and birth rates using human IVM oocytes. IVM and IVO oocytes showed separate clusters in principal component analysis, with higher inter-cell variability among IVM oocytes, and have little correlation between mRNA and protein changes during maturation. The patients with the most aberrantly expressed proteins in IVM oocytes had the lowest level of estradiol per mature follicle on trigger day. Our data provide a rich resource to evaluate effect of IVM on oocyte quality and study mechanism of oocyte maturation.

Mebendazole-Induced Blood-Testis Barrier Injury in Mice Testes by Disrupting Microtubules in Addition to Triggering Programmed Cell Death.

Mebendazole (MBZ) is a synthetic benzimidazole known for its antiparasitic properties. In recent years, growing evidence showed that MBZ was also used as an anti-tumor agent. However, whether (and to what extent) this drug treatment affected the male reproductive system was not well-understood. In this study, male C57BL/6 mice were injected with 40 mg/kg/day of MBZ. The treatment was for 3 and 7 days. Our results showed that the injected mice exhibited an abnormal spermatogenic phase with a significant decrease in sperm. We further detected microtubule disruption and transient functional destruction of the blood-testes barrier (BTB) in the MBZ-injected mice testes (BTB). Our data confirmed that MBZ suppressed the expression of the BTB junction-associated proteins and disrupted the Sertoli cells' function in vivo. Moreover, MBZ-treated mice demonstrated an aberrant caspase-3 signalling pathway, which resulted in the apoptosis of the germ cells. Here, we present our data, indicating that MBZ impairs BTB by reducing the expression of the microtubules' and BTB junction-associated proteins. The last leads to activating the caspase-3 pathway, which triggers extensive germ cell apoptosis.

Potential clinical value of in vitro spermatogenesis†.

Infertility has become the third most common disease threatening human health, immediately after tumors and cardiovascular diseases. Male infertility is primarily caused by spermatogenesis disorders that may be classified as either genetic or non-genetic. For part of non-genetic disorders, in vitro spermatogenesis can be induced by adjusting the microenvironment of the testis culture. Establishing the in vitro spermatogenic induction system helps to clarify the critical molecular mechanisms in spermatogonia self-renewal, spermatocyte meiosis, and sperm formation during spermatogenesis. In this review, we summarize recent advances in the field of in vitro sperm cells induction. Therefore, we hope to provide ideas and solutions for the clinical treatment of male infertility.

T-complex protein 1 subunit zeta-2 (CCT6B) deficiency induces murine teratospermia.

BACKGROUND: The CCT complex is an important mediator of microtubule assembly and intracellular protein folding. Owing to its high expression in spermatids, CCT knockdown can disrupt spermatogenesis. In the present report, we therefore evaluated the in vivo functionality of the testis-specific CCT complex component CCT6B using a murine knockout model system. METHODS: A CRISPR/Cas9 approach was used to generate Cct6b-/- mice, after which candidate gene expression in these animals was evaluated via qPCR and Western blotting. Testicular and epididymal phenotypes were assessed through histological and immunofluorescent staining assays, while a computer-assisted sperm analyzer was employed to assess semen quality. RESULTS: Cct6b-/- mice were successfully generated, and exhibited no differences in development, fertility, appearance, testis weight, or sperm counts relative to control littermates. In addition, no differences in spermatogenesis were detected when comparingCct6b+/+ and Cct6b-/- testes. However, when progressive motility was analyzed, the ratio of normal sperm was significantly decreased in Cct6b-/- male mice, with nuclear base bending being the primary detected abnormality. In addition, slight decreases in Cct4 and Cct7 expression were detected. CONCLUSION: These data indicated that CCT6B is an important regulator of murine spermatogenesis, with the loss of this protein resulting in CCT complex dysfunction, providing a foundation for further studies.

Single-cell RNA-Seq reveals a highly coordinated transcriptional program in mouse germ cells during primordial follicle formation.

The assembly of primordial follicles in mammals represents one of the most critical processes in ovarian biology. It directly affects the number of oocytes available to a female throughout her reproductive life. Premature depletion of primordial follicles contributes to the ovarian pathology primary ovarian insufficiency (POI). To delineate the developmental trajectory and regulatory mechanisms of oocytes during the process, we performed RNA-seq on single germ cells from newborn (P0.5) ovaries. Three cell clusters were classified which corresponded to three cell states (germ cell cyst, cyst breakdown, and follicle) in the newborn ovary. By Monocle analysis, a uniform trajectory of oocyte development was built with a series of genes showed dynamic changes along the pseudo-timeline. Gene Ontology term enrichment revealed a significant decrease in meiosis-related genes and a dramatic increase in oocyte-specific genes which marked the transition from a germ cell to a functional oocyte. We then established a network of regulons by using single-cell regulatory network inference and clustering (SCENIC) algorithm and identified possible candidate transcription factors that may maintain transcription programs during follicle formation. Following functional studies further revealed the differential regulation of the identified regulon Id2 and its family member Id1, on the establishment of primordial follicle pool by using siRNA knockdown and genetic modified mouse models. In summary, our study systematically reconstructed molecular cascades in oocytes and identified a series of genes and molecular pathways in follicle formation and development.

Association of assisted reproductive technology, germline de novo mutations and congenital heart defects in a prospective birth cohort study.

Emerging evidence suggests that children conceived through assisted reproductive technology (ART) have a higher risk of congenital heart defects (CHDs) even when there is no family history. De novo mutation (DNM) is a well-known cause of sporadic congenital diseases; however, whether ART procedures increase the number of germline DNM (gDNM) has not yet been well studied. Here, we performed whole-genome sequencing of 1137 individuals from 160 families conceived through ART and 205 families conceived spontaneously. Children conceived via ART carried 4.59 more gDNMs than children conceived spontaneously, including 3.32 paternal and 1.26 maternal DNMs, after correcting for parental age at conception, cigarette smoking, alcohol drinking, and exercise behaviors. Paternal DNMs in offspring conceived via ART are characterized by C>T substitutions at CpG sites, which potentially affect protein-coding genes and are significantly associated with the increased risk of CHD. In addition, the accumulation of non-coding functional mutations was independently associated with CHD and 87.9% of the mutations were originated from the father. Among ART offspring, infertility of the father was associated with elevated paternal DNMs; usage of both recombinant and urinary follicle-stimulating hormone and high-dosage human chorionic gonadotropin trigger was associated with an increase of maternal DNMs. In sum, the increased gDNMs in offspring conceived by ART were primarily originated from fathers, indicating that ART itself may not be a major reason for the accumulation of gDNMs. Our findings emphasize the importance of evaluating the germline status of the fathers in families with the use of ART.

Systematic analysis of the ubiquitome in mouse testis.

A growing body of evidence now supports the fact that protein ubiquitination is an important modification during the regulation of spermatogenesis. However, little is known about the ubiquitome of the testis. In this study, we created a large-scale mouse testis ubiquitome profile using di-glycine remnant antibodies and mass spectrometry and identified a total of 14,219 ubiquitination sites in 4217 proteins. Bioinformatics and phenotypic analyses showed that the ubiquitinated proteins were closely related to meiosis and spermiogenesis. And 512 ubiquitination regulatory enzymes were identified in testis that can exert regulatory functions over ubiquitination: the homologous to E6AP C-terminus (HECT) and multi-subunit RING-finger type E3 ligases were significantly enriched. In addition, we identified 22 new ubiquitination sites on testicular histones and 146 ubiquitinated epigenetic factors, thus demonstrating that ubiquitination plays an important role in epigenetic regulation. Collectively, this in-depth characterization of the ubiquitome in mouse testis could provide a rich resource for further studies of regulatory events at the protein level during spermatogenesis. All MS data are available via ProteomeXchange with the identifier PXD025866.

The testis-specifically expressed gene Trim69 is not essential for fertility in mice.

Protein ubiquitination is essential for diverse cellular functions including spermatogenesis. The tripartite motif (TRIM) family proteins, most of which have E3 ubiquitin ligase activity, are highly conserved in mammals. They are involved in important cellular processes such as embryonic development, immunity, and fertility. Our previous studies indicated that Trim69, a testis-specific expressed TRIM family gene, potentially participates in the spermatogenesis by mediating testicular cells apoptosis. In this study, we investigated the biological functions of Trim69 in male mice by established Trim69 knockout mice with CRISPR/Cas9 genomic editing technology. Here, we reported that the male Trim69 knockout mice had normal fertility. The adult knockout mice have shown that the appearance of testes, testis/body weight ratios, testicular histomorphology, and the number and quality of sperm were consistent with wild-type mice. These results indicated that the E3 ubiquitin ligase protein Trim69 was not essential for male mouse fertility, and it might be compensated by other TRIM family members such as Trim58 in Trim69-deficiency testis. This study would help to elucidate the functions of tripartite motif protein family and the regulation of spermatogenesis.

Generation of human induced pluripotent stem cell (SKLRMi001-A) from a patient with partial androgen insensitivity syndrome (PAIS).

Androgen receptor (AR) is essential for maintaining normal spermatogenesis and male fertility, and its mutation can cause complete or partial androgen insensitivity syndrome (CAIS or PAIS) in patients. We established an induced pluripotent stem cell line (SKLRMi001-A) from a PAIS patient with AR mutation (c.2710G > A; p. V904M). The iPSC line expressed pluripotency markers, retained normal karyotype, carried the mutation, showed capability of differentiating into three germ layers and was absence of mycoplasma infection. The iPSC line will help to further elucidate the pathogenic mechanisms of AR mutation, and benefit treatment for PAIS in the future.