Jiahao Sha

Generation of gene-modified cynomolgus monkey via Cas9/RNA-mediated gene targeting in one-cell embryos.

Monkeys serve as important model species for studying human diseases and developing therapeutic strategies, yet the application of monkeys in biomedical researches has been significantly hindered by the difficulties in producing animals genetically modified at the desired target sites. Here, we first applied the CRISPR/Cas9 system, a versatile tool for editing the genes of different organisms, to target monkey genomes. By coinjection of Cas9 mRNA and sgRNAs into one-cell-stage embryos, we successfully achieve precise gene targeting in cynomolgus monkeys. We also show that this system enables simultaneous disruption of two target genes (Ppar-γ and Rag1) in one step, and no off-target mutagenesis was detected by comprehensive analysis. Thus, coinjection of one-cell-stage embryos with Cas9 mRNA and sgRNAs is an efficient and reliable approach for gene-modified cynomolgus monkey generation.

A spermatogenesis-related gene expression profile in human spermatozoa and its potential clinical applications

Spermatogenesis is an essential stage in the human reproductive process. In a previously study aiming to determine which genes might be involved in spermatogenesis, we compared the gene expression profiles of adult and fetal testes by hybridizing cDNA probes prepared from adult and fetal testes to membranes dotted with gene clones derived from a commercial human testis library. We identified 266 differentially expressed genes that showed higher expression levels in adult testes, indicating their potential roles in spermatogenesis. In the present study, we applied the same cDNA microarray technique to the analysis of gene expression in the spermatozoa of normal fertile men and found 149 genes that were expressed at higher levels in adult testis. A further study of five sperm motility-related genes selected from this profile by real-time PCR revealed that there was significant difference in the expression levels of two genes (TPX-1, testis-specific protein 1 and LDHC, lactate dehydrogenase C, transcript variant 1) between normal (n=29) and motility impaired (n=24) semen samples, indicating that these genes are involved in sperm function. Our results demonstrated that spermatogenesis-related gene profiling could help to assess sperm quality in humans, and further study of these genes will help us to elucidate the mechanisms involved in spermatogenesis and diseases relating to human infertility.

A genome-wide association study in Chinese men identifies three risk loci for non-obstructive azoospermia

Non-obstructive azoospermia (NOA) is one of the most severe forms of male infertility. Its pathophysiology is largely unknown, and few genetic influences have been defined. To identify common variants contributing to NOA in Han Chinese men, we performed a three-stage genome-wide association study of 2,927 individuals with NOA and 5,734 controls. The combined analyses identified significant (P < 5.0 × 10−8) associations between NOA risk and common variants near PRMT6 (rs12097821 at 1p13.3: odds ratio (OR) = 1.25, P = 5.7 × 10−10), PEX10 (rs2477686 at 1p36.32: OR = 1.39, P = 5.7 × 10−12) and SOX5 (rs10842262 at 12p12.1: OR = 1.23, P = 2.3 × 10−9). These findings implicate genetic variants at 1p13.3, 1p36.32 and 12p12.1 in the etiology of NOA in Han Chinese men.

Complete Meiosis from Embryonic Stem Cell-Derived Germ Cells In Vitro

In vitro generation of functional gametes is a promising approach for treating infertility, although faithful replication of meiosis has proven to be a substantial obstacle to deriving haploid gamete cells in culture. Here we report complete in vitro meiosis from embryonic stem cell (ESC)-derived primordial germ cells (PGCLCs). Co-culture of PGCLCs with neonatal testicular somatic cells and sequential exposure to morphogens and sex hormones reproduced key hallmarks of meiosis, including erasure of genetic imprinting, chromosomal synapsis and recombination, and correct nuclear DNA and chromosomal content in the resulting haploid cells. Intracytoplasmic injection of the resulting spermatid-like cells into oocytes produced viable and fertile offspring, showing that this robust stepwise approach can functionally recapitulate male gametogenesis in vitro. These findings provide a platform for investigating meiotic mechanisms and the potential generation of human haploid spermatids in vitro.

Early second-trimester serum miRNA profiling predicts gestational diabetes mellitus.

Background Gestational diabetes mellitus (GDM) is one type of diabetes that presents during pregnancy and significantly increases the risk of a number of adverse consequences for the fetus and mother. The microRNAs (miRNA) have recently been demonstrated to abundantly and stably exist in serum and to be potentially disease-specific. However, no reported study investigates the associations between serum miRNA and GDM. Methodology/Principal Findings We systematically used the TaqMan Low Density Array followed by individual quantitative reverse transcription polymerase chain reaction assays to screen miRNAs in serum collected at 16–19 gestational weeks. The expression levels of three miRNAs (miR-132, miR-29a and miR-222) were significantly decreased in GDM women with respect to the controls in similar gestational weeks in our discovery evaluation and internal validation, and two miRNAs (miR-29a and miR-222) were also consistently validated in two-centric external validation sample sets. In addition, the knockdown of miR-29a could increase Insulin-induced gene 1 (Insig1) expression level and subsequently the level of Phosphoenolpyruvate Carboxy Kinase2 (PCK2) in HepG2 cell lines. Conclusions/Significance Serum miRNAs are differentially expressed between GDM women and controls and could be candidate biomarkers for predicting GDM. The utility of miR-29a, miR-222 and miR-132 as serum-based non-invasive biomarkers warrants further evaluation and optimization.

In-depth proteomic analysis of the human sperm reveals complex protein compositions

The male gamete (sperm) can fertilize an egg, and pass the male genetic information to the offspring. It has long been thought that sperm had a simple protein composition. Efforts have been made to identify the sperm proteome in different species, and only about 1000 proteins were reported. However, with advanced mass spectrometry and an optimized proteomics platform, we successfully identified 4675 human sperm proteins, of which 227 were testis-specific. This large number of identified proteins indicates the complex composition and function of human sperm. Comparison with the sperm transcriptome reveals little overlap, which shows the importance of future studies of sperm at the protein level. Interestingly, many signaling pathways, such as the IL-6, insulin and TGF-beta receptor signaling pathways, were found to be overrepresented. In addition, we found that 500 proteins were annotated as targets of known drugs. Three of four drugs studied were found to affect sperm movement. This in-depth human sperm proteome will be a rich resource for further studies of sperm function, and will provide candidate targets for the development of male contraceptive drugs.

Rapid evolution of mammalian X-linked testis microRNAs.

BackgroundMicroRNAs (miRNAs), which are small, non-coding RNAs approximately 21-nucleotides in length, have become a major focus of research in molecular biology. Mammalian miRNAs are proposed to regulate approximately 30% of all protein-coding genes. Previous studies have focused on highly conserved miRNAs, but nonconserved miRNAs represent a potentially important source of novel functionalities during evolution.ResultsAn analysis of the chromosome distribution of miRNAs showed higher densities of miRNAs on the X chromosome compared to the average densities on autosomes in all eight mammalian species analyzed. The distribution pattern did not, however, apply well to species beyond mammals. In addition, by comparing orthologous human and mouse miRNAs, we found that X-linked miRNAs had higher substitution rates than autosomal miRNAs. Since the highest proportion of X-linked miRNAs were found in mouse testis, we tested the hypothesis that testis miRNAs are evolving faster on the X chromosome than on autosomes. Mature X-linked testis miRNAs had an average substitution rate between mouse and human that was almost 25-fold higher than mature testis miRNAs on autosomes. In contrast, for mature miRNAs with precursors not expressed in testis, no significant difference in the substitution rate between the X chromosome and autosomes was found. Among mammals, the rapid evolution of X-linked testis miRNAs was also observed in rodents and primates.ConclusionThe rapid evolution of X-linked testis miRNAs implies possible important male reproductive functions and may contribute to speciation in mammals.

Human spermatogenic failure purges deleterious mutation load from the autosomes and both sex chromosomes, including the gene DMRT1.

Gonadal failure, along with early pregnancy loss and perinatal death, may be an important filter that limits the propagation of harmful mutations in the human population. We hypothesized that men with spermatogenic impairment, a disease with unknown genetic architecture and a common cause of male infertility, are enriched for rare deleterious mutations compared to men with normal spermatogenesis. After assaying genomewide SNPs and CNVs in 323 Caucasian men with idiopathic spermatogenic impairment and more than 1,100 controls, we estimate that each rare autosomal deletion detected in our study multiplicatively changes a mans risk of disease by 10% (OR 1.10 [1.04–1.16], p<2×10−3), rare X-linked CNVs by 29%, (OR 1.29 [1.11–1.50], p<1×10−3), and rare Y-linked duplications by 88% (OR 1.88 [1.13–3.13], p<0.03). By contrasting the properties of our case-specific CNVs with those of CNV callsets from cases of autism, schizophrenia, bipolar disorder, and intellectual disability, we propose that the CNV burden in spermatogenic impairment is distinct from the burden of large, dominant mutations described for neurodevelopmental disorders. We identified two patients with deletions of DMRT1, a gene on chromosome 9p24.3 orthologous to the putative sex determination locus of the avian ZW chromosome system. In an independent sample of Han Chinese men, we identified 3 more DMRT1 deletions in 979 cases of idiopathic azoospermia and none in 1,734 controls, and found none in an additional 4,519 controls from public databases. The combined results indicate that DMRT1 loss-of-function mutations are a risk factor and potential genetic cause of human spermatogenic failure (frequency of 0.38% in 1306 cases and 0% in 7,754 controls, p = 6.2×10−5). Our study identifies other recurrent CNVs as potential causes of idiopathic azoospermia and generates hypotheses for directing future studies on the genetic basis of male infertility and IVF outcomes.

Autophagy and apoptosis act as partners to induce germ cell death after heat stress in mice.

Testicular heating suppresses spermatogenesis which is marked by germ cell loss via apoptotic pathways. Recently, it is reported that autophagy also can be induced by heat treatment in somatic cells. In this study, the status of autophagy in germ cells after heat treatment, as well as the partnership between autophagy and apoptosis in these cells was investigated. The results demonstrated that besides initiating apoptotic pathways, heat also induced autophagic pathways in germ cells. Exposure of germ cells to hyperthermia resulted in several specific features of the autophagic process, including autophagosome formation and the conversion of LC3-I to LC3-II. Furthermore, the ubiquitin-like protein conjugation system was implicated as being likely responsible for heat-induced autophagy in germ cells since all genes involving this system were found to be expressed in the testes. In addition, the upstream protein in this system, Atg7 (Autophagy-related gene 7), was found to be expressed in all types of spermatogenic cells, and its expression level was positively correlated with the level of autophagy in germ cells. As a result, Atg7 was selected as the investigative target to further analyze the role of autophagy in heat-induced germ cell death. It was shown that down expression of Atg7 protein resulted in the notable decrease in the level of autophagy in heat-treated germ cells, and this down-regulation of autophagy caused by Atg7 knockdown further reduced the apoptotic rate of germ cells. These results suggest that autophagy plays a positive role in the process of germ cell apoptosis after heat treatment. In conclusion, this study demonstrates that heat triggers autophagy and apoptosis in germ cells. These two mechanisms might act as partners, not antagonist, to induce cell death and lead to eventual destruction of spermatogenesis.

Proliferating Cell Nuclear Antigen (PCNA) Regulates Primordial Follicle Assembly by Promoting Apoptosis of Oocytes in Fetal and Neonatal Mouse Ovaries

Primordial follicles, providing all the oocytes available to a female throughout her reproductive life, assemble in perinatal ovaries with individual oocytes surrounded by granulosa cells. In mammals including the mouse, most oocytes die by apoptosis during primordial follicle assembly, but factors that regulate oocyte death remain largely unknown. Proliferating cell nuclear antigen (PCNA), a key regulator in many essential cellular processes, was shown to be differentially expressed during these processes in mouse ovaries using 2D-PAGE and MALDI-TOF/TOF methodology. A V-shaped expression pattern of PCNA in both oocytes and somatic cells was observed during the development of fetal and neonatal mouse ovaries, decreasing from 13.5 to 18.5 dpc and increasing from 18.5 dpc to 5 dpp. This was closely correlated with the meiotic prophase I progression from pre-leptotene to pachytene and from pachytene to diplotene when primordial follicles started to assemble. Inhibition of the increase of PCNA expression by RNA interference in cultured 18.5 dpc mouse ovaries strikingly reduced the apoptosis of oocytes, accompanied by down-regulation of known pro-apoptotic genes, e.g. Bax, caspase-3, and TNFα and TNFR2, and up-regulation of Bcl-2, a known anti-apoptotic gene. Moreover, reduced expression of PCNA was observed to significantly increase primordial follicle assembly, but these primordial follicles contained fewer guanulosa cells. Similar results were obtained after down-regulation by RNA interference of Ing1b, a PCNA-binding protein in the UV-induced apoptosis regulation. Thus, our results demonstrate that PCNA regulates primordial follicle assembly by promoting apoptosis of oocytes in fetal and neonatal mouse ovaries.