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Objectives: Accumulating evidences show that the regulatory network of m6A modification is essential for mammalian spermatogenesis. However, as an m6A reader, the roles of YTHDF2 remain enigmatic due to the lack of a proper model. Here, we employed the germ cell conditional knockout mouse model and explored the function of YTHDF2 in spermatogenesis. Materials and methods: Ythdf2 germ cell conditional knockout mice were obtained by crossing Ythdf2‐floxed mice with Vasa‐Cre and Stra8‐Cre mice. Haematoxylin and eosin (HE) staining, immunofluorescent staining and Western blotting were used for phenotyping. CASA, IVF and ICSI were applied for sperm function analysis. RNA‐seq, YTHDF2‐RIP‐seq and quantitative real‐time PCR were used to explore transcriptome changes and molecular mechanism analysis. Results: Our results showed that YTHDF2 was highly expressed in spermatogenic cells. The germ cell conditional knockout males were sterile, and their sperm displayed malformation, impaired motility, and lost fertilization ability. During differentiated spermatogonia transiting to pachytene spermatocyte, most m6A‐modified YTHDF2 targets that were degraded in control germ cells persisted in pachytene spermatocytes of Ythdf2‐vKO mice. These delayed mRNAs were mainly enriched in pathways related to the regulation of transcription, and disturbed the transcriptome of round spermatid and elongated spermatid subsequently. Conclusion: Our data demonstrate that YTHDF2 facilitates the timely turnover of phase‐specific transcripts to ensure the proper progression of spermatogenesis, which highlights a critical role of YTHDF2 in spermatogenesis. [ABSTRACT FROM AUTHOR]

Aging has many effects on the female reproductive system, among which decreased oocyte quality and impaired embryo developmental potential are the most important factors affecting female fertility. However, the mechanisms underlying oocyte aging are not yet fully understood. Here, we selected normal reproductively aging female mice and constructed a protein expression profile of metaphase II (MII) oocytes from three age groups. A total of 187 differentially expressed (DE) proteins were identified, and bioinformatics analyses showed that these DE proteins were highly enriched in RNA splicing. Next, RNA‐seq was performed on 2‐cell embryos from these three age groups, and splicing analysis showed that a large number of splicing events and genes were discovered at this stage. Differentially spliced genes (DSGs) in the two reproductively aging groups versus the younger group were enriched in biological processes related to DNA damage repair/response. Binding motif analysis suggested that PUF60 might be one of the core splicing factors causing a decline in DNA repair capacity in the subsequent development of oocytes from reproductively aging mice, and changing the splicing pattern of its potential downstream DSG Cdk9 could partially mimic phenotypes in the reproductively aging groups. Taken together, our study suggested that the abnormal expression of splicing regulation proteins in aged MII oocytes would affect the splicing of nascent RNA after zygotic genome activation in 2‐cell embryos, leading to the production of abnormally spliced transcripts of some key genes associated with DNA damage repair/response, thus affecting the developmental potential of aged oocytes. [ABSTRACT FROM AUTHOR]

(E) Two spEV protein biomarker for noninvasive differential diagnosis of azoospermia (NOA vs. However, the choices for high sensitivity and specificity in the noninvasive differential diagnosis of azoospermia are limited.3 Extracellular vesicles (EVs) are increasingly being considered as a promising source of novel diagnostic biomarkers due to their noninvasive nature and high reproducibility.4 In this study, we revealed that two proteins SLC5A12 and HIST1H2BA from human seminal plasma extracellular vesicle (spEV) could differentially diagnose azoospermia with high sensitivity and specificity. Dear Editor, Azoospermia, which accounts for about 10%-15% of infertile men, is generally classified as obstructive azoospermia (OA) due to male reproductive tract obstruction and nonobstructive azoospermia (NOA) due to testicular failure.1 NOA has three major forms: hypospermatogenesis (HS), germ cell arrest (MA), and Sertoli cell-only (SCO) based on the histopathological examination of testicular tissue.2 An accurate diagnosis of the subtype of azoospermia is crucial and mandatory, as the treatment approach differs between NOA and OA. Thus, significant decrease of spEV SLC5A12 is expected in OA patients, but not NOA patients. [Extracted from the article]

Orderly and stage‐specifically expressed proteins are essential for spermatogenesis, and proteases play a key role in protein activation and function. The present study aimed to investigate serine protease 55 (PRSS55), which was reported to play a role in sperm‐uterotubal junction (UTJ) migration and sperm‐zona pellucida (ZP) binding. We found that PRSS55 was specifically expressed in testicular spermatids and epididymal spermatozoa. By constructing knockout mice targeting all transcripts of Prss55, we demonstrated that deletion of Prss55 resulted in a serious decline of male fertility, with significantly increased sperm malformation and decreased sperm motility. In Prss55−/− mice, increased structural abnormality, including deficient "9 + 2" microtubules, damaged peripheral dense fibre, and defective mitochondrial cristae, were found in sperm. In addition, sperm showed decreased expression of electron transfer chain molecules and lower ATP contents. These could be the potential causes of the astheno/teratozoospermia phenotype of the Prss55−/− mice, and provided new evidence for the previously reported impaired sperm‐UTJ migration. Moreover, preliminary studies allowed us to speculate that PRSS55 might function by activating type II muscle myosin in the testis, which is involved in many processes requiring motivation and cytoskeleton translocation. Thus, PRSS55 is essential for the structural differentiation and energy metabolism of sperm, and might be a potential pathogenic factor in astheno/teratozoospermia. Our results provide an additional explanation for the male sterility of Prss55−/− mice, and further reveal the role of PRSS55. [ABSTRACT FROM AUTHOR]

RNA modifications represent a novel layer of regulation of gene expression. Functional experiments revealed that N6‐methyladenosine (m6A) on messenger RNA (mRNA) plays critical roles in cell fate determination and development. m6A mark also resides in the decoding center of 18S ribosomal RNA (rRNA); however, the biological function of m6A on 18S rRNA is still poorly understood. Here, we report that methyltransferase‐like 5 (METTL5) methylates 18S rRNA both in vivo and in vitro, which is consistent with previous reports. Deletion of Mettl5 causes a dramatic differentiation defect in mouse embryonic stem cells (mESCs). Mechanistically, the m6A deposited by METTL5 is involved in regulating the efficient translation of F‐box and WD repeat domain‐containing 7 (FBXW7), a key regulator of cell differentiation. Deficiency of METTL5 reduces FBXW7 levels and leads to the accumulation of its substrate c‐MYC, thereby delaying the onset of mESC differentiation. Our study uncovers an important role of METTL5‐mediated 18S m6A in mESC differentiation through translation regulation and provides new insight into the functional significance of rRNA m6A. Synopsis: METTL5, the m6A methyltransferase for 18S rRNA, is critical for Fbxw7 translation and mESC differentiation. METTL5 is dispensable for mESC pluripotency.Orderly differentiation of mESCs requires METTL5.METTL5 promotes the translation efficiency of Fbxw7. [ABSTRACT FROM AUTHOR]

Oligoasthenozoospermia is a major cause of male infertility; however, its etiology and pathogenesis are unclear and may be associated with specific gene abnormalities. This study focused on Tppp2 (tubulin polymerization promoting protein family member 2), whose encoded protein localizes in elongating spermatids at stages IV‐VIII of the seminiferous epithelial cycle in testis and in mature sperm in the epididymis. In human and mouse sperm, in vitro inhibition of TPPP2 caused significantly decreased motility and ATP content. Studies on Tppp2 knockout (KO) mice demonstrated that deletion of TPPP2 resulted in male subfertility with a significantly decreased sperm count and motility. In Tppp2−/− mice, increased irregular mitochondria lacking lamellar cristae, abnormal expression of electron transfer chain molecules, lower ATP levels, decreased mitochondrial membrane potential and increased apoptotic index were observed in sperm, which could be the potential causes for its oligoasthenozoospermia phenotype. Moreover, we identified a potential TPPP2‐interactive protein, eEf1b (eukaryotic translation elongation factor 1 beta), which plays an important role in protein translation extension. Thus, TPPP2 is probably a potential pathogenic factor in oligoasthenozoospermia. Deficiency of TPPP2 might affect the translation of specific proteins, altering the structure and function of sperm mitochondria, and resulting in decreased sperm count, motility and fertility. [ABSTRACT FROM AUTHOR]

Spermatogonial stem cells ( SSCs) are undifferentiated cells that are required to maintain spermatogenesis throughout the reproductive life of mammals. Although SSC transplantation and culture provide a powerful tool to identify the mechanisms regulating SSC function, the precise signalling mechanisms governing SSC self-renewal and specific surface markers for purifying SSCs remain to be clearly determined. In the present study, we established a steady SSC culture according to the method described by Shinohara's lab. Fertile progeny was produced after transplantation of cultured SSCs into infertile mouse testis, and the red fluorescence exhibited by the culture cell membranes was stably and continuously transmitted to the offspring. Next, via advanced mass spectrometry and an optimized proteomics platform, we constructed the proteome profile, with 682 proteins expressed in SSCs. Furthermore bioinformatics analysis showed that the list contained several known molecules that are regulated in SSCs. Several nucleoproteins and membrane proteins were chosen for further exploration using immunofluorescence and RT- PCR. The results showed that SALL1, EZH2, and RCOR2 are possibly involved in the self-renewal mechanism of SSCs. Furthermore, the results of tissue-specific expression analysis showed that Gpat2 and Pld6 were uniquely and highly expressed in mouse testes and cultured SSCs. The cellular localization of PLD6 was further explored and the results showed it was primarily expressed in the spermatogonial membrane of mouse testes and cultured SSCs. The proteins identified in this study form the basis for further exploring the molecular mechanism of self-renewal in SSCs and for identifying specific surface markers of SSCs. [ABSTRACT FROM AUTHOR]

Acidithiobacillus ferrooxidans showed the compensate growth and oxidation after re-feeding with sufficient ferrous materials after starvation. Compensatory phenomena were first detected in chemoautotrophic organisms. Starvation stress of Acidithiobacillus ferrooxidans was achieved via culturing in low concentrations of iron. During compensation, growth and ferrous oxidation took place faster than in controls. In addition, some genes related to ferrous oxidation (such as rus) and carbon assimilation ( cbbR, csoS3) were expressed in different patterns in the low energy environments. Their expression patterns can account for this increased growth and oxidation. Other groups of genes ( cspAB, feoAB, fur) were suppressed in response to starvation stress. The presence of pyrite and joint cold stress can render compensation nearly undetectable. This may be why the compensation phenomena observed under these conditions was not the same as that observed under single starvation stress conditions. Gene expression reflected a possible mechanism of tolerance to starvation in Acidithiobacillus ferrooxidans, which would allow the organism to adapt and survive in ferrous-limited environments. [ABSTRACT FROM AUTHOR]

Aims Long-term outcome of patients after myocardial infarction (MI) largely depends on the extent of post-infarct remodelling. To explore the molecular mechanism of remodelling, comparative proteomic analysis was undertaken to identify differential myocardial proteome profiles expressed in the border zone of the post-MI heart. Methods and results Two-dimensional gel electrophoresis and matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometry were used to identify the differential protein profiles expressed in the border zone at specific time points (Days 0, 1, 4, and 10 post-infarction) in a permanent rat MI model. We identified 96 differential protein spots, corresponding to 69 proteins. Cluster analysis exhibited five main temporal expression patterns corresponding to the three phases of early stage remodelling. The alteration in expression was supported by reverse transcription-polymerase chain reaction, western blotting, and immunohistochemical analysis of three selected proteins. Bioinformatics analysis revealed that the proteins in each pattern were functionally related to specific cell processes in remodelling, such as ischaemia, inflammation, and proliferation. Conclusion A differential myocardial proteome profile was identified in the border zone during early stage post-infarct remodelling. Bioinformatics analysis indicated a possible role of these proteins in remodelling. Proteomics data provided the basis for further functional study of these proteins and for identifying potential molecular targets with therapeutic anti-remodelling effects. [ABSTRACT FROM PUBLISHER]

Phosphorylation is a key post-translational modification regulating protein function and biological outcomes. However, the phosphorylation dynamics orchestrating mammalian oocyte development remains poorly understood. In the present study, we apply high-resolution mass spectrometry-based phosphoproteomics to obtain the first global in vivo quantification of mouse oocyte phosphorylation. Of more than 8000 phosphosites, 75% significantly oscillate and 64% exhibit marked upregulation during meiotic maturation, indicative of the dominant regulatory role. Moreover, we identify numerous novel phosphosites on oocyte proteins and a few highly conserved phosphosites in oocytes from different species. Through functional perturbations, we demonstrate that phosphorylation status of specific sites participates in modulating critical events including metabolism, translation, and RNA processing during meiosis. Finally, we combine inhibitor screening and enzyme-substrate network prediction to discover previously unexplored kinases and phosphatases that are essential for oocyte maturation. In sum, our data define landscape of the oocyte phosphoproteome, enabling in-depth mechanistic insights into developmental control of germ cells. [ABSTRACT FROM AUTHOR]