Mouse PUMILIO1 (PUM1) and PUMILIO2 (PUM2) belong to the PUF (Pumilio/FBF) family, a highly conserved RNA binding protein family whose homologues play critical roles in embryonic development and germ line stem cell maintenance in invertebrates. However, their roles in mammalian embryonic development and stem cell maintenance remained largely uncharacterized. Here we report an essential requirement of the Pum gene family in early embryonic development. A loss of both Pum1 and Pum2 genes led to gastrulation failure, resulting in embryo lethality at E8.5. Pum-deficient blastocysts, however, appeared morphologically normal, from which embryonic stem cells (ESCs) could be established. Both mutant ESCs and embryos exhibited reduced growth and increased expression of endoderm markers Gata6 and Lama1, making defects in growth and differentiation the likely causes of gastrulation failure. Furthermore, ESC Gata6 transcripts could be pulled down via PUM1 immunoprecipitation and mutation of conserved PUM-binding element on 3′UTR (untranslated region) of Gata6 enhanced the expression of luciferase reporter, implicating PUM-mediated posttranscriptional regulation of Gata6 expression in stem cell development and cell lineage determination. Hence, like its invertebrate homologues, mouse PUM proteins are conserved posttranscriptional regulators essential for embryonic and stem cell development.

Circular RNAs (circRNAs) are a large family of newly identified transcripts, and their physiological roles and evolutionary significance require further characterization. Here, we identify circRNAs generated from a conserved reproductive gene, Boule, in species from Drosophila to humans. Flies missing circular Boule (circBoule) RNAs display decreased male fertility, and sperm of circBoule knockout mice exhibit decreased fertilization capacity, when under heat stress conditions. During spermatogenesis, fly circBoule RNAs interact with heat shock proteins (HSPs) Hsc4 and Hsp60C, and mouse circBoule RNAs in sperm interact with HSPA2. circBoule RNAs regulate levels of HSPs by promoting their ubiquitination. The interaction between HSPA2 and circBoule RNAs is conserved in human sperm, and lower levels of the human circBoule RNAs circEx3-6 and circEx2-7 are found in asthenozoospermic sperm. Our findings reveal conserved physiological functions of circBoule RNAs in metazoans and suggest that specific circRNAs may be critical modulators of male reproductive function against stresses in animals.

D-Alanine is a central component of the cell wall in most prokaryotes. D-Alanine synthesis in Escherichia coliis carried out by two different alanine racemases encoded by the alr and dadX genes. Deletion of alr and dadX from the E. coli genome results in a D-alanine auxotrophic phenotype. However, we have observed growth of prototrophic phenotypic revertants during routine culturing of a D-alanine auxotrophic strain. We present a detailed comparison of the proteome and transcriptome profiles of the D-alanine auxotroph and a prototrophic revertant strain. Most noticeably, a general upregulation of genes involved in methionine synthesis in the revertant strain was detected. The appearance of the revertant phenotype was genetically linked to point mutations in the methionine repressor gene (metJ). Our results reveal an alternative metabolic pathway which can supply essential D-alanine for peptidoglycan synthesis of alr-and dadX-deficient E. coli mutants and provide evidence for significant alanine racemase coactivity of the E. coli cystathionine beta-lyase (MetC).Alanine racemases (EC 5.1.1.1) are unique prokaryotic enzymes that catalyze the reversible racemization of L-and Dalanine, the latter one being an essential component in the biosynthesis of the bacterial peptidoglycan of Gram-positive and Gram-negative bacteria (47). The bacteria investigated to date have been found to possess either one or two distinct alanine racemase genes. The alr gene encodes a constitutively expressed alanine racemase, which provides D-alanine for sufficient cross-linking of adjacent peptidoglycan strands in the cell wall. The second gene encodes the so-called catabolic alanine racemase, which is essential for L-alanine catabolism (24,28,41,42,48). In Escherichia coli, the alr-encoded alanine racemase is constitutively expressed, whereas the dadX-encoded enzyme is essential only for L-alanine catabolism, providing a substrate for a D-alanine-specific dehydrogenase encoded by the dadA gene (51). The dadX gene product provides a secondary source of D-alanine for cell wall biosynthesis.D-Alanine auxotrophic E. coli, Bacillus subtilis, Corynebacterium glutamicum, Listeria monocytogenes, and Lactobacillus plantarum strains have been generated by inactivating genes encoding alanine racemases (15,17,24,42,43,45). A strong selective pressure for maintenance of an alanine racemase (Dal)-encoding plasmid in a chromosomal dal mutant of Bacillus subtilis was observed upon growth on rich medium. In Lactobacillus plantarum, plasmids encoding alanine racemase (Alr) were efficiently selected in an alr-deficient Lactobacillus plantarum strain (5). In Listeria monocytogenes, two genes, an alanine racemase gene (dal) and a D-amino acid aminotransferase gene (dat), which control the synthesis of D-alanine, had to be inactivated in order to achieve complete D-alanine auxotrophy (46).Under certain circumstances, the D-alanine auxotrophic phenotype was lost, indicating a redundancy of alanine racemase activity in bacteria. The D-alanine auxotrophic phenoty...

Background The side effects of busulfan on male reproduction are serious, so fertility preservation in children undergoing busulfan treatment is a major worldwide concern. Human placental mesenchymal stem cells (hPMSCs) have advantages such as stable proliferation and lower immunogenicity that make them an ideal material for stimulating tissue repair, especially restoring spermatogenesis. The protective effects of hPMSCs in busulfan-induced Sertoli cells and in busulfan-treated mouse testes have not been determined. Our study aimed to elaborate the protective effect and potential mechanisms of hPMSCs in busulfan-treated testes and Sertoli cells. Methods First, we developed a mouse model of busulfan-induced testicular toxicity in vivo and a mouse Sertoli cell line treated with busulfan in vitro to assess the protective effect and mechanisms of hPMSC treatment on spermatogenesis. Then, the length, width, and weight of the testes were monitored using Vernier calipers. Furthermore, at 1 week and 4 weeks after the transplantation of hPMSCs, histological sections of testes were stained with hematoxylin-eosin, and the seminiferous tubules with fluid-filled cavities were counted. Through ELISA analysis, testosterone levels and MDA, SOD, LDH, and CAT activities, which are associated with ROS, were detected. Markers of ROS, proliferation (Ki67), and apoptosis (Annexin V) were evaluated by FACS. Next, the fluorescence intensity of proliferation markers (BrdU and SCP3), an antioxidant marker (SIRT1), a spermatogenesis marker (PLZF), and autophagy-related genes (P62 and LC3AB) were detected by fluorescence microscopy. The mRNA expression of γ-H2AX, BRCA1, PARP1, PCNA, Ki67, P62, and LC3 was determined by qRT-PCR. Results hPMSCs restored disrupted spermatogenesis, promoted improved semen parameters, and increased testosterone levels, testis size, and autophagy in the testis toxicity mouse model induced by busulfan. hPMSCs suppressed the apoptosis of Sertoli cells and enhanced their rate of proliferation in vitro. Additionally, hPMSCs protected against oxidative stress and decreased oxidative damage in the testis toxicity mouse model induced by busulfan. Furthermore, hPMSCs increased the expression of proliferation genes (PCNA and KI67) and decreased the mRNA levels of apoptotic genes such as γ-H2AX, BRCA1, and PARP1. Conclusions This research showed that hPMSC injection ameliorated busulfan-induced damage in the testis by reducing apoptosis/oxidative stress and promoting autophagy. The present study offers an idea for a new method for clinical treatment of chemotherapy-induced spermatogenesis.

Fertilization is one of the fundamental biological processes, but so far, we still do not have a full understanding of the underlying molecular mechanism. We have identified a human acrosome protein, LY6/PLAUR domain containing 4 (LYPD4), expressed specifically in human testes and sperm, and conserved within mammals. Mouse Lypd4, also specific to the testis and sperm, is essential for male fertility. LYPD4 protein first appeared in round spermatids during acrosome biogenesis and became part of acrosomes during spermatogenesis and in mature sperm. Lypd4 knockout mice are infertile with normal sperm number and motility. Mutant sperm, however, failed to reach oviduct during sperm migration inside the female reproductive tract, leading to fertilization failure and infertility. In addition, Lypd4 mutant sperms were unable to fertilize denuded egg via IVF (in vitro fertilization) but could fertilize eggs within intact Cumulus-Oocyte Complex, supporting an additional role in sperm-zona interaction. Out of more than five thousand spermatozoa proteins identified by mass spectrometry analysis, only a small subset of proteins (26 proteins) was changed in the absence of LYPD4, revealing a whole proteome picture of mutant sperm defective in sperm migration and sperm-zona binding. ADAM3, a key component of fertilization complex, as well as other sperm ADAM proteins are significantly reduced. We hence propose that LYPD4 plays an essential role in mammalian fertilization, and further investigation of its function and its interaction with other sperm membrane complexes may yield insights into human fertilization and novel strategy to improve IVF success.