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Transposable elements constitute nearly half of the mammalian genome and play important roles in genome evolution. While a multitude of both transcriptional and post-transcriptional mechanisms exist to silence transposable elements, control of transpositionin vivoremains poorly understood. MOV10, an RNA helicase, is a potent inhibitor of mobilization of retrotransposons and retroviruses in cell culture assays. Here we report that MOV10 restricts LINE1 retrotransposition in mice. Although MOV10 is broadly expressed, its loss causes only incomplete penetrance of embryonic lethality, and the surviving MOV10-deficient mice are healthy and fertile. Biochemically, MOV10 forms a complex with UPF1, a key component of the nonsense-mediated mRNA decay pathway, and primarily binds to the 3’UTR of somatically expressed transcripts in testis. Consequently, loss of MOV10 results in an altered transcriptome and a modest upregulation of two LINE1 families in testis. Analyses using a LINE1 reporter transgene reveal that loss of MOV10 leads to increased LINE1 retrotransposition in somatic and reproductive tissues from both embryos and adult mice. Moreover, the degree of LINE1 retrotransposition inhibition is dependent on theMov10gene dosage. Furthermore, MOV10 deficiency reduces reproductive fitness over successive generations. Our findings demonstrate that MOV10 attenuates LINE1 retrotransposition in a dosage-dependent manner in mice.Author summaryTransposable elements (TEs), including L1 and SINEs, are abundant in the genome and play important roles in evolution, development, and diseases. While TEs propagate in individuals and across generations, the host organism needs to suppress them, resulting in an ongoing arms race between TEs and the host genome. L1, a retrotransposon, accounts for about 17% of the mammalian genome. L1 encodes two proteins, which bind to the L1 transcript to form L1 ribonucleoprotein particles. L1 proliferates in the genome via retrotransposition. A multitude of transcriptional and post-transcriptional mechanisms exist to suppress TEs, however, retrotransposition of TEs remains poorly understood. L1 ribonucleoprotein particles are associated with a large number of host proteins, one of which is the MOV10 RNA helicase. MOV10 exhibits anti-viral activities against retroviruses such as HIV-1. In cultured cells, MOV10 is a potent inhibitor of retrotransposition of L1, SINEs, and IAP. Although MOV10 is expressed in a broad range of tissues, loss of MOV10 causes only incomplete penetrance of embryonic lethality. The viable MOV10-deficient mice are grossly normal and fertile. Importantly, analyses using a L1 transgene reporter reveal that MOV10 inhibits L1 retrotransposition in both somatic tissues and reproductive tissues in a gene dosage-dependent manner. Therefore, MOV10 functions as a host restriction factor for L1 and possibly other transposable elementsin vivo.

Mice deficient in the piRNA pathway display sterility only in males. Taking advantage of a more representative piRNA pathway in golden hamsters, three studies now demonstrate that the piRNA pathway is essential for fertility in hamsters of both sexes and thus strongly link this small RNA pathway to fertility regulation in both men and women.

Homeostasis of meiotic DNA double strand breaks (DSB) is critical for germline genome integrity and homologous recombination. Here we demonstrate an essential role for SKP1, a constitutive subunit of the SCF (SKP1-Cullin-F-box) ubiquitin E3 ligase, in early meiotic processes. SKP1 restrains accumulation of HORMAD1 and the pre-DSB complex (IHO1-REC114-MEI4) on the chromosome axis in meiotic germ cells. Loss of SKP1 prior to meiosis leads to aberrant localization of DSB repair proteins and a failure in synapsis initiation in meiosis of both males and females. Furthermore, SKP1 is crucial for sister chromatid cohesion during the pre-meiotic S-phase. Mechanistically, FBXO47, a meiosis-specific F-box protein, interacts with SKP1 and HORMAD1 and targets HORMAD1 for polyubiquitination and degradation in HEK293T cells. Our results support a model wherein the SCF ubiquitin E3 ligase prevents hyperactive DSB formation through proteasome-mediated degradation of HORMAD1 and subsequent modulation of the pre-DSB complex during meiosis.

Piwi-interacting RNAs (piRNAs) play critical roles in protecting germline genome integrity and promoting normal spermiogenic differentiation. In mammals, there are two populations of piRNAs: pre-pachytene and pachytene piRNAs. Transposon-rich pre-pachytene piRNAs are expressed in fetal and perinatal germ cells and are required for retrotransposon silencing, whereas transposon-poor pachytene piRNAs are expressed in spermatocytes and round spermatids and regulate mRNA transcript levels. MOV10L1, a germ cell-specific RNA helicase, is essential for the production of both populations of piRNAs. Although the requirement of the RNA helicase domain located in the MOV10L1 C-terminal region for piRNA biogenesis is well known, its large N-terminal region remains mysterious. Here we report a novel Mov10l1 mutation in the Mov10l1 N-terminal region named yama. The yama mutation results in a single amino acid substitution V229E. The yama mutation causes meiotic arrest, de-repression of transposable elements, and male sterility because of defects in pre-pachytene piRNA biogenesis. Moreover, restricting the Mov10l1 mutation effects to later stages in germ cell development by combining with a postnatal conditional deletion of a complementing wild-type allele causes absence of pachytene piRNAs, accumulation of piRNA precursors, polar conglomeration of piRNA pathway proteins in spermatocytes, and spermiogenic arrest. Mechanistically, the V229E substitution in MOV10L1 reduces its interaction with PLD6, an endonuclease that generates the 5′ ends of piRNA intermediates. Our results uncover an important role for the MOV10L1-PLD6 interaction in piRNA biogenesis throughout male germ cell development.Author SummarySmall non-coding RNAs play critical roles in silencing of exogenous viruses, endogenous retroviruses, and transposable elements, and also play multifaceted roles in controlling gene expression. Piwi-interacting RNAs (piRNAs) are found in gonads in diverse species from flies to humans. An evolutionarily conserved function of piRNAs is to silence transposable elements through an adaptive mechanism and thus to protect the germline genome integrity. In mammals, piRNAs also provide a poorly understood function to regulate postmeiotic differentiation of spermatids. More than two dozen proteins are involved in the piRNA pathway. MOV10L1, a germ-cell-specific RNA helicase, binds to piRNA precursors to initiate piRNA biogenesis. Here we have identified a single amino acid substitution (V229E) in MOV10L1 in the yama mutant. When constitutively expressed as the only source of MOV10L1 throughout germ cell development, the yama mutation abolishes piRNA biogenesis, de-silences transposable elements, and causes meiotic arrest. When the mutant phenotype is instead revealed only later in germ cell development by conditionally inactivating a complementing wild-type copy of the gene, the point mutant abolishes formation of later classes of piRNAs and again disrupts germ cell development. Point mutations in MOV10L1 may thus contribute to male infertility in humans.

Background Our aim was to evaluate the clinical utility of non-invasive prenatal testing for pregnant women with different diagnostic indications. Methods In eight counties and districts of Yancheng, we studied 13,149 pregnant women with different indications who were offered NIPT for fetal screening, including for sex chromosomal aneuploidies (SCAs), rare autosomal trisomies (RATs), and subchromosomal copy number variations (CNVs). The purpose was to compare the detection of positive predictive values (PPVs) of different indications with the use of NIPT. The results were validated by karyotyping, chromosomal microarray analysis (CMA), or follow-up of pregnancy outcomes. Results 13,149 maternal plasma samples were sequenced, among which 28 samples (0.2%) failed the sequencing quality control. The remaining 13,121 samples were analyzed, and birth follow-up missed 2,192 samples (16.7%). The PPVs of NIPT results for trisomy 21 (T21) and trisomy 18 (T18) and SCAs were 96.67, 63.64, and 31.34%, respectively. Among the advanced maternal age (AMA), serum screening high risk (SSHR), serum screening intermediate risk (SSIR), and voluntary screening (VS) groups, the PPVs for the common trisomies were 81.25, 85.71, 100, and 70%, respectively; the PPVs for total chromosomal abnormalities were 55.82, 65.22, 23.08, and 36.59%, respectively. Conclusion NIPT for T21 and T18 and SCAs screening were ideal, and the PPVs for trisomy 13 (T13), RATs, and CNVs were low. For the AMA and VS groups, NIPT could be used as a first-line screening program; for SSHR and SSIR groups, NIPT could be used as a second-line supplementary screening program.

H1 histones bind to linker DNA. H1t (H1f6), a testis-specific linker histone variant, is present in pachytene spermatocytes and spermatids. The expression of H1t histone coincides with the acquisition of metaphase I competence in pachytene spermatocytes. Here we report the generation of H1t-GFP transgenic mice. The H1t-GFP (H1 histone testis-green fluorescence protein) fusion protein expression recapitulates the endogenous H1t expression pattern. This protein appears first in mid pachytene spermatocytes in stage V seminiferous tubules, persists in round spermatids and elongating spermatids, but is absent in elongated spermatids. The strong green fluorescence signal, due to the high abundance of H1t-GFP, is maintained in spermatocytes after induction towards metaphase I through treatment with okadaic acid. Therefore, H1t-GFP can be used as a visual marker for monitoring the progression of meiosis in vitro and in vivo, as well as fluorescence-activated cell sorting (FACS) sorting of germ cells.

The present study described the diagnosis of a fetus with sex chromosome mosaicism in three cell lines and two marker chromosomes. A 24‑year‑old woman underwent amniocentesis at 21 weeks and 4 days of gestation due to noninvasive prenatal testing identifying that the fetus had sex chromosome abnormalities. Amniotic cell culture revealed a karyotype of 45,X[13]/46,X,+mar1[6]/46,X,+mar2[9], and prenatal ultrasound was unremarkable. The woman underwent repeat amniocentesis at 23 weeks and 4 days of gestation for molecular detection. Single nucleotide polymorphism (SNP) microarray analysis on uncultured amniocytes revealed that the fetus had two Y chromosomes and 7.8‑Mb deletions in Yq11.222q12. The deletion regions included DAZ, RBMY and PRY genes, which could cause spermatogenesis obstacle and sterility. Interphase fluorescence in situ hybridization (FISH) using centromeric probes DXZ1/DYZ3/D18Z1 was performed on uncultured amniocytes to verify the two marker chromosomes to be Y chromosome derivatives. According to these examinations, the mar1 was identified as a derivative of the Y chromosome with a deletion in Yq11.222q12, and the mar2 was identified as a dicentric derivative of the Y chromosome. The molecular karyotype was therefore 45,X,ish(DXZ1+, DYZ3‑,D18Z1++)[5]/46,X,del(Y)(q11.222),ish(DXZ1+,DYZ3+,D18Z1++)[11]/46, X,idic(Y)(q11.222),ish(DXZ1+,DYZ3++,D18Z1++)[14]. The comprehensive use of cytogenetic, SNP array and FISH detections was advantageous for accurately identifying the karyotype, identifying the origin of the marker chromosome and preparing effective genetic counseling.

Piwi-interacting RNAs (piRNAs) play critical roles in protecting germline genome integrity and promoting normal spermiogenic differentiation. In mammals, there are two populations of piRNAs: pre-pachytene and pachytene. Transposon-rich pre-pachytene piRNAs are expressed in fetal and perinatal germ cells and are required for retrotransposon silencing, whereas transposon-poor pachytene piRNAs are expressed in spermatocytes and round spermatids and regulate mRNA transcript levels. MOV10L1, a germ cell-specific RNA helicase, is essential for the production of both populations of piRNAs. Although the requirement of the RNA helicase domain located in the MOV10L1 C-terminal region for piRNA biogenesis is well known, its large N-terminal region remains mysterious. Here we report a novel Mov10l1 mutation, named yama, in the Mov10l1 N-terminal region. The yama mutation results in a single amino acid substitution V229E. The yama mutation causes meiotic arrest, de-repression of transposable elements, and male sterility because of defects in pre-pachytene piRNA biogenesis. Moreover, restricting the Mov10l1 mutation effects to later stages in germ cell development by combining with a postnatal conditional deletion of a complementing wild-type allele causes absence of pachytene piRNAs, accumulation of piRNA precursors, polar conglomeration of piRNA pathway proteins in spermatocytes, and spermiogenic arrest. Mechanistically, the V229E substitution in MOV10L1 reduces its interaction with PLD6, an endonuclease that generates the 5′ ends of piRNA intermediates. Our results uncover an important role for the MOV10L1-PLD6 interaction in piRNA biogenesis throughout male germ cell development., Author summary Small non-coding RNAs play critical roles in silencing of exogenous viruses, endogenous retroviruses, and transposable elements, and also play multifaceted roles in controlling gene expression. Piwi-interacting RNAs (piRNAs) are found in gonads in diverse species from flies to humans. An evolutionarily conserved function of piRNAs is to silence transposable elements through an adaptive mechanism and thus to protect germline genome integrity. In mammals, piRNAs also provide a poorly understood function to regulate postmeiotic differentiation of spermatids. More than two dozen proteins are involved in the piRNA pathway. MOV10L1, a germ-cell-specific RNA helicase, binds to piRNA precursors to initiate piRNA biogenesis. Here we have identified a single amino acid substitution (V229E) in MOV10L1 in the yama mouse mutant. When constitutively expressed as the only source of MOV10L1 throughout germ cell development, the yama mutation abolishes piRNA biogenesis, de-silences transposable elements, and causes meiotic arrest. When the mutant phenotype is instead revealed only later in germ cell development by conditionally inactivating a wild-type copy of the gene, the point mutant abolishes formation of later classes of piRNAs and again disrupts germ cell development. Point mutations in MOV10L1 may thus contribute to male infertility in humans.

The microrchidia (MORC) family proteins are chromatin-remodelling factors and function in diverse biological processes such as DNA damage response and transposon silencing. Here, we report that mouse Morc2b encodes a functional germ cell-specific member of the MORC protein family. Morc2b arose specifically in the rodent lineage through retrotransposition of Morc2a during evolution. Inactivation of Morc2b leads to meiotic arrest and sterility in both sexes. Morc2b-deficient spermatocytes and oocytes exhibit failures in chromosomal synapsis, blockades in meiotic recombination, and increased apoptosis. Loss of MORC2B causes mis-regulated expression of meiosis-specific genes. Furthermore, we find that MORC2B interacts with MORC2A, its sequence paralogue. Our results demonstrate that Morc2b, a relatively recent gene, has evolved an essential role in meiosis and fertility., Author summary In sexually reproducing organisms, meiosis, a process unique to germ cells, produces haploid gametes. Abnormalities in meiosis can lead to infertility, loss of pregnancy, or genetic diseases such as Down syndrome. The meiotic processes are tightly regulated by a large number of genes including many meiosis-specific ones. The majority of meiosis-specific factors are conserved, however, species-specific factors have evolved. Here we report functional studies of a rodent lineage–specific gene named Morc2b. Morc2b belongs to a family of chromatin-remodelling factors. Morc2b is specifically expressed in germ cells. Disruption of Morc2b causes meiotic arrest and infertility in both sexes. Notably, MORC2B regulates the expression of a number of meiosis-specific genes. Interestingly, MORC2B interacts with its sequence homologue MORC2A. These functional studies have uncovered a new protein complex in the regulation of key meiotic processes and suggested the presence of continued selection pressure for evolution of new meiosis-specific factors.

This review explores the cellular and molecular mechanisms that regulate spermatogenesis in the post-pubertal testis that is regressing in response to mild undernutrition, using the sexually mature male sheep as a model. Testis regression leads to reductions in daily sperm production and in the quality of ejaculated spermatozoa (poorer movement, DNA damage). There is also a reduction in spermatogenic efficiency that appears to be caused, at least partially, by increases in germ cell apoptosis. Sertoli cell number does not change with testis regression, although about 1% of Sertoli cells do appear to retain proliferative ability after puberty. On the other hand, Sertoli cell function is disrupted during testis regression, as evidenced by a disorganization of tight junctions and indications that cell differentiation and maturation are reversed. Disrupted Sertoli cell function can explain, at least partially, the increase in germ cell apoptosis and any decrease in the rate of spermatogenesis, the two major contributors to spermatogenic efficiency. These outcomes seem to be mediated by changes in two RNA-based processes: (i) the expression of small non-coding RNAs that are involved in the regulation of Sertoli cell function, spermatogenesis and germ cell apoptosis and (ii) alternative pre-mRNA splicing that affects the regulation of spermatogenesis but does not appear to affect germ cell apoptosis, at least during testis progression induced by undernutrition in the male sheep. These research outcomes can be extended to other animal models and are relevant to issues in human male fertility.

Regulatory relationships for two pairs of miRNAs and mRNAs that were differentially expressed in sheep testis following nutritional treatment: oar-novel-miR-33 with 68 mRNAs, and oar-novel-miR-31 with 52 mRNAs. (PDF 937 kb)

qRT-PCR validation of differentially expressed genes. mRNA expressions from qRT-PCR are shown by line graphs on the top and values are shown on the right Y-axis as relative expression (2-ΔΔCt). mRNA expressions from RNA-Seq are shown by bar graphs on the bottom and values are shown on the left Y-axis as log2 (normalised reads number). a, b - indicate the significant difference in the relative expression of mRNAs detected via qRT-PCR at P

We tested whether the quality of spermatozoa from mature male sheep would be affected during nutrition-induced changes in testicular mass. Merino rams were fed for 65 days with diets that increased, maintained or decreased body and testis mass (n=8 per group). In semen collected on Days 56 and 63, underfed rams had less sperms per ejaculate than well-fed rams (P

A series of batch cultures were conducted to investigate the effects of oleic acid (OA) on in vitro ruminal dry matter degradability (IVDMD), gas production, methane (CH4) and hydrogen (H2) production, and proportion of fatty acids. Rumen fluid was collected from fistulated goats, diluted with incubation buffer, and then incubated with 500 mg Leymus chinensis meal supplemented with different amounts of OA (0, 20, 40, and 60 mg for the CON, OA20, OA40 and OA60 groups, respectively). Incubation was carried out anaerobically at 39°C for 48 h, and the samples were taken at 12, 24 and 48 h and subjected to laboratory analysis. Supplementation of OA decreased IVDMD, the cumulative gas production, theoretical maximum of gas production and CH4 production, but increased H2 production. However, no effect was observed on any parameters of rumen fermentation (pH, ammonia, production of acetate, propionate and butyrate and total volatile fatty acid production). The concentrations of some beneficial fatty acids, such as cis monounsaturated fatty acids and conjugated linoleic acid (CLA) were higher (P < 0.05) from OA groups than those from the control group at 12 h incubation. In summary, these results suggest that the OA supplementation in diet can reduce methane production and increase the amount of some beneficial fatty acids in vitro.

Background: The optimal serum level of 25-hydroxyvitamin D (25-(OH)D) for bone health is still unclear, especially in children. Hypovitaminosis D is also re-emerging in developed and developing countries. The purpose of the present study was therefore to determine optimal serum 25-(OH)D level and preliminarily identify the vitamin D nutritional status in Nanjing children. Methods: All subjects (76 healthy, 66 suffering from infectious diseases) aged 0–10 years were recruited during the period December 2007–March 2008. Venous blood samples were collected before breakfast and the levels of serum 25-(OH)D, parathyroid hormone (PTH), bone-specific alkaline phosphatase (BAP), calcium (Ca), phosphorus (P) were determined. The optimal level of serum 25-(OH)D was explored using the three response curves of 25-(OH)D versus PTH, 25-(OH)D versus BAP, and 25-(OH)D versus Ca×P product. Results: For 25-(OH)D ≤ 50 nmol/L, PTH and BAP were both inversely correlated with 25-(OH)D (PTH, r=−0.864, P 50–60 nmol/L, levels of PTH and BAP remained steady. With regard to the Ca×P product, for 25-(OH)D ≤ 50 nmol/L, Ca×P product increased as 25-(OH)D increased (r= 0.037, P > 0.05). For 25-(OH)D > 50–60 nmol/L, Ca×P product remained steady. The mean serum level of 25-(OH)D was 80.5 ± 29.3 nmol/L (mean ± SD) in the healthy children, and 65.7 ± 32.3 nmol/L in the sick children. Conclusion: The optimal 25-(OH)D level may be 50–60 nmol/L for bone health in Nanjing children. The vitamin D nutritional status of Nanjing children is relatively good in winter.

The molecular regulatory mechanisms of host responses to Mycobacterium avium subsp. paratuberculosis (MAP) infection during the early subclinical stage are still not clear. In this study, surgically isolated ileal segments in newborn calves (n = 5) were used to establish in vivo MAP infection adjacent to an uninfected control intestinal compartment. RNA-Seq was used to profile the whole transcriptome (mRNAs) and the microRNAome (miRNAs) of ileal tissues collected at one-month post-infection. The most related function of the differentially expressed mRNAs between infected and uninfected tissues was “proliferation of endothelial cells”, indicating that MAP infection may lead to the over-proliferation of endothelial cells. In addition, 46.2% of detected mRNAs displayed alternative splicing events. The pre-mRNA of two genes related to macrophage maturation (monocyte to macrophage differentiation-associated) and lysosome function (adenosine deaminase) showed differential alternative splicing events, suggesting that specific changes in the pre-mRNA splicing sites may be a mechanism by which MAP escapes host immune responses. Moreover, 9 miRNAs were differentially expressed after MAP infection. The integrated analysis of microRNAome and transcriptome revealed that these miRNAs might regulate host responses to MAP infection, such as “proliferation of endothelial cells” (bta-miR-196 b), “bacteria recognition” (bta-miR-146 b), and “regulation of the inflammatory response” (bta-miR-146 b).

We tested whether reductions in spermatozoal quality induced by under-nutrition are associated with increased germ cell apoptosis and disrupted spermatogenesis and whether these effects are mediated by small RNAs. Groups of 8 male sheep were fed for a 10% increase or 10% decrease in body mass over 65 days. Underfeeding increased the number of apoptotic germ cells (P TP53, CASP3, FASL). The proportion of miRNAs as a total of small RNAs was greater in well-fed males than in underfed males (P

We tested whether the reversible effects of nutrition on spermatogenesis in sexually mature sheep were mediated by Sertoli cells. Rams were fed with diets designed to achieve a 10% increase (High), no change (Maintenance) or a 10% decrease (Low) in body mass after 65 days. At the end of treatment, testes were lighter in the Low than the High group (P

SKP1 coordinates chromosome synapsis and meiotic recombination with progression through the first meiotic cell division in mouse., The meiotic prophase I to metaphase I (PI/MI) transition requires chromosome desynapsis and metaphase competence acquisition. However, control of these major meiotic events is poorly understood. Here, we identify an essential role for SKP1, a core subunit of the SKP1–Cullin–F-box (SCF) ubiquitin E3 ligase, in the PI/MI transition. SKP1 localizes to synapsed chromosome axes and evicts HORMAD proteins from these regions in meiotic spermatocytes. SKP1-deficient spermatocytes display premature desynapsis, precocious pachytene exit, loss of PLK1 and BUB1 at centromeres, but persistence of HORMAD, γH2AX, RPA2, and MLH1 in diplonema. Strikingly, SKP1-deficient spermatocytes show sharply reduced MPF activity and fail to enter MI despite treatment with okadaic acid. SKP1-deficient oocytes exhibit desynapsis, chromosome misalignment, and progressive postnatal loss. Therefore, SKP1 maintains synapsis in meiosis of both sexes. Furthermore, our results support a model where SKP1 functions as the long-sought intrinsic metaphase competence factor to orchestrate MI entry during male meiosis.

Background The effects of nutrition on testis mass in the sexually mature male have long been known, however, the cellular and molecular processes of the testis response to nutrition was not fully understood. Methods We tested whether the defects in spermatogenesis and increases in germ cell apoptosis in the testis that are induced by under-nutrition are associated with changes in mRNA expression and pre-mRNA alternative splicing using groups of 8 male sheep fed for a 10% increase or 10% decrease in body mass over 65 days. Results We identified 2,243 mRNAs, including TP53 and Claudin 11, that were differentially expressed in testis from underfed and well-fed sheep (FDR