piRNAs and Aubergine cooperate with Wispy poly(A) polymerase to stabilize mRNAs in the germ plasm

Piwi-interacting RNAs (piRNAs) and PIWI proteins play a crucial role in germ cells by repressing transposable elements and regulating gene expression. In Drosophila, maternal piRNAs are loaded into the embryo mostly bound to the PIWI protein Aubergine (Aub). Aub targets maternal mRNAs through incomplete base-pairing with piRNAs and can induce their destabilization in the somatic part of the embryo. Paradoxically, these Aub-dependent unstable mRNAs encode germ cell determinants that are selectively stabilized in the germ plasm. Here we show that piRNAs and Aub actively protect germ cell mRNAs in the germ plasm. Aub directly interacts with the germline-specific poly(A) polymerase Wispy, thus leading to mRNA polyadenylation and stabilization in the germ plasm. These results reveal a role for piRNAs in mRNA stabilization and identify Aub as an interactor of Wispy for mRNA polyadenylation. They further highlight the role of Aub and piRNAs in embryonic patterning through two opposite functions

Digital multiplexed mRNA analysis of functionally important genes in single human oocytes and correlation of changes in transcript levels with oocyte protein expression

Objective To investigate functionally important transcripts in single human oocytes with the use of NanoString technology and determine whether observed differences are biologically meaningful. Design Analysis of human oocytes with the use of NanoString and immunoblotting. Setting University-affiliated reproductive medicine unit. Patients Women undergoing in vitro fertilization. Intervention Human oocytes were analyzed with the use of NanoString or immunoblotting. Main Outcome Measures The abundance of transcripts for ten functionally important genes—AURKA, AURKC, BUB1, BUB1B (encoding BubR1), CDK1, CHEK1, FYN, MOS, MAP2K1, and WEE2—and six functionally dispensable genes were analyzed with the use of NanoString. BubR1 protein levels in oocytes from younger and older women were compared with the use of immunoblotting. Result(s) All ten functional genes but none of the six dispensable genes were detectable with the use of NanoString in single oocytes. There was 3- to 5-fold variation in BUB1, BUB1B, and CDK1 transcript abundance among individual oocytes from a single patient. Transcripts for these three genes—all players within the spindle assembly checkpoint surveillance mechanism for preventing aneuploidy—were reduced in the same oocyte from an older patient. Mean BUB1B transcripts were reduced by 1.5-fold with aging and associated with marked reductions in BubR1 protein levels. Conclusion(s) The abundance of functionally important transcripts exhibit marked oocyte-to-oocyte heterogeneity to a degree that is sufficient to affect protein expression. Observed variations in transcript abundance are therefore likely to be biologically meaningful, especially if multiple genes within the same pathway are simultaneously affected

Cell arrest and cell death in mammalian preimplantation development

The causes, modes, biological role and prospective significance of cell death in preimplantation development in humans and other mammals are still poorly understood. Early bovine embryos represent a very attractive experimental model for the investigation of this fundamental and important issue. To obtain reference data on the temporal and spatial occurrence of cell death in early bovine embryogenesis, three-dimensionally preserved embryos of different ages and stages of development up to hatched blastocysts were examined in toto by confocal laser scanning microscopy. In parallel, transcript abundance profiles for selected apoptosis-related genes were analyzed by real-time reverse transcriptase-polymerase chain reaction. Our study documents that in vitro as well as in vivo, the first four cleavage cycles are prone to a high failure rate including different types of permanent cell cycle arrest and subsequent non-apoptotic blastomere death. In vitro produced and in vivo derived blastocysts showed a significant incidence of cell death in the inner cell mass (ICM), but only in part with morphological features of apoptosis. Importantly, transcripts for CASP3, CASP9, CASP8 and FAS/FASLG were not detectable or found at very low abundances. In vitro and in vivo, errors and failures of the first and the next three cleavage divisions frequently cause immediate embryo death or lead to aberrant subsequent development, and are the main source of developmental heterogeneity. A substantial occurrence of cell death in the ICM even in fast developing blastocysts strongly suggests a regular developmentally controlled elimination of cells, while the nature and mechanisms of ICM cell death are unclear. Morphological findings as well as transcript levels measured for important apoptosis-related genes are in conflict with the view that classical caspase-mediated apoptosis is the major cause of cell death in early bovine development

Discovery of a novel oocyte-specific KRAb-containing zinc finger protein required for early embryogenesis in cattle

Much of the loss of potential offspring in cattle is concentrated in the early embryonic period. Maternal mRNAs that accumulate in the oocyte during oogenesis have important functional roles during the initial stages of embryonic development, before embryonic genome activation. It is well regarded that the oocyte plays an active role in regulation of key aspects of the reproductive process required for fertility. What is more, oocyte-specific transcription factors seem to be the controlling feature influencing germ cell success throughout oogenesis, fertilization, and early embryonic development. Of the remarkably diverse array of transcription factors encoded by mammalian genomes, about two-thirds encode C2H2 zinc-finger proteins.;Zinc finger proteins exclusively expressed in mammalian oocytes have not been reported. Deep sequencing of a bovine oocyte library revealed a highly abundant transcript that matches an uncharacterized gene in the NCBI database. cDNA cloning of the novel ZNFO gene revealed a transcript containing a 2,145 bp open reading frame that codes for a protein of 714 amino acids with a conserved KRAB domain at the N-terminus and nine zinc finger motifs at the C-terminus. The individual ZNF motifs fit the conserved two cysteine-two histidine sequence model. ZNFO mRNA was detectable in fetal ovaries and was undetectable in all somatic tissues analyzed, including granulosa and theca cells. Real-time PCR analysis revealed ZNFO mRNA is highly abundant in GV and MII stage oocytes as well as in pronuclear to 8-cell stage embryos but undetectable in blastocyst stage embryos. Immunohistochemical analysis detected ZNFO protein in oocytes throughout folliculogenesis. Identification and characterization revealed the novel ZNFO is a KRAB-containing maternal-effect gene found exclusively in bovine oocytes.;To elucidate the functional role of ZNFO, zygotes were generated by in vitro maturation and fertilization of oocytes and injected with small interfering RNA (siRNA) designed to knockdown ZNFO. Cleavage rates were not affected by ZNFO siRNA injection. However, embryonic development to 8- to 16-cell stage and blastocyst stage was reduced significantly relative to the uninjected and negative control siRNA-injected embryos. Furthermore, interaction of ZNFO with the highly conserved transcriptional repressor co-factor (KAP1) was demonstrated by GST pull-down, and evidence supporting transcriptional repression by ZNFO using a GAL4-luciferase assay. In addition, transfection studies verified that a ZNFO-GFP fusion protein localizes specifically to the nucleus, further supporting the proposed function in transcriptional regulation. These studies demonstrate that ZNFO is a maternally-derived oocyte-specific factor required for early embryonic development in cattle, which has a functional role as a transcriptional regulator required during early embryogenesis by repressing transcription, possibly controlling activation of the embryonic genome

STUDY OF THE IMPACT OF MICRONUTRIENT FOUND IN SPERAMAX® DURING PREGNANCY PERIODS ON EMBRYONIC DEVELOPMENT AND NEWBORN

Objective: This paper aims at evaluating the benefits of vitamins and minerals found in the Speramax® supplement and the risks to mother and infants of additional supplementation and possible adverse interactions between micronutrients in pregnancy.Method: A total of 30 male and 120 female albinos Swiss mice of 8–12 weeks of age weighing 25–35 g were used. Speramax was administrated orally for 1, 2, and 4 weeks. Fertile female mice were classified into four main groups: Group 1 is spontaneously (SPO); Group 2 is administrated with SperamaxÒ only; Group 3 is treated SperamaxÒ with superovulation (SUO); and Group 4 is superovulated only without Speramax.Result: The results indicated that treatment with Speramax® showed a positive effect on neonatal development and an increase in the number of newborn SPO and SUO treated with Speramax® after 1 week and 2 weeks. The results showed high significance (p˂0.000) compared with SPO and SUO mice not treated with Speramax® and with groups treated for 4 weeks.Conclusion: Good nutrition found in Speramax® may, therefore, be especially important to this group of infants and must be instituted alongside other nutritional supplements

Genome Editing Approach To Uncover Microtubule-Actin Crosslinking Factor (macf1) Essential Domains In Establishing Oocyte Polarity And Nuclear Positioning

The totipotent egg of most vertebrates is polarized in a so called animal-vegetal (AV) axis that is crucial for early embryonic development. AV polarity is established during early oogenesis through the formation and disassembly of the Balbiani Body (Bb) at the vegetal pole. The Bb is a non-membrane bound large mRNP granule, conserved from insects to humans and composed of mitochondria, RNAs and proteins. The Bb components, which include germ cell determinants, anchor to the vegetal cortex upon Bb dissociation in late stage I oocytes. Importantly, Bb dissociation at the oocyte cortex defines the future vegetal pole of the egg. Our lab discovered in zebrafish the only genes known to function in AV polarity formation in vertebrates: bucky ball and macf1. On one hand, Bucky ball is required for Bb formation, and is thought to act by the formation of amyloid-like fibers that capture Bb components. On the other hand, Macf1 is crucial for Bb dissociation. Macf1 is a conserved and giant multi-domain cytoskeletal linker protein that can interact with microtubules (MTs), actin filaments (AF) and intermediate filaments (IF). Macf1 is the only factor known to regulate Bb dissociation, however the Macf1 and cytoskeleton-dependent mechanism by which Macf1 regulates Bb mRNP granule dissociation and, thus, defines AV polarity in the egg is unknown. Here, we unravel Macf1 function via interrogating, for the first time, individual macf1-encoded domains from its endogenous locus to determine their requirement in Bb dissociation and ultimately in egg polarity establishment. Our results show that the Macf1 actin binding domain is essential for Bb dissociation, whereas the Macf1 plakin repeat domain, which interacts with IF, is dispensable for Macf1 function in this context. The method presented here is applicable to other cytolinkers involved in human diseases

ELUCIDATION OF THE ROLE OF AGOUTI-SIGNALING PROTEIN THROUGHOUT FOLLICULOGENESIS AND EARLY EMBRYONIC DEVELOPMENT IN CATTLE

The oocyte expresses certain genes during folliculogenesis to regulate the acquisition of oocyte competence. Oocyte competence, which refers to the presence of imperative molecular factors in the oocyte that are critical for high oocyte quality, is directly related to the ability of the oocyte to result in a successful pregnancy following fertilization. Over the past few decades, the development and optimization of assisted reproductive technologies, particularly in vitrofertilization, have enabled the beef and dairy industries to advance cattle genetics and productivity. However, only approximately 40% of bovine embryos will develop to the blastocyst stage in vitro. In addition, bovine embryos produced in vitro are developmentally inferior compared to in vivo derived embryos due to the lack of optimization of the oocyte and embryo culture conditions in vitro. Characterization of factors regulating these processes is crucial to improve the efficiency of bovine in vitro embryo production. RNA Sequencing data obtained by our laboratory demonstrated that the secreted protein, agouti-signaling protein (ASIP), is highly abundant in the bovine oocyte. Agouti-signaling protein (ASIP) has a characterized role in the distribution of melanin pigment in some mammalian species, including mice. In adipose tissue, ASIP expression is associated with insulin resistance and obesity. Recently, it was demonstrated that ASIP is crucial in regulating mammary epithelial cell lipid metabolism in cattle. However, the role of ASIP in the bovine oocyte and early embryo has not been previously elucidated. This research aimed to characterize the ASIP spatiotemporal expression profile in the ovary and throughout early embryonic development. Further, objectives included revealing the effects of supplementation of ASIP during in vitro oocyte maturation and embryo culture on subsequent embryonic development. In addition to oocyte expression, ASIP was detected in granulosa, cumulus, and theca cells isolated from antral follicles. Both ASIP mRNA and protein were found to decline with oocyte maturation, suggesting a prospective role for ASIP in achieving oocyte competence. Microinjection of presumptive zygotes using small interfering RNAs targeting ASIP led to a 13% reduction in the rate of development to the blastocyst stage. Additionally, we examined potential ASIP signaling mechanisms through which ASIP may function to establish oocyte developmental competence. Expression of melanocortin receptors 3 and 4 and the coreceptor attractin was detected in the oocyte and follicular cells. Interestingly, the addition of cortisol, which was previously determined to be beneficial for oocyte competence in cattle, during in vitro maturation significantly increased oocyte ASIP levels. Cumulus-oocyte complexes or presumptive zygotes were placed in culture medium containing either 0, 1, 10, or 100 ng/mL of recombinant ASIP, and effects on subsequent development, gene expression, lipid content, and blastocyst cell allocation were examined. Supplementation of ASIP during oocyte maturation improved the blastocyst development rate and produced blastocysts with an increased inner cell mass to trophectoderm cell ratio. Nile red staining revealed that adding ASIP during oocyte maturation increased oocyte but not embryo lipid levels. The expression of genes involved in lipid metabolism, including FASN, PPAR, SCD, CSL1, ELOVL5, and ELOVL6, were not found to be significantly altered in blastocysts due to treatment. Meanwhile, supplementation of ASIP during embryo culture did not affect blastocyst rates. These results support a functional role for ASIP in promoting oocyte maturation and subsequent embryonic development, potentially through signaling mechanisms involving cortisol. Additionally, these data further support the role of ASIP in acquiring oocyte competence and suggest that supplementing ASIP during oocyte maturation may lead to the production of blastocysts of increased quality. Future prospective applications of this work include optimizing bovine oocyte or embryo culture conditions to emulate better the in vivo maternal environment through normalizing lipid metabolism and, subsequently, minimizing stress. Further, future research should explore the utilization of ASIP in developing improved cryopreservation techniques for bovine embryos

High-throughput analysis reveals novel maternal germline RNAs crucial for primordial germ cell preservation and proper migration

During oogenesis, hundreds of maternal RNAs are selectively localized to the animal or vegetal pole, including determinants of somatic and germline fates. Although microarray analysis has identified localized determinants, it is not comprehensive and is limited to known transcripts. Here, we utilized high-throughput RNA sequencing analysis to comprehensively interrogate animal and vegetal pole RNAs in the fully grown Xenopus laevis oocyte. We identified 411 (198 annotated) and 27 (15 annotated) enriched mRNAs at the vegetal and animal pole, respectively. Ninety were novel mRNAs over 4-fold enriched at the vegetal pole and six were over 10-fold enriched at the animal pole. Unlike mRNAs, microRNAs were not asymmetrically distributed. Whole-mount in situ hybridization confirmed that all 17 selected mRNAs were localized. Biological function and network analysis of vegetally enriched transcripts identified protein-modifying enzymes, receptors, ligands, RNA-binding proteins, transcription factors and co-factors with five defining hubs linking 47 genes in a network. Initial functional studies of maternal vegetally localized mRNAs show that sox7 plays a novel and important role in primordial germ cell (PGC) development and that ephrinB1 (efnb1) is required for proper PGC migration. We propose potential pathways operating at the vegetal pole that highlight where future investigations might be most fruitful.Fil: Owens, Dawn A.. University of Miami; Estados UnidosFil: Butler, Amanda M.. University of Miami; Estados UnidosFil: Agüero, Tristán Horacio. University of Miami; Estados Unidos. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Investigaciones Biológicas. Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas; ArgentinaFil: Newman, Karen M.. University of Miami; Estados UnidosFil: Van Booven, Derek. University of Miami; Estados UnidosFil: King, Mary Lou. University of Miami; Estados Unido

Ovarian inflammation mediated by Toll-like receptor 4 increased transcripts of maternal effect genes and decreased embryo development

Obese women are subfertile and have reduced assisted reproduction success, which may be due to reduced oocyte competence. We hypothesize that consumption of a high-fat/high-sugar diet induces ovarian inflammation, which is a primary contributor to decreased oocyte quality and pre-implantation embryo development. To test this hypothesis, C57BL/6 (B6) mice with a normal inflammatory response and C3H/HeJ (C3H) mice with a dampened inflammatory response due to dysfunctional Toll-like receptor 4 were fed either normal chow or high-fat/high-sugar diet. In both B6 and C3H females, high-fat/high-sugar diet induced excessive adiposity and hyperglycemia compared to normal chow-fed counterparts. Conversely, ovarian CD68 levels and oocyte expression of oxidative stress markers were increased when collected from B6 high-fat/ high-sugar but not C3H high-fat/high-sugar mice. Following in vitro fertilization of in vivo matured oocytes, blastocyst development was decreased in B6-high-fat/high-sugar but not C3H high-fat/high-sugar mice. Expression of cumulus cell markers of oocyte quality were altered in both B6 high-fat/high-sugar and C3H high-fat/high-sugar. However, there were no diet-dependent differences in spindle abnormalities in either B6 or C3H mice, suggesting potential defects in cytoplasmic maturation. Indeed, there were significant increases in the abundance of maternal effect gene mRNAs in oocytes from only B6 high-fat/high-sugar mice. These differentially expressed genes encode proteins of the subcortical maternal complex and associated with mRNA metabolism and epigenetic modifications. These genes regulate maternal mRNA degradation at oocyte maturation, mRNA clearance at the zygotic genome activation, and methylation of imprinted genes suggesting a mechanism by which inflammation induced oxidative stress impairs embryo development