A method to quantitate maternal transcripts localized in sea urchin egg cortex by RT-qPCR with accurate normalization

The sea urchin egg cortex is a peripheral region of eggs consisting of cell membrane and adjacent cytoplasm, which contains actin and tubulin cytoskeleton, cortical granules and some proteins required for early development. Method for isolation of cortices from sea urchin eggs and early embryos has been developed in 70s of 20th Century. Since that time this method has been reliable tool to study protein localization and cytoskeletal organization in cortex of unfertilized eggs and embryos during first cleavages. This study is an estimation of reliability of RT-qPCR to analyze levels of maternal transcripts that are localized in egg cortex. Firstly, we selected seven potential reference genes, 28S, Cycb , Ebr1 , GAPDH , Hmg1 , Smtnl1 and Ubb , which transcripts are maternally deposited in sea urchin eggs. The candidate reference genes were ranked by five different algorithms (BestKeeper, CV, ΔCt, geNorm and NormFinder) upon calculated level stability in both eggs and isolated cortices. Our results show that gene ranking differs in total RNA and mRNA samples, though Ubb is most suitable reference gene in both cases. To validate feasibility of comparative analysis of eggs and isolated egg cortices by RT-qPCR, we selected Daglb-2 as a gene of interest, which transcripts potentially localized in cortex, and found increased level of Daglb -2 in egg cortices. This suggests that proposed RNA isolation method with subsequent quantitative RT-qPCR analysis can be used to approve cortical association of transcripts in sea urchin eggs.

A Quantitative Ultrastructural Study of Oocytes During the Early Stages of Ovarian Follicular Development in Booroola and Wild-Type Sheep

<p>In the sheep ovary, primordial follicles are formed as an oocyte surrounded by either a single layer of flattened granulosa cells (type 1) or a mixture of flattened and cuboidal granulosa cells (type 1a). Booroola sheep have a mutation in the growth factor receptor, activin-like kinase receptor 6 (ALK6) which is expressed in oocytes of follicles at the type 1 stage of development. In Booroola ewes homozygous for the ALK6 mutation (BB), oocytes undergo precocious maturation that appears to be initiated during the preantral growth phase. The aim of this study was to quantify the ultrastructural features of oocytes of ovarian follicles at the types 1/1a, 2 and 3 stages of development, from BB and wild-type (++) ewes. Ovaries from 6 ++ and 5 BB 4 week old ewe lambs were processed for both light microscopy (LM) and electron microscopy (EM). LM and stereological methods were used to estimate the mean volume of the oocytes of each follicular type and genotype. EM images and point counting or linear intercept counting were used to estimate the volume of smooth endoplasmic reticulum (SER), Golgi, mitochondria, vesicles, lipid, ribosomes, zona pellucida (ZP) and cortical granules (CG), and the surface area of the outer and inner mictochondrial membranes, microvilli and cell junctions. Oocytes of type 1/1a follicles of BB animals had a greater diameter than that in ++ animals (BB: 29.76 ± 0.58 μm vs ++: 27.05 ± 0.30 μm; P < 0.01) but there were no significant genotype differences in the oocyte diameters of type 2 or 3 follicles. As the follicles of ++ animals developed from the type 1 through to the type 3 stage, the volume and 3 surface areas of all sub-cellular structures measured within oocytes increased (P < 0.05). In oocytes of type 1/1a follicles of BB animals, the SER, mitochondria and ZP volumes were greater than in ++ animals (P < 0.05) as were the surface areas of the outer mitochondrial membranes, oocyte membrane, and zonula adherens type junctions (P < 0.05). At the type 2 stage of development the lipid volume was greater in oocytes of ++ animals, and at the type 3 stage of development the ribosomal volume was greater in oocytes of BB animals. These results suggest that the ALK6 mutation in BB animals has influenced the ultrastructural properties of oocytes in the type 1/1a follicle. This early genotype difference in follicular characteristics may influence the rate of follicular development during the early developmental stages.</p>

A Quantitative Ultrastructural Study of Oocytes During the Early Stages of Ovarian Follicular Development in Booroola and Wild-Type Sheep

<p>In the sheep ovary, primordial follicles are formed as an oocyte surrounded by either a single layer of flattened granulosa cells (type 1) or a mixture of flattened and cuboidal granulosa cells (type 1a). Booroola sheep have a mutation in the growth factor receptor, activin-like kinase receptor 6 (ALK6) which is expressed in oocytes of follicles at the type 1 stage of development. In Booroola ewes homozygous for the ALK6 mutation (BB), oocytes undergo precocious maturation that appears to be initiated during the preantral growth phase. The aim of this study was to quantify the ultrastructural features of oocytes of ovarian follicles at the types 1/1a, 2 and 3 stages of development, from BB and wild-type (++) ewes. Ovaries from 6 ++ and 5 BB 4 week old ewe lambs were processed for both light microscopy (LM) and electron microscopy (EM). LM and stereological methods were used to estimate the mean volume of the oocytes of each follicular type and genotype. EM images and point counting or linear intercept counting were used to estimate the volume of smooth endoplasmic reticulum (SER), Golgi, mitochondria, vesicles, lipid, ribosomes, zona pellucida (ZP) and cortical granules (CG), and the surface area of the outer and inner mictochondrial membranes, microvilli and cell junctions. Oocytes of type 1/1a follicles of BB animals had a greater diameter than that in ++ animals (BB: 29.76 ± 0.58 μm vs ++: 27.05 ± 0.30 μm; P < 0.01) but there were no significant genotype differences in the oocyte diameters of type 2 or 3 follicles. As the follicles of ++ animals developed from the type 1 through to the type 3 stage, the volume and 3 surface areas of all sub-cellular structures measured within oocytes increased (P < 0.05). In oocytes of type 1/1a follicles of BB animals, the SER, mitochondria and ZP volumes were greater than in ++ animals (P < 0.05) as were the surface areas of the outer mitochondrial membranes, oocyte membrane, and zonula adherens type junctions (P < 0.05). At the type 2 stage of development the lipid volume was greater in oocytes of ++ animals, and at the type 3 stage of development the ribosomal volume was greater in oocytes of BB animals. These results suggest that the ALK6 mutation in BB animals has influenced the ultrastructural properties of oocytes in the type 1/1a follicle. This early genotype difference in follicular characteristics may influence the rate of follicular development during the early developmental stages.</p>

Knockin’ on Egg’s Door: Maternal Control of Egg Activation That Influences Cortical Granule Exocytosis in Animal Species

Fertilization by multiple sperm leads to lethal chromosomal number abnormalities, failed embryo development, and miscarriage. In some vertebrate and invertebrate eggs, the so-called cortical reaction contributes to their activation and prevents polyspermy during fertilization. This process involves biogenesis, redistribution, and subsequent accumulation of cortical granules (CGs) at the female gamete cortex during oogenesis. CGs are oocyte- and egg-specific secretory vesicles whose content is discharged during fertilization to block polyspermy. Here, we summarize the molecular mechanisms controlling critical aspects of CG biology prior to and after the gametes interaction. This allows to block polyspermy and provide protection to the developing embryo. We also examine how CGs form and are spatially redistributed during oogenesis. During egg activation, CG exocytosis (CGE) and content release are triggered by increases in intracellular calcium and relies on the function of maternally-loaded proteins. We also discuss how mutations in these factors impact CG dynamics, providing unprecedented models to investigate the genetic program executing fertilization. We further explore the phylogenetic distribution of maternal proteins and signaling pathways contributing to CGE and egg activation. We conclude that many important biological questions and genotype–phenotype relationships during fertilization remain unresolved, and therefore, novel molecular players of CG biology need to be discovered. Future functional and image-based studies are expected to elucidate the identity of genetic candidates and components of the molecular machinery involved in the egg activation. This, will open new therapeutic avenues for treating infertility in humans.

New Contributions to the Diversity of Hypotrichous Ciliates: Description of a New Genus and Two New Species (Protozoa, Ciliophora)

Ciliated protists (ciliates) are extremely diverse and play important ecological roles in almost all kinds of habitats. In this study, two new hypotrichs, Wilbertophrya sinica n. g. and n. sp. and Bakuella xianensis n. sp., from China are investigated. Wilbertophrya n. g. can be separated from related genera mainly by the combination of lacking a buccal cirrus, pretransverse cirri, and caudal cirri, while possessing frontoterminal cirri. Analyses based on morphological and molecular data confirm the validity of the species, W. sinica n. sp., which is characterized as follows: body 50–115 μm × 15–35 μm in vivo; midventral complex comprises four or five cirral pairs only and terminates above mid-body; three frontal, two frontoterminal cirri, and two to four transverse cirri; about 15 macronuclear nodules; colorless cortical granules sparsely distributed. Another new species, B. xianensis n. sp., was isolated from a freshwater wetland and is defined as follows: body 115–150 μm × 40–65 μm in vivo; about 70 macronuclear nodules; dark-brownish cortical granules in groups; midventral complex comprises 8–12 cirral pairs forming a row that terminates posteriorly in mid-body region and two or three short midventral rows that are continuous with the row of midventral pairs; three frontal, four to six frontoterminal, and three to five fine transverse cirri; three bipolar dorsal kineties. Phylogenetic analyses based on small subunit ribosomal DNA (SSU rDNA) sequence data suggest that the new genus Wilbertophrya n. g. belongs to an isolated clade, which might represent an undescribed taxon at the family level, whereas B. xianensis n. sp. groups with several congeners and members of other related genera are within the core urostylids.

P–280 Changes in oolemma height during ICSI injection on day 0 is associated with day 5–6 blastocyst formation

Study question Does the oolemma response to ICSI injection on day 0 affect blastocyst formation on day 5–6 (d5/6)? Summary answer A large change in oolemma height during ICSI injection on day 0 is associated with lower blastocyst formation rates on d5/6. What is known already The oolemma changes in all dimensions (i.e. height, width and depth) and can exhibit different reactions in ICSI during needle injection. This is seen as instant rupture or with little needle pressure, normal rupture with the needle pushed approximately halfway through, or difficult rupture with repeated attempts or the needle passing 3/4 of the oocyte width. Previous studies have shown that these responses can affect degeneration and fertilisation rates on day 1, however, there is little research on its effect on blastocyst formation rates. Furthermore, most previous studies have used qualitative methods to assess oolemma response. Study design, size, duration This is a retrospective study using ICSI procedure videos conducted by four embryologists in a private clinic from 2013–2015. All videos of procedures which did not result in 2PN or in which the oocyte was not fully visible were excluded. Six operators categorised 455 videos (by majority vote) into four groups based on the oolemma response: oolemma breakage within 1/4, between 1/4 and 1/2, between 1/2 and 3/4 and beyond 3/4 of the oocyte’s width. Participants/materials, setting, methods A U-Net neural network model was trained to extract the frame of maximum oolemma indent from each video which were validated by a human operator; any in which maximum indent occured after breaking of the oolemma were excluded. The ratio of starting to maximal indent width/height were calculated automatically and human-validated. Chi-squared tests were performed for each ratio vs d5/6 blastocyst formation. These results were compared with those obtained from purely human annotations. Main results and the role of chance From the purely human annotations, the percentages of oocytes in groups 1–4 respectively were: 3.3%, 85.3%, 11.4% and 0%. This variation in oolemma response may be due to the arrangement of thick and thin microfilaments or cortical granules in the cytoskeleton. When analysed with d5/6 blastocyst formation, these showed no significant result (p = 0.12) which is consistent with findings using the model. The artificial intelligence (AI) model processed 26 frames per second. During human validation of the ratios calculated at maximal indentation, 36% of width ratios and 31% of height ratios were rejected. The proportion of blastocysts formed in the upper and lower quartile for each ratio was analysed. Both the upper (0.49) and lower (0.41) quartiles of the width ratios were not significant for d5/6 blastocyst formation. The lower (1.12) quartile of height ratios showed no significance, however there were significantly fewer blastocysts formed on d5/6 for the upper (1.18) quartile of height ratios (p &lt; 0.025). This subtle change in the height ratio, which was significant for d5/6 blastocyst formation was not taken into account when grouping oocytes any previous literature (and our human labelling). Limitations, reasons for caution This study was conducted at a single clinic so variations between clinics were not captured in the study and would need further collaborations to confirm the proportion of oocytes responses. Due to the small sample size, this study also did not identify any group 4 oocytes cultured until d5/6. Wider implications of the findings: The grouping criteria in this study were more quantitative than previous work yet indicated no correlation between the oolemma group and d5/6 blastocyst formation. However, changes in the height which are hard to assess in real-time (and which have been neglected in previous literature) were seen to be significant. Trial registration number NA

P–230 The NAD+ precursor nicotinamide riboside protects against postovulatary aging in vitro

Study question Can nicotinamide riboside, one of the NAD+ precursor, protect against postovulatary aging in vitro? Summary answer The NAD+ precursor nicotinamide riboside can protect against postovulatary aging in vitro. What is known already Postovulatory aging(POA) has been considered one of the most intractable challenges that limit the successful rate of ART. Multiple cellular and molecular changes have been involved during the process of POA. The NAD+ is a prominent redox cofactor which is indispensable to DNA repair, energy metabolism, autophagy, genomic stability as well as epigenetic homeostasis. Over the last several decades, increasingly studies have eported that NAD+ contents decline with age across multiple tissues and loss of it are implicated in various diseases associated to aging. As one of a precursor of NAD+, nicotinamide riboside play important role in regulating oxidative stress. Study design, size, duration In this study, we take advantage of in vitro aging model to explore the influences of NR administration on the postovulatory aged oocytes in mice. We analyzed the association of NR supplementation with the aging-related deterioration of oocyte quality, such as mitochondrial dysfunction, mislocalization of cortical granules, followed by embryonic development potential and the NAD+/SIRT1 signaling. We used 3582 oocytes totally. Participants/materials, setting, methods CD–1 mice oocytes/ in vitro culture/ UPLC-MS/MS for NAD+ contents measurement ,DCFH-DA staining for ROS detecting, γH2AX staining for DNA damage measurement, BODIPY FL ATP staining for ATP detecting, LCA-FITC staining to assess the distribution and dynamics of cortical granules (CGs), In vitro fertilization, Quantitative real time PCR Main results and the role of chance NR supplementation exerted protective effects on morphological defects of oocytes, and that these protective effects were concentration dependent. We detected a significantly decline in NAD+ levels in aging oocytes, however, NAD+ accumulation was present in aging oocytes after 200μM NR treatment, indicating that NR administration might be a feasible strategy to enhance the quality of aging oocytes. Furthermore, NR indeed elevated the embryonic development potential of POA oocytes after fertilization. Our findings revealed that NR administration effectively ameliorated ROS accumulation in POA oocytes.Then we tried to uncover the effects of NR on the meiotic apparatus in aging oocytes. NR supplementation can partially restores normal spindle assembly and chromosome alignment in postovulatory aging oocytes. Furthermore, we investigated the protective effects of NR on mitochondrial function through following expects: mitochondrial distribution, ATP production and mitochondrial membrane potential. NR treatment could promote mitochondrial function in oocytes during postovulatory aging in vitro. In addition, DNA damage and mislocalized CGs during postovulatory aging might be rescued by the supplementation of NR. Based on these evidences, we identified that NR improved the quality of aging oocytes by NAD+/SIRT1 axis. Limitations, reasons for caution Only in vitro, shown only in mice. Wider implications of the findings: Our work represents a clinically effective pharmacological chemical to improve infertility caused by POA process. Further studies are needed to define the related mechanisms of NR supplementation on oocyte quality as well as reproductive outcomes clinically. Trial registration number N

Luteolin Orchestrates Porcine Oocyte Meiotic Progression by Maintaining Organelle Dynamics Under Oxidative Stress

Increasing evidence has demonstrated that oxidative stress impairs oocyte maturation, but the underlying mechanisms remain largely unknown. Here, for the first time, we examined the antioxidant role of luteolin in meiotic progression and the underlying mechanisms. Supplementation of 5 μM luteolin increased the rates of first polar body extrusion and blastocyst formation after parthenogenetic activation, and the expression levels of oocyte competence (BMP15 and GDF9)-, mitogen-activated protein kinase (MOS)-, and maturation promoting factor (CDK1 and Cyclin B)-related genes were also improved. Luteolin supplementation decreased intracellular reactive oxygen species levels and increased the expression levels of oxidative stress-related genes (SOD1, SOD2, and CAT). Interestingly, luteolin alleviated defects in cell organelles, including actin filaments, the spindle, mitochondria, the endoplasmic reticulum, and cortical granules, caused by H2O2 exposure. Moreover, luteolin significantly improved the developmental competence of in vitro-fertilized embryos in terms of the cleavage rate, blastocyst formation rate, cell number, cellular survival rate, and gene expression and markedly restored the competencies decreased by H2O2 treatment. These findings revealed that luteolin supplementation during in vitro maturation improves porcine meiotic progression and subsequent embryonic development by protecting various organelle dynamics against oxidative stress, potentially increasing our understanding of the underlying mechanisms governing the relationship between oxidative stress and the meiotic events required for successful oocyte maturation.

Exposure to Copper Compromises the Maturational Competency of Porcine Oocytes by Impairing Mitochondrial Function

Copper (Cu) is an essential trace element for animals, and also an important nutritional component for the normal physiology and metabolism of animal reproductive systems. An excess or lack of Cu will directly or indirectly affect animal reproductive activities. However, the effect of Cu, in particular excessive Cu, on the reproductive performance of sows has not been studied. Here, we report that excessive Cu had negative effects on oocyte maturation and organelle functions. We showed that Cu exposure perturbed porcine oocyte meiotic maturation and impaired spindle/chromosome structure, resulting in a defective spindle assembly, as well as the abnormal distribution of actin dynamics and cortical granules. In addition, single-cell transcriptome analysis identified the target effectors of Cu actions in porcine oocytes, further demonstrating that Cu exposure affects the mitochondrial distribution and function, leading to the high levels of reactive oxygen species, DNA damage, and early apoptosis of porcine oocytes. These findings demonstrate that Cu exposure causes abnormalities in the mitochondrial distribution and function, resulting in the increased oxidative stress and levels of reactive oxygen species, DNA damage, and apoptosis, ultimately leading to a decreased porcine oocyte quality.

Antarctic population of Anteholosticha sigmoidea (Foissner, 1982) Berger, 2003 (Ciliophora: Urostylidae) with notes on its phylogenetic position

Anteholosticha sigmoidea (Foissner, 1982) Berger, 2003 was isolated from a wet soil sample collected on King George Island, Antarctica. Morphological observations and molecular phylogenetic analyses based on the gene sequences of small subunit ribosomal RNA (18S rRNA) were used to identify the species. Anteholosticha sigmoidea can be divided into two groups: group I (three populations described by Foissner 1982) and group II (described by Foissner 1984) based on the morphological differences. Group I differs from group II by the length of the midventral complex (65.1% vs. 52.5% of the cell length), the number of adoral membranelles (25–28 vs. 16–24), and the number of dorsal bristles in kinety 1 (16 bristles vs. nine bristles). Group I differs from the Antarctica population by the absence/presence of the collecting canals of the contractile vacuole and the number of macronuclear nodules (6–12 vs. 13–19). Group II differs from the Antarctica population by the number of macronuclear nodules (five to nine vs. 13–19); the arrangement of cortical granules (forming longitudinal rows vs. irregularly distributed); the length of the midventral complex (64.7% vs. 53.8% of cell length). In the phylogenetic analyses, A. sigmoidea was not nested with any species, and the gene tree indicated polyphyly of the genus Anteholosticha.