Zona Pellucida Proteins, Fibrils, and Matrix.

The zona pellucida (ZP) is an extracellular matrix that surrounds all mammalian oocytes, eggs, and early embryos and plays vital roles during oogenesis, fertilization, and preimplantation development. The ZP is composed of three or four glycosylated proteins, ZP1-4, that are synthesized, processed, secreted, and assembled into long, cross-linked fibrils by growing oocytes. ZP proteins have an immunoglobulin-like three-dimensional structure and a ZP domain that consists of two subdomains, ZP-N and ZP-C, with ZP-N of ZP2 and ZP3 required for fibril assembly. A ZP2-ZP3 dimer is located periodically along ZP fibrils that are cross-linked by ZP1, a protein with a proline-rich N terminus. Fibrils in the inner and outer regions of the ZP are oriented perpendicular and parallel to the oolemma, respectively, giving the ZP a multilayered appearance. Upon fertilization of eggs, modification of ZP2 and ZP3 results in changes in the ZP's physical and biological properties that have important consequences. Certain structural features of ZP proteins suggest that they may be amyloid-like proteins.

The Birth of the 3D Genome during Early Embryonic Development.

The 3D structure of chromatin in the nucleus is important for the regulation of gene expression and the correct deployment of developmental programs. The differentiation of germ cells and early embryonic development (when the zygotic genome is activated and transcription is taking place for the first time) are accompanied by dramatic changes in gene expression and the epigenetic landscape. Recent studies used Hi-C to investigate the 3D chromatin organization during these developmental transitions, uncovering remarkable remodeling of the 3D genome. Here, we highlight the changes described so far and discuss some of the implications that these findings have for our understanding of the mechanisms and functionality of 3D chromatin architecture.

Blastulation of a zygote to a hatched blastocyst without any clear cell division: an observational finding in a time-lapse system after in vitro fertilization.

PURPOSE: To describe an interesting not previously described morphokinetic finding. METHODS: Retrospective case report of a couple undergoing controlled ovarian stimulation (COS) followed by in vitro fertilization and blastocyst transfer. RESULTS: We identified a unique finding of blastulation of a fertilized human zygote after conventional in vitro fertilization. The fertilized zygote did not show any clear cytokinesis until approximately 107 h post insemination, when it started dividing into a blastocyst. By 113 h post insemination, inner cell mass and trophectoderm cells could be clearly distinguished and the blastocyst was completely hatched by 136 h post insemination. CONCLUSION: Time-lapse systems offer more detailed observations of embryonic development. Here, we report an atypical development of an embryo that was not described previously. We hope to become an insightful discussion among peers and incentive the publication of such findings in the future.

Removing the zona pellucida can decrease cytoplasmic fragmentations in human embryos: a pilot study using 3PN embryos and time-lapse cinematography.

PURPOSE: The aim of this study was to establish a new method of decreasing cytoplasmic fragmentation in early-stage human embryos. METHODS: The zona pellucida (ZP) of abnormally-fertilized oocytes (zygotes with three pronuclei (3PN)), which were donated by patients, was removed at the pronuclear stage. ZP-free embryos were observed in a time-lapse imaging and culturing system in order to examine developmental morphology and embryonic quality. RESULTS: Based on a modification of Veeck's criteria, 47 of 69 ZP-free 3PN embryos (68.1%) showed fragmentation of less than 20% of the total volume of cytoplasm at the first cleavage (grades 1 and 2), 17 (24.6%) showed 20-40% cytoplasmic fragments (grade 3), and only 5 (7.2%) showed more than 40% fragments (grade 4). These results suggest that the rate of fragmentation is decreased by ZP removal before the first cleavage, compared with normal (ZP-intact) 3PN and 2-pronuclear/2-polar body embryos. CONCLUSIONS: This study revealed that the ZP is not always necessary for normal development after the pronuclear stage because the ZP-free embryos studied herein developed normally, maintained their cell adhesion well, and showed a decreased rate of fragmentation. This innovative culture system might provide the major breakthrough needed for patients who have difficulty obtaining good-quality embryos.

Mitochondrial depolarization in yeast zygotes inhibits clonal expansion of selfish mtDNA.

Non-identical copies of mitochondrial DNA (mtDNA) compete with each other within a cell and the ultimate variant of mtDNA present depends on their relative replication rates. Using yeast Saccharomyces cerevisiae cells as a model, we studied the effects of mitochondrial inhibitors on the competition between wild-type mtDNA and mutant selfish mtDNA in heteroplasmic zygotes. We found that decreasing mitochondrial transmembrane potential by adding uncouplers or valinomycin changes the competition outcomes in favor of the wild-type mtDNA. This effect was significantly lower in cells with disrupted mitochondria fission or repression of the autophagy-related genes ATG8, ATG32 or ATG33, implying that heteroplasmic zygotes activate mitochondrial degradation in response to the depolarization. Moreover, the rate of mitochondrially targeted GFP turnover was higher in zygotes treated with uncoupler than in haploid cells or untreated zygotes. Finally, we showed that vacuoles of zygotes with uncoupler-activated autophagy contained DNA. Taken together, our data demonstrate that mitochondrial depolarization inhibits clonal expansion of selfish mtDNA and this effect depends on mitochondrial fission and autophagy. These observations suggest an activation of mitochondria quality control mechanisms in heteroplasmic yeast zygotes.

Could monopronucleated ICSI zygotes be considered for transfer? Analysis through time-lapse monitoring and PGS.

PURPOSE: The purpose of this study was to investigate the chromosomal constitution and the developmental potential of intracytoplasmic sperm injection (ICSI) deriving embryos displaying a single pronucleus at the zygote stage. METHODS: Eighty-eight embryos from single pronucleus (1PN) two polar bodies (2PB) ICSI zygotes from 64 preimplantational genetic screening (PGS) cycles (October 2012-December 2014), were retrospectively analyzed. Zygotes were cultured in a time-lapse incubator. Embryo biopsy was performed on day 3 and genetic analysis approached by array comparative genomic hybridization. RESULTS: Chromosomal analysis revealed that 17% (15/88) of embryos derived from 1PN 2PB zygotes were diagnosed as euploid. After blastomere biopsy at day 3, the blastocyst rate at day 5 was 3.4% (3/88). Only 2.3% (2/88) euploid blastocysts were obtained. In two couples and after counseling and patient agreement, the transfer of a euploid blastocyst from a 1PN 2PB ICSI zygote was performed resulting in the birth of a healthy child. CONCLUSIONS: These results open the possibility to consider embryos coming from 1PN 2PB ICSI zygotes for transfer when no other embryos from 2PN 2PB ICSI zygotes are available and if a PGS diagnosis of euploidy is obtained. Confirmation of biparental inheritance is strongly recommended.

Time-lapse observation and transcriptome analysis of a case with repeated multiple pronuclei after IVF/ICSI.

PURPOSE: The purpose of this study was to investigate the cause of repeated multipronucleus (MPN) formation in zygotes in a patient after both in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI). METHOD: This is a case study. A patient had unexplained primary infertility with recurring total MPN zygotes after IVF and ICSI cycles. Time-lapse monitoring of pronucleus formation was carried out. Embryos developed from MPN zygotes were analyzed by fluorescence in situ hybridization (FISH). Single-cell RNA-seq analysis was used to identify gene expression profiles of the patient's oocyte and zygote, and these were compared to the data from oocytes and zygotes from donors with normal fertilization (patient, n = 1; donors, n = 4). Oocyte-specific genes with differential expression were selected by the Amazonia! RESULTS: From time-lapse analysis, we observed the formation of multiple micronuclei near the site of the second polar body extrusion. These micronuclei migrated, expanded, and juxtaposed with the male pronucleus leading to a multipronucleus. None of these MPN zygotes could develop to the blastocyst stage, and FISH analysis revealed a chaotic chromosomal complement in the arrested embryos. RNA-seq analysis showed 113 differentially expressed genes (DEGs) between the patient and the donor oocytes and zygotes. Moreover, 25 of the 113 DEGs were unique or highly expressed in oocytes and early embryos. From 25 DEGs, three genes, DYNC2LI1, NEK2, and CCNH, which are involved in meiosis and the chromosome separation process, were further validated by real-time PCR. CONCLUSION: We identified several candidate genes affecting pronucleus formation as a new cause of infertility.

Spermatozoa telomeres determine telomere length in early embryos and offspring.

Offspring telomere length (TL) has been correlated with paternal TL, but the mechanism for this parent of origin-specific inheritance remains unclear. The objective of this study has been to determine the role of spermatozoa TL in embryonic telomere lengthening by using two mouse models showing dimorphism in their spermatozoa TL: Mus musculus vs Mus spretus and old vs young Mus musculus. Mus spretus spermatozoa displayed a shorter TL than Mus musculus. Hybrid offspring exhibited lower TL compared with Mus musculus starting at the two-cell stage, before the onset of telomerase expression. To analyze the role of spermatozoa telomeres in early telomere lengthening, we compared the TL in oocytes, zygotes, two-cell embryos and blastocysts produced by parthenogenesis or by fertilization with Mus musculus or Mus spretus spermatozoa. TL was significantly higher in spermatozoa compared with oocytes, and it increased significantly from the oocyte to the zygote stage in those embryos fertilized with Mus musculus spermatozoa, but not in those fertilized with Mus spretus spermatozoa or produced by parthenogenesis. A further increase was noted from the zygote to the two-cell stage in fertilized Mus musculus embryos, whereas hybrid embryos maintained the oocyte TL. Spermatozoa TL shortened with age in Mus musculus and the offspring from young males showed a significantly higher TL compared with that fathered by old males. These significant differences were already noticeable at the two-cell stage. These results suggest that spermatozoa telomeres act as a guide for telomerase-independent telomere lengthening resulting in differences in TL that persist after birth.

Stella controls chromocenter formation through regulation of Daxx expression in 2-cell embryos.

In mammals, the structure of the pericentromeric region alters from a ring structure to a dot-like structure during the 2-cell stage. This structural alteration is termed chromocenter formation (CF) and is required for preimplantation development. Although reverse transcripts of major satellite repeats at pericentromeric regions are known to play roles in CF, its underlying mechanism is not fully understood. We previously reported that Stella (also known as PGC7 and Dppa3) deficiency led to developmental arrest at the preimplantation stage, accompanied by frequent chromosome segregation. In this study, we further investigated the effect of Stella deficiency on chromatin reorganization. The Stella-null embryos exhibited impaired CF and reduced expression of the reverse strand of major satellite repeats. In addition, the accumulation of H3.3, a histone H3 variant associated with transcriptional activation, at the pericentromeric regions and expression of the H3.3-specific chaperone Daxx were reduced in Stella-null embryos. These abnormalities were restored by the enforced expression of Daxx in Stella-null embryos. Thus, Stella controls the expression of Daxx and ensures chromatin reorganization in early embryos.

Structural analysis of embryogenesis of Leiarius marmoratus (Siluriformes: Pimelodidae).

Embryological studies in fish species are useful to the understanding of their biology and systematics. The available biological data in Leiarius marmoratus are scarce and additional information about its reproductive biology is needed, mainly because this species has been commercially exploited and used in production of hybrid lineages. In order to evaluate the temporal-morphological embryonic modifications in L. marmoratus, samples of nearly 200 embryos were collected at random at different stages of development, starting from fecundation (time zero). Embryos were fixed in modified Karnovsk's solution and 2.5% glutaraldehyde, processed and analysed under optic and electron microscopy. The incubation period of L. marmoratus was equal to 14.42 h at a mean temperature of 28.3 ± 0.07°C. The following stages of embryonic development were established: zygote, cleavage, gastrula, organogenesis and hatching. These stages were divided into phases, as follows: cleavage - phases of 2, 4, 8, 16, 32 and 64 cells and morula; gastrula - phases of 25, 50, 75 and 90% of epiboly and blastopore closure; and organogenesis - neurula, segmentation and pre-larval phases. The embryogenesis of L. marmoratus was typical of neotropical teleosteans, with peculiarities in species development.

[ANONCHOCEPHALUS OLIGORHIS SP. NOV. (CESTODA: BOTHRIOCEPHALIDEA) FROM OPHIDIFORMES FISHES OF THE ATLANTIC OCEAN].

In presence article new species of genus Anonchocephalus Riggenbach Lühe, 1902 parasitazing on Hoplobrotula gnathopus Regan, 1921 from the south-eastern part of Atlan- tic Ocean is described. The morphometric analysis of species of genus Anonchocephalus is made, a description of new species is provided with emended of generic diagnosis.

Morphokinetic parameters of ICSI tripronucleated embryos observed using time lapse.

Time-lapse analysis of tripronucleated zygotes obtained in ICSI cycles showed that 75.4% cleaved into embryos. These embryos subsequently demonstrated slower developmental kinetics than normally fertilized embryos.

Envelope surface ultrastructure and specific gravity of artificially fertilized Pacific cod Gadus macrocephalus eggs.

The envelope surface ultrastructure and specific gravity of artificially fertilized eggs of the Pacific cod Gadus macrocephalus were examined. The unfertilized, demersal and slightly adhesive eggs of G. macrocephalus were almost spherical and had no oil globules. Wrinkled envelope surface with elaborated hexagonal reticulated patterns and type I micropyle were observed under a scanning electron microscope. The adhesiveness of the eggs was lost at the blastodermal-cap stage after fertilization. The micropylar canal was sealed by secretion of the perivitelline fluid, and the entire surface became rough. Numerous bacilli were deposited at the micropyle and the outer envelope surface at the late germ-ring stage and at the embryo five-eighths around the yolk stage. The micropyle was completely deformed at the embryo seven-eighths around the yolk stage. The specific gravity of the fertilized G. macrocephalus eggs ranged from c. 1·0316 to 1·0454. These values, however, sharply decreased towards the end stages of egg development to produce pelagic larvae. The ultrastructural changes in the micropyle and envelope surface of the G. macrocephalus eggs protected the embryo from microorganism infections and mechanical stress during the long incubation period. The adhesiveness and specific gravity of the eggs influenced their dispersion potential.

Irregularly calcified eggs and eggshells of Caiman latirostris (Alligatoridae: Crocodylia).

We describe irregularly calcified egg and eggshell morphologies for the first time in nests of the broad-snouted caiman, Caiman latirostris. Research is based on detailed descriptions of 270 eggs from a total sample of 46,800 collected between 2005 and 2011 in Santa Fe Province, Argentina, and encompasses animals from both natural habitats and held in captivity. We discuss possible reasons for the occurrence of eggs with different mineralisation patterns in our extensive C. latirostris field sample and its conservation significance; the chemistry of egg laying in amniotes is sensitive to environmental contamination which, in turn, has biological implications. Based on our egg sample, we identify two caiman eggshell abnormalities: (1) regularly calcified eggs with either calcitic nodules or superficial wrinkles at one egg end and (2) irregularly calcified eggs with structural gaps that weaken the shell. Some recently laid clutches we examined included eggs with most of the shell broken and detached from the flexible membrane. Most type 1 regularly calcified eggs lost their initial calcified nodules during incubation, suggesting that these deposits do not affect embryo survival rates. In contrast, irregularly calcified caiman eggs have a mean hatching success rate of 8.9% (range 0-38%) across our sample compared to a mean normal success of 75%. Most irregularly calcified caiman eggs probably die because of infections caused by fungi and bacteria in the organic nest material, although another possible explanation that merits further investigation could be an increase in permeability, leading to embryo dehydration.

Development, preimaginal phases and adult sensillar equipment in Aganaspis parasitoids (Hymenoptera: Figitidae) of fruit flies.

Aganaspis daci and Aganaspis pelleranoi (Hymenoptera: Figitidae) are important parasitoids of fruit flies. Here we studied, with light and scanning electron microscopy, aspects of their morphology that could help with plans to mass rear and thus contribute to improved pest control (preimaginal phases) and to shed light on parasitoid-pest relationships (sensillar equipment). The two species present a stalked egg, eucoiliform first and second-instar larvae and hymenopteriform third instar and mature larvae. The first instar presents tegumental differentiations in the mesoma and first metasomal segment in A. daci, but not in A. pelleranoi, while unlike other figitids, neither species displays setae in the mesosomal processes. Second and third instar and mature larvae present tegumental differentiations in A. daci, but not in A. pelleranoi. The moniliform (female) and filiform (male) antennae of A. daci and A. pelleranoi harbor seven types of sensilla, four of them (sensilla campaniformia, sensilla coeloconica type II, and two types of sensilla trichoidea) described here for the first time in Cynipoidea. The largest sensilla were the multiporous placoid sensilla, which were smaller and more numerous in A. pelleranoi. Species also differed to some extent in morphology of sensilla coeloconica. Observations on the ovipositor revealed the presence of coeloconic sensilla on Valva I in both species.

Nematode eggshells: A new anatomical and terminological framework, with a critical review of relevant literature and suggested guidelines for the interpretation and reporting of eggshell imagery.

A literature review for a recent ultrastructural study of a trichinelloid eggshell revealed consistently occurring errors in the literature on nematode eggshell anatomy. Examples included nematodes of medical, veterinary, and agricultural importance in several orders. Previous researchers had warned of some of these errors decades ago, but a comprehensive solution was not offered until 2012 when a clarifying new anatomical and developmental interpretation of nematode eggshells was proposed by members of the Caenorhabditis elegans Research Community. However, their findings were explained using arcane acronyms and technical jargon intended for an audience of experimental molecular geneticists, and so their papers have rarely been cited outside the C. elegans community. Herein we (1) provide a critical review of nematode eggshell literature in which we correct errors and relabel imagery in important historical reports; (2) describe common reporting errors and their causes using language familiar to researchers having a basic understanding of microscopy and nematode eggs; (3) recommend a new hexalaminar anatomical and terminological framework for nematode eggshells based on the 2012 C. elegans framework; and (4) recommend new unambiguous terms appropriate for the embryonated/larvated eggs regularly encountered by practicing nematodologists to replace ambiguous or ontogenetically restricted terms in the 2012 C. elegans framework. We also (5) propose a resolution to conflicting claims made by the C. elegans team versus classical literature regarding Layer #3, (6) extend the C. elegans hexalaminar framework to include the polar plugs of trichinelloids, and (7) report new findings regarding trichinelloid eggshell structure.

Title: La coque des œufs des nématodes : un nouveau cadre anatomique et terminologique, avec une revue critique de la littérature pertinente et des lignes directrices suggérées pour l'interprétation et la communication de l'imagerie des coques des œufs. Abstract: Une revue de la littérature pour une étude ultrastructurale récente de la coque de l'œuf d'un trichinelloïde a révélé des erreurs récurrentes dans la littérature sur l'anatomie de la coque de l'œuf des nématodes. Les exemples comprenaient des nématodes d'importance médicale, vétérinaire et agricole dans plusieurs ordres. Des chercheurs avaient mis en garde contre certaines de ces erreurs il y a des décennies, mais une solution complète n'a été proposée qu'en 2012, lorsqu'une nouvelle interprétation anatomique et développementale clarifiant la structure des coques des œufs de nématodes a été proposée par des membres de la communauté de recherche de Caenorhabditis elegans. Cependant, leurs découvertes ont été expliquées à l'aide d'acronymes mystérieux et d'un jargon technique destiné à un public de généticiens moléculaires expérimentaux, et leurs articles ont donc rarement été cités en dehors de la communauté de C. elegans. Ici, nous (1) fournissons une revue critique de la littérature sur les coques des œufs de nématodes dans laquelle nous corrigeons les erreurs et réétiquetons les images dans des rapports historiques importants; (2) décrivons les erreurs de description courantes et leurs causes en utilisant un langage familier aux chercheurs ayant une compréhension de base de la microscopie et des œufs de nématodes; (3) recommandons un nouveau cadre anatomique et terminologique hexalaminaire pour les coques des œufs de nématodes basé sur le cadre de C. elegans de 2012; et (4) recommandons de nouveaux termes non ambigus appropriés pour les œufs embryonnés/larvés régulièrement rencontrés par les spécialistes de nématodes en exercice pour remplacer les termes ambigus ou à restriction ontogénétique dans le cadre de C. elegans de 2012. Nous proposons également (5) une résolution des affirmations contradictoires de l'équipe C. elegans par rapport à la littérature classique concernant la couche 3, (6) étendons le cadre hexalaminaire de C. elegans pour inclure les bouchons polaires des trichinelloïdes, et (7) signalons de nouvelles découvertes concernant la structure de la coque des œufs des trichinelloïdes.

Unique pattern of ORC2 and MCM7 localization during DNA replication licensing in the mouse zygote.

In eukaryotes, DNA synthesis is preceded by licensing of replication origins. We examined the subcellular localization of two licensing proteins, ORC2 and MCM7, in the mouse zygotes and two-cell embryos. In somatic cells ORC2 remains bound to DNA replication origins throughout the cell cycle, while MCM7 is one of the last proteins to bind to the licensing complex. We found that MCM7 but not ORC2 was bound to DNA in metaphase II oocytes and remained associated with the DNA until S-phase. Shortly after fertilization, ORC2 was detectable at the metaphase II spindle poles and then between the separating chromosomes. Neither protein was present in the sperm cell at fertilization. As the sperm head decondensed, MCM7 was bound to DNA, but no ORC2 was seen. By 4 h after fertilization, both pronuclei contained DNA bound ORC2 and MCM7. As expected, during S-phase of the first zygotic cell cycle, MCM7 was released from the DNA, but ORC2 remained bound. During zygotic mitosis, ORC2 again localized first to the spindle poles, then to the area between the separating chromosomes. ORC2 then formed a ring around the developing two-cell nuclei before entering the nucleus. Only soluble MCM7 was present in the G2 pronuclei, but by zygotic metaphase it was bound to DNA, again apparently before ORC2. In G1 of the two-cell stage, both nuclei had salt-resistant ORC2 and MCM7. These data suggest that licensing follows a unique pattern in the early zygote that differs from what has been described for other mammalian cells that have been studied.

From embryo sac to oil and protein bodies: embryo development in the model legume Medicago truncatula.

• The cell and developmental biology of zygotic embryogenesis in the model legume Medicago truncatula has received little attention. We studied M. truncatula embryogenesis from embryo sac until cotyledon maturation, including oil and protein body biogenesis. • We characterized embryo development using light and electron microscopy, measurement of protein and lipid fatty acid accumulation and by profiling the expression of key seed storage genes. • Embryo sac development in M. truncatula is of the Polygonum type. A distinctive multicellular hypophysis and suspensor develops before the globular stage and by the early cotyledon stage, the procambium connects the developing apical meristems. In the storage parenchyma of cotyledons, ovoid oil bodies surround protein bodies and the plasma membrane. Four major lipid fatty acids accumulate as cotyledons develop, paralleling the expression of OLEOSIN and the storage protein genes, VICILIN and LEGUMIN. • Zygotic embryogenesis in M. truncatula features the development of a distinctive multicellular hypophysis and an endopolyploid suspensor with basal transfer cell. A clear procambial connection between the apical meristems is evident and there is a characteristic arrangement of oil bodies in the cotyledons and radicle. Our data help link embryogenesis to the genetic regulation of oil and protein body biogenesis in legume seed.

Egg activation in physiological polyspermy.

Fertilization is indispensable not only for restoring diploid genomes but also for the initiation of early embryonic cell cycles in sexual reproduction. While most animals exhibit monospermy, which is ensured by polyspermy blocks to prevent the entry of extra sperm into the egg at fertilization, several animals exhibit physiological polyspermy, in which the entry of several sperm is permitted but only one sperm nucleus participates in the formation of a zygote nucleus. Polyspermy requires that the sperm transmit the egg activation signal more slowly, thus allowing the egg to accept several sperm. An increase in intracellular Ca(2+) concentration induced by the fertilizing sperm is both necessary and sufficient for egg activation in polyspermy. Multiple small Ca(2+) waves induced by several fertilizing sperm result in a long-lasting Ca(2+) rise, which is a characteristic of polyspermic amphibian eggs. We introduced a novel soluble sperm factor for egg activation, sperm-specific citrate synthase, into polyspermic newt eggs to cause Ca(2+) waves. Citrate synthase may perform dual functions: as an enzyme in mitochondria and as a Ca(2+)-inducing factor in egg cytoplasm. We also discuss the close relationship between the mode of fertilization and the Ca(2+) rise at egg activation and consider changes in this process through evolution in vertebrates.