The abnormal expression of lncRNAs and miRNAs has been found in the placentas of patients with preeclampsia (PE). Therefore, we determined the role of lncRNA FOXD2-AS1/miR-3127 in trophoblast cells. The expression of lncRNA FOXD2-AS1 was detected by qRT-PCR. The proliferation, migration and invasion ability of trophoblast cells were evaluated using CCK-8, wound healing and transwell assays. The target gene of lncRNA FOXD2-AS1 was determined by StarBase and luciferase reporter assays. Western blotting was used to analyze the expression of matrix metalloproteinase 2 (MMP2) and matrix metalloproteinase 9 (MMP9). The results showed that FOXD2-AS1 affected trophoblast cell viability in vitro, while the expression of miR-3127 was decreased. FOXD2-AS1 silencing decreased the promotion effects on trophoblast cell induced by miR-3127 inhibition. In addition, FOXD2-AS1 and miR-3127 presented the same effect on MMP2 and MMP9 levels. lncRNA FOXD2-AS1 modulated trophoblast cell proliferation, invasion and migration through downregulating miR-3127 expression. Therefore, lncRNA FOXD2-AS1 could act as a latent therapeutic marker in preeclampsia.

As meiosis is initiated and the oogonium is transformed into a primary oocyte, the female germ cell becomes intimately invested by a single squamous layer of sex cord epithelium. As the follicle cell population expands during the initial stages of the ovarian cycle, oocyte and follicle cells become increasingly connected to one another by one of the most extensive populations of gap junctions documented in any epithelium (reviewed in Larsen & Wert, 1988).

Cryopreservation of mammal embryos has been technically feasible for many years, but morphological injuries still persist in fish embryos during cryopreservation. Thus, the objective of the present study was to describe these freezing injuries in Piaractus mesopotamicus embryos. Two hundred and twenty-five embryos were collected at the post-gastrula stage and assigned into four treatments of sucrose at 8.5, 17.0, 25.0 or 34.0% plus 9.0% methanol. The control was prepared with distilled water only. The gradual decrease in the temperature was 0.5°C/min. After the seeding stage, the fish embryos were stored in liquid nitrogen at −33°C. Thereafter, they were thawed for evaluating per cent hatching, and the samples collected from every treatment were submitted to scanning electron microscopy for morphological analysis. The micrographic images showed that there was substantial alterations in embryo morphology under the highest concentrations of sucrose. These solutions did not prevent the formation of ice crystals, which lead to deformities and killed the embryos, but the observed reduced level of morphological structure in these embryos when treated with 17.0% sucrose plus 9.0% methanol is a compelling argument for additional studies.

The objective of the present study was to describe ultrastructural changes in the nucleus and cytoplasmic organelles during in vitro maturation (IVM) of buffalo cumulus–oocyte complexes (COCs). The structures were collected by ovum pick-up (OPU). Some COCs, removed from maturation medium at 0, 6, 12, 18 and 24 h, were processed for transmission electron microscopy. The average number of COCs collected by OPU/animal/session was 6.4, and 44% of them were viable. Immature oocytes had a peripherally located nucleus, Golgi complex and mitochondrial clusters, as well as a large number of coalescent lipid vacuoles. After 6 h of IVM, the oocyte nucleus morphology changed from round to a flatter shape, and the granulosa cells (GC) lost most of their contact with zona pellucida (ZP). At 12 h the first polar body was extruded and the aspect of lipid droplet changed to dark, probably denoting lipid oxidation. Cortical granules were clearly visible at 18 h of maturation, always located along the oocyte periphery. At 24 h of IVM the number of cortical granules increased. Ultrastructure studies revealed that: (1) immature oocytes have a high lipid content; (2) the perivitelline space (PS) increases during IVM; (3) Golgi complexes and mitochondrial clusters migrate to oocyte periphery during IVM; (4) 6 h of IVM are enough to lose contact between GC and ZP; (5) the oocyte lipid droplets’ appearance changes between 6 and 12 h of IVM.

Initiation of sperm motility in urodeles, which is induced by a sperm motility-initiating substance (SMIS) in the sequestered granules on the surface of egg jelly, is mediated by the acrosome reaction (AR), which is triggered by an AR-inducing substance (ARIS) on a sheet-like structure. Details of the unique process of the interaction between egg jelly and sperm in these species is still unclear. The current study showed the fine structure of egg jelly in the newt Cynops pyrrhogaster, a urodele species, revealing that its outer surface was covered by a sheet-like structure of approximately 0.29 μm in thickness. Granules of approximately 2 μm in diameter with small particles of approximately 54 nm were attached to its surface and distributed inhomogeneously just beneath the sheet-like structure. Emission spectrometry revealed that the Ca2+ concentration was maintained at a high level compared with that of the blood plasma and the vas deferens fluid, suggesting that egg jelly is a reliable source of Ca2+ for the sperm–egg interaction. Blockers of the T-type voltage-dependent Ca2+ channel (VDCC), but not the L-type VDCC, inhibited both AR and initiation of sperm motility. Conversely, Ni+, which affects the α1 H subunit of T-type VDCC, only inhibited the initiation of sperm motility. These data suggest that, in response to ARIS and SMIS, sequential gating of distinct Ca2+ channels occurs in the AR, followed by the initiation of sperm motility on the surface of the egg jelly in C. pyrrhogaster at fertilization.

Low-density lipoproteins (LDL) is known to protect boar sperm during freezing–thawing, but little information is known about the effects of LDL extracted from different avian egg yolks on post-thaw boar semen quality. The purpose of this study was to compare and analyze the effects of LDL at various concentrations and different species on boar sperm quality after freezing–thawing. LDL extracted from the yolk of hen egg, duck egg, quail egg, pigeon egg or ostrich egg was added to the extender at the concentrations of 0.06, 0.07, 0.08, 0.09 and 0.1 g/ml, respectively, and their effects on frozen–thawed boar sperm quality were assessed. According to all measured parameters, the results showed that sperm motility, acrosome integrity and plasma membrane integrity were 43.20%, 52.57% and 48.13%, respectively, after being frozen–thawed with 0.09 g/ml LDL extracted from pigeon egg yolk. All these quality parameters were higher than that of other groups (P < 0.05). In conclusion, our results confirmed that LDL extracted from pigeon egg yolk had the best cryoprotective effects on frozen–thawed boar sperm among all of the groups supplemented with LDL from five kinds of avian egg in extender. The optimum concentration of LDL extracted from pigeon egg in boar semen freezing extender was 0.09 g/ml.

The addition of growth factors and vitamins enhances goat embryonic development in vitro. However, few attempts have been reported trying to identify supplementation regimens for oocyte maturation or embryo culture with additive properties. The present report was aimed to evaluate if retinoids [0.3 μM retinyl acetate (RAc) and 0.5 μM 9-cis-retinoic acid (RA)] supplementation during goat oocyte maturation and retinoids and/or 50 ng mL–1 IGF-I during embryo culture synergically enhanced embryonic development while diminishing the incidence of apoptosis. All combinations of RAc and RA treatment produced blastocysts with similar efficiencies, while IGF-I enhanced embryos yields irrespectively of retinoid addition. Moreover, retinoids and IGF-I supplementation showed similar caspase activity or DNA fragmentation indexes in blastocysts. In conclusion, supplementation with retinoids and IGF-I during goat embryo culture enhances blastocysts development without synergic reduction of apoptosis.

Ion-sensitive fluorophores are commonly used for quantitative measurements of intracellular ion concentrations. However, both the method of intracellular loading — which for many fluorophores involves endogenous esterase-mediated removal of hydrophobic groups such as acetoxymethyl esters (AM) — and fluorescence excitation of fluorophores in the cell, can produce toxic metabolites and reactive species. Techniques used to measure intracellular ion concentrations in mammalian eggs and embryos are being increasingly employed, yet little information is available about any detrimental effects of the use of fluorophores. We have therefore used in vitro fertilisation (IVF) to assess potential fluorophore toxicity in mouse eggs, and whole cell patch-clamp recordings to detect fluorophore-associated membrane damage in zygotes. Four fluorophores were examined: SNARF-1 and BCECF (pH indicators), Fura-2 (Ca2+) and MQAE (Cl−). Cleavage of AM groups alone had no effect either on the success of IVF or on membrane electrical properties of mouse zygotes. Intracellularly loaded BCECF, SNARF-1 and Fura-2 followed by fluorescence excitation were not cell-toxic under the conditions examined. In contrast, MQAE demonstrated significant toxicity both alone and in combination with fluorescence excitation.

Unequal cleavage requires a highly organised cytoskeleton. We investigated the localisation of both tubulins and microtubular arrays in Dreissena eggs during and after fertilisation using confocal laser scanning microscopy. Freshly spawned eggs are arrested in metaphase I. A maternal pool of γ-tubulin is found mainly in the centre of the asters of the meiotic spindle. The paternal pool of γ-tubulin, present in the fertilising sperm, could not be traced within the egg, but a microtubule-organising centre forms near the male pronucleus at anaphase II. Male and female pronuclei grow as they migrate in the wake of their aster and rendezvous. First cleavage is unequal and starts without pronuclear fusion. At metaphase the two equal-sized asters span the entire egg in a symmetrical arrangement. At late metaphase the spindle shifts along its longitudinal axis into an eccentric position and the peripheral aster takes on an umbrella-like appearance, whereas the central aster remains spherical. The cleavage furrow becomes determined in the circumferential overlap of the asters. The inequality at second cleavage, however, is due to the unequal size of the asters. The third cleavage spindle also has asymmetrical asters and spindle shift was only observed in the D-cell. The spiral character is a result of an asymmetrical organisation of the larger, vegetal aster. Our results show that the arrangement of the γ-tubulin clusters and of microtubules changes and develops during early development of Dreissena in a way that can explain the axis-generating asymmetries in cell pattern and the spiral sense of cleavage. The major cytological characters expected to direct pattern formation in this phase of development are: size, position, and symmetry or asymmetry of both spindle and asters.

Fusion of nucleoli or nucleolus precursor bodies (NPBs) has been observed during somatic cell interphase and pronuclear development of human zygotes; however, the underlying mechanism is unknown. NPB fusion and its regulation by mitogen-activated protein kinase (MAPK) and maturation-promoting factor (MPF) were studied in activated mouse oocytes. Small NPBs appeared about 4 h after ethanol activation, and took about 1.5 h to fuse into a large NPB, which persisted for about 10 h before disappearance. Analysis of the temporal windows for kinase action indicated that a high MAPK activity during the first 2 h and a low MPF activity during the first 3–4 h after activation were essential for subsequent NPB fusion. A preactivation decline in MAPK activity was associated with decreased NPB fusion following activation of aged oocytes. While MAPK inactivation by regulator U0126 prevented NPB fusion in oocytes activated by ethanol or 5 min Sr2+ treatments, it had no effect on oocytes fertilized or activated by 6 h Sr2+ treatment. In most cases, while rates of pronuclear formation did not differ, rates of NPB fusion differed significantly between different treatments. Our results suggest that: (i) the MAPK and MPF activities at the initial stage of activation regulate NPB fusion after pronuclear formation; (ii) pronuclear assembly and NPB fusion are two separable events that might be controlled by different mechanisms; and (iii) high MAPK activity and low MPF activity at the initial stage of activation is essential for NPB fusion when only one calcium rise is induced by ethanol, while inhibition of MAPK activity does not affect NPB fusion when the repetitive intracellular Ca2+ rises are induced after fertilization.

Emerging organic contaminants have been monitored in stream waters, raw and finished waters and wastewater effluents. Most of these contaminants, such as epigenetic substances, have been detected at very low levels. Unfortunately, their complete monitoring and/or removal are very difficult, given the increasing presence of new contaminants and due to analytical and economic considerations. For this reason, bioindicators are used as an alternative to monitor their presence. To this end, zebrafish is being used to assess certain contaminants in water quality studies.

As our long-term aim is to determine if zebrafish (Danio rerio) can be used to detect environmental epigenetic factors in drinking waters with effects on human reproduction, an initial question is whether the chorion could interfere with the possible action of epigenetic factors in two reproductive events: genital ridge formation and migration of the primordial germ cells (PGCs) to these genital ridges.

In the first experiment, we attempted to partially degrade the chorion of mid blastula transition (MBT) embryos with pronase, with acceptable survival rates at 5 days post fertilisation (dpf), with the group exposed for 15 min giving the best survival results. As denuded early embryos require a specific culture medium, in the next experiment embryo survival was evaluated when they were cultured up to 5 dpf in drinking waters from six different sources. Results showed a negative effect on embryo survival at 5 dpf from several waters but not in others, thus distorting the survival outcomes. These results suggest using embryos with the chorion intact from the outset when drinking waters from different sources are to be tested.

It has been demonstrated in Bufo arenarum that fully grown oocytes are capable of meiotic resumption in the absence of a hormonal stimulus if they are deprived of their follicular envelopes. This event, called spontaneous maturation, only takes place in oocytes collected during the reproductive period, which have a metabolically mature cytoplasm.

In Bufo arenarum, progesterone acts on the oocyte surface and causes modifications in the activities of important enzymes, such as a decrease in the activity of adenylate cyclase (AC) and the activation of phospholipase C (PLC). PLC activation leads to the formation of diacylglycerol (DAG) and inositol triphosphate (IP3), second messengers that activate protein kinase C (PKC) and cause an increase in intracellular Ca2+. Recent data obtained from Bufo arenarum show that progesterone-induced maturation causes significant modifications in the level and composition of neutral lipids and phospholipids of whole fully grown ovarian oocytes and of enriched fractions in the plasma membrane. In amphibians, the luteinizing hormone (LH) is responsible for meiosis resumption through the induction of progesterone production by follicular cells.

The aim of this work was to study the importance of gap junctions in the spontaneous and LH-induced maturation in Bufo arenarum oocytes. During the reproductive period, Bufo arenarum oocytes are capable of undergoing spontaneous maturation in a similar way to mammalian oocytes while, during the non-reproductive period, they exhibit the behaviour that is characteristic of amphibian oocytes, requiring progesterone stimulation for meiotic resumption (incapable oocytes).

This different ability to mature spontaneously is coincident with differences in the amount and composition of the phospholipids in the oocyte membranes. Capable oocytes exhibit in their membranes higher quantities of phospholipids than incapable oocytes, especially of PC and PI, which are precursors of second messengers such as DAG and IP3.

The uncoupling of the gap junctions with 1-octanol or halothane fails to induce maturation in follicles from the non-reproductive period, whose oocytes are incapable of maturing spontaneously. However, if the treatment is performed during the reproductive period, with oocytes capable of undergoing spontaneous maturation, meiosis resumption occurs in high percentages, similar to those obtained by manual defolliculation.

Interestingly, results show that LH is capable of inducing GVBD in both incapable oocytes and in oocytes capable of maturing spontaneously as long as follicle cells are present, which would imply the need for a communication pathway between the oocyte and the follicle cells. This possibility was analysed by combining LH treatment with uncoupling agents such as 1-octanol or halothane. Results show that maturation induction with LH requires a cell–cell coupling, as the uncoupling of the gap junctions decreases GVBD percentages. Experiments with LH in the presence of heparin, BAPTA/AM and theophylline suggest that the hormone could induce GVBD by means of the passage of IP3 or Ca2+ through the gap junctions, which would increase the Ca2+ level in the oocyte cytoplasm and activate phosphodiesterase (PDE), thus contributing to the decrease in cAMP levels and allowing meiosis resumption.

The objective of the study was to investigate the effect of l-ergothioneine (l-erg) (5 mM or 10 mM) supplementation in maturation medium on the developmental potential and OCTN1-dependant l-erg-mediated (10 mM) change in mRNA abundance of apoptotic (Bcl2, Bax, Casp3 and PCNA) and antioxidant (GPx, SOD1, SOD2 and CAT) genes in sheep oocytes and developmental stages of embryos produced in vitro. Oocytes matured with l-erg (10 mM) reduced their embryo toxicity by decreasing intracellular ROS and increasing intracellular GSH in matured oocytes that in turn improved developmental potential, resulting in significantly (P < 0.05) higher percentages of cleavage (53.72% vs 38.86, 46.56%), morulae (34.36% vs 20.62, 25.84%) and blastocysts (14.83% vs 6.98, 9.26%) compared with other lower concentrations (0 mM and 5 mM) of l-erg without change in maturation rate. l-Erg (10 mM) treatment did not influence the mRNA abundance of the majority of apoptotic and antioxidant genes studied in the matured oocytes and developmental stages of embryo. A gene expression study found that the SLC22A4 gene that encodes OCTN1, an integral membrane protein and specific transporter of l-erg was not expressed in oocytes and developmental stages of embryos. Therefore it was concluded from the study that although there was improvement in the developmental potential of sheep embryos by l-erg supplementation in maturation medium, there was no change in the expression of the majority of the genes studied due to the absence of the SLC22A4 gene in oocytes and embryos that encode OCTN1, which is responsible for transportation of l-erg across the membrane to alter gene expression.

Intracytoplasmic sperm injection (ICSI) is an important technique in male infertility treatment. Currently, sperm selection for ICSI in human assisted reproductive technology (ART) is subjective, based on a visual assessment by the operator. Therefore, it is desirable to develop methods that can objectively provide an accurate assessment of the shape and size of sperm heads that use low-magnification microscopy available in most standard fertility clinics. Recent studies have shown a correlation between sperm head size and shape and chromosomal abnormalities, and fertilization rate, and various attempts have been made to establish automated computer-based measurement of the sperm head itself. For example, a dictionary-learning technique and a deep-learning-based method have both been developed. Recently, an automatic algorithm was reported that detects sperm head malformations in real time for selection of the best sperm for ICSI. These data suggest that a real-time sperm selection system for use in ICSI is necessary. Moreover, these systems should incorporate inverted microscopes (×400–600 magnification) but not the fluorescence microscopy techniques often used for a dictionary-learning technique and a deep-learning-based method. These advances are expected to improve future success rates of ARTs. In this review, we summarize recent reports on the assessment of sperm head shape, size, and acrosome status in relation to fertility, and propose further improvements that can be made to the ARTs used in infertility treatments.

The asteroidal sperm-activating peptides (asterosaps) from the egg jelly bind to their sperm receptor, a membrane-bound guanylate cyclase, on the tail to activate sperm in sea stars. Asterosaps are produced as single peptides and then cleaved into shorter peptides. Sperm activation is followed by the acrosome reaction, which is subfamily specific. In order to investigate the molecular details of the asterosap–receptor interaction, corresponding cDNAs have been cloned, sequenced and analysed from the Asteriinae subfamily including Asterias amurensis, A. rubens, A. forbesi and Aphelasterias japonica, as well as Distolasterias nipon from the Coscinasteriinae subfamily. Averages of 29% and 86% identity were found from the deduced amino acid sequences in asterosap and its receptor extracellular domains, respectively, across all species examined. The phylogenic tree topology for asterosap and its receptor was similar to that of the mitochondrial cytochrome c oxidase subunit I. In spite of a certain homology, the amino acid sequences exhibited speciation. Conservation was found in the asterosap residues involved in disulphide bonding and proteinase-cleaving sites. Conversely, similarities were detected between potential asterosap-binding sites and the structure of the atrial natriuretic peptide receptor. Although the sperm-activating peptide and its receptor share certain common sequences, they may serve as barriers that ensure speciation in the sea star A. amurensis and closely related species.

Information on the dynamics of gamete interaction in marsupials is very limited and not available for any species from the major Australian Order Diprotodontia which includes most of the more familiar animals such as kangaroos, possums and the koala. This study addressed this deficiency by examining the ultrastructure of in vivo fertilised eggs from common brushtail possums (Trichosurus vulpecula). Females were superovulated by treatment with 15 IU PMSG and then 4 mg porcine LH 3 days later, and inseminations were performed 910-13 h after LH) using epididymal spermatozoa. Between 33 and 39 h after LH injection females were killed, reproductive tracts excised and the oviduct ampulla segment flushed for eggs. Three of the six eggs examined were fertilised as judged by the presence of sperm remnants in the cytoplasm. On the basis of these eggs it was found that sperm penetration left a large hole in the zona pellucida (ZP), suggesting that sperm zona penetration occurs primarily by the enzymatic action of acrosomal enzymes. Sperm lying within the perivitelline space were lacking both an outer acrosomal membrane and the associated acrosomal contents, while both these structures were found on sperm embedded within the mucoid layer, which is consistent with induction of the acrosome reaction by binding to the ZP. Once inside the egg cytoplasm, the sperm head travelled only a short distance before chromatin decondensation occurred. Fertilised eggs showed signs of cytoplasmic activation including cytoskeleton association with apparently dividing mitochondria and prominent rough endoplasmic reticulum. Unfertilised eggs appeared to be undergoing degenerative changes and lacked any evidence of activation. This study has demonstrated that superovulation and laparoscopic intravaginal artificial insemination provide a system through which perifertilisation events in the possum and other monovular Australian marsupials can be examined experimentally.

The conditions for transfer of human mitochondria into fertilised mouse ova were elaborated. Species-specific primers were designed to discriminate human mitochondrial DNA (mtDNA) and the endogenous mtDNA in the preimplantation embryos. Human mitochondria isolated from the HepG2 cell line were microinjected into murine zygotes, and the latter cultured for 96 h to the blastocyst stage. The polymerase chain reaction allowed the detection of human mtDNA at every stage of embryo cleavage. In some cases a clear disparity in distribution of human mtDNA among blastomeres was evident.

Global warming (GW) and ocean acidification (OA) have been recognized as severe threats for reef-building corals that support coral reef ecosystems, but these effects on the early life history stage of corals are relatively unknown compared with the effects on calcification of adult corals. In this study, we evaluated the effects of thermal stress and CO2-driven acidified seawater on fertilization in a reef-building coral, Acropora digitifera. The fertilization rates of A. digitifera decreased in response to thermal stress compared with those under normal seawater conditions. In contrast, the changes of fertilization rates were not evident in the acidified seawater. Generalized Linear Mixed Model (GLMM) predicted that sperm/egg crosses and temperature were explanatory variables in the best-fitted model for the fertilization data. In the best model, interactions between thermal stress and acidified seawater on the fertilization rates were not selected. Our results suggested that coral fertilization is more sensitive to future GW than OA. Taking into consideration the previous finding that sperm motility of A. digitifera was decreased by acidified seawater, the decrease in coral cover followed by that of sperm concentration might cause the interacting effects of GW and OA on coral fertilization.

During human in vitro culture, a morphological microscope analysis is normally performed to select the best embryo to transfer, with the hope of obtaining a successful pregnancy. The morphological evaluation may combine number and size of blastomeres, fragmentation, multinucleation, blastocyst expansion, inner-cell mass and trophectoderm appearance. However, standard microscopy evaluation involves the removal of the embryos from the incubator, exposing them to changes in pH, temperature, and oxygen level. Additionally, morphological assessments might include high inter-observer variability. Recently, continuous embryo culture using time-lapse monitoring (TLM) has allowed embryologists to analyse the dynamic and morphokinetic events of embryo development and, based on that, the embryologist is able to scrutinize the complete sequence of embryonic evolution, from fertilization to the blastocyst formation. Therefore, TLM allows an uninterrupted culture condition, reducing the need to remove embryos from the incubator. The monitoring system is normally composed of a standard incubator with an integrated microscope coupled to a digital camera, which is able to collect images at regular times, and subsequently processed into video. These data can be annotated and analyzed using an integrated software, therefore this allows embryologists to facilitate the process of embryo selection for transfer. The main aim of this paper is to discuss the potential benefits and uses of the TLM in the embryology laboratory.

We report full-term development of nuclear transfer embryos following nuclear exchange at the 2-cell stage. Nuclei from 2-cell rat embryos were transferred into enucleated 2-cell embryos and developed to term after transfer to recipients (NT2). Pronuclear exchange in zygotes was used for comparison (NT1). Zygotes and 2-cell embryos were harvested from 4-week-old female Sprague-Dawley rats. Nuclear transfer was performed by transferring the pronuclei or karyoplasts into the perivitelline space of recipient embryos followed by electrofusion to reconstruct embryos. Fused couplets were cultured for 4 or 24 h before being transferred into day 1 pseudopregnant recipients (Hooded Wistar) at the 1- or 2-cell stage. In vitro culture was also carried out to check the developmental competence of the embryos. In vitro development to the blastocyst stage was not significantly different between the two groups (NT1, 34.3%; NT2, 45.0%). Two of three recipients from NT1 and two of five recipients from NT2 became pregnant. Six pups (3 from NT1, 3 from NT2) were delivered from the four foster mothers. Three female pups survived; 2 from NT1 and 1 from NT2. At 2 months of age these pups appeared healthy, and were mated with Sprague-Dawley males. One rat derived from NT1 delivered 15 pups (5 males, 10 females) as did the rat from NT2 (7 males, 8 females). Our results show that by using karyoplasts from 2-cell stage embryos as nuclear donors and reconstructing them with enucleated 2-cell embryos, healthy rats can be produced.