Apoptotic and developmental effects of bovine Herpesvirus type-5 infection on in vitro-produced bovine embryos

Bovine Herpesvirus type-5 (BoHV-5), which is potentially neuropathogenic, was recently described to be related with reproductive disorders in cows. The objective was to elucidate mechanisms involved in propagation of BoHV-5 in embryonic cells. For this purpose, bovine embryos produced in vitro were assayed for apoptotic markers after experimental infection of oocytes, in vitro fertilization, and development. Host DNA fragmentation was detected with a TUNEL assay, expression of annexin-V was measured with indirect immunofluorescence, and viral DNA was detected with in situ hybridization. Infective BoHV-5 virus was recovered from embryos derived from exposed oocytes after two consecutive passages on Madin-Darby bovine kidney (MDBK) cells. The viral DNA corresponding to US9 gene, localized between nucleotides 126243 to 126493, was detected in situ and amplified. There was no significant difference between the ratio of TUNEL stained nuclei and total cells in good quality blastocysts (0.87 +/- 0.05, mean SD), but there were differences (P < 0.05) between infected (0.18 +/- 0.05) and uninfected blastocysts (0.73 +/- 0.07). The Annexin-V label was more intense in uninfected embryos (0.79 +/- 0.04; P < 0.05). The quality of infected and uninfected embryos was considered equal, with no significant effect on embryonic development. In conclusion, we inferred that BoHV-5 infected bovine oocytes, replicated, and suppressed some apoptotic pathways, without significantly affecting embryonic development. (C) 2010 Elsevier Inc. All rights reserved.

Insulin-like growth factor-1 protects preimplantation embryos from anti-developmental actions of menadione

Menadione is a naphthoquinone used as a vitamin K source in animal feed that can generate reactive oxygen species (ROS) and cause apoptosis. Here, we examined whether menadione reduces development of preimplantation bovine embryos in a ROS-dependent process and tested the hypothesis that actions of menadione would be reduced by insulin-like growth factor-1 (IGF-1). Menadione caused a concentration-dependent decrease in the proportion of embryos that became blastocysts. All concentrations tested (1, 2.5, and 5.0 mu M) inhibited development. Treatment with 100 ng/ml IGF-1 reduced the magnitude of the anti-developmental effects of the two lowest menadione concentrations. Menadione also caused a concentration-dependent increase in the percent of cells positive for the TUNEL reaction. The response was lower for IGF-1-treated embryos. The effects of menadione were mediated by ROS because (1) the anti-developmental effect of menadione was blocked by the antioxidants dithiothreitol and Trolox and (2) menadione caused an increase in ROS generation. Treatment with IGF-1 did not reduce ROS formation in menadione-treated embryos. In conclusion, concentrations of menadione as low as 1.0 mu M can compromise development of bovine preimplantation embryos to the blastocyst stage of development in a ROS-dependent mechanism. Anti-developmental actions of menadione can be blocked by IGF-1 through effects downstream of ROS generation.

Evaluation of Trypsin Treatment on the Inactivation of Bovine Herpesvirus Type 1 on In Vitro Produced Pre-implantation Embryos

The aim of this study was to evaluate the efficiency of trypsin treatment on the inactivation of bovine herpesvirus type 1 (BoHV-1) on in vitro produced by fertilization and artificially infected bovine embryos. Bovine embryos on day 7 were exposed with 10 mu l of BoHV-1, Los Angeles strain 10(7.5) TCID. These embryos and control embryos were divided in two groups: submitted to the sequential washes or to the trypsin treatment according to the International Embryo Transfer Society (IETS) guidelines. The embryos and the last washing drop of each group were used as inoculum to infect Madin Darby bovine kidney (MDBK) cells and submitted to nested PCR reaction using the primer that encodes the gene conserved region of virus glycoprotein gB. The data have shown that the control embryos and their last washing drop were negative. The exposed embryos that were treated with trypsin have shown positive results on the n-PCR and MDBK culture, and their last washing drop were negative. Our data have demonstrated that the trypsin treatment was not able to eliminate the BHV-1 of the embryos, suggesting an interaction between virus and embryo.

Human immature dental pulp stem cells` contribution to developing mouse embryos: production of human/mouse preterm chimaeras

In this study, we aimed at determining whether human immature dental pulp stem cells (hIDPSC) would be able to contribute to different cell types in mouse blastocysts without damaging them. Also, we analysed whether these blastocysts would progress further into embryogenesis when implanted to the uterus of foster mice, and develop human/mouse chimaera with retention of hIDPSC derivates and their differentiation. hIDPSC and mouse blastocysts were used in this study. Fluorescence staining of hIDPSC and injection into mouse blastocysts, was performed. Histology, immunohistochemistry, fluorescence in situ hybridization and confocal microscopy were carried out. hIDPSC showed biological compatibility with the mouse host environment and could survive, proliferate and contribute to the inner cell mass as well as to the trophoblast cell layer after introduction into early mouse embryos (n = 28), which achieved the hatching stage following 24 and 48 h in culture. When transferred to foster mice (n = 5), these blastocysts with hIDPSC (n = 57) yielded embryos (n = 3) and foetuses (n = 6); demonstrating presence of human cells in various organs, such as brain, liver, intestine and hearts, of the human/mouse chimaeras. We verified whether hIDPSC would also be able to differentiate into specific cell types in the mouse environment. Contribution of hIDPSC in at least two types of tissues (muscles and epithelial), was confirmed. We showed that hIDPSC survived, proliferated and differentiated in mouse developing blastocysts and were capable of producing human/mouse chimaeras.

A comparison of post-thaw results between embryos arising from intracytoplasmic sperm injection using surgically retrieved or ejaculated spermatozoa

Objective: To study the effect of freeze-thaw on embryos derived from intracytoplasmic sperm injection (ICSI) using surgically retrieved and ejaculated spermatozoa. Design: Retrospective study. Setting: Private IVF center. Patient(s): Three hundred eighty-three patients undergoing frozen-thawed ET cycles. Intervention(s): Testicular sperm aspiration (TESA) or percutaneous epididymal sperm aspiration (PESA) were the sperm surgical retrieval methods used for ICSI. Embryos resulting from ICSI using Surgically retrieved and ejaculated spermatozoa were frozen, thawed, and transferred. Main Outcome Measure(s): Post-thaw survival, implantation, and pregnancy rates. Result(s): No differences were found between the ejaculated sperm and TESA/PESA groups in terms of post-thaw survival rate (68.4% vs. 66.1%, respectively), pregnancy rate (20.1% vs. 16.1%), and implantation rate (10.6% vs. 12.7%). Similar results were found for those variables when comparing TESA and PESA groups. Conclusion(s): Cleavage embryos arising from ICSI cycles using testicular and epididymal spermatozoa can be frozen with survival, pregnancy,and implantation rates comparable to those obtained with ejaculated spermatozoa. (Fertil Steril (R) 2009;91:727-32. (C) 2009 by American Society for Reproductive Medicine.)

Evolution and developmental expression of nuclear receptor genes in the ascidian Herdmania

Nuclear receptors are a superfamily of metazoan transcription factors that have been shown to be involved in a wide range of developmental and physiological processes. A PCR-based survey of genomic DNA and developmental cDNAs from the ascidian Herdmania identifies eight members of this multigene family. Sequence comparisons and phylogenetic analyses reveal that these ascidian nuclear receptors are representative of five of the six previously defined nuclear receptor subfamilies and are apparent homologues of retinoic acid [NR1B], retinoid X [NR2B], peroxisome proliferator-activated [NR1C], estrogen related [NR3B], neuron-derived orphan (NOR) [NR4A3], nuclear orphan [NR4A], TR2 orphan [NR2C1] and COUP orphan [NR2F3] receptors. Phylogenetic analyses that include the ascidian genes produce topologically distinct trees that suggest a redefinition of some nuclear receptor subfamilies. These trees also suggest that extensive gene duplication occurred after the vertebrates split from invertebrate chordates. These ascidian nuclear receptor genes are expressed differentially during embryogenesis and metamorphosis.

A sponge allelochemical induces ascidian settlement but inhibits metamorphosis

Secondary metabolites synthesised by sessile invertebrates appear to play a role in creating and maintaining space on hard substrata by repelling competitors. In this study, we investigated the responses of the larvae of the ascidian Herdmania curvata to haliclonacyclamine A (HA), the major component of a suite of cytotoxic alkaloids extracted from the sponge Haliclona sp. 628. Both Haliclona sp. 628 and Herdmania curvata inhabit the crest and slope of Heron Island Reef. High rates of settlement were induced in competent H. curvata larvae by a range of concentrations of HA, all lower than that naturally occurring in the sponge. HA did not induce precompetent larvae to settle. Although early metamorphosis of HA-induced larvae was normal, larvae exposed to all but the lowest concentration of HA were developmentally arrested after completion of tail resorption, at about 4 h after the initiation of metamorphosis. These postlarvae underwent extensive cellular necrosis within 24 h. We also demonstrate that the addition of a transcriptional inhibitor, actinomycin D, to larvae also causes inhibition of metamorphosis after tail resorption is completed. Analyses of incorporation of radiolabelled nucleotides to measure levels of transcription during normal development and after the addition of the transcriptional inhibitor indicate that there is a significant burst of transcriptional activity just after tail resorption is completed. Despite inhibiting metamorphosis at the same stage as actinomycin D, HA increases initial rates of RNA synthesis after induction of metamorphosis in a manner similar to that observed in normal postlarvae until the onset of cellular necrosis. We conclude that HA initially induces H. curvata larvae to settle and progress through early metamorphosis possibly by engaging the same pathway as other artificial and environmental cues but subsequently inhibits completion of metamorphosis, resulting in death of the postlarvae. Since HA does not affect overall transcription rates, it appears to disrupt another important developmental process during early metamorphosis.

Exogenous induction of ovarian activity and ovulation and transfer of fresh embryos of domestic cat (Felis catus)

The objective of the present study was the exogenous stimulation of ovarian activity and definition of embryo collection, and transfer protocols, in the domestic cat for potential application in non-domestic endangered species. Sixteen adult queens and two adult male reproducers kept in the experimental cat house at the Morphology sector at the Veterinary Department (DVT), UFV, were used in this study. All the queens received a single application of 150 IU Equine Chorionic Gonadotropin (eCG) in the post estrus to induce ovarian activity and 80 to 84 hours later, received a single application of 100 UI Human Chorionic Gonadotropin (hCG) to induce ovulation. After hCG application, only the donor queens were naturally mated. The receptor queens received extra stimulus for induction of ovulation through manipulation of an intravaginal swab. Five to six days after hCG application, the donor queens were subjected to a laparotomy for embryo collection that was performed by trans-horn uterine washing. On average, six embryos were surgically inovulated. They were classified as type I and III compact morula and blastocysts in four receptor queens. Three animals presented pregnancy confirmed by ultrasound at day 36 and two of these animals gave birth to litters of two and four offsprings, respectively, at 66 and 63 days after induction of ovulation. Except for one still birth, all the offspring developed normally.

Primary screening of the bioactivity of brackishwater cyanobacteria: toxicity of crude extracts to artemia salina larvae and paracentrotus lividus embryos

Cyanobacteria are a diverse group of Gram-negative bacteria that produce an array of secondary compounds with selective bioactivity against vertebrates, invertebrates, plants, microalgae, fungi, bacteria, viruses and cell lines. The aim of this study was to assess the toxic effects of aqueous, methanolic and hexane crude extracts of benthic and picoplanktonic cyanobacteria isolated from estuarine environments, towards the nauplii of the brine shrimp Artemia salina and embryos of the sea urchin Paracentrotus lividus. The A. salina lethality test was used as a frontline screen and then complemented by the more specific sea urchin embryo-larval assay. Eighteen cyanobacterial isolates, belonging to the genera Cyanobium, Leptolyngbya, Microcoleus, Phormidium, Nodularia, Nostoc and Synechocystis, were tested. Aqueous extracts of cyanobacteria strains showed potent toxicity against A. salina, whereas in P. lividus, methanolic and aqueous extracts showed embryo toxicity, with clear effects on development during early stages. The results suggest that the brackishwater cyanobacteria are producers of bioactive compounds with toxicological effects that may interfere with the dynamics of invertebrate populations.

Cilia motility studies in zebrafish embryos

A thesis submitted in fulfilment of the requirements for the degree of Masters in Molecular Genetics and Biomedicine

Zebrafish embryos as in vivo model for toxicity evaluation: screening of nanoparticle formulations DODAB:MO and DODAC:MO

Dissertação de mestrado em Molecular Genetics

Factors associated with willingness to donate embryos for research among couples undergoing IVF

Between 2011 and 2012, 213 heterosexual couples undergoing fertility treatments in a Portuguese public fertility centre were systematically recruited to assess factors associated with willingness to donate embryos for research. Data were collected by questionnaire. Most couples (87.3%; 95% CI 82.1 to 91.5) were willing to donate embryos for research, citing benefits for science, health and infertile patients. Almost all couples (94.3%; 95% CI 89.8 to 96.7) reached consensus about the decision. Willingness to donate was more frequent in women younger than 36 years (adjusted OR 3.06; 95% CI 1.23 to 7.61) and who considered embryo research to be very important (adjusted OR: 6.32; 95% CI 1.85 to 21.64), and in Catholic men (adjusted OR 4.16; 95% CI 1.53 to 11.30). Those unwilling to donate reported conceptualizing embryos as children or living beings and a lack of information or fears about embryo research. Men with higher levels of trait anxiety (adjusted OR 0.90; 95% CI 0.84 to 0.96) were less frequently willing to donate. Future research on embryo disposition decision-making should include the assessment of gender differences and psychosocial factors. Ethically robust policies and accurate information about the results of human embryo research are required.

Couple's willingness to donate embryos for research: a longitudinal study

Introduction. Decision-making on embryo disposition is a source of distress and is subject to change over time. This paper analyses the willingness of couples undergoing in vitro fertilization to donate cryopreserved embryos for research from 15 days after embryo transfer to 12 months later, taking into account the influence of psychosocial, demographic, and reproductive factors. Materials and methods. Prospective longitudinal study, with 74 heterosexual couples undergoing in vitro fertilization in a public fertility centre in Portugal, recruited between 2011 and 2012. Participants were evaluated twice: 15 days after embryo transfer and 12 months later. Results. A significant decrease in patients’ willingness to donate embryos for research over time was observed [86.5% to 73.6%; relative risk (RR) = 0.85; 95% CI 0.76–0.95]. A higher education level (>12 years) [adjusted RR (RRadj) = 0.79; 95% CI 0.64–0.96], considering research on human embryos to be important (vs. very important) (RRadj = 0.59; 95% CI 0.39–0.85) and practicing a religion less than once a month (vs. at least once a month) (RRadj = 0.73; 95% CI 0.53–1.00) seemed associated with unwillingness to donate embryos for research over time. Change towards non-donation happened mainly among couples who first considered that it was better to donate than wasting the embryos. Change towards donation occurred mostly among those stating that their priority at time 1 was to have a baby and who became pregnant in the meantime. Conclusions. Quality of care guided by patients’ characteristics, values, preferences, and needs calls for considering the factors and reasons underlying couples’ willingness to donate embryos for research over time as a topic in psychosocial guidelines for infertility and medically assisted reproductive care.

Factors associated with the donation and non-donation of embryos for research: a systematic review

Background: Systematic knowledge on the factors that influence the decisions of IVF users regarding embryo donation for research is a core need for patient-centred policies and ethics in clinical practice. However, no systematic review has been provided on the motivations of patients who must decide embryo disposition. This paper fills this gap, presenting a systematic review of quantitative and qualitative studies, which synthesizes the current body of knowledge on the factors and reasons associated with IVF patients’ decisions to donate or not to donate embryos for research. Methods: A systematic search of studies indexed in PubMed, ISIWoK and PsycINFO, published before November 2013, was conducted. Only empirical, peer-reviewed, full-length, original studies reporting data on factors and reasons associated with the decision concerning donation or non-donation of embryos for research were included. Eligibility and data extraction were performed by two independent researchers and disagreements were resolved by discussion or a third reviewer, if required. The main quantitative findings were extracted and synthesized and qualitative data were assessed by thematic content analysis. Results: A total of 39 studies met the inclusion criteria and were included in the review. More than half of the studies (n ¼ 21) used a quantitative methodology, and the remaining were qualitative (n ¼ 15) or mixed-methods (n ¼ 3) studies. The studies were derived mainly from European countries (n ¼ 18) and the USA(n ¼ 11). The proportion of IVF users who donated embryos for research varied from 7% in a study in France to 73% in a Swiss study. Those who donate embryos for research reported feelings of reciprocity towards science and medicine, positive views of research and high levels of trust in the medical system. They described their decision as better than the destruction of embryos and as an opportunity to help others or to improve health and IVF treatments. The perception of risks, the lack of information concerning research projects and the medical system and the conceptualization of embryos in terms of personhood were the most relevant motives for not donating embryos for research. Results relating to the influence of sociodemographic characteristics and reproductive and gynaecological history were mostly inconclusive. Conclusions: Three iterative and dynamic dimensions of the IVF patients’ decision to donate or not to donate embryos for research emerged from this review: the hierarquization of the possible options regarding embryo disposition, according to the moral, social and instrumental status attributed to embryos; patients’ understanding of expectations and risks of the research on human embryos; and patients’ experiences of information exchange and levels of trust in the medical-scientific institutions.