Imaging motility during fertilization

Studying reproduction in domestic species is now possible at the cellular and molecular level due to advances in the production of large numbers of zygotes and embryos in these species. In this paper we review the microtubule patterns during fertilization in domestic species. These results indicate that domestic species accomplish fertilization in a similar fashion to one another but in a far different fashion from rodents. Recent results indicate that human fertilization is similar to that of domestic species. We discuss the significance this has on the use of domestic species as a model system for human studies and possible consequences for the alleviation of human infertility.     

Comparison of histone modifications in in vivo and in vitro fertilization mouse embryos

Histone modifications are thought to play important roles in various cellular functions. In this article, the distribution patterns of acetylation on histone H4, methylation on histone H3 lysine 9, and phosphorylation on histone H3 serine 10 were examined in in vivo and in vitro fertilization (IVF) preimplantation mouse embryos by using indirect immunofluorescence and scanning confocal microscopy. We desired to know whether the IVF, which has been widely used as a routine assisted reproductive technology in animal and human, was safe at the epigenetic level. As results, we found that there was no difference in these histone modification patterns in in vivo and IVF mouse embryos from zygote to blastocyst stage. Moreover, these histone modifications had different distributions at all examined stages, but they were consistent with the mouse embryo developmental stages.     

Aneuploidy in the human blastocyst

Studies of human cleavage stage embryos, 3 days after fertilization of the oocyte, have revealed remarkably high levels of chromosome abnormality. In addition to meiotic errors derived from the gametes, principally the oocyte, mitotic errors occurring after fertilization are also common, leading to widespread chromosomal mosaicism. The prevalence of chromosome anomalies in embryos may explain the relatively poor fertility and fecundity in humans and the low success rates of assisted reproductive treatments (e.g., IVF). While much is known concerning the incidence of aneuploidy during the first 3 days following fertilization, it is only in the last couple of years that large numbers of embryos at the final stage of preimplantation development, the blastocyst stage, 5 days after fertilization, have been subjected to detailed analysis. Here we discuss the latest data from the comprehensive cytogenetic analysis of blastocysts. These findings indicate that the majority of selection against chromosome abnormalities does not occur until the time of implantation or shortly after, with aneuploidy typically affecting more than 50% of blastocysts. Additionally, clinical results presented suggest that screening of blastocyst stage embryos for chromosome abnormality, with preferential transfer to the uterus of those found to be euploid, may help to improve the success rates of assisted reproductive treatments.     

Preventing polyspermy in mammalian eggs—Contributions of the membrane block and other mechanisms

The egg's blocks to polyspermy (fertilization of an egg by more than one sperm) were originally identified in marine and aquatic species with external fertilization, but polyspermy matters in mammalian reproduction too. Embryonic triploidy is a noteworthy event associated with pregnancy complications and loss. Polyspermy is a major cause of triploidy with up to 80% of triploid conceptuses being the result of dispermic fertilization. The mammalian female reproductive tract regulates the number of sperm that reach the site of fertilization, but mammals also utilize egg‐based blocks to polyspermy. The egg‐based blocks occur on the mammalian egg coat (the zona pellucida) and the egg plasma membrane, with apparent variation between different mammalian species regarding the extent to which one or both are used. The zona pellucida block to polyspermy has some similarities to the slow block in water‐dwelling species, but the mammalian membrane block to polyspermy differs substantially from the fast electrical block that has been characterized in marine and aquatic species. This review discusses what is known about the incidence of polyspermy in mammals and about the mammalian membrane block to polyspermy, as well as notes some lesser‐characterized potential mechanisms contributing to polyspermy prevention in mammals.     

Assisted reproductive technologies in rhesus macaques

The assisted reproductive technologies (ARTs) have been used in the production of rhesus monkey offspring at the Oregon National Primate Research Center (ONPRC) and that experience is summarized here. Additionally these technologies serve as a source of oocytes/embryos for monozygotic twinning, embryonic stem (ES) cell derivation and cloning. High fertilization efficiencies were realized with conventional insemination or following the use of intracytoplasmic sperm injection (ICSI) and approximately 50% of the resulting embryos grew in vitro to blastocysts. Both fresh and frozen sperm were employed in fertilization by ICSI and the resulting embryos could be low temperature stored for subsequent thawing and transfer when a synchronized recipient female was available or after shipment to another facility. Following the transfer of up to 3 embryos, an overall pregnancy rate of 30% was achieved with increasing rates dependent upon the number of embryos transferred. Singleton pregnancy outcomes following the transfer of ART produced embryos were similar to those observed in a control group of animals in the timed mated breeding colony at ONPRC. ICSI produced embryos were used in efforts to create monozygotic twins by blastomere separation or blastocyst splitting. While pregnancies were achieved following the transfer of demi-embryos, only one was a twin and it was lost to spontaneous abortion. ICSI produced embryos have also served as the source of blastocysts for the derivation of embryonic stem cells. These pluripotent cells hold potential for cell based therapies and we consider the monkey an important translational model in which to evaluate safety, efficacy and feasibility of regenerative medicine approaches based on the transplantation of stem cell-derived progeny. Finally, efforts to produce genetically-identical monkeys by nuclear transfer have been briefly summarized.     

Cryopreservation of mammalian oocytes and embryos: current problems and future perspectives

Cryopreservation techniques for mammalian oocytes and embryos have rapidly progressed during the past two decades,emphasizing their importance in various assisted reproductive technologies.Pregnancies and live births resulting from cryopreserved oocytes and embryos of several species including humans have provided proof of principle and led to the adoption of cryopreservation as an integral part of clinical in vitro fertilization.Considerable progress has been achieved in the development and application of the cryopreservation of mammalian oocytes and embryos,including preservation of the reproductive potential of patients who may become infertile,establishment of cryopreserved oocyte banks,and transport of oocytes and embryos internationally.However,the success rates are still far lower than those obtained with fresh oocytes and embryos,and there are still obstacles that need to be overcome.In this review,we address the major obstacles in the development of effective cryopreservation techniques.Such knowledge may help to eliminate these hurdles by revealing which aspects need improvement.Furthermore,this information may encourage further research by cryobiologists and increase the practical use of cryopreservation as a major part of assisted reproductive technologies for both humans and animal species.     

Molecular origin of mitotic aneuploidies in preimplantation embryos

Mitotic errors are common in human preimplantation embryos. The occurrence of mitotic errors is highest during the first three cleavages after fertilization and as a result about three quarters of human preimplantation embryos show aneuploidies and are chromosomally mosaic at day three of development. The origin of these preimplantation mitotic aneuploidies and the molecular mechanisms involved are being discussed in this review.At later developmental stages the mitotic aneuploidy rate is lower. Mechanisms such as cell arrest, apoptosis, active correction of the aneuploidies and preferential allocation of the aneuploid cells to the extra-embryonic tissues could underlie this lower rate.Understanding the mechanisms that cause mitotic aneuploidies in human preimplantation embryos and the way human preimplantation embryos deal with these aneuploidies might lead to ways to limit the occurrence of aneuploidies, in order to ultimately increase the quality of embryos and with that the likelihood of a successful pregnancy in IVF/ICSI. This article is part of a Special Issue entitled: Molecular Genetics of Human Reproductive Failure.     

Buffalo and cattle hybrid embryo development is decreased by caffeine treatment during in vitro fertilization

Water buffalo are renowned for difficulties in the implementation of assisted reproductive technologies, with both males and females being problematic. In this study, we used cattle oocytes to assess the effect of treatments with heparin and caffeine on buffalo spermatozoa and subsequent fertilization and embryo development in vitro. There was no significant difference between buffalo and bovine spermatozoa in the events associated with fertilization. Fertilization of cattle oocytes with buffalo spermatozoa resulted in 7.8% of oocytes developing into hybrid embryos. A difference in the developmental capability of hybrid embryos compared with the cattle control was observed. This has not been previously reported. The subsequent transfer of a limited number of hybrid embryos did not produce a viable pregnancy. However, control treatments in this experiment also failed to achieve pregnancy, so objective data is not available to provide conclusions about the developmental competence of the buffalo and cattle hybrid embryos. Optimal spermatozoa capacitation treatments achieved 61% fertilization and 21% zygote cleavage into two cell embryos. There was no significant difference in fertilization or development due to heparin or spermatozoa concentrations. However, treatment of buffalo and cattle spermatozoa with caffeine significantly decreased embryo cleavage but also tended to decrease embryo development to the blastocyst stage. These studies suggest that problems with reproduction in buffalo may reside with biological mechanisms associated with the oocyte that are often complicated by poor male reproductive performance. Selection for bull fertility would prevent some of these complications.     

Evidence for genetic etiology of heteroploidy in embryos of the Japanese quail (Coturnix coturnix japonica).

The Japanese quail was used as an experimental system to detect the effects of genes that affect chromosome behavior and distribution. From a random-bred population, three inbred generations were produced by full-sib matings in 36 families. The expectation from such a breeding scheme was that embryos bearing aberrations induced by recessive mutant genes would cluster within families and recur in particular lineages. Chromosomal aberrations caused by errors during fertilization, cleavage mitosis, and gametogenesis were scored in 2,037 16- to 18-h embryos from 107 families. Comparisons of the observed frequencies among families and lineages and pedigree analysis indicated that four types of chromosome aberrations had a genetic basis: (1) triploidy and triploid chimerism; (2) haploidy and haploid chimerism; (3) diploid/tetraploid mosaicism; and (4) a new aberration, referred to as "atypical mitotic metaphase." Analysis of the sex-chromosome complements of the embryos indicated that triploidy resulted predominantly from diploid ova, haploid cell lines originated from supernumerary sperm nuclei, and tetraploid cell lines resulted from endoreduplication or failure of cytokinesis. Clustering of triploidy in particular lineages was due to dispermy or recurrent suppression of one or both meiotic divisions during oogenesis.     

Effects of maternal age on oocyte developmental competence

The widespread use of a variety of assisted reproductive technologies has removed many of the constraints that previously restricted mammalian reproduction to the period between onset of puberty and reproductive senescence. In vitro embryo production systems now allow oocytes from very young animals to undergo fertilization and form embryos capable of development to normal offspring, albeit at somewhat reduced efficiencies compared to oocytes from adult females. They also can overcome infertility associated with advanced age of animals and women. This review examines oocyte developmental competence as the limiting factor in applications of assisted reproductive technologies for both juvenile and aged females. Age of oocyte donor is a significant factor influencing developmental competence of the oocyte. Age-related abnormalities of oocytes include a) meiotic incompetence or inability to complete meiotic maturation resulting in oocytes incapable of fertilization; b) errors in meiosis that can be compatible with fertilization but lead to genetic abnormalities that compromise embryo viability; and c) cytoplasmic deficiencies that are expressed at several stages of development before or after fertilization. In general, oocytes from juvenile donors and the embryos derived therefrom appear less robust and may be less tolerant to suboptimal handling and in vitro culture conditions than are adult oocytes. Research to identify specific cytoplasmic deficiencies of juvenile oocytes may enable modifications of culture conditions to correct such deficiencies and thus enhance developmental competence. Use of oocytes from aged donors for assisted reproduction can have a variety of applications such as extending the reproductive life of individual old females whose offspring still have high commercial value, and conservation of genetic resources such as rare breeds of livestock and endangered species. In general, female fertility decreases with advancing age. Studies of women in oocyte donation programs have established reduced oocyte competence as the major cause of declining fertility with age, although inadequate endometrial function can also be a contributing factor. Most research has emphasized the importance of chromosomal abnormalities because of the well established increase in aneuploidy with increasing maternal age but little is known about the underlying cellular and molecular mechanisms. Research aimed at identifying the specific developmental deficiencies of oocytes from juvenile donors and abnormalities of oocytes from aged females will assist in overcoming present bottlenecks that limit the efficiency of assisted reproduction technologies. Such research will also be crucial to the development of new oocyte-based technologies for overcoming infertility and possibly subverting chromosomal abnormalities in women approaching menopause.     

被子植物雌雄配子及早期胚胎的分离与研究应用

被子植物的有性生殖过程复杂精巧并深藏于母体组织内进行,一直以来难以对该过程进行直接的细胞学和分子生物学研究。如今在多种植物中获得了有活力而无污染的配子细胞和早期胚胎,结合少量细胞m RNA提取技术、基因组深度测序以及离体授精系统等技术,人们已能对被子植物受精过程中的配子识别、配子融合、合子激活等重要发育事件进行深入分析。本文对有代表性的被子植物配子和早期胚胎的分离技术及其在受精作用研究中的应用、存在的问题和前景进行了总结,旨在为植物生殖发育研究提供帮助。     

Limiting dilution bisulfite (pyro)sequencing reveals parent-specific methylation patterns in single early mouse embryos and bovine oocytes

To detect rare epigenetic effects associated with assisted reproduction, it is necessary to monitor methylation patterns of developmentally important genes in a few germ cells and individual embryos. Bisulfite treatment degrades DNA and reduces its complexity, rendering methylation analysis from small amounts of DNA extremely challenging. Here we describe a simple approach that allows determining the parent-specific methylation patterns of multiple genes in individual early embryos. Limiting dilution (LD) of bisulfite-treated DNA is combined with independent multiplex PCRs of single DNA target molecules to avoid amplification bias. Using this approach, we compared the methylation status of three imprinted (H19, Snrpn and Igf2r) and one pluripotency-related gene (Oct4) in three different groups of single mouse two-cell embryos. Standard in vitro fertilization of superovulated oocytes and the use of in vitro matured oocytes were not associated with significantly increased rates of stochastic single CpG methylation errors and epimutations (allele methylation errors), when compared with the in vivo produced controls. Similarly, we compared the methylation patterns of two imprinted genes (H19 and Snrpn) in individual mouse 16-cell embryos produced in vivo from superovulated and non-superovulated oocytes and did not observe major between-group differences. Using bovine oocytes and polar bodies as a model, we demonstrate that LD even allows the methylation analysis of multiple genes in single cells.     

<Emphasis Type="Italic">In vitro</Emphasis> fertilization and preimplantation development in the primate

Nonhuman primates represent a strong model for examining the chromosomal, biochemical, and temporal normality of embryos produced byin vitro fertilization. Morein vitro fertilized embryos from the squirrel monkey(Saimiri sciureus) have been produced and examined than with all other primate species combined. In studies over a 13 year period a fertilization rate approximating 60 % has been developed in this species with 30% of these embryos proceeding to the two cell stage and 50% of these to the three-four cell stage. Chromosomal abnormalities (primarily missing or extra chromosomes) at a level of nine to 16% have been found, a value corresponding to that found inin vivo mating andin vitro fertilization in other species. An incidence of triploidy of 16.7% was observed. RNA and protein synthetic rates appear comparable with those of laboratory species subjected toin vitro fertilization and indicate the initial stages of metabolic activity of the newly formed embryo. Similarly, increases in estrogen incorporation appear after fertilization but no effect is observed in progesterone incorporation. Utilizing 2-deoxy-glucose and insulin, it was determined that the glucose requirement as an energy source for early preimplantationin vitro fertilized primate embryos is very low.Of very great importance is the temporal relationship of the development ofin vitro fertilized squirrel monkey embryos compared with similar development in other primates (including humans) afterin vivo andin vitro fertilization. An analysis of over a decade of work with the squirrel monkey embryos demonstrates a pattern of temporal development that is comparable with all other primate species that have been examined (including the human) and comparable with development afterin vivo fertilization.     

小鼠体内外受精植入前胚胎全基因组甲基化模式比较

为考察体外受精、操作及培养环境对体外受精的小鼠植入前胚胎全基因组DNA甲基化模式的影响,本研究以体内受精的植入前胚胎作为对照,采用间接免疫荧光法检测小鼠体内外受精植入前胚胎基因组DNA甲基化模式.实验结果表明,体外受精各期植入前胚胎呈现出与之相应时期的体内受精植入前胚胎不同的DNA甲基化模式和水平,原核期甲基化水平较高,2-4-、8-细胞期明显降低,而桑葚胚和囊胚期又略有升高.各期体外受精植入前胚胎的基因组DNA甲基化水平都比同时期体内受精胚胎的甲基化水平低.本实验结果部分显示了体外受精、操作及培养环境可能对正常的DNA甲基化模式产生影响,造成体外受精植入前胚胎甲基化模式异常.     

Extended culture up to the blastocyst stage: a strategy to avoid multiple pregnancies in assisted reproductive technologies

The aim of this study was to review the experience and outcomes of assisted reproduction cycles with embryos grown up to day 5 of development, comparing different parameters according to the ages of the patients. We retrospectively studied 1,874 assisted reproduction cycles where embryo culture was extended up to the fifth or sixth day of development. All IVF and ICSI cycles were included, comparing, according to patient age, the following rates: blastocyst formation, pregnancy, implantation and abortion. As control, we analyzed cycles with donated oocytes from young donors (OD). The number of embryos reaching the blastocyst stage is similar in all groups of patients. Only the OD group was different in terms of blastocyst formation, pregnancy and implantation rates. Patients over 39 years of age had an abortion rate of 59.1 %, which is significantly higher than the other groups. Extended embryo culture up to the blastocyst stage can be implemented in programs of assisted reproduction in order to increase the pregnancy rate. The potential of blastocyst implantation is high, allowing us to transfer fewer embryos and reduce the probability of multiple pregnancies.     

Embryo technology in conservation efforts for endangered felids

Most of the 36 species of wild cats are classified as threatened, vulnerable or endangered due to poaching and habitat loss. The important role of assisted reproduction techniques (ART) as part of a multifaceted captive breeding program for selected wild cat species is gradually gaining acceptance. This recognition is a result of the progress made during the last decade in which the feasibility of oocyte recovery from gonadotropin-treated females, in vitro fertilization, embryo cryopreservation and embryo transfer (ET) was demonstrated in the domestic cat (Felis catus). Additionally, embryos have been produced in vitro from oocytes matured in vitro after recovery from ex situ ovaries of both domestic and non-domestic cat species and domestic kittens have been born following transfer of these embryos. In vitro fertilization has been successful in at least one-third of wild cat species and kittens were born after transfer of Indian desert cat (Felis sylvestris ornata) embryos into a domestic cat and con-specific transfer of tiger (Panthera tigris) embryos. The domestic cat is not only a valuable model for development of in vitro techniques but may serve as a recipient of embryos from several species of small wild cats.     

Preimplantation genetic diagnosis

Preimplantation genetic diagnosis (PGD) is an evolving technique that provides a practical alternative to prenatal diagnosis and termination of pregnancy for couples who are at substantial risk of transmitting a serious genetic disorder to their offspring. Samples for genetic testing are obtained from oocytes or cleaving embryos after in vitro fertilization. Only embryos that are shown to be free of the genetic disorders are made available for replacement in the uterus, in the hope of establishing a pregnancy. PGD has provided unique insights into aspects of reproductive genetics and early human development, but has also raised important new ethical issues about assisted human reproduction.     

Physiologie de l’interaction gametique: Revue de la litterature

Our knowledge of gamete interaction in vitro and in vivo has grown rapidly over the last century. Its importance has been reinforced by the development of in vitro fertilization and assisted conception procedures on one hand, and by research on the development of contraceptive vaccines on the other. This literature review synthesizes our current understanding of the successive steps in the fertilization process from sperm capacitation allthrough gamete fusion, including sperm interaction with the cumulus oophorus and the zona pellucida, as well as consideration on the mechanisms and timing of the acrosome reaction.     

Synthetic profiles of polypeptides of human oocytes and normal and abnormal preimplantation embryos

There is considerable variation in the rate of development in vitro of individual preimplantation human embryos. The relationship between the rate of development and patterns of polypeptide synthesis in individual embryos was examined using SDS-PAGE and autoradiography. After incubation in [35S]methionine, 19 polypeptide bands were identified that change between fertilization and the morula stage. Although changes in two of the bands occurred in embryos that were developing normally and in ageing oocytes, and are thus independent of fertilization, the changes identified in the remaining 17 bands occurred only after fertilization. In embryos that were developing abnormally, as assessed by delayed cleavage, cleavage arrest or extensive fragmentation, the alteration in polypeptide synthetic profiles increased with increasing abnormality.