In vitro gametogenesis from embryonic stem cells

Many insights into mammalian germ cell development have been gained through genetic engineering and in vivo studies, but the lack of an in vitro system for deriving germ cells has hindered potential advances in germ cell biology. Recent studies have demonstrated embryonic stem cell differentiation into germ cells and more mature gametes, although significant unanswered questions remain about the functionality of these cells. The derivation of germ cells from embryonic stem cells in vitro provides an invaluable assay both for the genetic dissection of germ cell development and for epigenetic reprogramming, and may one day facilitate nuclear transfer technology and infertility treatments.     

Prospects for sexing mammalian sperm edited by Rupert P. Amman and George E. Seidel, Jr.; 288 pages; Cloth - $32.50, Paper - $22.50. Available from Colorado Associated University Press, 1338 Grandview Avenue, Box 480, University of Colorado, Boulder, Colorado, 80309

Progress has been made toward developing conditions to enable bull sperm to penetrate and fertilize ova in vitro. Efforts toward understanding oocyte maturation also hold promise. A procedure for bovine in vitro fertilization (IVF) involving recovery of oocytes near the time of ovulation, fertilization with in vitro capacitated sperm, embryo culture and transfer has led to development of normal offspring. Additional research is needed to facilitate recovery of mature oocytes and to improve in vitro conditions to insure development of viable embryos that can continue normal development following non-surgical uterine transfer.Bovine IVF promises a means to overcome certain types of infertility, to produce large numbers of half-siblings simultaneously, to greatly extend valuable semen, a better way to assess functional performance of male and female gametes, to provide synchronously developing pronuclear stage ova for nuclear transfer and/or gene injection, and many possibilities in research and for future applications.     

Human parthenogenetic embryonic stem cells: one potential resource for cell therapy

Pluripotent stem cells derived from somatic cells through such processes as nuclear transfer or induced pluripotent stem (iPS) cells present an important model for biomedical research and provide potential resources for cell replacement therapies. However, the overall efficiency of the conversional nuclear transfer is very low and the safety issue remains a major concern for iPS cells. Embryonic stem cells (ESCs) generated from parthenogenetic embryos are one attractive alternative as a source of histocompatible cells and tissues for cell therapy. Recent studies on human parthenogenetic embryonic stem cells (hPG ESCs) have revealed that these ESCs are very similar to the hESCs derived from IVF or in vivo produced blastocysts in gene expression and other characteristics, but full differentiation and development potential of these hPG ESCs have to be further investigated before clinical research and therapeutic interventions. To generate various pluripotent stem cells, diverse reprogramming techniques and approaches will be developed and integrated. This may help elucidate the fundamental mechanisms underlying reprogramming and stem cell biology, and ultimately benefit cell therapy and regenerative medicine. Supported by the National High Technology Research and Development Program of China (Grant No. 2006AA02A101).     

利用IVF废弃胚胎为核移植受体构建人体细胞克隆胚胎

目的：探讨利用IVF废弃胚胎构建人体细胞克隆胚胎的发育潜能及其在人治疗性克隆应用的可能性。方法：收集2008年7-12月在广州医学院第三附属医院进行体外受精-胚胎移植周期中的多精受精胚胎和MII期体外受精失败卵母细胞,运用显微操作技术构建人体细胞克隆胚胎,观察胚胎发育情况。结果：多精受精胚胎为核移植受体的克隆胚胎能够发育到8-细胞期,受精失败MII期卵母细胞为核移植受体的克隆胚胎能够激活,但不能够卵裂。两种IVF废弃的胚胎构建的人体细胞克隆胚胎在去核成功率,注核成功率上无显著差异（P＆gt;0.05）,但卵裂率和8细胞率上具有显著差异（P＆lt;0.05）。结论：多精受精胚胎比MII期体外受精失败卵母细胞更适合作为人核移植受体细胞。     

Protocol for the production of viable bimaternal mouse embryos

A reliable nuclear transfer method was first reported in 1983; it provided definite evidence that parthenogenetic embryos are lethal at early postimplantation in mammals. Subsequently, nuclear transfer has been extensively used as an important and versatile tool for investigating embryo and somatic-cell cloning and nucleo-cytoplasmic interactions. Further development of this technique has enabled the generation of bimaternal embryos containing two haploid sets of maternal genomes from female germ cells of different origins. By using a 2-d nuclear transfer system for oocyte reconstruction, viable mice can be produced solely from maternal genomes, without the participation of the paternal genome. This oocyte reconstruction system, as described in this protocol, could provide valuable guidelines for exploring the potential endowments of gametes and for conferring novel properties to them.     

In vitro fertilization as a tool for investigating sexual reproduction of angiosperms

In vitro fertilization (IVF) of isolated male and female gametes of flowering plants was first accomplished in the last decade. Successful isolation of male and female gametes, and culturing of in vitro zygotes to form new plants, is a prelude to the use of IVF for research into the cellular and molecular control of fertilization in higher plants and its application as a tool in biotechnology. Genes unique to male and female gametes and zygotes of higher plants, although currently incompletely characterized, are expected to permit direct molecular dissection of fertilization. By applying IVF and microculture to zygotes and endosperm obtained by both in vivo and in vitro methods, newly activated fusion products may be observed and manipulated in media where they are directly accessible to the techniques of molecular cell biology. IVF and zygote culture may also offer potential for creating new hybrid plants by fusing isolated gametes from different species to produce unique zygotes and ultimately plants that would be impossible to obtain using typical crossing techniques. Transformation and regeneration frequencies using IVF may also be high enough to avoid the necessity of adding controversial antibiotic and herbicide resistant genes to screen transformed products. This review describes advances using IVF in plant sexual reproduction and discusses its potential in the genetic improvement of flowering plants.     

Intrafallopian transfer of gametes and early stage embryos for in vivo culture in cattle

It may be possible to avoid inadequate in vitro culture conditions by incubating gametes or embryos in the oviducts for a short time. Ideally, an optimized procedure should be devised, combining in vitro and in vivo systems, in order to achieve synchronization in cattle. We transferred gametes as well as embryos in various stages of development and placed them into the oviducts. Embryos were recovered on Day 7 by flushing of oviducts and uterine horns. Blastocyst rates were determined on Day 7 and on Day 8. Experimental designs included transfer of in vitro matured cumulus oocyte complexes into previously inseminated heifers (COCs group), transfer of in vitro matured COCs simultaneously with capacitated spermatozoa (GIFTs group), transfer of four to eight cell stage embryos developed in vitro after IVM/IVF (Cleaved Stages group) and a group of solely in vitro produced embryos (IVP control group). Our results indicate that in vivo culture of IVM/IVF embryos in the homologous bovine oviduct has a positive influence on subsequent pre-implantation development. In addition, we have evidence that in vitro maturation and in vivo fertilization cannot be synchronized.     

Advances in livestock nuclear transfer

Cloning and transgenic animal production have been greatly enhanced by the development of nuclear transfer technology. In the past, genetic modification in domestic animals was not tightly controlled. With the nuclear transfer technology one can now create some domestic animals with specific genetic modifications. An ever-expanding variety of cell types have been successfully used as donors to create the clones. Both cell fusion and microinjection are successfully being used to create these animals. However, it is still not clear which stage(s) of the cell cycle for donor and recipient cells yield the greatest degree of development. While for the most part gene expression is reprogrammed in nuclear transfer embryos, all structural changes may not be corrected as evidenced by the length of the telomeres in sheep resulting from nuclear transfer. Even after these animals are created the question of "are they really clones?" arises due to mitochondrial inheritance from the donor cell versus the recipient oocyte. This review discusses these issues as they relate to livestock.     

Nuclear transfer technologies: between successes and doubts.

Cloning of mammals by nuclear transfer can lead to the birth of healthy adult animals but more often compromises the development of the reconstructed embryos. A high incidence of fetal and postnatal losses has been observed in several species, revealing the existence of long-lasting effects induced by the nuclear transfer procedures. Remodeling of donor chromatin by the recipient cytoplasm after nuclear transfer is frequently associated with the deregulation of specific genes, and recent observations point to the potential importance of time-dependent DNA methylation events in the occurrence of these alterations. Screening strategies to design nuclear transfer procedures that would mimic the epigenetic remodeling occurring in normal embryos are being designed, and improvement in the efficiency of procedures could imply a pre-conditioning of donor cells. Early mammalian development appears to be rather tolerant to epigenetic abnormalities, raising the possibility that even a fully functional reprogrammed genome may have been subjected to some epigenetic alterations. Bringing nuclear transfer to routine practice requires greater knowledge and understanding of the basic biological processes underlying epigenetic controls of nuclear activities. An important issue at present is to limit the production of those aberrant phenotypes that may result in significant insult to the nature and welfare of animals.     

体细胞基因打靶制备动物乳腺生物反应器的策略与应用

在转基因动物研究中,由于基因表达调控元件的人工拼接和外源基因在动物基因组中随机整合所带来的“位置效应”,致使转基因动物外源基因的表达水平不高并且差异较大。为此,利用定位整合优势,对以基因同源重组为基础的基因打靶技术进行了大量研究。介绍了就利用体细胞基因打靶和核移植技术制备动物乳腺生物反应器的策略和应用情况做一综述,并对提高基因打靶效率的各种策略,打靶细胞的选择,转基因细胞核移植的低融合事件以及基因打靶制备乳腺生物反应器的优越性进行分析。     

In vitro fertilization from co-cultured pollen tubes and female gametophytes of Douglas fir (Pseudotsuga menziesii)

 Our previous attempt on in vitro fertilization (IVF) in conifers resulted in pollen tube penetration of female gametophytes, but because of the rapid decline in egg viability, no further interaction occurred. In this report, we describe for the first time that IVF has been achieved in conifers. Using Douglas fir (Pseudotsuga menziesii), we describe a two-step process which involved induction of pollen tubes in culture followed by introduction of isolated female gametophytes at the tips of growing pollen tubes. Pollen tubes penetrated the introduced isolated female gametophytes at various places, but a number of tubes entered the egg cell through the neck cells similar to the in vivo condition. Under our current culture conditions, longevity of pollen tubes and eggs has been improved resulting in the release of sperms, fusion of gametes, and initial formation of the proembryo. Continued plasmolysis of the egg limited the number of successful gametic interactions. IVF has been accomplished in flowering plants in several ways, but the gametophyte-gametophyte IVF system described in this paper is unique. IVF offers a novel breeding technology that takes advantage of the sexual reproductive route. When coupled with hybridization and genetic transformation, IVF could result in the development of stable novel genotypes of economically superior trees. Received: 28 October 1997 / Accepted: 9 December 1997     

Prospects for transgenesis in the chick

Research to develop a useful method for genetic modification of the chick has been on-going since the first demonstrations in the mouse in the 1980s that genetic modification is an invaluable tool for the study of gene function. Manipulation of the chick zygote is possible but inefficient. Considerable progress has been made in developing potentially pluripotent embryo stem cells and their contribution to somatic chimeric birds well-established. Germ line transmission of gametes derived from genetically modified embryo cells has not been described. Transfer of primordial germ cells from a donor embryo to a recipient and production of functional gametes from the donor-derived cells is possible. Genetic modification of primordial germ cells before transfer and their recovery through the germ line has not been achieved. The first transgenic birds described were generated using retroviral vectors. The use of lentiviral vectors may make this approach a feasible method for transgenic production, although there are limitations to the applications of these vectors. It is likely that a method will be developed in the next few years that will enable the use of transgenesis as a tool in the study of development in the chick and for many other applications in basic research and biotechnology.     

Global gene expression analysis of bovine blastocysts produced by multiple methods

Reproductive efficiency using somatic cell nuclear transfer (SCNT) technology remains suboptimal. Of the various efforts to improve the efficiency, chromatin transfer (CT) and clone-clone aggregation (NTagg) have been reported to produce live cloned animals. To better understand the molecular mechanisms of somatic cell reprogramming during SCNT and assess the various SCNT methods on the molecular level, we performed gene expression analysis on bovine blastocysts produced via standard nuclear transfer (NT), CT, NTagg, in vitro fertilization (IVF), and artificial insemination (AI), as well as on somatic donor cells, using bovine genome arrays. The expression profiles of SCNT (NT, CT, NTagg) embryos were compared with IVF and AI embryos as well as donor cells. NT and CT embryos have indistinguishable gene expression patterns. In comparison to IVF or AI embryos, the number of differentially expressed genes in NTagg embryos is significantly higher than in NT and CT embryos. Genes that were differentially expressed between all the SCNT embryos and IVF or AI embryos are identified. Compared to AI embryos, more than half of the genes found deregulated between SCNT and AI embryos appear to be the result of in vitro culture alone. The results indicate that although SCNT methods have altered differentiated somatic nuclei gene expression to more closely resemble that of embryonic nuclei, combination of insufficient reprogramming and in vitro culture condition compromise the developmental potential of SCNT embryos. This is the first set of comprehensive data for analyzing the molecular impact of various nuclear transfer methods on bovine pre-implantation embryos.     

The birth of mice from testicular spermatozoa retrieved from frozen testicular sections

Male gamete cryopreservation has been widely used for both human reproduction and animal breeding. We investigated whether testicular spermatozoa retrieved from frozen testicular sections (10 or 25 mum thick) could support the full-term development of normal progeny. For this purpose, frozen testicular sections were prepared from two genetic backgrounds (BDF1 or B6 GFP transgenic mice), and the functional ability of testicular spermatozoa after preservation for 1 day, 1 mo, and 3 mo was assessed by intracytoplasmic sperm injection (ICSI). Testicular spermatozoa were successfully retrieved from frozen testicular sections for the use of ICSI, regardless of the preservation period. The ICSI technique revealed that oocytes (BDF1 or B6 background) injected with testicular spermatozoa prepared from frozen testicular sections developed into normal progeny, even though the sections had been cryopreserved for 3 mo at -30 degrees C. Approximately 15% and 5% of the embryos preserved for 3 mo developed to full term if the testicular spermatozoa were prepared from the 25- and 10-mum sections, respectively. These results clearly indicate that male gametes can be viably preserved in frozen testicular sections. The technique described herein will allow the preservation of male gametes in the form of a "book" or "file" by mounting the sections on a paper-thin sheet. Furthermore, this technique may be of value in the clinical treatment of severe male infertility, since testicular spermatozoa can easily be found through examination of testicular cross sections rather than by attempts to identify them in testicular cell suspension.     

Factors Associated with Failed Treatment: an Analysis of 121,744 Women Embarking on Their First IVF Cycles

Background

In-vitro fertilization (IVF) is the treatment of choice for unresolved infertility. It comprises a number of key steps, each of which has to be negotiated before the next is attempted, but the factors which are associated with failure at each stage have not been reported.

Methods and Findings

We analyzed anonymised national data on women undergoing their first fresh autologous IVF and intracytoplasmic sperm injection (ICSI) cycle in the United Kingdom between 2000 and 2007 to predict factors associated with overall lack of livebirth as well as the chance of non-progress at different stages of an IVF cycle. A total of 121,744 women were included in this analysis. Multivariable models underlined the importance of increased female age and duration of infertility, lack of previous pregnancy, and a diagnosis of tubal or male factor infertility in predicting the risk of not having a live birth in an IVF treatment. At each stage, a woman’s chance of proceeding to the next stage of IVF treatment is affected by increased age and duration of infertility. The intention to use intra-cytoplasmic sperm injection (ICSI) is associated with a decreased risk of treatment failure in women starting an IVF cycle (RR 0.93, 99% CI 0.92, 0.94) but this association is reversed at a later stage once fertilisation has been confirmed (RR=1.01, 99%CI 1.00, 1.03).

Conclusions

Female age is a key predictor of failure to have a livebirth following IVF as well as the risk of poor performance at each stage of treatment. While increased duration of infertility is also associated with worse outcomes at every stage, its impact appears to be less influential. Women embarking on ICSI treatment for male factor infertility have a lower chance of treatment failure but this does not appear to be due to increased chances of implantation of ICSI embryos.     

A novel isolator-based system promotes viability of human embryos during laboratory processing

In vitro fertilisation (IVF) and related technologies are arguably the most challenging of all cell culture applications. The starting material is a single cell from which one aims to produce an embryo capable of establishing a pregnancy eventually leading to a live birth. Laboratory processing during IVF treatment requires open manipulations of gametes and embryos, which typically involves exposure to ambient conditions. To reduce the risk of cellular stress, we have developed a totally enclosed system of interlinked isolator-based workstations designed to maintain oocytes and embryos in a physiological environment throughout the IVF process. Comparison of clinical and laboratory data before and after the introduction of the new system revealed that significantly more embryos developed to the blastocyst stage in the enclosed isolator-based system compared with conventional open-fronted laminar flow hoods. Moreover, blastocysts produced in the isolator-based system contained significantly more cells and their development was accelerated. Consistent with this, the introduction of the enclosed system was accompanied by a significant increase in the clinical pregnancy rate and in the proportion of embryos implanting following transfer to the uterus. The data indicate that protection from ambient conditions promotes improved development of human embryos. Importantly, we found that it was entirely feasible to conduct all IVF-related procedures in the isolator-based workstations.     

Anomalies génétiques et infertilité masculine

Fifteen percent of couples are infertile and in about 50% of cases the cause is of male origin. The aetiology is still unknown in more than 90% of cases and there may be genetic or environmental causes. Three approaches are used to detect genetic causes for male infertility: 1) cytogenetics, resulting in particular from progress made in molecular cytogenetics and the direct analysis of gametes by in situ molecular hybridation techniques. When a chromosome anomaly, the most common cause of infertility, including deletion of the Y chromosome, is discovered, it is not easy to distinguish between gene anomalies resulting from change and mechanical anomalies that are an integral part of meiosis; 2) the analysis of candidate genes, which often uses data obtained from animal, usually murine, models. This approach, frequently described in the literature, tends to be lengthy, expensive and rarely results in the discovery of an abnormal gene, as is the case, for example, with meiotic genes; 3) Mendel’s approach is clearly the preferred choice, studying as it does cases of inherited infertility, which is much more widespread than we might think.