Utero-tubal Embryo Transfer and Vasectomy in the Mouse Model

The transfer of preimplantation embryos to a surrogate female is a required step for the production of genetically modified mice or to study the effects of epigenetic alterations originated during preimplantation development on subsequent fetal development and adult health. The use of an effective and consistent embryo transfer technique is crucial to enhance the generation of genetically modified animals and to determine the effect of different treatments on implantation rates and survival to term. Embryos at the blastocyst stage are usually transferred by uterine transfer, performing a puncture in the uterine wall to introduce the embryo manipulation pipette. The orifice performed in the uterus does not close after the pipette has been withdrawn, and the embryos can outflow to the abdominal cavity due to the positive pressure of the uterus. The puncture can also produce a hemorrhage that impairs implantation, blocks the transfer pipette and may affect embryo development, especially when embryos without zona are transferred. Consequently, this technique often results in very variable and overall low embryo survival rates. Avoiding these negative effects, utero-tubal embryo transfer take advantage of the utero-tubal junction as a natural barrier that impedes embryo outflow and avoid the puncture of the uterine wall. Vasectomized males are required for obtaining pseudopregnant recipients. A technique to perform vasectomy is described as a complement to the utero-tubal embryo transfer.     

小鼠胚胎体外发育培养基中氨基酸含量变化

通过检测哺乳动物早期胚胎体外发育过程中可以消耗或生成某些氨基酸的含量,可以了解胚胎的发育潜能。利用反相高效液相色谱法(RP-HPLC)检测KSOMaa培养基中17种氨基酸含量的变化,了解昆明小白鼠(Mus musculus)植入前胚胎体外培养过程中氨基酸含量的变化,旨在寻找一种能有效支持昆明小鼠胚胎体外发育的培养基氨基酸组成,优化小鼠胚胎体外培养体系。将180枚原核胚分为9组,体外培养至囊胚,分别于胚胎发育不同时期取样做高效液相色谱分析。这些氨基酸在胚胎发育不同时期的培养基中含量变化可分为5种类型:①在2细胞期增加但在4细胞期、8～16细胞期减少,囊胚期又增加的氨基酸(甘氨酸、亮氨酸、苏氨酸、缬氨酸、苯丙氨酸、酪氨酸);②在胚胎发育各个时期均下降(谷氨酸、甲硫氨酸、精氨酸、组氨酸);③在胚胎发育各个时期均增加(丝氨酸、赖氨酸、丙氨酸);④2细胞期含量减少而在其他时期持续增加(天冬氨酸、脯氨酸、色氨酸);⑤囊胚期减少,其他时期都有增加(异亮氨酸)。     

Human embryonic stem cells carrying mutations for severe genetic disorders

Human embryonic stem cells (HESCs) carrying specific mutations potentially provide a valuable tool for studying genetic disorders in humans. One preferable approach for obtaining these cell lines is by deriving them from affected preimplantation genetically diagnosed embryos. These unique cells are especially important for modeling human genetic disorders for which there are no adequate research models. They can be further used to gain new insights into developmentally regulated events that occur during human embryo development and that are responsible for the manifestation of genetically inherited disorders. They also have great value for the exploration of new therapeutic protocols, including gene-therapy-based treatments and disease-oriented drug screening and discovery. Here, we report the establishment of 15 different mutant human embryonic stem cell lines derived from genetically affected embryos, all donated by couples undergoing preimplantation genetic diagnosis in our in vitro fertilization unit. For further information regarding access to HESC lines from our repository, for research purposes, please email dalitb@tasmc.health.gov.il.     

Lysophosphatidic acid accelerates development of porcine embryos by activating formation of the blastocoel

Culture media modifications, including the addition of various factors, are important for the in vitro production of oocytes and embryos. In this study, we investigated the effects of lysophosphatidic acid (LPA) on porcine embryo development. Porcine parthenogenetic embryos were cultured with 0, 0.1, 1, and 10 μM LPA for 7 days, or cultured in basic medium until Day 4 and then treated with LPA from Days 4 to 7. No difference in the in vitro development of embryos cultured with LPA for 7 days was observed. Conversely, rates of blastocyst and over‐expanded blastocyst formation were higher in the 0.1 and 1 µM LPA‐treated versus the other groups of embryos treated from Days 4 to 7. Moreover, formation of early blastocysts occurred earlier and embryo size was larger in LPA‐treated compared to control embryos. Expression of Connexin 43 and gap junction and cell adhesion‐related genes (GJC1 and CDH1, respectively) was also higher in LPA‐treated compared to control embryos. Despite no difference in the blastocyst total cell number between groups, the apoptotic index was lower in the LPA‐treated group than in the control group; indeed, BCL2L1 (B‐cell lymphoma 2‐like protein 1) expression increased while BAK (Bcl‐2 homologous antagonist killer) decreased in the LPA‐treated group. Thus, addition of LPA to the medium from Days 4 to 7 of culture improves blastocyst formation and aids the development of preimplantation embryos.     

Swine in Biomedical Research: Creating the Building Blocks of Animal Models

Abstract

Sex of preimplantation porcine embryos was determined by DNA amplification using porcine male(Y chromosome)‐specific DNA primers in the polymerase chain reaction (PCR). In order to determine the sensitivity of this sexing method, single porcine embryos ranging from unfertilized ova to the blastocyst stage were amplified in the PCR using the Y‐specific primers, and analyzed by ethidium bromide‐staining of polyacrylamide gels. The 192 bp product which denotes the presence of the Y chromosome was seen in the embryos. The unfertilized ova which is of female origin gave no product. These results are representative of PCR analysis of a total of 34 swine embryos.

Results obtained using the PCR for sexing were validated by karyotyping and confirmed by in situ hybridization with the porcine Y‐chromosome‐specific probe. In order to confirm the sex of the embryos determined by PCR, 10 day‐old porcine preimplantation embryos were biopsied to produce a small number of cells for sex determination via PCR, while the remainder of the embryo was prepared for in situ hybridization using the biotinylated probe. In situ hybridization performed on embryos shown to be male by PCR, showed pinpoint fluorescence within the nuclei, similar to that obtained when male porcine lymphocytes were hybridized. No evidence of fluorescence was seen when in situ hybridization was performed in parallel on embryos determined to be female by the PCR.

The PCR was found to be a relatively fast, accurate and reproducible means of sex determination of swine preimplantation embryos. This capability could have significant impact on animal breeding and production programs by using PCR as a screening tool for traits of economic importance.     

Autocrine embryotropins revisited: how do embryos communicate with each other in vitro when cultured in groups?

In the absence of the maternal genital tract, preimplantation embryos can develop in vitro in culture medium where all communication with the oviduct or uterus is absent. In several mammalian species, it has been observed that embryos cultured in groups thrive better than those cultured singly. Here we argue that group‐cultured embryos are able to promote their own development in vitro by the production of autocrine embryotropins that putatively serve as a communication tool. The concept of effective communication implies an origin, a signalling agent, and finally a recipient that is able to decode the message. We illustrate this concept by demonstrating that preimplantation embryos are able to secrete autocrine factors in several ways, including active secretion, passive outflow, or as messengers bound to a molecular vehicle or transported within extracellular vesicles. Likewise, we broaden the traditional view that inter‐embryo communication is dictated mainly by growth factors, by discussing a wide range of other biochemical messengers including proteins, lipids, neurotransmitters, saccharides, and microRNAs, all of which can be exchanged among embryos cultured in a group. Finally, we describe how different classes of messenger molecules are decoded by the embryo and influence embryo development by triggering different pathways. When autocrine embryotropins such as insulin‐like growth factor‐I (IGF‐I) or platelet activating factor (PAF) bind to their appropriate receptor, the phosphatidylinositol‐4,5‐bisphosphate 3‐kinase (PI3K) pathway will be activated which is important for embryo survival. On the other hand, the mitogen‐activated protein kinase (MAPK) pathway is activated when compounds such as hyaluronic acid and serotonin bind to their respective receptors, thereby acting as growth factors. By activating the peroxisome‐proliferator‐activated receptor family (PPAR) pathway, lipophilic autocrine factors such as prostaglandins or fatty acids have both survival and anti‐apoptotic functions. In conclusion, considering different types of messenger molecules simultaneously will be crucial to understanding more comprehensively how embryos communicate with each other in group‐culture systems. This approach will assist in the development of novel media for single‐embryo culture.     

BMP4 plays a role in apoptosis during human preimplantation development

Comprehensive understanding of lineage differentiation and apoptosis processes is important to increase our knowledge of human preimplantation development in vitro. We know that BMP signaling is important for different processes during mammalian development. In mouse preimplantation embryos, BMP signaling has been shown to play a role in the differentiation into extra‐embryonic trophectoderm (TE) and primitive endoderm (PE). In this study, we aimed to investigate the effect of bone morphogenetic protein 4 (BMP4) supplementation on human preimplantation embryos cultured in vitro. The BMP4 treatment impaired human blastocyst formation. No differences in the expression of the early lineage markers NANOG, CDX2, GATA3, and GATA6 were found between BMP4‐treated embryos and controls. Instead, BMP4 supplementation triggered apoptosis in the human blastocyst. We focused on P53, which is known to play a major role in the apoptosis. In BMP4‐treated embryos, the P53 responsive gene expression was not altered; however, the P53 deacetylase SIRT1 was downregulated and acetylated P53 was increased in mitochondria. Altogether, our findings suggest that BMP4 plays a role in the apoptosis during human preimplantation development.     

Animal reproduction biotechnology in Poland

The key research areas of the Department are: in vitro production of embryos, embryo cryopreservation, animal transgenesis, cloning, cytometric semen sexing and evaluation. Research has been focused on the in vitro production of animal embryos, including the development of complex methods for oocyte maturation, fertilization and embryo culture. Moreover, experiments on long-term culturing of late preantral and early antral bovine ovarian follicles have been developed. Studies on the cloning of genetically modified pigs with "humanized" immunological systems have been undertaken. A cloned goat was produced from oocytes reconstructed with adult dermal fibroblast cells. The novel technique of rabbit chimeric cloning for the production of transgenic animals was applied; additionally, the recipient-donor-cell relationship in the preimplantation developmental competences of feline nuclear transfer embryos has been studied. Regarding transgenic animal projects, gene constructs containing growth hormone genes connected to the mMt promoter were used. Modifications of milk composition gene constructs with tissue-specific promoters were performed. Moreover, pigs for xenotransplantation and animal models of human vascular diseases have been produced. Over the last 15 years, our flow cytometry research group has focused its work on new methods for sperm quality assessment and sex regulation. In the 1970s, our team initiated studies on embryo cryopreservation. As a result of vitrification experiments, the world's first rabbits and sheep produced via the transfer of vitrified embryos were born.     

Radical solutions and cultural problems: Could free oxygen radicals be responsible for the impaired development of preimplantation mammalian embryos in vitro?

A major obstacel to the study of mammalian development, and to the practical application of knowledge gained from it in the clinic during therapeutic in vitro fertilisation and embryo transfer (IVF-ET), is the propensity of embryos to become retarded or arrested during their culture in vitro. The precise developmental cell cycle in which embryos arrest or delay is characteristic for the species and coincides with the earliest period of embryonic gene expression. Much evidence reviewed here implicates free oxygen radicals (FORs) in the process of arrest. Thus, studies on the development of mouse preimplantation embryos in vitro have shown that (i) FORs are elevated in vitro, but not in vivo, at the time at which embryos become arrested or delayed, (ii) systems for removing reactive oxygen species to limit the formation of hydroxy radicals are present, although they have not yet been assessed quantitatively and may differ qualitatively from those in adult cells, (iii) metabolic and possibly genetic adaptations to oxidative damage are evident, (iv) published procedures for overcoming in vitro arrest are explicable in terms of FOR-mediated damage or responses and (v) the arrest or delay of most embryos in vitro can be reduced or prevented experimentally by addition of metal chelators to limit hydroxy radical formation and lipid hydroperoxidation.     

Cell engineering and genetic approaches to development of human embryonic stem cell models for genetic disorders

We introduced a novel approach for the establishment of genetically modified hESC lines, and have shown that mutant hESC may be derived from affected embryos after preimplantation genetic diagnosis (PGD) screening for a particular single gene disorder. Here we describe the procedure of embryo and cell manipulation, their diagnostic layout, and the analysis of the efficiency of embryo development and hESC establishment, as well as the developments for hESC derivation in animal-product-free conditions. Our study shows that a high efficiency of hESC derivation (50%) is especially crucial when working with rare and unique resources such as genetically screened embryos necessary for the derivation of hESC lines that represent specific genetic diseases.     

Amino acid metabolism of bovine blastocysts: a biomarker of sex and viability

The ratio of male/female embryos may be modified by environmental factors such as maternal diet in vivo and the composition of embryo culture media in vitro. We have used amino acid profiling, a noninvasive marker of developmental potential to compare the effect of sex on the metabolism of bovine blastocysts conceived in vivo and in vitro. Blastocysts were incubated individually for 24 hr in a close‐to‐physiological mixture of amino acids and the depletion or appearance of 18 amino acids measured using HPLC. Blastocysts were then sexed by PCR. Amino acid depletion by in vitro‐produced blastocysts and expanded blastocysts was higher than in embryos conceived in vivo (P = 0.02). When cultured in vitro, female embryos exhibited increased depletion of arginine, glutamate, and methionine and appearance of glycine, while male embryos displayed increased depletion of phenylalanine, tyrosine, and valine. Overall, in vitro‐produced blastocysts exhibited sex‐specific differences in metabolic profiles of 7 out of 18 amino acids; in vivo‐produced, in 2 out of 18. These differences had disappeared by the expanded blastocyst stages. We have also shown that amino acid metabolism can predict the ability of bovine zygotes to develop to the blastocyst stage, providing “proof of principle” for the use of this technology in clinical IVF to select single embryos for transfer and thereby avoid the problem of multiple births. Mol. Reprod. Dev. 77: 285–296, 2010. © 2010 Wiley‐Liss, Inc.     

Platelet activating factor (PAF) production by mouse embryos in vitro and its effect on embryonic metabolism

Factors affecting the production of platelet activating factor (PAF) by mouse embryos during culture in vitro were investigated. Detectable levels of embryo-derived PAF were produced within 1-4 hr with maximum PAF activity being observed after 6 hr of culture in vitro. The amount of PAF detected in media after 24 hr of culture of two-cell embryos was equivalent to 12.8 ng PAF/embryo. However, differences in activity were apparent with increased time in culture. Reduced synthesis of PAF during culture in vitro was supported by the observation that morulae stage embryos collected fresh from the reproductive tract displayed more PAF activity than morulae resulting from the 48 hr culture of two-cell embryos. In addition to determining production characteristics of PAF by embryos, we also show that the production of CO2 from carbon-1 position of lactate is positively correlated with the ability of embryos to develop during subsequent culture in vitro and therefore could be used as a measure of embryo viability. Furthermore, culture of embryos in media supplemented with PAF resulted in an increase in lactate utilization demonstrating a direct effect of PAF on the embryo. As PAF is produced by preimplantation embryos, an autocoid role of PAF in regulating embryo development is implicated. Therefore, the reduced production of PAF by embryos in vitro may explain the decreased viability of embryos commonly observed following their culture in vitro.     

Quiet please,do not disturb: a hypothesis of embryo metabolism and viability

This review uses nutritional markers of normal and impaired development to address the question; what makes a viable mammalian preimplantation embryo? Resolution of this question is important to ensure the long‐term safety of embryo‐based biotechnologies in man and domestic animals, the optimisation of embryo production and culture conditions and the development of methods to select viable embryos for replacement. After considering the nutrition of embryos and somatic cells, and the phenomenon of caloric restriction, it is concluded that preimplantation embryo survival is best served by a relatively low level of metabolism; a situation achieved by reducing the concentrations of nutrients in culture media and encouraging the use endogenous resources. BioEssays 24:845–849, 2002. © 2002 Wiley Periodicals, Inc.     

Interspecies somatic cell nuclear transfer is dependent on compatible mitochondrial DNA and reprogramming factors

Interspecies somatic cell nuclear transfer (iSCNT) involves the transfer of a nucleus or cell from one species into the cytoplasm of an enucleated oocyte from another. Once activated, reconstructed oocytes can be cultured in vitro to blastocyst, the final stage of preimplantation development. However, they often arrest during the early stages of preimplantation development; fail to reprogramme the somatic nucleus; and eliminate the accompanying donor cell's mitochondrial DNA (mtDNA) in favour of the recipient oocyte's genetically more divergent population. This last point has consequences for the production of ATP by the electron transfer chain, which is encoded by nuclear and mtDNA. Using a murine-porcine interspecies model, we investigated the importance of nuclear-cytoplasmic compatibility on successful development. Initially, we transferred murine fetal fibroblasts into enucleated porcine oocytes, which resulted in extremely low blastocyst rates (0.48%); and failure to replicate nuclear DNA and express Oct-4, the key marker of reprogramming. Using allele specific-PCR, we detected peak levels of murine mtDNA at 0.14±0.055% of total mtDNA at the 2-cell embryo stage and then at ever-decreasing levels to the blastocyst stage (<0.001%). Furthermore, these embryos had an overall mtDNA profile similar to porcine embryos. We then depleted porcine oocytes of their mtDNA using 10 μM 2',3'-dideoxycytidine and transferred murine somatic cells along with murine embryonic stem cell extract, which expressed key pluripotent genes associated with reprogramming and contained mitochondria, into these oocytes. Blastocyst rates increased significantly (3.38%) compared to embryos generated from non-supplemented oocytes (P<0.01). They also had significantly more murine mtDNA at the 2-cell stage than the non-supplemented embryos, which was maintained throughout early preimplantation development. At later stages, these embryos possessed 49.99±2.97% murine mtDNA. They also exhibited an mtDNA profile similar to murine preimplantation embryos. Overall, these data demonstrate that the addition of species compatible mtDNA and reprogramming factors improves developmental outcomes for iSCNT embryos.     

In vitro-produced equine embryos: production of foals after transfer, assessment by differential staining and effect of medium calcium concentrations during culture

Viability of equine embryos produced by oocyte maturation, intracytoplasmic sperm injection and embryo culture to the blastocyst stage in vitro was evaluated after transfer of embryos to recipient mares. No pregnancies were produced after transfer of five blastocysts that had been cultured in G media. Transfer of 10 blastocysts cultured in modified DMEM/F-12 medium produced five pregnancies and three live foals; the two lost pregnancies developed only trophoblast (based on transrectal ultrasonography). To evaluate the status of the inner cell mass, equine blastocysts produced in vivo and in vitro were assessed after differential staining. A discrete inner cell mass could not be appreciated in blastocysts of either source after staining; this was attributed to the presence of a network of cells within the trophoblastic vesicle. Because increased medium calcium concentrations have been reported to decrease the incidence of trophoblast-only pregnancy after transfer of equine nuclear transfer embryos, we investigated the effect of increased calcium concentrations during oocyte maturation or during embryo culture. Increasing calcium concentration of culture medium from 2 to 5.6mM during in vitro oocyte maturation did not affect maturation rate (75 and 68%, respectively) or blastocyst development after fertilization (23 and 27%). However, increasing calcium concentration (from 1.3 to 4.9 mM) of medium used for embryo culture significantly decreased blastocyst development (27% versus 13%, respectively) and adversely affected embryo morphology. More work is needed to optimize culture systems for in vitro production of equine embryos.     

Inhibition of phosphorylated Ser473‐Akt from translocating into the nucleus contributes to 2‐cell arrest and defective zygotic genome activation in mouse preimplantation embryogenesis

Phosphorylated Ser473‐Akt (p‐Ser473‐Akt) is extensively studied as a correlate for the activity of Akt, which plays an important role in mouse oogenesis and preimplantation embryogenesis. However, little progress has been made about its effect on the mouse zygotic genome activation (ZGA) of 2‐cell stage in mouse preimplantation embryos. In this study, we confirmed its localization in the pronuclei of 1‐cell embryos and found that p‐Ser473‐Akt acquired prominent nucleus localization in 2‐cell embryos physiologically. Akt specific inhibitors API‐2 and MK2206 could inhibit the development of mouse preimplantation embryos in vitro, and induce 2‐cell arrest at certain concentrations. 2‐cell embryos exposed to 2.0 μmol/L API‐2 or 30 μmol/L MK2206 displayed attenuated immunofluorescence intensity of p‐Ser473‐Akt in the nucleus. Simultaneously, qRT‐PCR results revealed that 2.0 μmol/L API‐2 treatment significantly downregulated the mRNA pattern of MuERV‐L and eIF‐1A, two marker genes of ZGA, suggesting a defect in ZGA compared with that of control group. Collectively, our work demonstrated the nuclear localization of p‐Ser473‐Akt during major ZGA, and Akt specific inhibitors API‐2 and MK2206 which led to 2‐cell arrest inhibited p‐Ser473‐Akt from translocating into the nucleus of 2‐cell embryos with defective ZGA as well, implying p‐Ser473‐Akt may be a potential player in the major ZGA of 2‐cell mouse embryos.