Effect of culture environment on gene expression and developmental characteristics in IVF-derived embryos

It is generally accepted that mammalian preimplantation embryos are sensitive to their environment and that conditions of culture can affect future growth and developmental potential both pre- and postnatally. Evidence suggests that while culture conditions during bovine in vitro embryo production can impact somewhat on the developmental potential of the early embryo, the intrinsic quality of the oocyte is the key factor determining the proportion of oocytes developing to the blastocyst stage. In addition, evidence suggests that the period of post fertilization embryo culture is the most critical period affecting blastocyst quality assessed in terms of cryotolerance, gene expression pattern and ability to establish a pregnancy. This paper reviews the current literature, with emphasis on the bovine model, demonstrating evidence for an effect of post fertilization culture environment on embryo gene expression and quality.     

Effects of oocyte quality, oxygen tension, embryo density, cumulus cells and energy substrates on cleavage and morula/blastocyst formation of bovine embryos

Various factors, such as quality of the oocyte, oxygen tension, embryo density, and kind of energy substrate during in vitro production of embryos may affect the rate of preimplantation embryo development. In the present study we used 12553 bovine oocytes aspirated from slaughterhouse ovaries to evaluate various culture conditions that would increase in vitro production of advanced stages of preimplantation embryos. The morphological quality of the oocyte based on the compactness and number of layers of cumulus cells had significant positive effects on the rates of in vitro maturation, fertilization and development to the morula and blastocyst stages. None of the corona-enclosed or nude oocytes progressed beyond the 8- to 16-cell stage. The level of oxygen (5 or 20%) did not affect the proportion of one-cell embryos undergoing cleavage or progressing to morula and blastocyst stages. The rate of development of one-cell embryos originating from inferior quality oocytes was significantly improved when cultured in groups of 40 instead of 20 embryos per 0.5 mL medium. In the presence of cumulus cells, glucose had beneficial effects on in vitro maturation and subsequent development of IVM-IVF zygotes. The presence of serum improved the rate of in vitro development of one-cell embryos. Minimum Essential Medium supplemented with energy substrates according to the findings of metabolic studies was less effective in supporting in vitro maturation and subsequent development than TCM-199. In conclusion, morphological grading of immature oocytes is an appropriate selection criterion for their developmental ability. Embryo yields from low quality oocytes can be increased by culturing them in large groups. Serum is not essential for in vitro generation of embryos but its addition improves rates of success.     

In vitro production of embryos in swine.

In recent years, progress has been achieved in the production of pig embryos through IVM and IVF techniques. Cytoplasmic maturation of oocytes has been improved by modifications to IVM procedures. However, the historical problem of polyspermic penetration still remains a major issue to be solved. Recent studies indicate that the type of IVF medium and certain modifications to that medium can reduce polyspermy. Efforts should be directed to increase the developmental competence and quality of embryos. At present, many embryo culture (EC) media are available that can overcome the historical 4-cell block and support development of early in vivo derived embryos to the blastocyst stage. In contrast, blastocyst development of in vitro produced embryos in these culture media varies significantly. Furthermore, morphology and cell numbers in in vitro produced blastocysts are inferior to their in vivo counterparts. However, several modifications to EC techniques have improved embryo quality and developmental competence. Testing embryo viability through surgical transfer to recipient animals has resulted in acceptable pregnancy rates with moderate litter sizes. Although reliable in vitro systems are available for the generation of pig embryos, the problem of polyspermy and poor embryo development hamper their large-scale implementation. Further research efforts should be directed to improve oocyte/embryo quality and the methods to minimize polyspermy through development of novel IVM, IVF, and EC techniques.     

Timing of the first zygotic cleavage as a marker of developmental potential of mammalian embryos

Embryo quality related to its developmental potential is now one of the most important issues in modern embryology. It has been demonstrated that some in vitro produced blastocysts fail to hatch and implant after transfer despite a normal morphology. Although embryos are able to adjust to sub-optimal culture conditions, significant changes in expression profiles of developmentally important genes have been noticed. Timing of the first zygotic cleavage is considered a non-invasive marker of embryo developmental potential and has been successfully used in human IVF programs for identifying embryos of superior quality. Early-cleaving zygotes are more likely to develop to the blastocyst stage than their late-cleaving counterparts. The timing of the first zygotic cleavage has been associated with several parameters that may affect developmental potential of the resulting embryos. The mechanism causing variation in the timing of the first zygotic cleavage has not been identified. It may be related to culture environment or to some intrinsic factors within the oocyte, the sperm or both. In this paper we discuss some of the important aspects related to the timing of the first zygotic cleavage and its influence on the developmental competence of resulting embryos.     

In vitro and in vivo developmental capacity of oocytes from prepubertal and adult sheep

Development to the blastocyst stage and survival following embryo transfer were assessed for oocytes obtained from prepubertal and adult sheep matured and fertilized in vitro. The rates of maturation, fertilization and cleavage in vitro did not differ significantly between oocytes from prepubertal and adult sheep. The proportion of cleaved zygotes reaching the blastocyst stage was significantly lower for oocytes derived from prepubertal than for those from adult sheep (15.4% and 34.1% respectively). There were no differences in the pregnancy rate and number of lambs born following transfer of blastocyst stage embryos derived from prepubertal and adult sheep to adult recipients. These data show that embryos derived from prepubertal lamb oocytes have reduced developmental potential in vitro but, of those which do reach the blastocyst stage, they have equal capacity to develop to term as embryos derived from adult sheep.     

Fertilization in vitro with spermatozoa from different mice increased variation in the developmental potential of embryos compared to artificial parthenogenetic activation

Although successful embryo development is dependent upon genetic and epigenetic contributions from both the male and female, the male potential to adversely affect embryo development has been scarcely studied. It is unclear whether the sperm variation among different males would affect the outcome of oocyte evaluation by embryo development following fertilization. In the present study, variation in the developmental potential of mouse embryos was first compared between in vitro fertilization with epididymal spermatozoa from different males and Sr(2+) parthenogenetic activation using oocytes of different qualities, and then the effect of male on fertilization and embryo development was examined using randomly chosen oocytes and spermatozoa from cauda epididymidis, vas deferens or electro-ejaculates. Rates of fertilization and blastocyst formation were significantly higher with spermatozoa from cauda epididymidis or vas deferens than with ejaculated spermatozoa. Rates of embryonic development differed significantly between different males, but not between different ejaculates of the same male. Analysis of standard errors of means and coefficients of variance indicated that as long as multiple males were involved, the variation in oocyte fertilization/activation and blastocyst formation was always higher after fertilization than after Sr(2+) parthenogenetic activation whether spermatozoa were collected from epididymidis, vas deferens or ejaculates and regardless of oocyte qualities. It is concluded that (1) epididymal mouse spermatozoa fertilize more oocytes than ejaculated spermatozoa under identical experimental conditions; (2) like farm animals, the mice also show a remarkable male effect on the developmental potential of in vitro produced embryos although they are supposed to be less genetically diverse; (3) parthenogenetic activation is recommended for assessment of oocyte quality to exclude the effect of male.     

Maternal age effect on early human embryonic development and blastocyst formation

In humans, age-related decline in female fertility can be explained by a reduction in quality either of the older uterus or of the embryos arising from aging oocytes. The aim of this study was to examine the latter hypothesis, using in vitro fertilization (I.V.F.) and coculture of embryos until the blastocyst stage. We determined the blastocyst formation rate ([expanded blastocysts/blastocysts]*100) according to the patient's age the day of I.V.F. With increase in age, the number of retrieved oocytes decreased, without alteration of the cleavage rate. In patients above age 30 years, preimplantation development to blastocysts declined due to an increase in embryo arrest at the morula stage. If blastocyst stage was reached, a negative linear relationship between blastocyst expansion rate and patient age was observed. Drops in gamete production and embryo development with increasing age led to a drastic decrease in patients having at least one expanded blastocyst (<30 years, 82%; ≥40 years, 36%). A high delivery rate per oocyte retrieval (25.8%) was observed in patients above age 40 years after embryo transfer at the blastocyst stage. These results give a clear indication of decline in the quality of human embryos arising from aging oocytes. The origin of this alteration is discussed in terms of chromosome abnormalities, role of maternally-inherited products from the oocyte, timing of genomic activation, and temporal pattern of gene expression during initial development of the human embryo. © 1996 Wiley-Liss, Inc.     

Responses of oocytes and embryos to the culture environment

Embryo development is strongly influenced by events occurring during oocyte maturation. Although many immature oocytes are capable of completing meiosis in vitro, only a small percentage of the original pool of immature oocytes is competent to continue development to the blastocyst stage and subsequently result in a pregnancy. This indicates that maturation of oocytes in vitro may not be occurring in an entirely normal manner. Cytoplasmic changes occurring during maturation, collectively termed cytoplasmic maturation, are essential for embryonic development. The cytoplasm of the oocyte may play a crucial role in assembling the correct metabolic machinery for production of sufficient energy for cellular functions during maturation, cleavage and blastocyst formation. A better understanding of the structural, functional and metabolic characteristics of the oocyte during maturation, and the consequence of changes in these parameters on developmental competence is needed. Understanding the role of cytoplasmic changes during oocyte maturation will help increase the efficiency of in vitro embryo production. Better embryo production strategies will facilitate basic research into the control of early development, improve implementation in endangered species, provide a source of high quality oocytes for nuclear transfer and transgenic technologies and benefit the commercial embryo transfer industry.     

The contribution of the male to ovine embryogenesis in an in vitro embryo production system

The differences in the embryo production potential of four rams used in a commercial embryo transfer program were examined in both in vivo and in vitro embryo production systems. Processing frozen-thawed spermatozoa through Percoll density gradients prior to in vitro insemination eliminated differences in the estimates of sperm viability between the four rams, and yet, differences in embryo production persisted throughout the in vitro culture period. However, there was no effect of ejaculate within ram on embryo production rates. In addition, the timing of the onset of the differences between the rams at each stage of in vitro embryo development were revealed. Ram 2 differed from ram 4 in the proportion of fertilized oocytes at 17 h post-insemination (pi) and by 52 h, ram 3 differed from ram 4 in the proportion of cleaved embryos, and the observed differences between ram 1 and ram 2 in their blastocyst production were initiated prior to activation of the embryonic genome. Once differences in embryo development rates were detected among the four rams, they persisted throughout the in vitro culture period. The reduced in vitro fertilization (IVF) rates from ram 2 compared with the other rams was paralleled in vivo by the significantly lower proportion of embryos recovered from ewes mated to ram 2, and this was further exacerbated by a significantly lower embryo survival rate after transfer. However, the subtle differences observed in the timing of the contribution of each sire to embryo development during in vitro culture were not able to be detected in vivo. However, the higher proportions of transferable quality blastocysts obtained from ewes mated to ram 4 did not result in increased embryo survival throughout the remainder of gestation. Therefore, in this study, the blastocyst production potential for a particular sire, either in vitro or in vivo, does not necessarily reflect the potential for the production of live offspring.     

In vitro production of bovine embryos using individual oocytes

The development of a bovine in vitro embryo production system where individual oocytes could be followed through to the morula or blastocyst stage would be of interest to several fields of study and would allow us to characterise developmentally competent oocytes and their corresponding follicular environment. Several studies have, however, reported significantly reduced embryo development when oocytes or embryos were cultured individually compared to in groups. The aim of this study was to establish such an embryo production system, with embryo development rates similar to that observed under control (grouped) conditions. This study showed that conservation of the oocyte/embryo medium densities generally employed for grouped culture does not facilitate embryo development if oocytes/embryos are cultured individually. However, individual oocytes could effectively undergo IVM/IVF/IVC to the expanded blastocyst stage with some small modifications to the standard protocol. Individual IVF was effective if carried out in either 100 μl of medium in wells or in 50 μl droplets. Individual IVC, if carried out in 10 or 20 μl droplets of SOF with FCS added at either 0 or 24 hr, was effective in terms of blastocyst yields but 20 μl droplets did yield significantly fewer hatched blastocysts compared to grouped controls (P < 0.05). An entirely individual embryo production system was effective when it included individual IVM in 10 μl droplets of M199 + 10 ng/ml EGF resulting in day 8 blastocyst yields not significantly different from controls (38% vs. 35% respectively). The use of 10% FCS during individual IVM appeared, at least under our experimental conditions, to be detrimental to subsequent development. The uses of an individual system for embryo production are many and varied. The results of this study show clearly that a large proportion of bovine oocytes can develop to the blastocyst stage when matured, fertilized, and cultured individually. This opens the way for studies regarding the quality of specific oocytes in such a way as will greatly improve our understanding of the events of late folliculogenesis. © 1996 Wiley-Liss, Inc.     

Development of sheep androgenetic embryos is boosted following transfer of male pronuclei into androgenetic hemizygotes

Androgenetic embryos are useful model for investigating the contribution of the paternal genome to embryonic development. Little work has been done with androgenetic embryo production in domestic animals. The aim of this study was the production of diploid androgenetic sheep embryos. In vitro matured sheep oocytes were enucleated and fertilized in vitro; parthenogenetic and normally fertilized embryos were also produced as a control. Fifteen hours after in vitro fertilization (IVF), presumptive zygotes were centrifuged and scored for the number of pronucleus. IVF, parthenogenetic, and androgenetic embryos (haploid, diploid, and triploid) were cultured in SOFaa medium with bovine serum albumin (BSA). The proportion of oocytes with polyspermic fertilization increased linearly with increasing sperm concentration. After IVF, there was no significant difference in early cleavage and morula formation rates between the groups, while there was a significant difference on blastocyst development between IVF, parthenogenetic, and androgenetic embryos, the last ones displaying poor developmental potential (IVF, parthenogenetic, and haploid, diploid, and triploid androgenetic embryos: 43%, 38%, 0%, 2%, and 2%, respectively). In order to boost androgenetic embryonic development, we produced diploid androgenetic embryos through pronuclear transfer. Single pronuclei were aspirated with a bevelled pipette from haploid or diploid embryos and transferred into the perivitelline space of other haploid embryos, and the zygotes were reconstructed by electrofusion. Fusion rates approached 100%. Pronuclear transfer significantly increased blastocyst development (IVF, parthenogenetic, androgenetic: Diploid into Haploid, and Haploid into Haploid: 42%, 42%, 19%, and 3%, respectively); intriguingly, the Haploid + Diploid group showed the highest development to blastocyst stage. The main findings of our study are: (1) sheep androgenetic embryos display poor developmental ability compared with IVF and parthenogenetic embryos; (2) diploid androgenetic embryos produced by pronuclear exchange developed in higher proportion to blastocyst stage, particularly in the Diploid-Haploid group. In conclusion, pronuclear transfer is an effective method to produce sheep androgenetic blastocysts.     

Oocyte-secreted factors enhance oocyte developmental competence

The capacity of fully grown oocytes to regulate their own microenvironment by paracrine factors secreted by the oocyte (oocyte-secreted factors, OSFs) may in turn contribute to oocyte developmental competence. Here, we investigated if OSFs have a direct influence on oocyte developmental competence during in vitro maturation (IVM). Bovine cumulus-oocyte complexes (COCs) were aspirated from abattoir-derived ovaries and matured in serum-free medium. COCs were either co-cultured with denuded oocytes (DOs) or treated with specific OSFs: recombinant bone morphogenetic protein 15 (BMP15) and/or growth differentiation factor 9 (GDF9). Following maturation, embryos were fertilized and cultured in vitro and blastocyst development and cell number were assessed on day 8. Co-culturing intact COCs with DOs did not affect cleavage rate, but increased (P<0.001) the proportion of cleaved embryos that reached the blastocyst stage post-insemination from 39% to 51%. OSFs also altered blastocyst cell allocation as co-culture of COCs with DOs significantly increased total and trophectoderm cell numbers, compared to control COCs. BMP15 alone, GDF9 alone or the two combined all (P<0.05) increased the proportion of oocytes that reached the blastocyst stage post-insemination from 41% (controls) to 58%, 50% and 55%, respectively. These results were further verified in neutralization experiments of the exogenous growth factors and of the native OSFs. Follistatin and the kinase inhibitor SB-431542, which antagonize BMP15 and GDF9, respectively, neutralized the stimulatory effects of the exogenous growth factors and impaired the developmental competence of control COCs. These results demonstrate that OSFs, and particularly BMP15 and GDF9, enhance oocyte developmental competence and provide evidence that OSF regulation of the COC microenvironment is an important determinant of oocyte developmental programming.     

The mycotoxin beauvericin induces oocyte mitochondrial dysfunction and affects embryo development in the juvenile sheep

Beauvericin (BEA) is a mycotoxin produced by Beauveria bassiana and Fusarium species recently reported as toxic on porcine oocyte maturation and embryo development. The aim of this study was to assess, in the juvenile sheep, whether its effects are due to alterations of oocyte and/or embryo bioenergetic/oxidative status. Cumulus‐oocyte‐complexes (COCs) were exposed to BEA during in vitro maturation (IVM), evaluated for cumulus cell (CC) apoptosis, oocyte maturation and bioenergetic/oxidative status or subjected to in vitro fertilization (IVF) and embryo culture (IVEC). Oocyte nuclear maturation and embryo development were assessed after Hoechst staining and CC apoptosis was analysed by terminal deoxynucleotidyl transferase‐mediated dUTP nick‐End labeling assay and chromatin morphology after Hoechst staining by epifluorescence microscopy. Oocyte and blastocyst bioenergetic/oxidative status were assessed by confocal microscopy after mitochondria and reactive oxygen species labelling with specific probes. BEA showed various toxic effects, that is, short‐term effects on somatic and germinal compartment of the COC (CCs and the oocyte) and long‐term carry‐over effects on developing embryos. In detail, at 5 µM, it significantly reduced oocyte maturation and immature oocytes showed increased late‐stage (Type C) CC apoptosis and DNA fragmentation while matured oocytes showed unaffected CC viability but abnormal mitochondrial distribution patterns. At lower tested concentrations (3–0.5 µM), BEA did not affect oocyte maturation, but matured oocytes showed reduced mitochondrial activity. At low concentrations, BEA impaired embryo developmental capacity and blastocyst quality after IVF and IVEC. In conclusion, in the juvenile sheep, COC exposure to BEA induces CC apoptosis and oocyte mitochondrial dysfunction with negative impact on embryo development.     

Improving successful pregnancies after embryo transfer

Over the past 20 years the rate of blastocyst development in vitro has improved through the development of sequential defined media, refining the oxygen concentrations during culture and providing substrates to ameliorate free radical accumulation. Despite these advances there has been little progress in improving calving rates after the transfer of in vitro produced embryos. This suggests that the culture conditions have been very effective in enabling those fertilised oocytes to reach the blastocyst stage that otherwise would not occur in vivo.We suggest that the next advance by which the embryo transfer technology gains more acceptance in cattle production will be identifying those cows which are intrinsically superior recipients. This must be coupled to the development of non-invasive assessments of the developmental competence of both the oocyte and the blastocyst. Until these two goals are achieved the ET industry will remain static and unable to overcome the economic loss caused by embryo mortality occurring 7-10 days after transfer.     

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.     

Effect of individual heifer oocyte donors on cloned embryo development in vitro

The aim of this study was to determine the effect of individual oocyte donors on cloned embryo development in vitro. Five Holstein heifers of varied genetic origins were subject to ovum pick up (OPU) once weekly. In total, 913 oocytes were recovered from 1304 follicles. A mean of 7.7+/-0.4 oocytes was recovered per session per animal. Individual mean oocyte production varied significantly in quantity but not in quality (morphological categories) among heifers. Oocytes from individual heifers were used as recipient cytoplasm for somatic cell nuclear transfer (SCNT). Cumulus cells, collected from a single Holstein cow genetically unrelated to the oocyte donor, were used as donor cells. Although the percentage of reconstructed embryos that started to cleave was nearly constant, the percentage of cleaved embryos that developed into blastocysts showed clear individual heifer variation (61%, 51%, 31%, 28% and 24%, respectively), with a mean of 38% showing blastocyst formation. In vitro fertilization (IVF) was also conducted with oocyte from the same heifers used in SCNT. A variation of blastocyst production among individual heifers was also shown in the IVF experiment, but the rank of oocyte donor based on the blastocyst rate was changed. In conclusion, individual oocyte donor may have an effect on cloned embryo development in vitro, which differed from the effect on IVF embryos.     

Noninvasive bovine oocyte quality assessment: possibilities of a single oocyte culture

Although bovine embryos are routinely produced in vitro for several decades, there still exists a critical need for techniques to accurately predict the oocyte's developmental competence in a noninvasive way, before the in vitro embryo production procedure. In this review, several noninvasive methods to evaluate oocyte quality are discussed, such as morphological assessment of the cumulus oocyte complex and the use of brilliant cresyl blue. Because an individual oocyte and embryo culture method can possibly generate additional insights into the factors that determine oocyte quality, the second part of this review summarizes the state of the art of bovine single oocyte culture. The optimization of individual in vitro embryo production can obviously accelerate the quest for better noninvasive oocyte quality markers, because more information about the oocyte's requirements and intrinsic quality will be revealed. Although each step of in vitro culture has to be re-examined in light of the hampered production of single embryos, the reward at the end will be substantial. Individual scored oocytes will be traceable along the in vitro embryo production procedure and the final blastocyst outcome can be linked to the original oocyte quality and follicular environment without the bias caused by simultaneously developing embryos.     

激光辅助制作小鼠嵌合体胚胎的研究

目的：探索激光辅助体外制作小鼠嵌合体胚胎的方法。方法：激光辅助去除不同发育阶段体外受精胚胎的透明带,随机组合卵裂球体外培养,观察胚胎融合情况。结果：没有透明带的卵裂球体外能够“自发”融合,并且融合胚胎在体外培养环境中,能够发育至囊胚期,显微镜观察其形态基本与二倍体胚胎无差别。结论：激光辅助方法获得裸露的卵裂球能够在体外培养环境制作嵌合体胚胎。