Development of preimplantation rabbit embryos in vivo and in vitro

Qualitative patterns of protein synthesis in preimplantation rabbit embryos grown in vivo and in vitro were examined by SDS polyacrylamide gel electrophoresis followed by autoradiography. The results demonstrate that (1) most qualitative changes in the pattern of protein synthesis occur during cleavage, (2) the blastocyst period of development is characterized by a remarkably uniform and constant pattern of protein synthesis, and (3) the qualitative pattern of protein synthesis in embryos cultured in vitro from the 1-cell to the blastocyst stage is essentially identical to the pattern of protein synthesis in embryos grown to a comparable stage in vivo.These results indicate that no “special” maternal factors, such as uterine proteins, are required in vitro either for the qualitative changes in the pattern of protein synthesis during cleavage, or for the initial expression of a pattern of protein synthesis characteristic of the entire blastocyst period. From these studies we conclude that, once fertilized, the rabbit egg proceeds through cleavage and blastocyst formation on its own endogenous developmental program.     

Development retardation in cultured preimplantation rabbit embryos

Day 3 to Day 5 preimplantation rabbit embryos were cultured for 24 h in chemically defined media which are widely used in early embryo culture (BSM II and Ham's F-10) supplemented with BSA or homologous serum. For the next 24 h, the embryos were left in the same culture medium, placed in freshly made medium, or cultured in medium which was supplemented with uterine flushings. In addition, 24-h cultured embryos were transferred into uteri of synchronous recipients for 1 day. After culture or transfer, development was assessed by cell proliferation evaluated by incorporation of tritiated thymidine. In comparison to non-cultured controls, thymidine incorporation demonstrated a considerably impaired cell proliferation after culture in defined media irrespective of medium, supplement, or replenishment with fresh medium. For Day 3 embryos, there was a developmental retardation amounting to about 1 day after 2 days in culture. Compared to Day 3 embryos, delay was clearly more pronounced in Day 4 and Day 5 blastocysts, i.e. in stages which had been retrieved from the uterus before culture. Supplementation with uterine flushings markedly promoted blastocyst cell proliferation (P less than 0.001). Incorporation data examined after transfer showed that impairment of cell proliferation caused by 1 day in culture had been compensated for to a large extent within 1 day in utero.     

Development of preimplantation goat (Capra hircus) embryos in vivo and in vitro

Preimplantation goat embryos were cultured with or without goat oviduct epithelial cells in Earle's 199 medium + 10% goat serum (E199 + 10%GS), and in three different simple chemically defined media. In-vivo development was characterized by an extended 8- to 16-cell stage followed by a rapid cleavage rate in the next 3 cell cycles. Culture of 1-8-cell embryos in Medium E199 + 10%GS led to cleavage arrest at the 8-16-cell stage, while in the chemically defined media embryos developed poorly and a high percentage failed to pass the 8-16-cell stage. In co-culture, however, a high percentage (77% and 96%) of 1-2-cell and 4-8-cell embryos, respectively, developed beyond the 16-cell stage. In co-culture, 1-2-cell embryos maintained cleavage rates equivalent to those in vivo until the 8-cell stage, but thereafter cell numbers lagged behind those in vivo, and by 168 h after ovulation, cell numbers (+/- s.e.) in vitro were 47.6 +/- 7.9 compared to 238 +/- 27.2 in vivo (t = 6.93, P less than 0.001). The results demonstrate that co-culture of embryos with oviduct cells allows a high percentage of embryos to develop through the period of cleavage arrest, providing a favourable environment for development through the 1-16-cell stages but a less adequate environment for development to the blastocyst stage.     

Development and DNA polymerase activities in cultured preimplantation mouse embryos: comparison with embryos developed in vivo

Embryos from superovulated female mice that developed in vitro from the two-cell stage were compared with in vivo embryos with respect to yield of blastocytes, number and types of cells, morphology in histologic section, and DNA polymerase activities. Significantly more embryos developed into blastocytes in vitro (93%) than in vivo (18%). Inner cell mass (ICM) cells comprised approximately 30% of total cells in late morula/early blastocyst stage embryos developed either in vitro or in vivo. However, the in vitro embryos developed approximately half the number of total cells as in vivo embryos, did not develop endoderm, and did not develop abembryonic trophoblast cells with morphologic characteristics of late preimplantation in vivo embryos. DNA-dependent DNA polymerase activities in in vitro embryos decreased in correspondence with the decrease in cell number resulting in per cell levels comparable to in vivo embryos. In contrast, the poly (A).oligo(dT)-dependent DNA polymerase activity was the same in embryos developing either in vitro or in vivo, indicating different regulatory mechanisms for the two enzyme activities. A variety of nutrients and growth factors in the culture medium did not increase cell numbers or DNA polymerase activities in embryos cultured for 3 days; extending the culture an additional 24 hours resulted in a loss of ICM cells and decreases in both DNA polymerase activities. These results show that the retarded growth of embryos in vitro is equally distributed between ICM and trophoblast, is not reversed by culture conditions that include serum growth factors, and is not due to decreased cellular levels of DNA polymerase activities.     

Development of preimplantation rabbit embryos in uterine flushing-supplemented culture media

The development of cultured rabbit preimplantation embryos grown in standard media (Ham's F-10 or BSM II supplemented with bovine serum albumin (BSA) or homologous serum) or in Ham's medium supplemented with uterine flushings was compared. The uterine flushings derived from donors of 0.5-6 years of age. Uterine flushing supplemented media were used natively or after treatments like sterilization by filtration, lyophilization, three times freezing/thawing, heat denaturation, dialysis, or ultrafiltration. Compared with in vivo controls, embryonic growth was substantially reduced during in vitro culture, demonstrably by smaller diameters and impaired cell proliferation (measured by thymidine incorporation). The growth retardation was more pronounced in blastocysts (recovered at day 4 post coitum [p.c.]) than in morulae (recovered at day 3 p.c.). Development in uterine flushing media was notably better than in standard media but did not comply with in vivo development. Highest thymidine incorporation was observed in media with increased concentrations of uterine secretions and after sequential supplementation of flushings from subsequent progestational stages. Advanced donor ages, heating up to 80 degrees C, freezing, and lyophilizing did not affect incorporation data statistically significantly, whereas sterilization by filtration, ultrafiltration, and dialysis led to a significantly reduced thymidine incorporation in the cultured embryos. The positive effects of uterine flushing supplementation are attributed to the supply of components more adjusted to the needs of the cultured embryos and/or to a reduction of pathological effects in vitro like washing out of nutritive and regulatory components from the embryo into the surrounding culture medium.     

Lactate dehydrogenase in preimplantation rabbit embryos

Unfertilized eggs and early embryos up to the 2-day (16-cell) cleavage stage of development in the rabbit contain predominantly the most cathodal lactate dehydrogenase isoenzyme made up of A-type subunits. Following early cleavage there is a progressing increase in total LDH activity in the embryo as development proceeds through 4- and 6-day blastocyst stages. This is accompanied by an increase in the amount of B-type subunits and a concomitant shift in the lactate dehydrogenase isoenzyme electrophoretic pattern toward the anodal isoenzyme types.     

The effect of hyperthermia in vitro on vitality of rabbit preimplantation embryos

The aim of our study was to test the influence of short exposure (6 h) of preimplantation rabbit embryos to elevated temperatures (41.5 degrees C or 42.5 degrees C) in vitro on their developmental capacity. Fertilized eggs recovered from female oviducts at the pronuclear stage (19 hpc) were cultured at standard temperature (37.5 degrees C) until the morula stage (72 hpc). Afterwards, the embryos were divided into two groups, cultured for 6 h either at hyperthermic (41.5 degrees C or 42.5 degrees C) or standard temperature (control 37.5 degrees C), post-incubated overnight (16-20 h) at 37.5 degrees C and then evaluated for developmental stages, apoptosis (TUNEL), proliferation (cell number), actin cytoskeleton and presence of heat-shock proteins Hsp70. It was observed that hyperthermia at 41.5 degrees C did not alter progression of embryos to higher preimplantation stages (expanded and hatching/hatched blastocysts), rate of apoptosis, total cell number of blastocysts and structure of actin filament compared to 37.5 degrees C. Western-blotting revealed the presence of heat stress-induced 72 kDa fraction of Hsp70 proteins in granulosa cells (exposed to 41 degrees C) and embryos (exposed to 41.5 degrees C). Following the elevation of temperature to 42.5 degrees C embryo development was dramatically compromised. The embryos were arrested at the morula or early blastocyst stage, showed an increased rate of apoptosis and decreased total cell number compared to control. The structure of actin filaments in most of blastomeres was damaged and such blastomeres often contained apoptotic nuclei. In this group a presence of heat-stress-induced fraction of Hsp70 proteins had not been confirmed. This is the first report demonstrating a threshold of thermotolerance of rabbit preimplantation embryos to hyperthermic exposure in vitro. A detrimental effect of higher temperature on the embryo is probably associated with the loss of their ability to produce Hsp70 de novo, which leads to cytoskeleton alterations and enhanced apoptosis.     

Methylation reprogramming and chromosomal aneuploidy in in vivo fertilized and cloned rabbit preimplantation embryos

Active demethylation of the paternal genome but not of the maternal genome occurs in fertilized mouse, rat, pig, and bovine zygotes. To study whether this early demethylation wave is important for embryonic development, we have analyzed the global methylation patterns of both in vivo-fertilized and cloned rabbit embryos. Anti-5-methylcytosine immunofluorescence of in vivo-fertilized rabbit embryos revealed that the equally high methylation levels of the paternal and maternal genomes are largely maintained from the zygote up to the 16-cell stage. The lack of detectable methylation changes in rabbit preimplantation embryos suggests that genome-wide demethylation is not an obligatory requirement for epigenetic reprogramming. The methylation patterns of embryos derived from fibroblast and cumulus cell nuclear transfer were similar to those of in vivo-fertilized rabbit embryos. Fluorescence in situ hybridization with chromosome-specific BACs demonstrated significantly increased chromosomal aneuploidy rates in cumulus cell nuclear transfer rabbit embryos and embryos derived from nuclear transfer of rabbit fibroblasts into bovine oocytes compared with in vivo-fertilized rabbit embryos. The incidence of chromosomal abnormalities was correlated with subsequent developmental failure. We propose that postzygotic mitotic errors are one important explanation of why mammalian cloning often fails.     

Sex-related growth rate differences in mouse preimplantation embryos in vivo and in vitro

Sex-related growth rate differences in preimplantation mouse embryos were investigated. In experiment I, Day 3 embryos were recovered from reproductive tracts, classified according to developmental stage, and cultured for 24 hr in CZB medium containing glucose. Each embryo was then reclassified and stained for measurement of number of nuclei and finally sexed using the polymerase chain reaction. In experiment II, Day 4 embryos were recovered, classified, stained, and sexed as in experiment I immediately after recovery. Morphologically, there were no differences between the sexes in either of the experiments on Day 4. However, based on number of nuclei, the data showed that in vitro conditions support the development of male embryos to the blastocyst stage compared to female embryos. Furthermore, growth rate differences were observed in vivo on Day 3, as females compacted earlier than males. These results suggest that the increased cell proliferation in cultured male embryos is an artifact caused by the in vitro environment. The variation may be due to sex differences in embryonal energy metabolism during the preimplantation stage. The growth difference implies different in vitro requirements of male and female embryos. © 1995 Wiley-Liss, Inc.     

Energy metabolism in preimplantation bovine embryos derived in vitro or in vivo

This study was an investigation of metabolism during bovine preimplantation development from the oocyte up to the hatched blastocyst derived in vitro or in vivo. Metabolism was determined by estimating the consumption of radiolabeled glucose, pyruvate, or lactate during a 4-h incubation period in a closed noninvasive system with NaOH as trap for the continuous collection of CO(2). The postincubation medium was analyzed for the presence of lactate. Embryonic metabolism from the matured oocyte to the 12-cell stage was more or less constant, with pyruvate being the preferred substrate. The first marked increase in oxidation of glucose occurred between the 12- and 16-cell stage. Compaction of morula and blastocyst expansion was accompanied by significant increases in oxidation of all three energy substrates. The incorporation of glucose increased steadily 15-fold from the 1-cell to the blastocyst stage. In general, the pattern of metabolism was similar between the embryos derived in vitro and in vivo but with some distinct differences. The most apparent feature of glucose metabolism by in vitro-produced embryos was a 2-fold higher rate of aerobic glycolysis as compared to that in their in vivo counterparts. In vitro-matured oocytes produced measurable amounts of lactate, whereas in vivo-matured oocytes exhibited a significantly lower metabolic activity and did not produce any lactate. When in vivo-collected embryos were preexposed to culture conditions, lactate production increased significantly and at the hatched blastocyst stage matched that of their in vitro counterparts. In vitro-produced embryos up to the 8-cell stage oxidized significantly higher amounts of lactate and had a lower ratio of pyruvate-to-lactate oxidation than the in vivo-obtained embryos. The results of this study show that under our culture conditions, important differences exist at the biochemical level between bovine embryos produced in vitro and those generated in vivo that may well affect the developmental capacity.     

RNA and protein synthesis in preimplantation rabbit embryos

A phenol-sodium dodecyl sulfate procedure has been used to extract DNA, RNA, and protein from 3-, 4.5-, and 6.5-day-old preimplantation rabbit embryos that were incubated for different lengths of time in the presence of labeled precursors. The results suggest that the newly-synthesized RNA and protein are more stable in the growing blastocyst than in the trophoblast at the time of implantation.This conclusion was derived from the results of embryo incorporation kinetics. The results also suggest that the increase in protein and RNA content in preimplantation rabbit blastocysts was preceded by an increase in protein synthesis accompanied by low catabolism.     

Expression of adipokines in preimplantation rabbit and mice embryos

Recent studies point to a role for adipokines in reproduction. Leptin is involved in embryo metabolism and may participate in embryo-maternal crosstalk. Little is known about potential roles of other adipokines in reproduction. We therefore studied the expression of adiponectin and pathway members during the pre- and periimplantation period in rabbits and mice. Adiponectin protein is localized in glandular epithelium of the rabbit endometrium on day 6 and 8 p.c. and in mouse endometrium on day 3.5 and 5 p.c. Rabbit, but not mice blastocysts express adiponectin mRNA. Adiponectin receptors one and two, adiponectin paralogues and PPARs were found in both species. Both, trophoblast and embryoblast were adiponectin positive. Real time PCR for adipoR1 and adipoR2 in rabbit blastocysts of different gastrulation stages at day 6 p.c. revealed a specific switch in expression: Expression was high in the trophoblast in early stages and in the embryoblast shortly prior to implantation. In conclusion, during the pre- and periimplantation period, members of the adiponectin pathway are expressed in endometrium and blastocysts, with a specific expression pattern in the embryonic disk of the gastrulating rabbit blastocyst, giving support to a role of the adipokine network in blastocyst differentiation and embryo-maternal interactions.     

Distribution patterns of rabbit embryos during preimplantation stage

The pattern of transport and distribution of rabbit embryos in the oviduct and uterus was studied 15 to 168 hours post coitum (p. c.). The reproductive tract was frozen in liquid nitrogen, thawed, and cleared in benzyl-benzoate solution using Orsini's technique. The location of the eggs and the ampullary-isthmic junction were identified using transmitted light from a dissecting microscope. Accumulation of the eggs in the oviduct occured in two phases. In the first phase the eggs were retained above the ampullaryisthmic junction, 3–12 hours after ovulation. In the second phase, the eggs were retained 36–60 hours after ovulation, above the uterotubal junction (at a distance approximately 12 % of the oviductal length). The rate of transport of individual eggs in the oviduct, and the time of the entry of eggs into the uterus were variable. Au 78 hours p. c. most blastocysts occupied the proximal half of the uterine horn, although some appeared very close to the internal os of the cervix. Spacing of blastocysts in the uterus, 114 to 120 hours p. c., involved movement of blastocysts away from the cervix. Unfertilized eggs remained in the uterus, along with developing blastocysts 168 hours p. c. Few eggs were retained in the oviduct at 108 and 115 hours p. c.