PCR sexing and developmental rate differences in preimplantation mouse embryos fertilized and cultured in vitro

A two-step polymerase chain reaction (PCR) assay was used to determine the sex of mouse preimplantation embryos obtained from oocytes fertilized and cultured in vitro, to investigate the differences in the developmental rates of mouse embryos according to the sex. All the in vitro developed embryos could be analyzed by this method. When the embryos were classified according to the time of morula to blastocyst transition as fast-intermediate- and slow-growing embryos, a significantly high percentage (78.0%) of the fast-developing embryos were identified as males; while a significantly lower percentage (42.5%) of slow-developing embryos were identified as males. The intermediate-developing embryos presented a sex ratio not significantly different from the total (57.5%). The deviation of sex ratio was further confirmed by embryo transfer experiment, where fast- and slow-developing embryos gave 76.2% and 25.7% male fetuses, respectively. We concluded that male mouse embryos fertilized and cultured in vitro develop faster than female embryos. © 1993 Wiley-Liss, Inc.     

Selection of mouse preimplantation embryos carrying exogenous DNA by polymerase chain reaction

A polymerase chain reaction (PCR) system to detect transgenes in mouse preimplantation embryos was employed so that transgenic embryos could be selected before they were transferred to recipient mice. The selection system involves bisection of morulae, selection of the half-morulae containing target sequences within 7 hr, and culture and transfer of the sister half-morulae. PCR analysis of morulae derived from transgenic mice confirmed that the PCR system was reliable. However, five of 41 implanted embryos derived from PCR-positive morulae did not contain the transgenes. Also, one of 28 implanted embryos from PCR-negative morulae were transgenic. The selection system was applied to fertilized mouse eggs into which pSV2-gpt-gE1A DNA was injected. The injected DNA was detected in 30 of 84 morulae derived from the microinjected eggs. All seven implanted embryos developed from PCR-negative morulae had no detectable amount of transgenes, and one of two successfully implanted embryos from PCR-positive morulae was transgenic.     

Development of cytoskeletal connections between cells of preimplantation mouse embryos

Summary Observations by scanning electron microscopy of mouse cleaving embryos reveal the presence of long microvilli around cell contact regions that often bridge the gap between blastomeres. These microvilli correspond, in detergent-extracted morulae, to strings connecting the cortical cytoskeletons of adjoining cells. They appear about 4 h after compaction in synchronized cultures. Transmission electron microscopy, heavy meromyosin decoration and DNase I digestion show that cytoskeletal connections contain bundles of actin microfilaments. The establishment of cytoskeletal connections does not require immediate protein synthesis, as shown by incubation with cycloheximide. Diverse treatments that interfere with compaction were tested for the development of cytoskeletal connections: culture media with low Ca²⁺ and/or Mg²⁺, or EGTA, or -lactalbumin, do not prevent the establishment of connections, while colchicine delays their appearance and cytochalasin D suppresses it. The relation between cytoskeletal connections, compaction and blastulation is discussed.     

Cell allocation to the inner cell mass and the trophectoderm in rat embryos during in vivo preimplantation development

Summary The number of trophectoderm (TE) and inner cell mass (ICM) cells was determined by complementmediated lysis and differential staining in rat embryos collected at different times during in vivo preimplantation development. At 90 h after fertilization, two groups of morulae were discriminated according to the presence or absence of detectable ICM cells, and the analysis of their total cell number indicated that acquisition of a permeability seal between TE cells begins at the 14-cell stage. On the other hand, our data confirmed that blastocoele formation occurs after the fourth cleavage division in the rat. The total cell number increased exponentially with time in blastocysts recovered between 90 h and 127 h but the cell kinetics of TE and ICM cells were different. The proportion of ICM cells consequently varied throughout blastocyst development, with a peak value for expanded blastocysts at 103 h. Finally, a linear-quadratic relationship was found between the numbers of TE and ICM cells when all the embryos with a detectable ICM were analysed together.     

Insulin-like growth factor-1 stimulates growth of mouse preimplantation embryos in vitro.

Because recent studies have particularly implicated the insulin growth factor family in early development, the effects of insulin-like growth factor (IGF-1) on the development of mouse embryos in vitro were investigated in detail. When added to the medium for culture of two-cell embryos, IGF-1 stimulated the number of cells in the resultant blastocysts after 54 hr, entirely by increasing the number of cells in the inner cell mass (ICM) (16.0 +/- 0.5 vs. 12.6 +/- 0.5 cells/ICM). This stimulation was also achieved when ICMs were isolated from blastocysts prior to culture for 24 hr with IGF-1 (22.3 +/- 1.0 vs. 17.5 +/- 0.8 cells/ICM). There was no effect on IGF-1 on trophectoderm (TE) cell proliferation. In morphology studies, IGF-1 also increased the proportion of blastocysts (62% +/- 3% vs. 49% +/- 4%) while decreasing the number of embryos remaining as morulae (32% +/- 3% vs. 38% +/- 2%) or in the early cleavage stages (7% +/- 3% vs. 13% +/- 3%) after 54 hr culture from the two-cell stage. All these effects were achieved with EC50s of approximately 60 pM IGF-1, which is in the range for IGF-1 receptor mediation; however, cross reaction with insulin, IGF-2, or other unknown receptors is not excluded. Nonetheless, the results show that physiological concentrations of IGF-1 (17-170 pM, 0.1-1 ng/ml), which have been observed in the reproductive tract, affect the early embryo, suggesting a normal role for this factor in the regulation of growth of the developing conceptus before implantation.     

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.     

Sex determination of in vitro developed buffalo (Bubalus bubalis) embryos by DNA amplification

This study was conducted to determine the sex of buffalo embryos produced in vitro by amplifying male specific DNA sequences using the polymerase chain reaction (PCR). This method uses three different pairs of bovine Y-chromosome specific primers and a pair of bovine satellite specific primers. Buffalo in vitro fertilized embryos at the 4-cell to blastocyst stage were collected at days 3, 4, 6, and 8 postinsemination, and the sex of each embryo was determined using all three different Y-chromosome specific primers. The bovine satellite sequence specific primers recognize similar sequences in buffalo and are amplified both in males and in females. Similarly, Y-chromosome specific primers amplify the similar Y-chromosome specific sequences in male embryos of buffalo. Upon examining genomic DNA from lymphocytes of adult males and females, and embryos, the results demonstrate the feasibility of embryo sexing in buffaloes. Furthermore, sex determination by PCR was found to be a rapid and accurate method. © 1993 Wiley-Liss, Inc.     

Sex-related differences in developmental rates of bovine embryos produced and cultured in vitro.

The classical concept of sex determination in mammals is that a Y chromosomal gene controls the development of the indifferent gonad into a testis. Subsequent divergence of sexual phenotypes is secondary to this gonadal determination. The most likely candidate gene is SRY (sex-determining region Y) in humans, and Sry in mouse. However, several lines of evidence indicate that sexual dimorphism occurs even before the indifferent gonad appears. Here we present evidence that bovine male embryos generally develop to more advanced stages than do females during the first 8 days after insemination in vitro. Corresponding relationships between both cell numbers and mitotic indices and sex were also seen. Although it is not clear whether this phenomenon involves factors originating before or after fertilization, these findings suggest that sex-related gene expression affects the development of embryos soon after activation of the embryonic genome and well before gonadal differentiation.     

Effect of Shigella toxin on preimplantation mouse embryos in vitro

Preimplantation mouse embryos at the 4-cell to 8-cell stage were exposed to Shigella dysenteriae toxin at concentrations of 0.001-100 pg/ml in vitro. The effect of the toxin was studied by morphological observation of the embryos to the blastocyst stage, by assessing protein synthesis with 14C-leucine incorporation, and by measuring embryonic adenosine triphosphate (ATP) content. Preimplantation mouse embryos were highly sensitive to the toxin. All variables investigated were adversely influenced by the toxin. After a lag period of 24 hr, 0.01 pg/ml toxin inhibited development to the blastocyst stage and protein synthesis. Toxin concentrations of 1.0 pg/ml resulted in a significant decrease in ATP content.     

Effects of methylmercury and mercuric chloride on preimplantation mouse embryos in vivo

This report compares the effects of methylmercuric chloride (MMC) and mercuric chloride (MC) on the development of mouse preimplantation embryos in vivo. Female mice were injected with a single intravenous dose of 0.5-20.0 mg Hg/kg MMC or 0.5-2.5 mg Hg/kg MC on day 0 of gestation. The embryos were recovered by flushing excised oviduct and uterus on day 3.5 of pregnancy, and were examined for abnormalities. In the groups treated with doses of 0.5 and 1.0 mg Hg/kg of both compounds, the rates of abnormal embryos were not significantly different from that in the control group. The 50% effective dose of MMC was twice as great as that of MC. With increasing dose, the difference became more obvious; the 80% effective doses differed by a factor of ten. The body weight of dams decreased in terms of the dose of mercury in MC-treated groups, but did not vary in MMC-treated groups. The sensitive developmental stage for mercury toxicities could not be determined clearly, although the high sensitivity was reported in the blastocyst stage in vitro. The embryos treated in vivo were less sensitive than those reported in vitro.     

Effects of selected plant essential oils on the growth and development of mouse preimplantation embryos in vivo

Plant essential oils (EOs) have been reported to have health benefit properties and their preventive and therapeutic use in animals is expected to increase in the future. We evaluated the influence of five essential oils obtained from plant species which are known to have positive antimicrobial, antioxidative and anti-inflammatory effects--sage EO from Salvia officinalis L. (Lamiaceae), oregano EO from Origanum vulgare L. (Lamiaceae), thyme EO from Thymus vulgaris L. (Lamiaceae), clove EO from Syzygium aromaticum L. (Myrtaceae) and cinnamon EO from Cinnamomum zeylanicum Blume (Lauraceae) on the growth and development of mouse preimplantation embryos in vivo. Essential oils were added to commercial diet at concentrations of 0.25% for sage EO, thyme EO, clove EO, cinnamon EO and 0.1% for oregano EO, and fed to ICR female mice for 2 weeks ad libitum. Females were then mated with males of the same strain. Embryos obtained on Day 4 of pregnancy at the blastocyst stage were stained by morphological triple staining (Hoechst, PI, Calcein-AM) and evaluated using fluorescent microscopy. The effects of essential oils were estimated by the viability of embryos, number of nuclei and distribution of embryos according to nucleus number. Cinnamon EO significantly decreased the number of nuclei and the distribution of embryos according to nucleus number was significantly altered. Sage EO negatively influenced the distribution of embryos according to nucleus number. Clove and oregano EOs induced a significantly increased rate of cell death. Only thyme EO had no detectable effects on embryo development. In conclusion, none of the essential oils had any positive effect on embryo development, but some of them reduced the number of cells and increased the incidence of cell death.     

Lectin-induced blockage of developmental processes in preimplantation mouse embryos in vitro

This study attempts to assess the developmental importance of cell surface glycoconjugates of preimplantation mouse embryos. This was done by incubating early embryos in various lectins and analyzing subsequent development. If specific cell surface glycoconjugates (lectin receptors) are linked to specific developmental processes, such as cell division, compaction, and blastocyst formation, then different lectins should block these different developmental processes. The results show that wheat-germ agglutinin (WGA; N-acetyl-D-glucosamine-specific) at 50 μg/ml prevents the cell division of four-cell embryos. However, this effect of WGA occurs only in embryos with intact zonae pellucidae. Concanavalin A (Con A; α-D-glucose and α-D-mannose-specific) treatment, 20 μg/ml, of four-cell or early eight-cell embryos prevents compaction, the first major change in cell shape in early mouse embryogenesis. Divalent succinly Con A does not affect development, suggesting that the Con A effect is due to crosslinking of cell surface glycoconjugates. Exposure of four-cell or early eight-cell embryos to 10 μg/ml Lotus Tetragonolobus puprureas agglutinin (LTA; α-L-fucose-specific) or 25 μg/ml Limulus polyphemus agglutinin (LPA; sialic acid-specific) allows compaction or development to the morula stage, but blocks blastocyst formation. All lectins tested retard cell division to some extent. Late morulae and early blastocysts are more resistant than earlier stages to all of the lectins studied. This study demonstrates that very low concentrations of these lectins affect different developmental processes, presumably based upon their sugar specificities.     

Morphological development and sex of bovine in vitro-fertilized embryos.

Bovine in vitro-fertilized embryos at the blastocyst stage were collected at days 7, 8, and 10 postinsemination and sex was determined via the polymerase chain reaction (PCR) to compare the embryonic development with the sex of the embryos. The percentages of males (sex ratio) after division of the embryos into three developmental groups were 68%, 48%, and 35% in the fast, intermediate, and slow groups, respectively (P = 0.014). The percentages of males on days 7, 8, and 10 were 60%, 40% and 33%, respectively (P = 0.043). The average sex ratio for the whole material was 50%. It is thus concluded that male bovine preimplantation embryos develop faster than female embryos.     

Divergent selection for in vitro developmental capacity of preimplantation mouse embryos

Summary Replicated divergent selection was conducted for two generations in ICR mice for in vitro developmental capacity (IVDC; percentage of fertilized one-cell zygotes developing to blastocysts in vitro per female donor). Realized heritabilities based on high and low selection were 0.03±0.08 and –0.11±0.09 in replicate 1, and 0.10±0.11 and 0.08±0.10 in replicate 2. No differences were detected between selection lines (P>0.2) or replicates (P>0.1). Estimate of heritability in the base population based on 332 daughter-dam pairs was 0.14±0.18. These results indicate that additive genetic variance contributes little to the phenotypic variance in this trait. Considerable phenotypic variation in IVDC was observed (mean=49.3; SD=31.0), with a range of IVDC from 0%–100%. Utilization of donor female as a blocking factor is suggested for designs of experiments with preimplantation embryos to increase precision and power of statistical analyses.     

Comparison of preimplantation developmental competence after mouse oocyte growth and development in vitro and in vivo

Culture systems for oocytes are essential for the experimental analysis of the basic mechanisms of oocyte development and, moreover, they will eventually find wide application in agriculture, the clinic, and wildlife preservation. Here, progress in mouse oocyte growth and development in vitro using oocyte-granulosa cell complexes from preantral follicles is reviewed. Oocyte-granulosa cell complexes were isolated from preantral (secondary) follicles of 12 day old mice, grown in vitro for 10 days, then matured and fertilized in vitro. The developmental competence of these oocytes was compared with oocytes grown in vivo and isolated from 22 day old mice, then matured and fertilized in vitro. In vitro-grown oocytes did not achieve the same size as their in vivo-grown counterparts. However, when oocytes were grown in medium containing fetal bovine serum, their preimplantation developmental competence was equivalent to that of in vivo-grown oocytes. Surprisingly, more blastocysts per animal were produced when oocytes were grown in vitro than in vivo. There was no correlation between oocyte size and either preimplantation developmental competence or number of cells per blastocyst. Oocytes grown in serum-free medium did not achieve the same developmental competence as oocytes grown in medium supplemented with serum. Lastly, the health status as an adult of the only animal born after complete oocyte development in vitro is described and discussed.