Early Morula Research Articles

Observation of some key stages of the embryonic development of Biomphalaria straminea (Dunker, 1848) (Molluska, Planorbidae)

Summary Biomphalaria straminea is a freshwater snail and one of the intermediate hosts of the trematode parasite which causes schistosomiasis in Brazil. The main stages of embryonic development were analyzed with confocal laser scanning microscopy (CLSM), using the fluorescent probe DiOC and Nile Red as a vital stain in in vivo preparations. In fixed preparations nuclei were stained by the Feulgen reaction or the fluorescent DNA probe, Höechst 33342. Results obtained from the analysis of embryos at the stages of early cleavage, morula, blastula, gastrula, early trocophore and veliger showed that these stages are similar to those described for Biomphalaria glabrata and Biomphalaria tenagophila. CLSM optical sections of early trochophore showed important morphological structures such as the blastopore, stomodeum and shell gland; and in early veliger, internal organs such as the esophagus, stomach and male and female ducts were also clearly identified.

Transfer of blastocysts and morulae on day 5

Objective To limit the number of embryos transferred and reduce high-order multiple pregnancies without compromising a patient's opportunity to become pregnant. Design Retrospective, nonrandomized analysis of embryo development and patient outcome when embryos were transferred on day 5. Setting Private practice. Patient(s) Women undergoing in vitro fertilization (IVF) treatment. Intervention(s) Extend embryo culture to day 5 before embryo transfer (ET) to reduce the number of embryos transferred and minimize high-order multiple pregnancies. Main outcome measure(s) Clinical pregnancy rate (CPR), implantation rate (IR), and live born rate (LBR) from expanding (expanded blastocysts and blastocysts) and nonexpanding (early blastocysts and morulae) embryos transferred on day 5. Result(s) Approximately 60% of patients had expanding embryos (EXE) on day 5. Forty percent of patients having an ET had transferable non-expanding embryos (NEE). Patients with EXE had higher CPR and LBR compared to patients with NEE. Implantation rate and multiple pregnancy rate (MPR) were also higher for patients with EXE. The miscarriage rate (MCR) for patients with EXE and NEE was not different. Approximately 5.5% of patients did not have an ET, with most (>98%) of the ET failures from patients with ≤3 two pronuclei (2PN) embryos. The number of 2PN embryos had an effect on CPR, LBR, MPR, and the number of patients having cryopreservation. Conclusion(s) Day 5 ET allows for the selection of embryos with the highest implantation potential as evidenced by acceptable pregnancy rates for patients with either EXE or NEE, without the need to transfer more than two embryos.

Onchocerca volvulus: expression and immunolocalization of a nematode cathepsin D-like lysosomal aspartic protease

The N-terminal region of the cathepsin D-like aspartic protease from the human filarial parasite Onchocerca volvulus was expressed as His-tag fusion protein. Light and electron microscopic immunohistology using antibodies against the recombinant protein showed labeling of lysosomes in the hypodermis and epithelia of the intestine and the reproductive organs of Onchocerca. While developing oocytes were negative, mature oocytes and early morulae showed strong labeling. In older embryos and mature microfilariae, stained lysosomes were only found in a few cells. Cell death in degenerating microfilariae of patients untreated and treated with microfilaricidal drugs was associated with strong expression of aspartic protease. IgG1, IgG4, and IgE antibodies reactive with the recombinant protein were demonstrated in sera from onchocerciasis patients indicating exposure and recognition of the enzyme by the host’s defence system. The aspartic protease of O. volvulus appears to function in intestinal digestion and tissue degradation of the filaria.

Production of dromedary ( Camelus dromedarius) embryos by IVM and IVF and co-culture with oviductal or granulosa cells

The general objective of this work was to produce dromedary embryos from cumulus-oocyte complexes (COCs) that were matured, fertilized and co-cultured in vitro. A total of 1598 COCs were recovered from 457 ovaries; 1308 were deemed suitable for IVM and were cultured at 38.5 °C, 5% CO 2, and >95% humidity for 36 h in TCM-199 supplemented with 10% heat-treated fetal calf serum (FCS), 10 ng/ml epidermal growth factor (EGF), 1 μg/ml FSH, and 500 μM cysteamine. Matured COCs ( n = 88) were denuded, fixed, and stained to determine nuclear status; 63% (56/88) had reached metaphase II (MII) at 36 h. Overall, 1135 COCs were inseminated with ejaculated fresh semen (0.5 × 10 6 spermatozoa/ml in modified TALP-solution). Inseminated oocytes ( n = 155) were examined for evidence of fertilization; 68% (106/155) were penetrated by spermatozoa, including 52% (55/106) with two pronuclei and 34% (36/106) with polyspermy. Inseminated, denuded oocytes ( n = 819) were co-cultured with dromedary oviductal epithelial or granulosa cells in TCM-199 supplemented with 10% heat-treated FCS. Although the rate of first cleavage (two to eight cells) was similar for the two co-culture systems (32 versus 33%, respectively), more embryos (two-cell to blastocyst stage) were obtained from oocytes co-cultured with oviductal versus granulosa cells (61 versus 45%; P < 0.05). The proportions of fertilized oocytes developing to the early morula stage were 19% (80/417) and 12% (48/402) for oocytes co-cultured for 7 days with oviductal or granulosa cells, respectively ( P > 0.05). However, development to the blastocyst stage (10% of fertilized oocytes) occurred only in oocytes co-cultured with oviductal cells. In conclusion, dromedary embryos were produced in vitro using abattoir-derived oocytes, fresh (ejaculated) semen, and oviductal cell co-culture.

232SEARCH FOR THE BOVINE HOMOLOG OF THE MURINE PED GENE

The aim of the current study was to identify the bovine homolog of the murine Ped (preimplantation embryo development) gene, which regulates mouse preimplantation embryonic growth, including cleavage rate and embryo survivability, and to characterize the expression pattern of this gene during bovine preimplantation embryo development. Experiment (I): The National Center for Biotechnology Information (NCBI) GenBank/EBI EST databases were searched for bovine-expressed sequence tags (EST) that were homologous with the murine Ped gene (Accession number: NM_010394). The resulting ESTs were aligned and assembled in to one complete sequence (841bp), which was shown to be homologous with the Murine Ped gene and the Bovine Major Histocompatibility Complex class I 4221.1 gene (Accession No.: AJ010865, length 1090bp). The expression of the protein product of the Ped gene by bovine tissue was confirmed using Western Blot analysis. Experiment (II): The expression pattern of the Ped gene homolog during in vivo and in vitro bovine preimplantation embryo development was characterized using real time PCR. Embryos at the same stage for age were compared (Day 1: 2-cell; Day 2: 4-cell; Day 3: 8-cell; Day 4: 16-cell; Day 5: early morula;; Day 6, compact/late morula;; Day 7: blastocyst). The relative transcript abundance was consistently lower in the in vitro-cultured embryos at all stages of preimplantation development the differences were significant on Days 2, 4, 5, 6, and 7. The relative transcript abundance was significantly lower on Days 1, 2, and 3 of in vivo culture than on Days 4, 5, 6, and 7 and was significantly higher in Day 7 blastocysts than in Day 5 early morula. In in vitro-cultured embryos the relative transcript abundance was significantly higher in Day 7 blastocysts compared to all other stages of the preimplantation period. Experiment (III): A quantitative analysis of the Ped gene homolog was carried out on replicates of pools of ten 2-cell embryos collected at 25, 28, 32, and &amp;gt;36hpi from three different fertilizations. Transcript relative abundance was highest in those embryos that had cleaved by 25hpi. By 28hpi abundance had decreased slightly;; as time to cleavage increased further to 32 and &amp;gt;36hpi, the relative abundance decreased significantly. In conclusion, we have successfully identified a potential bovine homolog of the murine Ped gene. Furthermore, we have characterized the expression pattern of this gene during preimplantation embryo development in cattle and we have shown that a greater relative abundance of the gene transcript is associated with embryos of higher quality and greater developmental potential.

243TEMPORAL DIVERGENCE IN THE PATTERN OF MRNA EXPRESSION IN BOVINE EMBRYOS CULTURED FROM THE ZYGOTE TO BLASTOCYST STAGE IN VITRO OR IN VIVO

The objective of this study was to examine the time during the post-fertilization culture period that gene expression patterns of in vitro cultured bovine embryos diverge from those of their in vivo cultured counterparts. Presumptive bovine zygotes were produced by IVM/IVF of immature oocytes collected from the ovaries of slaughtered animals. At approximately 20h post-insemination (hpi), presumptive zygotes were randomly divided into two culture groups, either in vitro in synthetic oviduct fluid or in vivo, and transferred into the ewe oviduct. Embryos were recovered from both systems at approximately 30hpi (2-cell), two (4-cell), three (8-cell), four (16-cell), five (early morula), six (compact morula) or seven (blastocyst) days pi and snap-frozen for the analysis of transcript abundance using real-time PCR. The transcripts studied were interferon-tau, apoptosis regulator box-a (Bax), connexin 43, sarcosine oxidase, glucose transporter 5, mitochondrial Mn-superoxide dismutase, insulin-like growth factor II, and insulin-like growth factor-I receptor, most of which are known from our previous work to be differentially transcribed in blastocysts derived from culture in vitro or in vivo. Analysis was done on pools of 10 embryos. Data were analyzed using one-way repeated measures ANOVA. The relative abundance of the transcripts studied varied throughout the preimplantation period and was strongly influenced by the culture environment. For example, transcripts for interferon-tau were detected from the 8-cell stage onwards in in vitro-cultured embryos but not until the early morula stage in those cultured in vivo. Levels of this transcript increased significantly at the compact morula and blastocyst stages in both groups but were significantly higher (P&amp;lt;0.05) in in vitro-cultured embryos at both stages. mRNA for Bax was not detected before the 8-cell stage in in vitro cultured embryos and not until the 16-cell stage in in vivo cultured embryos. The abundance of this transcript increased significantly thereafter up to the blastocyst stage in both groups. The level of expression was significantly higher (P&amp;lt;0.05) at all stages of development in in vitro-cultured embryos than those cultured in vivo. The relative abundance of Cx43 transcripts decreased in both in vitro- and in vivo-cultured embryos at the 8- to 16-cell stage. Levels remained low thereafter in the in vitro-cultured embryos but significantly increased in those cultured in vivo. Transcript abundance was significantly higher in in vivo cultured embryos from Day 4 onwards with a ten-fold difference presence at the blastocyst stage. Differences also existed for the other transcripts studied. These data demonstrate that changes in transcript abundance in blastocyst stage embryos are in many cases a consequence of perturbed transcription earlier in development. Depending on the transcript, these differences may be evident in as short as 10h of culture.

The effect of divergent selection for uterine capacity on prenatal survival in rabbits: maternal and embryonic genetic effects.

The aim of this work was to determine whether prenatal survival depends on the genotype of the mother or of the embryo and to identify the critical periods for prenatal mortality in two lines of rabbits divergently selected by high (H) and low (L) uterine capacity. Does from H (n = 124) and L (n = 115) lines were slaughtered at 72 h of gestation. Embryos recovered at 72 h of gestation were transferred to the oviducts of recipient does from the H (n = 23) and L (n = 19) lines. Each recipient does received eight embryos from the H line into one oviduct and eight embryos from the L line into the other. Recipient does were slaughtered on d 28 of gestation. No differences were found between lines in the embryo recovery either in ovulation rate (OR) or in fertilization rate of ova recovered. Recovery rate was higher for the H line (0.80 vs. 0.72, P < 0.01). The number of embryos recovered, fitting ovulation rate as a covariate, was also higher for the H line (9.74 vs. 8.78, P < 0.05). The H line showed a more advanced embryonic stage of development, having a higher percentage of blastocysts (PB) and a lower percentage of compact morulae (PCM) (38% vs. 20%, P < 0.001 for PB, and 51% vs. 64%, P < 0.01 for PCM). The percentage of early morulae was low and similar in both lines. Neither donor nor recipient lines affected embryonic survival from 72 h to 7 d of gestation. Fetal survival was affected by the recipient line (P < 0.05). An interaction between donor and recipient was found. Embryos from the H donor line had a better fetal survival rate than embryos from the L donor line (P < 0.05) in H recipient females. Within L recipient females, embryos from H and L donor lines showed similar fetal survival. Fetal survival was divided into early (from d 7 to 17 of gestation) and late (from d 17 to 28 of gestation). The high recipient line showed a higher early fetal survival than the L recipient line (P < 0.05). The same effect was observed for late fetal survival, but the difference between H and L recipient lines was lower (P < 0.10). Thus, fetal survival depends mainly on the maternal genotype, and the embryo genotype only affects fetal survival when embryo transfer is performed to a favorable maternal environment. Selection for uterine capacity in rabbits leads to modification of early embryonic survival and of early and late fetal survival, but differences are higher for early than for late fetal survival.

Temporal divergence in the pattern of messenger RNA expression in bovine embryos cultured from the zygote to blastocyst stage in vitro or in vivo.

The objective of this study was to examine the time during the postfertilization period that gene expression patterns in in vitro-cultured bovine embryos diverge from those of their in vivo-cultured counterparts. Presumptive bovine zygotes were produced by in vitro maturation and fertilization of immature oocytes collected from the ovaries of slaughtered animals. Approximately 20 h post insemination (hpi), zygotes were denuded and randomly divided into two groups for culture either in vitro, in synthetic oviduct fluid medium, or in vivo, in the ewe oviduct. Embryos were recovered from both systems at approximately 30 hpi (2-cell), 2 (4-cell), 3 (8-cell), 4 (16-cell), 5 (early morula), 6 (compact morula), or 7 (blastocyst) days post insemination. On recovery, they were examined for stage of development and snap frozen in liquid nitrogen for the analysis of transcript abundance using real-time polymerase chain reaction. The transcripts studied were glucose transporter 5, sarcosine oxidase, mitochondrial Mn-superoxide dismutase, connexin 43, interferon tau, insulin-like growth factor II, apoptosis regulator box-alpha and insulin-like growth factor-I receptor, most of which are known from our previous work to differ in terms of transcript abundance in blastocysts derived from culture in vitro or in vivo. The results demonstrate that the relative abundance of the transcripts studied varies throughout the preimplantation period and is strongly influenced by the culture environment. In addition, the data demonstrate that changes in transcript abundance in blastocyst stage embryos are in many cases a consequence of perturbed transcription earlier in development. Depending on the transcript, these differences may be evident by as little as 10 h of initiation of culture. Such information has implications not only for basic biology but also for human assisted reproduction in which there is a move toward culturing embryos to the blastocyst stage, necessitating prolonged culture in vitro under potentially deleterious conditions.

Inositol transport in preimplantation rabbit embryos: effects of embryo stage, sodium, osmolality and metabolic inhibitors

The preimplantation period in the rabbit consists of a 3 day cleavage stage during which the number of cells increases with little change in embryo size, followed by a 3-4 day blastocyst stage during which the inner cell mass, the blastocoel and the trophectodermal layer are formed and the embryo grows rapidly in size and protein content. This study used [(3)H]inositol to investigate the transport of inositol, an essential component of the phosphatidylinositol signal transduction system, over the 6 days of preimplantation development by rabbit embryos. In the presence of 15 micromol inositol l(-1) in the incubation medium, there was a small linear increase in inositol uptake from 0.07 pmol per embryo per h at the one-cell stage (day 1) to 0.135 pmol at the late morula (day 3) stage. Inositol uptake increased to 0.58 pmol per embryo per h for early blastocysts (day 4) and 23.7 pmol for late blastocysts (day 6). There was a significant linear relationship between inositol uptake and blastocyst diameter and surface area. Efflux of inositol from early morulae was minimal (about 1.25% of embryo content per h), whereas efflux from mid-blastocysts (day 5) was much greater (about 15.6% of embryo content per h). Efflux of inositol from both early morulae and mid-blastocysts was increased by decreasing the osmolality of the incubation medium. Varying the osmolality had no effect on inositol uptake up to 2 h. Inositol uptake was dependent on sodium in cleavage-stage embryos but independent of sodium in blastocyst stages. In early morulae, inositol uptake was inhibited by glucose and the sodium-dependent hexose transport inhibitor, phloridzin, but not by the facilitated transport inhibitor, phloretin. Inositol uptake in early morulae was saturable; estimates of 0.227 and 0.288 pmol per morula per h for V(max) and 0.045 and 0.038 micromol l(-1) for K(m) were obtained for sodium-dependent transport in two separate experiments. All of these results are consistent with the hypothesis that transport in cleavage stages occurs via a sodium myo-inositol transporter (SMIT) protein. Uptake in blastocysts was non-saturable. Uptake into blastocysts appeared to take place by a transcellular rather than a paracellular route.

Inositol transport in preimplantation rabbit embryos: effects of embryo stage, sodium, osmolality and metabolic inhibitors

The preimplantation period in the rabbit consists of a 3 day cleavage stage during which the number of cells increases with little change in embryo size, followed by a 3-4 day blastocyst stage during which the inner cell mass, the blastocoel and the trophectodermal layer are formed and the embryo grows rapidly in size and protein content. This study used [(3)H]inositol to investigate the transport of inositol, an essential component of the phosphatidylinositol signal transduction system, over the 6 days of preimplantation development by rabbit embryos. In the presence of 15 micromol inositol l(-1) in the incubation medium, there was a small linear increase in inositol uptake from 0.07 pmol per embryo per h at the one-cell stage (day 1) to 0.135 pmol at the late morula (day 3) stage. Inositol uptake increased to 0.58 pmol per embryo per h for early blastocysts (day 4) and 23.7 pmol for late blastocysts (day 6). There was a significant linear relationship between inositol uptake and blastocyst diameter and surface area. Efflux of inositol from early morulae was minimal (about 1.25% of embryo content per h), whereas efflux from mid-blastocysts (day 5) was much greater (about 15.6% of embryo content per h). Efflux of inositol from both early morulae and mid-blastocysts was increased by decreasing the osmolality of the incubation medium. Varying the osmolality had no effect on inositol uptake up to 2 h. Inositol uptake was dependent on sodium in cleavage-stage embryos but independent of sodium in blastocyst stages. In early morulae, inositol uptake was inhibited by glucose and the sodium-dependent hexose transport inhibitor, phloridzin, but not by the facilitated transport inhibitor, phloretin. Inositol uptake in early morulae was saturable; estimates of 0.227 and 0.288 pmol per morula per h for V(max) and 0.045 and 0.038 micromol l(-1) for K(m) were obtained for sodium-dependent transport in two separate experiments. All of these results are consistent with the hypothesis that transport in cleavage stages occurs via a sodium myo-inositol transporter (SMIT) protein. Uptake in blastocysts was non-saturable. Uptake into blastocysts appeared to take place by a transcellular rather than a paracellular route.

Double vitrification of rat embryos at different developmental stages using an identical protocol

The aim of the present investigation was to test the effectiveness of a method of vitrifying rat embryos at different stages of development (from early morula to expanding blastocyst) in a double vitrification procedure. Wistar rat embryos were vitrified and warmed in super-fine open-pulled straws (SOPS). Before being plunged into liquid nitrogen, the embryos were exposed to 40% ethylene glycol+0.75 M sucrose in TCM-199+20% fetal calf serum (FCS) for 20 s at 38 °C. Subsequent warming and direct rehydration of the embryos was conducted in culture medium (TCM-199+20% FCS) at 38 °C. Early morula stage (7–10 blastomeres) embryos ( n=358) were vitrified, warmed and cultured in vitro (EM group). Batches of these embryos were then cryopreserved again (revitrified) at the early blastocyst (EB group, n=87), blastocyst (B group, n=93) or expanding blastocyst stage (ExpB group, n=73). After the first (EM group) and repeated (EB, B, and ExpB groups) vitrification procedures, developmental rates of 81, 83, 34 and 76%, respectively were achieved (for EM–EB–ExpB P>0.1; for EM, EB, ExpB-B P<0.005). Our data demonstrate the possibility of using the described identical protocol for the SOPS vitrification of rat early morulae, early blastocysts and expanding blastocysts. The low survival rate of blastocysts subjected to double vitrification requires further investigation.

Effects of BSA and fetal bovine serum in culture medium on development of rat embryos.

Rat 1-cell embryos, recovered from naturally mated females, were cultured in a chemically defined medium (mR1ECM) or in mR1ECM supplemented with BSA (4 mg/ml; mR1ECM-BSA) or fetal bovine serum (FBS; 10%, v:v; mR1ECM-FBS) instead of polyvinylalcohol. There was no difference in percentages of embryos that developed to the 2-cell to blastocyst stages between mR1ECM and mR1ECM-BSA, but in mR1ECM-FBS, no development beyond the 2-cell stage was observed. When embryos were transferred to mR1ECM-FBS from mR1ECM after 24 to 64 h of culture, development of embryos to and beyond the 4-cell stage was inhibited. However, when transferred after 80 h of culture, more embryos developed to blastocysts and hatching or hatched blastocysts than in embryos cultured in mR1ECM. When 8-cell embryos and early morulae obtained after 72 and 80 h of culture in mR1ECM, respectively, were cultured in mR1ECM-FBS, a higher proportion of early morulae developed to the blastocyst stage than did 8-cell embryos. When morulae obtained after culture in mR1ECM or mR1ECM-BSA were transferred to recipient females, there was no difference in proportions of fetuses obtained. However, a higher proportion of blastocysts cultured in mR1ECM-FBS developed to fetuses compared with those obtained in mR1ECM. These results indicate that BSA has neither deleterious nor beneficial effects on development of rat 1-cell embryos. In contrast, FBS has deleterious effects on early cleavage of embryos but it promotes more rapid development of morulae to blastocysts, resulting in better quality blastocysts.

Factors affecting the survival of sheep embryos after transfer within a MOET program

Multiple ovulation and embryo transfer (MOET) has the potential to increase the rate of genetic improvement in sheep. However, better realization of this potential requires maximum survival rates of transferred embryos of high genetic merit after transfer into recipient ewes. These studies were therefore conducted to investigate the effect of both embryonic and recipient ewe factors on the survival rate of transferred embryos. Survival rate was similar after transfer of morula or blastocyst stage embryos, and these were higher ( P<0.05) than for very early morulae and early morulae. Advanced embryos (Day 5 blastocyst) had an advantage ( P<0.05) in survival rate over retarded embryos (Day 6 morula). Grades 1 and 2 embryos survived significantly ( P<0.05) better than Grades 3 or 4 embryos. There was no difference in embryo survival rate following transfer to recipients with different numbers of corpora lutea. In general, age or parity of recipient ewes did not affect embryo survival rate, although a higher ( P<0.05) embryo survival rate was observed for yearling recipients. Buserelin (GnRH agonist) treatment of recipient ewes 5 or 6 days after transfer of embryos (Day 12 of the cycle) did not improve embryo survival rate. These results confirm that both embryonic and recipient factors can play an important role in the success of a MOET program in sheep.

Ovulation of aged follicles does not affect embryo quality or fertility after a 14-day progestagen estrus synchronization protocol in ewes

The aim was to examine the effect of ovulation of aged follicles on embryo quality and fertility in ewes. In Experiment 1, ewes (n = 39) received a prostaglandin analogue on Day 6 of the cycle and then received either a progestagen sponge from Day 6 to 20 after estrus (Single sponge) or a progestagen sponge on Day 6 that was replaced on Day 11 and 16 and removed on Day 20 (Multiple sponges). In a subgroup of ewes, the growth of ovarian follicles was characterised using ultrasonography. Fertile rams were introduced 48 hours after sponge withdrawal; we slaughtered the ewes on Day 5 of pregnancy and recovered the embryos. The mean age of the ovulatory follicles was greater in ewes that received a single sponge compared with multiple sponges (8.7±0.8 days, range 4 to 14, versus 4.5±0.7 days, range 3 to 6; P<0.05). However, the groups did not differ (P>0.05) in ovulation rate (2.4±0.3 corporal lutea per ewe) or the proportion of good quality embryos recovered (71 to 82%; developed to the early morula stage or further). In Experiment 2, ewes (570 in total) received treatments similar to those in Experiment 1 but were kept until lambing. Ewes that received a single sponge came into heat earlier (P<0.05) than those that received multiple sponges, but ≥97% of ewes in all groups (P>0.05) were bred by 48 to 72 hours after ram introduction. There was no difference (P>0.05) between groups for the proportion of ewes that lambed to first service (80 to 86%) or the number of lambs per ewe (1.94±0.08 lambs). We conclude that when luteolysis occurs at the beginning of progestagen synchronisation, ewes will ovulate aged follicles, but that compared to shorter duration follicles, these follicles produce oocytes that are equally competent to be fertilised and develop into good quality embryos and full-term lambs.

Effects of dioxin, an environmental pollutant, on mouse blastocyst development and apoptosis

Objective: To evaluate the effects of 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD; dioxin) on mouse embryo development and apoptosis. Design: Controlled animal study. Setting: Academic research environment. Animal(s): Female mice (CB6F1) at 3 to 6 weeks of age and proven breeders (C578B46). Intervention(s): Mouse embryos were obtained at the morula stage and cultured to the blastocyst stage in a pharmacologic dose of TCDD (3.1 μM) or a control medium. The morphology was assessed, and staining for apoptosis was performed. Immunohistochemistry for the presence of aromatic hydrocarbon receptor (AhR) was performed in another set of morula-stage embryos. Main Outcome Measure(s): The number of embryos developing from the morula to the blastocyst stage and number of apoptotic blastomeres in control vs. TCDD culture conditions. Result(s): No statistically significant differences were observed in the percentage of embryos reaching the blastocyst stage: 80.9% (115 of 142) in the TCDD-treated group, vs. 82.9% (121 of 146) in the control group. There was also no difference in the degree of apoptosis: 22.6 ± 7.3% apoptotic cells (TCDD) vs. 25.3 ± 9.7% (controls). Staining indicated the slight presence of aromatic hydrocarbon receptor in the morula-stage mouse embryos. Conclusion(s): TCDD at 3.1 μM did not alter the development of early mouse morula to blastocysts and did not significantly induce apoptosis in vitro.

Successful in vitro production of embryos in the red deer ( [formula omitted][formula omitted]) and the sika deer ( [formula omitted][formula omitted

The aim of our study was to define the conditions for IVM and IVF of oocytes in 2 common deer species as models for endangered related subspecies. Immature oocytes were recovered during the breeding season from postmortem ovaries (red deer) or by repeated laparoscopic follicular aspiration (sika deer). Oocytes were cultured for 24 h in IVM medium supplemented with EGF or FSH and follicular fluid. Stag semen was collected by electroejaculation (both species) or by epididymal flushing (red deer) and cryopreserved. For IVF, oocytes were exposed to different concentrations of thawed spermatozoa in a modified Tyrode albumin lactate pyruvate medium supplemented with 20% (v / v) estrus sheep serum for 18 h. After IVF, presumptive zygotes were allowed to develop in vitro for 7 days in synthetic oviduct fluid (SOF) supplemented with fetal calf serum (10%, v / v). In both species, the presence of ovine FSH and follicular fluid improved the in vitro maturation rate. In the sika deer, the optimal sperm concentration for IVF was 10 6 / mL and some fertilized oocytes reached the early morula stage (20 to 25 cells). In the red deer, after IVF with ejaculated or epididymal spermatozoa (2.0 x 10 6 / mL), 20% of zygotes developed to the blastocyst stage (50 to 80 cells).

Alterations in preimplantation in vivo development after preconceptional chronic moderate alcohol consumption in female mice.

Although many studies have explored the effects of acute or chronic ethanol exposure during the postimplantation period on embryo/fetal development, few reports have described the ethanol effects on preimplantation embryo development. Little is known about the effects of ethanol consumption prior to gestation on embryo growth. Recently, we have shown that chronic moderate ethanol intake by prepubertal female mice reduces the ovulatory response and impairs in vitro fertilization and in vitro embryo preimplantation development. The purpose of the present work was to evaluate the effects of preconceptional chronic moderate ethanol ingestion on preimplantation embryo morphology and differentiation, the timing of cleavage and embryo growth in vivo, and to determine the time pattern in which alterations appear. Prepubertal female mice were treated with 10% (w/v) ethanol for 30 days prior to conception. After inducing ovulation on day 27 and 29 of the ethanol treatment, females were mated with control males and the day of presence of vaginal plug was day 1. On day 1, a decreased percentage of normal fertilized oocytes, elevated parthenogenetic oocyte activation and unfertilized eggs with abnormal metaphase II were found in ethanol-treated, compared to control females. On day 2, while any differences in the total percentage of 2-cell embryos were observed, the treated females had a significantly higher percentage of morphologically abnormal embryos, compared to control females. On day 3, the preconceptional consumption of ethanol produced significantly reduced percentages of compacted morulae and an increased percentage of uncompacted morulae. The total percentage of morulae in the treated females was lower than in controls. On day 4, ethanol-treated females showed significantly decreased percentages of hatched attached blastocysts and increased early blastocyst and morula percentages, compared to controls. Thus, preconceptional chronic moderate ethanol ingestion by prepubertal female mice produced retarded development, impaired blastocyst hatching, abnormal embryo morphology and embryo loss by fragmentation due to alterations induced in the female gamete.

Production of transgenic rabbits for the human glutamic acid decarboxylase

We have previously reported an inhibitory effect of gossypol and its metabolite on bovine and mouse early embryonic development. In the present study, eggs were collected from oviducts of superovulated hamsters. Epididymal sperm were used for in vitro fertilization (IVF). Gossypol at 5, 10, and 30 μg/ml significantly inhibited the formation of 2 pronuclei by 45, 65 and 95%, respectively. On the first day of pregnancy, hamsters were given an intrauterine treatment of 200 μg of gossypol in 100 μl of corn oil per uterine horn. On day 3, embryos from controls were in morula (65%) and early morula (17%) stages, while less than 2% of embryos from the gossypol-treated hamsters were in the morula stage The numbers of embryo implantation sites on day 8 and pups in controls (14 ± 2.0 and 12 ± 1.5, respectively) were significantly higher than those in the gossypol-treated hamsters (8.5 ± 20 and 4.0 ± 1.5, respectively). Our results suggest that gossypol is able to affect fertilization, embryonic development, embryo implantation, and the number of pups in hamsters through a not-yet-defined mechanism.

Developmental kinetics of the first cell cycles of bovine in vitro produced embryos in relation to their in vitro viability and sex

The development of bovine IVP-embryos was observed in a time-lapse culture system to determine cell cycle lengths of 1) embryos that developed into compact morulae (CM) or blastocysts (BL) within 174 h after insemination (viable), 2) embryos that arrested during earlier stages (nonviable) and 3) male and female embryos. In 4 replicates, inseminated oocytes were cultured on a microscope stage in 3 to 4 groups on a granulosa cell monolayer in supplemented TCM 199. Images were sequentially recorded and stored at 30-min intervals. All embryos that could be identified throughout the culture period were included (n=392), and the times of cleavage events noted. After culture, 100 CM or BL were randomly selected for sexing by PCR. BL developed equally well in the time-lapse and control culture systems (36 vs 38%). The respective lengths of the first 4 cell cycles of viable embryos were 32.0 ± 3.9, 8.8 ± 1.6, 10.8 ± 4.7 and 47.7 ± 11.8 h. The subsequent intervals between the 9- to 16-cell, early morula, CM and BL stages lasted 16.2 to 18.2 h. Blastomeres of 2-,4- and 8-cell embryos cleaved asynchronously with <1, 2.6 ± 2.5 and 9.2 ± 4.5 h intervals, respectively, between the first and last blastomere to cleave. The interval from insemination to tight compaction and formation of a blastocoel was 128.4 ± 10.7 and 145.8 ± 12.5 h, respectively. The first 3 cell cycles were approximately 3 h shorter (P < 0.1) while the fourth cycle was 5 h shorter (P = 0.06) for the viable vs nonviable embryos. On this basis it was possible to define time windows in which the proportion of viable 2-, 3- to 4-, 5- to 8- and 9- to 16-cell embryos were at their highest. No differences were found between the cleavage intervals of male and female embryos. We conclude 1) that the time-lapse culture system allows for detailed observation of the developmental kinetics of several embryo groups at the same time, and 2) that these embryos can be manipulated at the end of culture, thus allowing a linkage between early cleavage events and other developmental parameters such as embryo sex or viability after transfer.

Activation of embryonic genome in chick.

The earliest stages of development in most animals are under the control of maternally inherited information. The initiation of embryonic gene expression has been reported at the mid-blastula in amphibians and the mid-2-cell stage to the early morula in mammals. In chick embryos, embryonic gene expression was detectable at stage X (morula) and showed marked activation at stage XIII (blastula) with a gradual increase thereafter. Synthesis of rRNA and tRNA was low at stage X and was already the major class of RNA at stage XIII in chick embryos. The observed upregulation of RNA synthesis seems to coincide with a period of extensive fine structural differentiation when the first major cellular migrations start and signal the formation of the primitive streak in the chick embryo.