Early Morula Stage Research Articles

118 EXPRESSION PROFILES OF CIRCADIAN CLOCK GENES IN MOUSE OOCYTES AND PRE-IMPLANTATION EMBRYOS

Matsuo et al. reported that circadian clock genes regulate the timing of cell division in mouse regenerating liver cells (2003). Their results suggested the importance of circadian clock genes for organs or tissues for which functions are characterized by cell division, such as pre-implantation embryos. To obtain basic information on the molecular functions of circadian clock genes in pre-implantation embryos, we investigated the expression profiles of transcripts and proteins of some circadian clock genes, clock, bmal1, cry1, and per2, in mouse germinal vesicle oocytes (GV), MII oocytes (MII), and pre-implantation embryos using real-time PCR and immunocytochemistry (ICC). Germinal vesicle oocytes were collected from ICR females at 48 h after PMSG priming. The mouse at 48 h after PMSG priming was primed with hCG, and MII were collected at 15 h after hCG priming. The pre-implantation embryos were collected at 6, 12, 24, 36, 48, 60, 72, 84, and 96 h after insemination, and they corresponded to early 1-cell, late 1-cell, early 2-cell, late 2-cell, 4-cell, 8-cell, early morula, late morula, and blastocyst stages, respectively. cDNA was produced by mRNA isolated from 20 oocytes or embryos using oligo dT and was subjected to real-time PCR using a TaqMan Probe system (ABI). Three sets of 20 oocytes or embryos at each developmental stage were applied to mRNA extraction and real-time PCR analysis to ensure equal mRNA extraction efficiency between samples. The level of mRNA of each clock gene contained in 3 samples from each developmental stage was almost the same. Statistical analysis of the transcripts of each gene were done by ANOVA. Germinal vesicles, MII and embryos collected at each time point were subjected to ICC using antibodies of CLOCK, BMAL1, CRY1, and PER2. The oocytes or embryos treated with only secondary antibody did not produce any signal. All of the examined genes except per2 were expressed in oocytes and pre-implantation embryos. The transcript level of clock, bmal1, and cry1 in MII were significantly lower than those in GV (P < 0.05). After fertilization, transcript levels of clock, bmal1, and cry1 significantly decreased from early 1-cell stage to late 2-cell stage (P < 0.05). These decreased transcript levels were maintained until the blastocyst stage after the late 2-cell stage. Immunocytochemistry analysis showed the nuclear localization of CLOCK and BMAL1 in early and late 2-cell embryos and of CRY1 in early 2-cell embryos but no signals of PER2 in oocytes or pre-implantation embryos. Because mouse oocytes and 1- to 2-cell embryos are transcriptionally inert, the abundant transcripts of clock, bmal1, and cry1 in these stages seemed to indicate that they were synthesized and stored during the oocyte growth phase. Moreover, the nuclear localization of CLOCK, BMAL1, and CRY1 in the oocytes and 1- to 2-cell stage embryos suggested that some clock genes were translated and worked for oocyte maturation and early embryogenesis. This study was supported by a Grant-in-Aid for the 21st Century COE Program of the Japan Mext and by a grant for the Wakayama Prefecture Collaboration of Regional Entities for the Advancement of Technology Excellence of the JST.

196 IN VITRO DEVELOPMENT OF RECONSTRUCTED WATER BUFFALO (BUBALUS BUBALIS) OOCYTES AFTER FETAL SKIN FIBROBLAST CELL NUCLEAR TRANSFER

The present study was undertaken to explore the feasibility of using buffalo fetal skin fibroblasts as donor nuclei and to determine the developmental competence of embryos following transfer of these nuclei to in vitro-matured enucleated buffalo oocytes. Skin cells were isolated from 1–2-month-old fetuses, obtained from an abattoir, by enzymatic digestion (0.5% w/v trypsin + 0.05% w/v collagenase in Dulbecco's PBS) for 15–20 min. The cells were washed four times with Dulbecco's PBS and then once with RPMI-1640 medium + 10% FBS by centrifugation at 600g. The cells were then cultured in the same medium in a CO2 incubator (5% CO2 in air) at 38.5°C for 2–3 days. COCs collected from slaughterhouse buffalo ovaries were subjected to IVM in the IVM medium (TCM-199 + 1 μg mL−1 E-β + 5 μg mL−1 FSH-P + 10 μg mL−1 LH + 10% FBS) for 22–24 h in a CO2 incubator (5% CO2 in air) at 38.5°C. Oocytes were denuded with 0.1% trypsin followed by repeated pipetting and then enucleated by aspirating the first polar body and 10–15% of nearby cytoplasm with a micromanipulator. Two different types of donor cells (growing cells and those arrested with cytochalasin-B) were used for reconstruction of oocytes. The reconstructs were electrofused and incubated in the activation medium (TCM-199 + 8 μg mL−1 cytochalasin-B + 10% FBS) for 4 h. These were then cultured in IVC medium (TCM-199 + 10% FBS) in a CO2 incubator (5% CO2 in air) at 38.5°C for 48 h. Next, the cleaved embryos were co-cultured with buffalo oviductal cells in embryo development medium. Out of 119 denuded matured oocytes which were enucleated and reconstructed with growing cells, 78 (65.5%) were electrofused, activated and cultured, out of which 4 (5.1%) reconstructs cleaved and developed to the 2-cell stage, 3 (3.8%) reached the 4-cell stage, and 1 (1.3%) reached the 8-cell stage. In the synchronized group, out of 100 denuded matured oocytes which were reconstructed with cytochalasin-B blocked cells, 40 (40%) were electrofused, activated, and cultured, out of which 4 (10%) developed to the 2-cell stage, 3 (7.5%) to the 4-cell stage, 2 (5.0%) to early morula stage, and 1 (2.5%) to blastocyst stage. These results suggest that buffalo fetal skin fibroblasts could be used as donor nuclei for the production of buffalo embryos after nuclear transfer to enucleated in vitro-matured buffalo oocytes.

58 HANDMADE CLONING IN TRANS-SPECIES NT: CULTURE MEDIUM HAS AN EFFECT ON THE ABILITY OF RECONSTRUCTED BOVINE-MURINE EMBRYOS TO DEVELOP BEYOND THE 8-CELL STAGE

The objectives of studies of trans-species nuclear transfer (NT) include epigenetic reprogramming and stem cell technology. The present study evaluated the effect of culture media on the development of reconstructed bovine-murine embryos. Bovine NT served as a technical control. The NT embryos were produced with the hand made cloning (HMC) technique (Vajta G et al. 2003 Biol. Reprod. 68, 571–578), and to our knowledge, this is the first report on the application of HMC in trans-species NT. Abattoir-derived bovine oocytes were matured for 21 h and enucleated by hand as described (Vajta G et al. 2003). Bovine cytoplasts were fused with either bovine granulosa cells or murine fetal fibroblasts. The bovine NT embryos were cultured for 7 days in modified SOFaaci (Holm P et al. 1999 Theriogenology 52, 683–700) containing either 5% FBS (8 trials) or 4 mg mL−1 fatty acid free albumin (FAFBSA, 6 trials). Bovine-murine NT embryos were cultured for 4.5 days in SOFaaci + FAFBSA (5 trials), or for the first 12 h in SOFaaci + FAFBSA and until 4.5 days in KSOMaa (Biggers JD 1991 J. Reprod. Fertil. 91, 543) containing 1 mg mL−1 embryo-tested BSA (4 trials). The results are shown in Table 1. In bovine NT embryos, both cleavage (day 2) and day 7 blastocyst rates were significantly improved was SOFaaci + FAFBSA was used as culture medium. Culture medium did not affect the cleavage rate of bovine-murine NT embryos at 12–16 h after start of culture. The development of reconstructed bovine-murine embryos beyond the 8-cell stage was significantly improved when SOFaaci + FAFBSA was replaced with KSOMaa + BSA after 12 h culture. Fourteen of a total of 464 (3.0%) Day 4.5 bovine-murine reconstructed embryos reached early morula stage with signs of compaction. The study showed that the development of the reconstructed NT embryos was significantly affected by the culture medium. Contrary to earlier findings (Park SH et al. 2004 Mol. Reprod. Dev. 68, 25–34), the bovine-murine reconstructed embryos developed beyond 8-cell stage, even until early compaction. The gene expression of species-specific and development-related genes of the reconstructed embryos is under characterization. Table 1. Effect of culture medium on cleavage and development of reconstructed NTt embryos Professor Gabor Vajta is greatly acknowledged for his contribution in establishing the HMC technique in our laboratory.

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.

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.

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.

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.

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).

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.

Gene targeting in C57BL/6 ES cells. Successful germ line transmission using recipient BALB/c blastocysts developmentally matured in vitro.

There are significant advantages to the production of gene-knockout mice directly in mouse strains other than 129. The availability now of ES cells derived from the C57BL/6 mouse strain presents workers with a valuable alternative. A major difficulty, however, is the requirement for BALB/c blastocysts as recipients for ES cell injection. Using standard procedures, few BALB/c blastocysts can be obtained. This limitation has now been resolved by harvesting BALB/c embryos at the early morula stage and maturing these to blastocysts by in vitro culture. Of early morulae harvested and cultured, over 70% were recovered as fully expanded and injectable blastocysts. C57BL/6 ES cell injection of these blastocysts has enabled the production of a number of gene-knockout mice with a success rate similar to that reported for ES cells derived from the 129 mouse strains.

Flow cytometric sorting of sperm: influence on fertilization and embryo/fetal development in the rabbit.

Viable, intact rabbit sperm, prepared, processed, and flow cytometrically sorted, were used in this study to determine the influence of flow sorting on fertilization and embryo development. In experiment I, flow-sorted or control (unstained and unsorted) sperm were surgically inseminated into the uterine horn of hormonally primed does (10 to 12 does per time point). At 42 hr postsurgical insemination, flushed embryos were assessed for development. Fetal development was determined at day 7, day 14, and day 21 post-surgical insemination. Embryos resulting from does surgically inseminated with control sperm at 42 hr post-insemination were observed to be at the early morula stage of development (> 16 cell), whereas embryos from does inseminated with flow sorted sperm were at the 8- to 16-cell stage. No difference was observed between treatments at day 7, 14, or 21, however, there was a significant decrease in fetus number per doe inseminated with flow-sorted sperm over time. In experiment II, mature oocytes were flushed from the oviducts of superovulated does and coincubated in vitro (IVF) with flow-sorted or control rabbit sperm. Oocytes observed at 6 hr post-coincubation exhibited swollen sperm heads in the cytoplasm, demonstrating that fertilization had occurred (2 PN + T). There was a higher percentage of fertilized oocytes by 8 hr post-coincubation for both control (31%) and flow-sorted sperm (31%) when used for IVF. By 10 and 12 hr post-coincubation, little difference was observed in the number of fertilized oocytes between sperm treatments (52% and 66% for control vs. 57 and 54% for flow-sorted, respectively). These studies demonstrate that flow-sorted sperm are capable of fertilizing mature oocytes under in vitro conditions. In addition they show that flow sorting may not negatively influence fertilization events, but likely interferes during early embryonic and fetal development.

Effects of amino acids on development of IVM/IVF bovine embryos in a simple protein-free medium

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% CO2, 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 × 106 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.

Cryopreservation of fish embryos and embryonic cells

Embryonic cells of dechorionated fish embryos dissociated in Ca*+, Mg’+ free medium were successfylly integregated to recipient embryos using a novel aggregation method for fish resulting in survival of fish chimeras. Embryonic cells were successfully cultured in sterile cell culture for up to 1 month. Experiments were carried out on the cryopreservation of pluripotent diploid embryonic cells derived from dechorionated embryos of African catfish (Clurias gariepinus) using two-step freezing and programmable freezer. Cells were equilibrated in media containing DMSO and methanol as cryoprotectants. A high percentage (84-95%) of survival has been achieved upon thawing unrelated to seeding applied. Survival was tested using fluorescein diacetate (FDA) vital staining in order to examine the membrane integrity of the cells upon thawing. In teleosts, the cryopreservation of eggs and embryos has had very limited success so far. In some cases eggs tolerated temperatures below 0°C but no one succeeded in prolonged preservation below -60°C. Several species of teleost, mostly Salmonidae were studied by other groups. We were the first to choose the African catfish as a suitable model for the examination of cryobehaviour of fish ova and embryos. In order to find the stages with the highest penetration rate we examined the permeability of the eggs towards cryoprotectants (CPAs) in different developmental stages. Following exposure of the ova to concentrated CPA solutions, the penetration of the CPAs was detected by visual observation of vitrification of the eggs plunged into liquid nitrogen in straws. Our results indicated that the permeability of unfertilized eggs is much higher than that of fertilized eggs. The permeability is sharply increased in the early morula stage and remains high later on. The vitrification is thought to be a combined effect of dehydration and penetration of CPAs. The percent of eggs vitrified following lo,30 and 60 min exposure at 23°C to 6.5 M DMSO was 4%, 87% and 100%; 6.5 M propylene glycol was O%, 90% and 100%; 6.5 M glycerol was O%, 0% and 90%; 6.0 M ethylene glycol was 0%, 0% and 83%, respectively. None of the eggs survived more than 5 min exposure to the CPA solutions studied.

Induction of cyclooxygenase and prostaglandin E2 production by the bovine pre-embryo

A study was conducted to determine the induction of cyclooxygenase production in the cleaved and non-cleaved oocyte and to measure the secretion of prostaglandin E2 (PGE2), PGF2 alpha and tumour necrosis factor-alpha (TNF alpha) in the conditioned medium of non-mature oocytes (denuded), mature oocytes (oocyte-cumulus complex), cleaved oocytes, and non-cleaved oocytes (mature oocytes incubated in the absence of sperm). Cyclooxygenase was readily detectable in 48-h cleaved oocytes whereas the signal for the enzyme in the non-cleaved oocytes was weak or undetectable. Cyclooxygenase expression in the cleaved oocytes was transient and enzyme concentrations at the early morula stage (72 h) were low. Measurable amounts of both PGE2 and TNF alpha were secreted during the first 24 h of maturation and 6 h after fertilization, whereas PGF2 alpha was undetectable at these times. There was a dramatic enhancement in PGE2, PGF2 alpha and TNF alpha secretion 48 h after fertilization. At 72 h after fertilization, PGE2 concentrations were much lower and PGF2 alpha was again undetectable which corresponded to a low concentration of cyclooxygenase. In contrast, high TNF alpha activity continued to be detected even 72 h after fertilization. It is possible that the induction of the production of cyclooxygenase and the secretion of PGs and TNF alpha by fertilized oocytes at specific times after fertilization plays a role in the regulation of embryonic development, maternal immunological recognition of pregnancy, and the maintenance of a suitable hormonal environment for embryonic viability.

Inhibition of in vitro fertilization and early embryonic development in hamsters by gossypol

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.

In vitro maturation and fertilization of sheep oocytes collected by laparoscopic folliculocentesis

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% CO2, 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 × 106 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.

Tripronuclear human zygotes: the first cell cycle and subsequent development.

About 6.6% of human oocytes fertilized in vitro were tripronuclear. The first cell cycle and developmental ability of these triploid zygotes were studied by autoradiographic analysis of pronuclear [3H]thymidine incorporation and by morphological observations of living zygotes at various times after insemination. Weak labelling over the pronuclei was first detected at 10 h after insemination and in many zygotes the pronuclei were heavily saturated with radioactive grains by 11-13 h. Thymidine incorporation was not apparent in the majority of tripronuclear zygotes by 21-22 h. At this time, 96% of living triploids (65/68) had visible pronuclei while the others entered the first mitosis and their pronuclei disappeared. By 25 h, 70% of triploid zygotes (37/58) still exhibited visible pronuclei and 26% were already in mitosis. The first divided embryos were seen by 25 h. The results suggest that the length of the complete cell cycle of human 1-cell stage triploids is approximately 25 h. Pronuclear DNA synthesis starts approximately 9-10 h after insemination and seems to last a minimum of 4 h (the precise time of S phase could not be determined). The minimal duration times of G2 phase and mitosis are estimated in the range of 4-6 h and 3.5-4 h, respectively. Tripronuclear zygotes were capable of substantial in-vitro development. About half of examined triploids attained at least the 4-6-cell stage of development (14/32; 44%); however, approximately 19% of these exhibited multinuclear cells. The remaining triploids had reached early morula (8 and 13 cells; 25 and 19%) or blastocyst (6%) stage.

Differences in the effects of treatment of uncompacted and compacted mouse embryos with phorbol esters on pre- and postimplantation development

Abstract Differences are described in the effects of treatment of preimplantation mouse embryos with low levels (0.01–1 n M) of phorbol myristate acetate (PMA), during three different periods of a 48-h culture from the 2-cell stage, on pre- and postimplantation development. Treatment of embryos with PMA for 48 h (first group) or 24 h (second group) from the 2-cell stage caused premature cavitation (prior to the 16-cell stage) and it also reduced the size and alkaline phosphatase (ALPase) activity of inner cell masses (ICMs), as well as the numbers of cells in blastocysts, in a dose-dependent manner. Treatment of early morulae with PMA for 24 h (third group) did not have the abovementioned effects on embryos but inhibited the formation and subsequent enlargement of the blastocoel. The blastocysts that were allowed to develop in the three treatment groups were examined for postimplantation development. Implantation was unaffected in all groups. The survival rate after implantation was low in the first and second groups but relatively high in the third group. The results indicate that an embryo exposed to PMA for 24 h from the 2-cell stage forms a premature blastocoel, and, in such an embryo, quantitative and qualitative differentiation into the ICM is blocked but qualitative differentiation into trophectoderm is uninhibited. Consequently, the embryo can implant but does not survive for a long time. When embryos were exposed to PMA for 24 h from the early morula stage, the formation and enlargement of the blastocoel were inhibited even though the treatment had a minimal effect on other developmental events. It is suggested that the effects of PMA on early mouse development are specific to each period at which the drug is applied.

Incorporation of [3H]inositol into phosphoinositides and inositol phosphates by rabbit blastocysts

Preimplantation rabbit embryos collected at the early morula stage were cultured to blastocysts in the presence of [3H]inositol. The blastocysts were lysed, and both the aqueous and lipid portions were analysed for incorporated radioactivity. Thin-layer chromatographic separation of the lipid portion indicated that [3H]inositol was incorporated into phosphatidylinositol, phosphatidylinositol 4-phosphate, and phosphatidylinositol 4,5-bisphosphate. HPLC anion-exchange chromatography indicated that [3H]inositol was incorporated into inositol phosphates, including the two second messengers, inositol 1,4,5-triphosphate and inositol 1,3,4,5-tetrakisphosphate, and also inositol monophosphate and inositol 1,4-bisphosphate. These results provide evidence that rabbit blastocysts may have an active phosphatidylinositol second messenger system, which may be responsive to intrauterine factors or intraembryonic paracrine factors.