Effect of Post-IVF Developmental Kinetics on In Vitro Survival of Vitrified-warmed Domestic Cat Blastocysts

A limited number of reports is available on cryopreservation of in vitro fertilization (IVF)‐derived cat blastocysts. In the present study, IVF‐derived domestic cat embryos which reached the blastocyst stage either on day 6 or day 7 were cryopreserved by vitrification using Cryotop as a cryodevice. Fresh control and post‐warm surviving blastocysts were examined by differential cell staining with Hoechst 33342 and propidium iodide to determine total cell number and inner cell mass (ICM) ratio, and the post‐warm survival rate was determined by re‐expansion of the blastocoel during 24 h of in vitro culture. In fresh control, the mean number of total cells of day 7 blastocysts (61.4 cells) tended to be smaller than that of day 6 blastocysts (81.9 cells, p = 0.096). The post‐warm survival rates of day 6 and day 7 blastocysts were not statistically different (73.8%; 31 of 42 vs 66.7%; 18 of 27). There were no significant differences in the total cell number and ICM ratio between fresh control and vitrified blastocysts, although the ICM ratio of surviving day 7 blastocysts was significantly smaller than that of fresh controls (stained at day 8, 18.9% vs 28.9%, p < 0.05). These results indicate that IVF‐derived domestic cat embryos that reached the blastocyst stage earlier can survive the Cryotop vitrification without a reduction in the parameters studied.     

注入嵌合法制作黄牛和水牛种间嵌合胚的初步研究

采用注入嵌合法初步建立了一套黄牛和水牛种间嵌合的程序与方法。采用机械剥离法或免疫外科法分离胚胎内细胞团(ICM),然后注入到已去除ICM的受体囊胚中构建形成水牛和黄牛的嵌合胚。结果发现,在用免疫外科法分离ICM时,抗血清的灭活温度从57℃升至63.5℃,ICM的获得数显著升高(0%vs100%,P<0.01),如若在分离培养液中添加6%的胎牛血清(FCS),ICM的获得数大大降低(97.6%vs0%,P<0.01)。采用免疫外科法分离得到的黄牛ICM进行水牛囊胚的ICM置换重组,重组胚的存活率与机械剥离法得到的ICM无显著差异(91.4%vs87.5%,P>0.05);但囊胚孵化率则显著提高(80%vs43.8%;P<0.05)。以上结果表明,⑴水牛和黄牛胚胎通过ICM置换获得的种间嵌合胚胎能继续发育;⑵用于黄牛ICM分离的兔抗牛抗血清需在63.5℃灭活30min,且分离需在无血清的培养液中进行;⑶通过分离ICM置换进行胚胎嵌合时,免疫外科法优于显微手术法。     

ICR小鼠胚胎干细胞建系初步研究

实验旨在探讨消化方式和胚胎发育阶段对ICR小鼠胚胎干细胞(ES细胞)建系效率的影响。ICR小鼠3.5 d囊胚在饲养层上贴壁后采用单一酶消化或机械化与酶消化法相结合分离隆起的细胞集落,进行传代培养;然后选择二者中较优消化方式对不同发育时期囊胚所形成的细胞集落进行处理。结果表明:采用机械化与胰酶消化相结合的方式,形成的类ES细胞超过7代的比率(85.0%)要显著高于单一的胰酶消化(15.0%)(P<0.05);当用二者相结合的方式对ICR小鼠3.5 d(早期囊胚)、4.0 d(扩张囊胚)和4.5 d(孵化囊胚)所形成的细胞集落进行消化传代培养,三者在贴壁率和形成原代细胞集落率上均无显著差别(P>0.05),但传代超过7代的效率上早期囊胚和扩张囊胚均高于孵化囊胚(P<0.05)。结果提示,采用机械化与酶消化法相结合更适合于3.5～4.0 d ICR小鼠囊胚的ES细胞建系。     

Comparison the effects of 5-Aza-2′-deoxycytidine and zebularine on the in vitro development,blastocyst quality,methylation pattern and conception rate on handmade cloned buffalo embryos

In this study we treated the handmade cloned (HMC) buffalo embryos with the DNA methylation inhibitors; 5-aza-2′-deoxycytidine (AzadC) or Zebularine individually after post-fusion and during in vitro culture till eighth day. The blastocysts production rate significantly improved (p < .01) after treating embryos independently with 5 nM AzadC and 5 nM zebularine compared with 2 and 10 nM AzadC or zebularine groups, respectively. The highest cleavage rates were obtained for 5 nM treatment of AzadC and zebularine compared with other treatments and untreated control group. Quality of blastocysts were evaluated using total cell number (TCN) and the ratio of number of inner cell mass (ICM) cells/total cell number (ICM/TCN). Zebularine treatments (2/5/10 nM) significantly improved both TCN and ICM/TCN ratio compared with AzadC treatments (2/5/10 nM); however, control group TCN and ICM/TCN ratio was found lower. The methylation percentage of pDS4.1 and B. bubalis satellite DNA were comparatively more attenuated with 5 nM zebularine than 5 nM AzadC treatment. The increased in vitro development rates of the treated embryos were correlated with the decreased level of DNA methylation and the improved blastocyst quality. Following transfer of 5 nM zebularine treated embryos to 6 recipients, 4 were found to be pregnant, though the pregnancies were not carried to full term.     

Improved isolation and culture of embryonic stem cells from Chinese miniature pig

Pigs serve as a better research model for human beings than other species. The Chinese laboratory miniature pig is a new laboratory animal and is expected to be applicable in many medical research fields. This study was to establish effective technologies to isolate and culture ES cells in Chinese miniature pigs. For isolation of the inner cell mass from blastocysts, an enzyme-digestive method was compared with the traditional immunosurgery. Isolated ICM were cultured in three feeder cell layers: mouse embryonic fibroblasts (MEF), porcine embryonic fibroblasts (PEF) and a continuous cell line of mouse embryonic fibroblasts (STO). Microtubule activity of the three feeder cells was further examined by immunofluorescence. ICM were successfully isolated from 85% of blastocysts by the enzyme-digestive method, compared to only 40% by immunosurgery. When ICM were cultured in three feeder layers for two to three days, 75%, 65% and 20% of ICMs formed primary cell colonies in MEF, PEF and STO, respectively. Colonies were also formed during subcultures after 9, 5 and 1 passage in MEF, PEF and STO, respectively. Microtubules in STO cells were significantly fewer than those in MEF and PEF. When the ES-like cells were cultured in a differentiation medium, they differentiated to neuron-like cells and other types of cells. These results indicate that healthier ICM can be obtained with the enzyme-digestive method. Successful culture of ICM to ES-like cells has been achieved not only in MEF, but also in homologous (pig) feeder layer. The ES cells obtained in the present study were pluripotent.     

In vitro culture of bovine embryos in murine ES cell conditioned media negatively affects expression of pluripotency-related markers OCT4, SOX2 and SSEA1

Despite extensive efforts, establishment of bovine embryonic stem (ES) cell lines has not been successful. We hypothesized that culture conditions for in vitro-produced (IVP) embryos, the most used source of inner cell mass (ICM) to obtain ES cells, might affect their undifferentiated state. Therefore, the aim of this work was to improve pluripotency of IVP blastocysts to produce suitable ICM for further culturing. We tested KSR and foetal calf serum (FCS) supplements in SOF medium and ES cell conditioned medium (CM) on IVC (groups: KSR, KSR CM, FCS and FCS CM). Cleavage and blastocyst rates were similar between all groups. Also, embryonic quality, assessed by apoptosis rates (TUNEL assay), total cell number and ICM percentage did not differ between experimental groups. However, expression of pluripotency-related markers was affected. We detected down-regulation of OCT3/4, SOX2 and SSEA1 in ICM of FCS CM blastocysts (p < 0.05). SOX2 gene expression revealed lower levels (p < 0.05) on KSR CM blastocysts and a remarkable variation in SOX2 mRNA levels on FCS-supplemented blastocysts. In conclusion, pluripotency-related markers tend to decrease after supplementation with ES cell CM, suggesting different mechanisms regulating mouse and bovine pluripotency. KSR supplementation did not differ from FCS, but FCS replacement by KSR may produce blastocysts with stable SOX2 gene expression levels.     

Development of large and small blastomeres from 2‐cell embryos produced in vitro in pigs

In the present study, we examined the development to blastocysts of large and small blastomeres from unevenly cleaved 2‐cell embryos (uneven 2‐cell embryos) in pigs. Proportion of blastocysts derived from large blastomeres (52.8 ± 6.4%) was significantly higher (P < 0.05) compared with small ones (32.1 ± 4.6%). However, there were no differences in total cell number, inner cell mass (ICM) cell number and ICM/total cells ratio between them. Of 53 sister blastomere pairs in the same embryos examined there were 12 pairs (22.6%) in which both blastomeres developed to blastocysts, 16 pairs (30.2%) in which only large blastomeres developed to blastocysts, and five pairs (9.4%) in which only small blastomeres developed to blastocysts. Relative total amount of active mitochondria in small blastomeres were lower (P < 0.05) than that of large blastomeres and blastomeres from evenly cleaved 2‐cell embryos. However, there was no difference in relative density of active mitochondria in these three types of blastomeres. In conclusion, blastocysts derived from small and large blastomeres in uneven 2‐cell embryos had comparable quality in terms of cell number, ICM number, ICM/total cell ratio and distribution of active mitochondria. The results suggest that these blastomeres may contribute multiple offspring production in pigs.     

山羊类ES细胞的分离与克隆

采集山羊交配后6～8d的桑椹胚、囊胚和孵化囊胚,将桑椹胚和囊胚分别放在小鼠原代胎儿成纤维细胞(PMEF)饲养层和同源原代胎儿成纤维细胞(PGEF)饲养层上比较其脱带时间及脱带率。脱带后,将各自一半胚胎切割,把含ICM的半胚分别放在相应饲养层上进行培养,另一半整胚在各自饲养层上继续培养,而孵化囊胚直接于PGEF饲养层上培养。当ICM增殖一定程度时进行传代,以比较其类ES细胞分离与克隆的效果。结果表明,在2种不同饲养层上,囊胚的脱带时间均短于桑椹胚,囊胚的脱带率均高于桑椹胚,而饲养层的种类对胚胎的脱带时间以及脱带率影响不大。脱带切割囊胚不论在PMEF还是在PGEF饲养层上,其贴壁时间均短于脱带整胚及孵化囊胚,而贴壁率高于脱带整胚,与孵化囊胚相似。脱带整胚及脱带切割胚在PMEF饲养层上所获类ES细胞只能维持3代,而在PGEF饲养层上,脱带切割半胚和孵化囊胚所获类ES细胞传至5代。由此认为,对脱带后的胚胎进行切割处理,有利于ICM的贴壁和增殖;应用同源原代胎儿成纤维细胞饲养层培养系统,有利于类ES细胞的分离与克隆。     

Effects of Retinol on the in vitro Development of Bos Indicus Embryos to Blastocysts in Two Different Culture Systems

The objective of this study was to evaluate the effect of retinol on the in vitro development of early embryos of cultured Bos indicus (Expt 1) to the blastocyst stage in medium simplex of optimization (KSOM) or sintetic fluid of oviduct (SOF) or co-cultured (Expt 2) with an oviduct cell monolayer (OCM) in KSOM or SOF. A total of 3149 cumulus-oocyte complexes obtained by aspirating follicles (2-5 mm diameter) from ovaries of slaughtered animals were selected for IVM and incubated in TCM 199 supplemented with 25 mM HEPES at 39 degrees C in air with 5% CO(2) and maximum humidity for 24 h. In vitro fertilization (IVF) was performed in modified defined medium (mDM) medium. Eighteen hours after IVF, cumulus cells were removed and presumptive zygotes were randomly allocated to the experimental groups. Zygotes cultured (Expt 1) in KSOM + retinol, KSOM, SOF + retinol and SOF were incubated in maximum humidity at 39 degrees C, 5% CO(2), 5% O(2) and 90% N(2). Zygotes co-cultured (Expt 2) in KSOM + retinol + OCM, KSOM + OCM, SOF + retinol + OCM and SOF + OCM were incubated at 39 degrees C, 5% CO(2). In both experiments media were partially changed 48 h after IVF and unfertilized ova were removed. Afterwards embryos were kept in culture or co-culture for further 9 days. In Expt 1, blastocyst rates (day 7) were 14.6% (KSOM + retinol), 15.8% (KSOM), 16.4% (SOF + retinol) and 15.9% (SOF). In Expt 2, the blastocyst rates (day 7) were 25.4% (KSOM + retinol + OCM) 14.2% (KSOM + OCM), 24.3% (SOF + retinol + OCM) and 15.9% (SOF + OCM). The same influence profile of retinol was observed in the formation of the expanded (day 9) and hatched (day 11) blastocysts. The results obtained in Expt 2 demonstrated that the addition of 0.28 microg/ml retinol to the embryo culture media used in this study had a significant (p < 0.05) positive effect on bovine early embryonic development, under the conditions tested, and can be used to enhance in vitro embryo production.     

猪孤雌胚胎干细胞建系的研究

以猪孤雌激活囊胚为材料,囊胚透明带消化后采用全胚培养,培养液中添加不同培养成分或因子（如FGF2,LIF,2i等）,以及选择不同的初始培养液体积来筛选猪胚胎干细胞（embryonic stem cell,ES细胞）建系的优化培养体系。囊胚内细胞团形成的细胞集落采用胰酶消化传代。结果显示：透明带消化后,囊胚贴壁率显著升高（19．4％VS．8．8％）（P〈0．05）;初始培养液体积比平常培养液体积（0．30mL／孔,24孔培养板）减半条件下,能显著提高其贴壁率（91．7％VS 20．0％）（P〈0．01）,而且获得了可传至7代的类ES细胞系2株,碱性磷酸酶染色成阳性;当用2i因子（CHIR99021和PD03025901）去替代培养液中的FGF2,囊胚贴壁率（29．400VS53．3％）和原代集落形成率（20．0％VS 87．5％）反而显著下降（P〈0．01）。这表明培养液添加了FGF2和LIF（不舍2i因子）,用24孔板培养,最初培养体积为0．15mL,透明带消化的培养体系比较适合猪孤雌激活胚的ES细胞建系。     

Total Cell Number and its Allocation to Trophectoderm and Inner Cell Mass in In Vitro Obtained Cats' Blastocysts

The aim of this study was to evaluate the developmental kinetics of cats' blastocysts in connection with their morphology and blastomeres allocation to trophoblast or embryoblast cells. We examined gross blastocyst morphology and the total number of blastomeres together with its allocation to inner cell mass (ICM) or trophectoderm (TE) cells in pre‐implantation feline embryos obtained from 6th to 9th day of in vitro culture. From all the investigated embryos, 61.8% developed to early blastocyst, 37.4% to full and 7.6% to hatching blastocyst stage. The total cell number (TCN) varied form 58 cells in early day 6 to 245 in hatching day 8 blastocyst, with the mean 84.9 cells in early, 156.7 in full, and 204.4 in hatching ones. Day 8 blastocyst had the highest number of total cells, together with the highest mean number of ICM regardless of its morphological assessment. Early blastocyst (apart from day 6) had the highest number of arrested cells, while dead cells were the highest in full day 9 blastocyst. More data about the relationship between blastocyst development and morphology would facilitate the selection of optimal blastocysts for further procedures.     

Improved development of ICR mouse 2-cell embryos by the addition of amino acids to a serum-, phosphate-and glucose-free medium

This study was conducted to evaluate how exogenous amino acids could affect preimplantation development of ICR mouse embryos. Two-cell embryos collected from naturally mated mice were cultured in amino acid-, glucose- and phosphate-free preimplantation (P)-1 medium. In Experiments 1, 19 amino acids (aa; 1% and 0.5% of MEM essential and nonessential amino acid solutions, respectively) were added to P-1 medium supplemented with either fatty acid-free bovine serum albumin (BSA; 3 mg/mL) or human follicular fluid (hFF; 10%). Regardless of BSA or hFF addition, embryo development to the morula (84 to 86% vs. 97 to 100%) and the blastocyst (54% vs. 93 to 94%) stages was significantly (P<0.05) enhanced by the addition of aa compared with no addition. In Experiment 2, the cell number of blastomeres and inner cell mass (ICM) cells in blastocysts and the ratio of ICM cell to trophectodermal cell (TE) were evaluated after aa addition. In both BSA- and hFF-containing P-1 medium, a significant increase in total blastomere number were found after aa addition (47 to 52 vs. 62 to 63 cells) compared with no addition. However, the ICM/TE ratio was not significantly affected by aa supplementation in both media, while ICM cell number was greatly increased after aa addition in hFF-containing medium (12 vs. 17 cells). When blastocysts were further cultured up to 162 hr post-hCG injection, development to the hatched blastocyst stage was significantly promoted by aa addition (0% vs. 11 to 20%) in both BSA- and hFF-containing media. In conclusion, aa significantly promote the preimplantation development to the hatched blastocyst stage and such effect mainly exerted on supporting blastomere proliferation.     

Immunolocalization of Androgen Receptor in the Boar Epididymis: the Effect of GnRH Agonist Deslorelin

Epididymides from nine crossbred male pigs [Polish Landrace × (Duroc × Pietrain)] (n = 3 per each group) were used in this study to show whether there are any differences between androgen receptor (AR) distribution along epididymal duct of a GnRH agonist deslorelin-treated boars when compared to the control tissues. The active agent was administered by way of a subcutaneous controlled-release implant containing 4.7 mg deslorelin at 91 or 147 days of age respectively. Boars from two experimental groups and the control group were slaughtered at 175 day of age. Immunolocalization was performed using a polyclonal rabbit antiserum against the AR. In control boars, strong staining for AR was detected in nuclei of the epithelial (principal and basal) and stromal cells, whereas in boars treated with deslorelin the staining was confined to the principal cell nuclei. In those treated for 84 days, AR-immunostaining was weak or the principal cells were negative for the AR. Irrespective of the time from deslorelin insertion all stromal cells were immunonegative. The results demonstrate for the first time the effect of deslorelin on the distribution of the AR in the three regions of the boar epididymis. It is likely that stromal cells are more sensitive than epithelial cells to the regulation of AR expression by androgen. The morphological and functional alterations along the epididymal duct and lack of spermatozoa within the lumen after deslorelin treatment indicate that a potent GnRH agonist is likely responsible for an impairment of the microenvironment created by epididymal cells for sperm maturation and their storage.     

Effect of co‐culture with intact embryos on development of bovine separated blastomeres

To improve embryo development in bovine separated blastomeres, we evaluated applicability of co‐culture with intact embryos. The morphological quality of blastocysts derived from separated blastomeres and rate of blastocyst formation were only slightly increased when the cells were co‐cultured with intact embryos, which did not provide significant differences when statistically analyzed. However, the cell count of inner cell mass (ICM), trophectoderm (TE) and total number of cells in Day 8 blastocysts were significantly higher when the cells were co‐cultured with the intact embryos than those with the cells cultured individually (P < 0.05). Transfer of four monozygotic pairs of blastocysts derived from the cells co‐cultured with intact embryos led to three pregnancies even when the blastomeres were produced by in vitro maturation and in vitro fertilization of oocytes collected by ovum pick‐up from elite cows. These results suggest that co‐culturing with intact embryos may enhance development of bovine separated blastomere.     

Effects of bovine serum albumin and estrous cow serum on development and ultrastructure of in vitro-produced porcine embryos

This study evaluated the effects of bovine serum albumin (BSA; 4 mg/ml) and estrous cow serum (ECS; 10%) in North Carolina State University (NCSU) 23 medium on the development of in vitro-matured and in vitro-fertilized porcine oocytes. Early cleavage rate was significantly (P < 0.05) higher in NCSU/ECS (71.3 +/- 14.7%) vs. NCSU/BSA (60.6 +/- 4.7%). Cleavage beyond the four-cell stage was not different between the two culture media (43.5 +/- 9.5% and 41.4 +/- 17.7%, respectively). The proportion of development to blastocysts was--with borderline significance (P = 0.05)--higher in NCSU/BSA (28.0 +/- 4.4%) than in NCSU/ECS (20.4 +/- 7.3%). Blastocysts produced in NCSU/BSA had significantly (P < 0.001) higher cell numbers than those cultured in NCSU/ECS (29.5 +/- 20.1 vs. 16.9 +/- 10.8). The ultrastructure of in vitro-produced blastocysts from both culture systems was compared vs. in vivo-derived blastocysts. The latter showed a clear differentiation between trophectoderm (TE) and inner cell mass (ICM) cells. The TE cells were anchored to other TE cells or ICM cells by long, well-developed junctional complexes. The apical membrane of trophoblast cells was covered with numerous microvilli. Mitochondria were abundant, round to elongated in shape, and showed clear transverse cristae. The ultrastructure of blastocysts cultured in NCSU/BSA mimicked that of in vivo-derived embryos closely. In contrast, blastocysts from the NCSU/ECS culture system displayed an irregular ultrastructure with reduced numbers of organelles and numerous cytoplasmic inclusions, such as lipid-yolk-vacuoles and vacuoles with lipid content. In some sections of these embryos, cellular debris was detected in cytoplasm. The shape of mitochondria was more ovoid and cristae were not visible. In summary, our results demonstrate a beneficial influence of ECS in the culture medium on initial cleavage of in vitro-produced porcine embryos. Clearly negative effects of ECS in the subsequent culture period are associated with marked ultrastructural changes of embryonic cells.     

Comparison of prophylactic or therapeutic dietary administration of capsaicin for reduction of Salmonella in broiler chickens

In three experiments the effects of prophylactic or therapeutic dietary inclusion of capsaicin, the pungent component of peppers, were evaluated as a nonantibiotic alternative for reduction of Salmonella in broiler chickens through culture and morphologic assessment of cecal tissue. Expt. 1 evaluated the effects of 0 or 10 ppm purified capsaicin (CAP) in the starter phase (days 1-16) on chicks challenged with Salmonella Enteritidis (SE) on day of age. Therapeutic inclusion of 10 ppm purified CAP increased (P < 0.05) liver/spleen (L/S) and ceca positive results for SE. In Expt. 2, capsaicin oleoresin (CO) was included in the finisher diet (days 30-37) at 0, 5, or 20 ppm with SE challenge on day 31. Inclusion of 5 ppm CO increased ceca positive results for SE, and a linear decrease in cecal lamina propria thickness of SE-challenged birds was observed with increased CO concentration in the diet. Expt. 3 evaluated prophylactic CO treatment at 0, 5, or 20 ppm in starter, grower, and finisher diets for resistance to SE or Salmonella Typhimurium (ST) challenge on day 14 or 29. With challenge on day 14, 5 and 20 ppm prophylactic CO feeding reduced ceca SE positive results by 37% and 26%, respectively, and ST culture rate was reduced similarly with 5 ppm CO. Lamina propria thickness of the ceca increased with 5 ppm CO feeding in SE-challenged birds, whereas a decrease was observed in nonchallenged birds fed 5 ppm CO. Challenge on day 29 of birds fed 20 ppm CO resulted in reduced L/S positive results for SE. Lamina propria thickness decreased with 5 ppm CO and SE or ST challenge compared with nonchallenged birds fed 5 ppm. An increase was observed in ST- or SE-challenged birds fed 20 ppm CO compared with nonchallenged birds fed 20 ppm CO. No differences were observed in mast cell number in either Expt. 2 or 3. These data provide evidence that prophylactic or therapeutic dietary capsaisin differentially affects broiler susceptibility to Salmonella.     

Naloxone increases maturation rate and ratio of inner cell mass to total cells in blastocysts in pigs

The purposes of the present study were to examine the effect of naloxone, a mu‐opioid receptor (MOR) antagonist, on porcine oocyte maturation and embryo development. MOR gene was expressed in germinal vesicle (GV) and metaphase II (M‐II) porcine oocytes, one‐, four‐cell stage embryos and blastocysts. In blastocysts, MOR gene was mainly expressed in inner cell mass (ICM) cells. Supplementation of 10^?8 mol/L naloxone in in vitro maturation (IVM) medium increased the maturation rate (P < 0.05). However, 10^?4 mol/L naloxone reduced the maturation rate (P < 0.05) compared with the control. The presence of naloxone during IVM had no effects on fertilization status and subsequent embryonic development after in vitro culture (IVC). The addition of 10^?3 mol/L dibutyryl cyclic adenosine monophosphate (dbcAMP), and 10^?8 mol/L naloxone together into IVM medium increased nuclear maturation (P < 0.05) compared with the addition of either dbcAMP or naloxone alone. Supplementation with naloxone in IVC medium did not improve embryonic development. However, at the concentrations of 10^?6 mol/L and 10^?8 mol/L, naloxone increased the ratio of ICM to total cells in blastocysts (P < 0.05). In conclusion, at low concentration, naloxone increases maturation rate and the ratio of ICM to total cells in blastocysts. Naloxone and cAMP have a synergistic effect on oocyte maturation.     

Stress exposure during the preimplantation period affects blastocyst lineages and offspring development

We found retardation of preimplantation embryo growth after exposure to maternal restraint stress during the preimplantation period in our previous study. In the present study, we evaluated the impact of preimplantation maternal restraint stress on the distribution of inner cell mass (ICM) and trophectoderm (TE) cells in mouse blastocysts, and its possible effect on physiological development of offspring. We exposed spontaneously ovulating female mice to restraint stress for 30 min three times a day during the preimplantation period, and this treatment caused a significant increase in blood serum corticosterone concentration. Microscopic evaluation of embryos showed that restraint stress significantly decreased cell counts per blastocyst. Comparing the effect of restraint stress on the two blastocyst cell lineages, we found that the reduction in TE cells was more substantial than the reduction in ICM cells, which resulted in an increased ICM/TE ratio in blastocysts isolated from stressed dams compared with controls. Restraint stress reduced the number of implantation sites in uteri, significantly delayed eye opening in delivered mice, and altered their behavior in terms of two parameters (scratching on the base of an open field test apparatus, time spent in central zone) as well. Moreover, prenatally stressed offspring had significantly lower body weights and in 5-week old females delivered from stressed dams, fat deposits were significantly lower. Our results indicate that exposure to stress during very early pregnancy can have a negative impact on embryonic development with consequences reaching into postnatal life.     

Buffalo (Bubalus bubalis) Embryonic Stem Cell‐Like Cells and Preimplantation Embryos Exhibit Comparable Expression of Pluripotency‐Related Antigens

In this study, inner cell mass (ICM) cells were isolated from in vitro produced buffalo blastocysts and were cultured on mitomycin‐C treated buffalo foetal fibroblast feeder layer for producing embryonic stem (ES) cells. Among different sources (hatched vs expanded blastocysts) or methods (enzymatic vs mechanical), mechanical isolation of ICM from hatched blastocysts resulted in the highest primary colony formation rate and the maximum passage number up to which ES cells survived. Putative ES cells expressed alkaline phosphatase and exhibited a normal karyotype up to passage 7. Putative ES cells and embryos at 2‐ to 4‐cell, 8‐ to 16‐cell, morula and blastocyst stages strongly expressed stage‐specific embryonic antigen (SSEA)‐4 but lacked expressions of SSEA‐1 and SSEA‐3. Putative ES cells also expressed tumour rejection antigen (TRA)‐1‐60, TRA‐1‐81 and Oct4. Whereas in all early embryonic stages, TRA‐1‐60 was observed only in the periplasmic space, and TRA‐1‐81 expression was observed as small spots at a few places inside the embryos, both these markers were expressed by ICM. Oct4 expression, which was observed at all the embryonic stages and also in the trophectoderm, was the strongest in the ICM. Buffalo putative ES cells possess a unique pluripotency‐related surface antigen phenotype, which resembles that of the ICM.