Effects of steroidal glycoalkaloids from potatoes (Solanum tuberosum) on in vitro bovine embryo development

alpha-Solanine and alpha-chaconine are two naturally occurring steroidal glycoalkaloids in potatoes (Solanum tuberosum), and solanidine-N-oxide is a corresponding steroidal aglycone. The objective of this research was to screen potential cyto-toxicity of these potato glycoalkaloids using bovine oocyte maturation, in vitro fertilization techniques and subsequent embryonic development as the in vitro model. A randomized complete block design with four in vitro oocyte maturation (IVM) treatments (Experiment 1) and four in vitro embryo culture (IVC) treatments (Experiment 2) was used. In Experiment 1, bovine oocytes (n=2506) were matured in vitro in medium supplemented with 6 microM of alpha-solanine, alpha-chaconine, solanidine-N-oxide or IVM medium only. The in vitro matured oocytes were then subject to routine IVF and IVC procedures. Results indicated that exposure of bovine oocytes to the steroidal glycoalkaloids during in vitro maturation inhibited subsequent pre-implantation embryo development. Potency of the embryo-toxicity varied between these steroidal glycoalkaloids. In Experiment 2, IVM/IVF derived bovine embryos (n=2370) were cultured in vitro in medium supplemented with 6 microM of alpha-solanine, alpha-chaconine, solanidine-N-oxide or IVC medium only. The results showed that the pre-implantation embryo development is inhibited by exposure to these glycoalkaloids. This effect is significant during the later pre-implantation embryo development period as indicated by fewer numbers of expanded and hatched blastocysts produced in the media containing these alkaloids. Therefore, we conclude that in vitro exposure of oocytes and fertilized ova to the steroidal glycoalkaloids from potatoes inhibits pre-implantation embryo development. Furthermore, we suggest that ingestion of Solanum species containing toxic amounts of glycoalkaloids may have negative effects on pre-implantation embryonic survival.     

Analysis of post-implantation mouse embryos after maternal heat stress during meiotic maturation

The effects of heat stress during oocyte maturation were studied in post-implantation mouse embryos. Virgin ICR mice were exposed to 35 +/- 1 degree C and 65 +/- 3% RH for 12.5 h beginning immediately after synchronization of ovulation with PMSG and hCG. Embryos of heat-stressed dams were developmentally heterogeneous and showed significant delays in development with as much as 48 h delayed development. Nearly 6% of these embryos were triploid, and another 2% were hyper-diploid. Development of triploid embryos was delayed more than 24 h. Nine embryos with severe developmental delay had heterogeneous chromosome constitutions. Embryo mortality before and after implantation was higher in heat-stressed dams than in controls.     

Susceptibility of bovine germinal vesicle-stage oocytes from antral follicles to direct effects of heat stress in vitro

Delineation of maternal versus direct effects of heat stress in reducing development at the germinal vesicle (GV) stage is challenging, because oocytes spontaneously resume meiosis after removal from antral follicles. The use of S-roscovitine (inhibitor of p34(cdc2)/cyclin B kinase) to hold bovine oocytes at the GV stage without compromising early embryo development was previously validated in our laboratory. The objective of the present study was to assess the direct effects of an elevated temperature commonly seen in heat-stressed dairy cows on cumulus-oocyte complexes (COCs) held at the GV stage using 50 microM S-roscovitine. During roscovitine culture, GV-stage COCs (antral follicle diameter, 3-8 mm) were cultured at 38.5 or 41 degrees C. Thereafter, oocytes were removed from roscovitine medium and allowed to undergo in vitro maturation, fertilization, and culture. Zona pellucida hardening (solubility to 0.5% pronase), nuclear stage (Hoechst 33342), cortical granule type (lens culinaris agglutinin-fluorescein isothiocyanate [FITC]), and early embryo development were evaluated. Culture of GV-stage COCs at 41 degrees C increased the proportion that had type III cortical granules and reduced the proportion that progressed to metaphase II after in vitro maturation. Effects of 41 degrees C on zona pellucida hardening, fertilization (penetration, sperm per oocyte, pronuclear formation, and monospermic and putative embryos), and cleavage of putative zygotes were not noted. However, culture of GV-stage COCs at 41 degrees C for 6 h decreased the proportion of 8- to 16-cell embryos, whereas 41 degrees C for 12 h reduced blastocyst development. In summary, antral follicle COCs are susceptible to direct effects of elevated body temperature, which may account in part for reduced fertility in heat-stressed cows.     

Heat stress on oocyte or zygote compromises embryo development,impairs interferon tau production and increases reactive oxygen species and oxidative stress in bovine embryos produced in vitro

Interferon tau (IFNT) is the cytokine responsible for the maternal recognition of pregnancy in ruminants and plays a role modulating embryo–maternal communication in the oviduct inducing a local response from immune cells. We aimed to investigate IFNT production, reactive oxygen species, and oxidative stress under the influence of heat stress (HS) during different stages of bovine in vitro embryo production. HS was established when the temperature was gradually raised from 38.5°C to 40.5°C in laboratory incubator, sustained for 6 hr, and decreased back to 38.5°C. To address the HS effects on IFNT production, reactive oxygen species, and oxidative stress, ovaries from a slaughterhouse were used according to treatments: control group (38.5°C); oocytes matured under HS; oocytes fertilized under HS; zygotes cultured in the first day under HS; and cells submitted to HS at oocyte maturation, fertilization, and the first day of zygote culture. The HS negatively affected cleavage and blastocyst rates, in all HS groups. On Day 7, all HS‐treated embryos showed decrease IFNT gene and protein expressions, whereas reactive oxygen species were increased in comparison to the control. In conclusion, the compromised early embryo development due to higher temperatures during in vitro oocyte maturation, fertilization, and/or zygote stage have diminished IFNT expression and increased reactive oxygen species in bovine.     

Expression of Bcl-2 and Bax proteins in relation to quality of bovine oocytes and embryos produced in vitro

The mechanisms underlying the visual assessment and selection of immature oocytes resulting in optimum embryonic development following in vitro maturation, fertilization and culture (in vitro maturation (IVM)/in vitro fertilization (IVF)/in vitro embryo culture (IVC)) are unknown. Also, the reasons for the more frequent occurrence of cytoplasmic fragmentation in in vitro produced bovine embryos, resulting in poor survival following cryopreservation and decreased pregnancy rates following embryo transfer are not clear. The objectives of this study are: (1) to investigate whether differences in the quality of immature oocytes and embryo fragmentation are associated with apoptosis; and (2) to study the pattern of Bcl-2 and Bax expression in oocytes and embryos to help elucidate their potential roles in the regulation of apoptosis during development. Bovine oocytes were obtained from slaughterhouse ovaries and divided into four grades (grades I–IV) based on their morphology. Oocytes of different grades were cultured in serum-free medium for 48 h. Embryos were produced only from grade I oocytes (highest quality) via IVM, IVF and IVC procedures. The morphological analysis of apoptosis in oocytes and embryos was carried out using propidium iodide staining and terminal deoxynucleotidyl transferase mediated dUTP nick end labeling. The expression of Bcl-2 and Bax in oocytes and embryos of different qualities and stages was determined using western blotting. The results showed that the number of morphologically abnormal oocytes with shrinkage and/or fragmentation of the ooplasm, which are typical features of apoptosis, was significantly higher in grade IV oocytes (denuded oocytes, the lowest quality) than in grade I oocytes after 48 h in vitro culture (P<0.05). DNA fragmentation, a hallmark of the biochemical changes seen in apoptotic cell death, was observed in morphologically fragmented oocytes and embryos. The expression of Bcl-2 was high in good quality oocytes and embryos, low in fragmented embryos, and hardly detectable in denuded oocytes. In contrast, the expression of Bax was found in all types of oocytes and embryos with the highest expression in the denuded oocytes. This implies that the ratio of Bcl-2 to Bax may be used to gauge the tendency of oocytes and embryos towards either survival or apoptosis. Overall, our results show that apoptosis appears to be an underlying mechanism of bovine oocyte degeneration and embryo fragmentation. Interactions between the Bcl-2 family of proteins may play a critical role in pre-implantation embryo development. These findings could have important implications for improving IVF and related techniques.     

Redox status of the oviduct and CDC2 activity in 2-cell stage embryos in heat-stressed mice

Mammalian preimplantation embryos are vulnerable to heat stress. However, the mechanisms by which maternal heat stress compromises embryonic development are unclear. We hypothesized that the loss of developmental competence in maternally heat-stressed embryos results from enhanced oxidative stress in the oviducts. In experiment 1, oviducts and zygotes were collected from mice that were heat-stressed at 35 degrees C and 60% relative humidity for 12 h on the day of pregnancy as well as from control mice. The zygotes were cultured for 84 h to assess their development, and the H(2)O(2) level, glutathione concentration, and free radical scavenging activity (FRSA) were measured in the oviduct. In experiment 2, zygotes were cultured for 22 h to reach the late G(2) phase in the 2-cell stage, and Cdc2 activity was assessed using immunoblotting. A high percentage (87.6%) of control embryos developed to morulae or blastocysts, whereas the majority (67.4%) of the heat-stressed group arrested at the 2-cell stage. Although heat stress did not alter the FRSA or glutathione concentration in the oviducts, the H(2)O(2) level (P < 0.01) and its ratio to the FRSA (P < 0.05) significantly increased in the heat-stressed group. The Cdc2 activation at the 2-cell stage, as shown by the ratio of the dephosphorylated form to the phosphorylated form, was evident in control embryos but absent in heat-stressed embryos, and the level was similar to that in embryos blocked at the 2-cell stage (positive control). These results indicate that maternal heat stress enhances oxidative stress in the oviducts and that loss of developmental competence in maternally heat-stressed embryos correlates with a defect in Cdc2 activity at the 2-cell stage.     

In Vitro maturation and fertilization of domestic cat follicular oocytes

The time course and conditions necessary for oocyte maturation and subsequent fertilization in vitro were studied in the domestic cat. Darkly pigmented oocytes surrounded by cumulus cells and a tight corona radiata were collected from ovaries removed at ovariohysterectomy. After culture in Eagle's minimum essential medium, oocytes were evaluated for nuclear maturation by analyzing chromosomal spreads. Oocytes achieved metaphase II after intervals of 40–48 hr of in vitro incubation. The incidence of maturation was enhanced (P<0.05) when oocytes were recovered from inactive (54%) or follicular (56%) stage donors compared to those recovered from luteal phase (29%) or pregnant (35%) cats. The proportion of oocytes successfully maturing in vitro in medium containing no hormone supplementation (37%) was less (P<0.01) than counterparts cultured in follicle-stimulating hormone (FSH) only (48%) or FSH and luteinizing hormone (LH) (54%). The efficiency of maturation was not influenced (P >0.05) by either maintenance/transport temperature (4°C vs. 22°C) or delaying recovery of oocytes from antral follicles (2–8 hr vs. 24–32 hr). Approximately 36% of the in vitro matured oocytes cocultured with spermatozoa demonstrated evidence of fertilization; however, there appeared to be a critical development period for maximizing the incidence of fertilization. These results demonstrate that domestic cat antral oocytes are capable of maturing in vitro, and maturation is influenced by the reproductive status of the donor and the presence of gonadotropins in the culture medium. These oocytes are capable of forming embryos and developing to at least the 16-cell stage in vitro.     

继代核移植能成倍提高来自奶山羊卵胞质体的异种间核移植重构胚的发育率

为了提高异种间核移植重构胚的发育率,本研究以体内排放的奶山羊成熟卵为供胞质的受体细胞,以人、兔、波尔山羊等的异种或亚种体细胞的原代核移植（Primary Somatic Cell Nuclear Transfer,PSCNT）重构早胚（8-16细胞期）的卵裂球作供核体,观察经亚种或异种卵胞质体短期“修饰”的核再移植产生的继代（Secondary SCNT,SSCNT）重构胚的着床前发育潜能。结果：人、兔、波尔山羊的继代桑椹／囊胚发育率均显著地高于其PSCNT胚胎（人,14．81％VS．7．79％;兔,23．53％VS．12．50％;波尔羊,55．35％VS．24．53％）;这些早胚的各阶段发育时程仍遵循供核体动物正常受精卵的发育时程。结果启示：奶山羊成熟卵胞质对异种体细胞核亦具一定的去分化能力,能支持重构胚发育到囊胚;异种重构胚的发育特征是由供体核所决定的;继代核移植几乎能够成倍提高异种间重构胚的着床前发育率,提示核的去分化完全是在母型信息主导的调控之下完成的,而进一步发育的时序似乎是由核决定的：成倍延长在含母型信息主导调控环境中的时间能成倍提高SCNT重构胚的着床前发育率。     

Embryology in the era of proteomics

Currently, relatively little is known regarding the protein production of mammalian embryos. Unlike the genome, the proteome itself is dynamic reflecting both internal and external environmental stimuli. Until now the lack of sensitivity has remained a stumbling block for the global introduction of proteomics into the field of mammalian embryology. However, new developments in mass spectrometry have been revolutionary, utilizing protein profiling and peptide sequencing to elucidate underlying biological processes. The sensitivity of these platforms have allowed for the development of new protocols that are capable of profiling the proteome of individual mammalian oocytes and embryos. This information is fundamental to unravelling the complexity of embryo physiology including the dialogue between the developing embryo and its maternal environment. Such proteomic approaches are also assisting in the optimization of ART techniques, including oocyte cryopreservation and in vitro maturation. Embryo selection for transfer is another area of ART that should benefit in this era of proteomics. Currently, mammalian embryos are selected for transfer based on morphological grading systems. Although of great value, analysis of morphology alone cannot determine the embryo's physiological state or chromosomal complement. Subsequently, there is a need to identify in culture those embryos with the highest implantation potential. Proteomic analysis of the embryonic secretome (proteins produced by the embryo and secreted into the surrounding medium) followed by the identification of specific proteins critical for implantation, may lead to the development of a non-invasive viability assay to assist in the selection of embryos for transfer.     

Role of PTEN in mouse pre-implantation development

The PI3K/Akt signal transduction pathway plays an important role in pre-implantation embryonic development. The tumor suppressor gene PTEN negatively regulates the PI3K/Akt pathway. Although PI3K is constitutively activated during pre-implantation embryonic development, currently no evidence shows the presence and possible involvement of PTEN in early embryo development. We investigated the expression of PTEN protein in germinal vesicle (GV) stage oocytes as well as in pre-implantation embryos. The activated form of PTEN was distributed in the peripheral of GV oocytes and compact morula. Treatment of GV oocytes with PTEN inhibitor bpV(pic) did not affect the maturation of the oocyte, but significantly impaired embryonic development. Thus, our study suggests the necessary role of PTEN in pre-implantation embryonic development.     

Ghrelin在绵羊体内卵母细胞和早期胚胎的表达

为了明确ghrelin是否参与了卵母细胞成熟及胚胎早期发育进程,本研究利用免疫荧光技术和实时定量RT-PCR技术检测了绵羊卵母细胞和体内早期胚胎中ghrelin蛋白的表达定位和ghrelin mRNA水平相对表达变化规律。免疫荧光染色结果表明,ghrelin蛋白主要分布于卵母细胞胞质内;实时定量RT-PCR结果揭示绵羊卵母细胞和早期胚胎ghrelin mRNA的相对表达量依据发育阶段的不同而呈现一定变化规律,即在成熟卵母细胞,2细胞胚胎期和8细胞胚胎期显著高于未成熟卵母细胞和4细胞胚胎期(P<0.05),囊胚期表达量最高。卵母细胞和早期胚胎中ghrelin蛋白的表达及ghrelin mRNA特定的表达模式,揭示这一新型分子在绵羊卵母细胞成熟以及胚胎早期发育过程中具有潜在的调控作用。     

Leukaemia inhibitory factor and the regulation of pre-implantation development of the mammalian embryo

This paper reviews the evidence that certain growth factors, particularily leukaemia inhibitory factor (LIF), play a crucial role in regulating the development of the pre-implantation mammalian embryo. LIF was originally implicated in regulating the early development of the mouse embryo because it inhibited the differentiation of embryonic stem (ES) cells, pluripotential cells derived from the inner cell mass of the blastocyst. Subsequent studies on its role in vivo revealed, surprisingly, that it is essential for the growth rather than the differentiation of the blastocyst. In vivo, overtly normal blastocysts can be produced in a LIF-deficient environment that are capable of forming viable fertile adults. However, in the absence of LIF, they fail to implant and enter into a state resembling that exhibited by blastocysts undergoing delayed implantation, which is characterized by a cessation of cell proliferation. This failure to implant occurs because the principle sites of LIF production are the endometrial glands of the uterus. These synthesize and secrete LIF at implantation, with LIF synthesis essential for implantation. Preliminary evidence indicates that LIF synthesis is required both by the uterus for it to undergo decidualization and by the blastocyst for implantation. These data indicate that the maternal environment plays a crucial role in the development and growth of the pre-implantation embryo, by supplying factors that regulate these processes in the embryo. © 1994 Wiley-Liss, Inc.     

The characterization of bovine embryos obtained from prepubertal calf oocytes and their viability after non surgical embryo transfer

We have previously shown that the addition of epidermal growth factor (EGF) during in vitro maturation was capable of stimulating the cytoplasmic maturation of cow and calf oocytes. The aim of the present study was to compare calf and cow blastocysts produced in the presence of EGF in terms of total cell number and cell distribution between trophectoderm (TE) and inner cell mass (ICM), pattern of protein synthesis, and ability to establish pregnancy after embryo transfer to recipients. For all experiment, embryos at Day 7 were obtained from IVM/IVF/IVC oocytes. No significant differences were noted in total cell number (cow= 138±46 vs CALF= 142±59; mean±SD) or ICM and TE cell number between calf (ICM= 35 ± 19, TE= 107± 52) and cow (ICM= 38± 21, TE= 99 ± 32) blastocysts, nor in the ICM/total cell number ratio (cow= 0.27± 11, CALF= 0.25 ± 12). No differences were noted in the constitutive and the neosynthetic protein profiles between cow and calf embryos obtained in vitro. The results of embryo transfer, showed that there was higher pregnancy loss following transfer of calf compared with cow embryos. After Day 35, the rate of pregnancy decreases, with only 22% of calf embryos maintaining pregnancy until calving compared with 39% for cow embryos. In conclusion, it would seem that embryos originating from calf oocytes are less capable of establishing pregnancies than embryos obtained from adult oocytes, althrough this difference was not significant. This low viability cannot be explained by differences in cell number or by the protein profiles identifed between these 2 groups of embryos.     

Maternal hyperthermia disrupts developmental competence of follicle-enclosed oocytes: in vivo and ex vivo studies in mice

Mammalian oocytes are susceptible to thermal stress at various stages of follicular development. We examined whether the ovarian pool of oocytes is susceptible to maternal hyperthermia and if so, whether hyperthermia at the germinal vesicle (GV) stage further affects the developmental competence of preimplantation embryos and offspring quality. Synchronized female mice were exposed to thermal stress (40 degrees C, 65% RH) for 1.5-2h or maintained under normothermal conditions (25 degrees C, 45% RH). Thereafter, mice were paired with stud males. In the first experiment, mated mice were sacrificed 20h post hCG administration, and in vivo-derived zygotes were recovered and cultured in vitro. Maternal hyperthermia decreased the percentage of putative zygotes of apparent normal morphology in the heat-stressed group (81+/-1.3%) as compared to the control group (86+/-1.2%). Developmental competence was also compromised as expressed by the disruption in cleavage timing pattern, resulting in a reduced developmental rate to the blastocyst stage (57+/-2.6% versus 84+/-1.9%). In the second experiment, both groups were left with stud males until litter delivery. Litter size in the first delivery cycle was lower for the heat-stressed group (7.7+/-1.1 pups), followed by a slight increase throughout consecutive cycles as compared to the control group (11.3+/-1.0 pups). Behavioral examinations of 8-week-old pups revealed similar locomotor activity and learning potential between the groups. In summary, the findings indicate that a subpopulation of the ovarian pool of follicles is highly sensitive to thermal stress and that maternal hyperthermia disrupts developmental competence of GV-stage oocytes. Pups that developed from oocytes that survived thermal stress exhibited a developmental potential similar to that of the of control pups.     

Effect of the addition of insulin-transferrin-selenium and/or L-ascorbic acid to the in vitro maturation of prepubertal bovine oocytes on cytoplasmic maturation and embryo development

This study examines the effects of adding insulin-transferrin-selenium (ITS) and/or L-ascorbic acid (ASC) to a conventional medium for maturing prepubertal calf oocytes on chromosome organization, cortical granule (CG) distribution, and embryo development to the blastocyst stage. Cumulus-oocyte complexes (COCs) were matured in medium TCM 199 containing PVA and EGF (control), and supplemented with ITS and/or ASC for 12 or 24 h at 38.5 °C in a 5% CO₂ atmosphere. Calf oocytes matured with ITS + ASC or ASC for 12 h showed significantly higher percentages of peripherally distributed CG (83.3% and 86.2% respectively) than control oocytes (71.4%) or those matured with ITS alone (71.4%). No effects on chromosome organization were detected. Conversely, 24 h of supplementation did not affect CG distribution patterns, while the addition of ASC gave rise to significantly higher percentages of oocytes showing a normal alignment of their chromosomes (72.9%) compared to controls (58.7%). At 48 hpi, similar cleavage rates were observed among treatments regardless of the treatment time. However, the presence of ITS + ASC for 12 h rendered significantly higher blastocyst rates than those recorded in the remaining groups. Supplementation for 24 h with ITS or ITS + ASC had no significant effects on the percentage of blastocysts obtained, while the presence of ASC significantly reduced the proportions of embryos developing to the blastocyst stage. Our data suggest that ITS plus L-ascorbic acid supplementation during the first 12 h of in vitro maturation improves cytoplasm maturation and the developmental competence of embryos produced from prepubertal calf oocytes.     

Effect of prematuration, meiosis activating sterol and enriched maturation medium on the nuclear maturation and competence to development of calf oocytes

New strategies were proposed to improve the developmental competence of calf oocytes through in vitro technologies. Cumulus-oocyte complexes were first prematured for 24 h in the presence of meiosis inhibitors. Both Roscovitine alone (50 microM) or in combination with Butyrolactone-I (12.5 microM Rosco+6.25 microM BL-I) prevented the progression of meiosis. Their effect on nuclear maturation was reversible after a further 17 or 24 h maturation step. However, a dramatic decrease in embryo development was observed after fertilization (abattoir oocytes: 4-9% blastocyst rate versus 14-17% for control embryos). Similar results were obtained with oocytes collected by Ovum Pick Up from living donors. No pregnancy was obtained after single transfer of two blastocysts obtained from prematured oocytes (0/2 versus 4/12 for control embryos). Adding low concentrations (1, 3 or 10 microM) of follicular fluid-meiosis activating sterol (FF-MAS) during the maturation step had a beneficial effect on nuclear maturation (73-86% metaphase II versus 58% for control oocytes). However, subsequent embryo development was not improved. Enriching the maturation medium, namely with hormones, growth factors and precursors of glutathione, induced a sixfold increase in glutathione in the oocyte and had a beneficial effect on embryo development (38% increase in blastocyst rate). In conclusion, in opposition to the results reported with adult oocytes, prematuring calf oocytes had a negative impact on their developmental potential. Although FF-MAS improved nuclear maturation, its addition in the maturation medium did not increase embryo development. However, enriching the maturation medium had a positive effect on embryo development, indicating that cytoplasmic maturation was improved.     

Culture media for mouse oocyte maturation affect subsequent embryonic development

These experiments were done to determine whether the culture medium used for the spontaneous maturation of mouse oocytes can affect the subsequent capacity of the ova to become fertilized and complete preimplantation development in vitro and development to live young. Oocytes obtained from antral follicles of gonadotropin-primed immature mice underwent spontaneous maturation in control medium, i.e. Eagle's Minimum Essential Medium (MEM) supplemented with 5% fetal bovine serum, or in one of eight different media which were also supplemented with serum. All of the ova were fertilized in Whitten's medium and were assessed for cleavage to the 2-cell stage and for further preimplantation development to blastocysts during culture in Whitten's medium. Three of the eight media used for oocyte maturation improved the capacity of the ova to develop to the blastocyst stage when compared with the control: Waymouth MB 752/1, MEM with non-essential amino acids, and MEM Alpha; Waymouth medium promoted the highest frequency of development of ova to the blastocyst stage. Moreover, the blastocysts derived from oocytes that matured in Waymouth medium contained more cells than blastocysts derived from oocytes that matured in control medium. Although BGJb medium promoted the cleavage of eggs to the 2-cell stage when present during oocyte maturation, it had a detrimental effect on their subsequent preimplantation developmental capacity. Following transfer to foster mothers, more 2-cell stage embryos developed to live young after oocyte maturation in Waymouth medium (21%) than in control medium (13%).(ABSTRACT TRUNCATED AT 250 WORDS)