A third of transcripts present in the early bovine embryos have sex-specific expression patterns, which might contribute to a sexual dimorphic development of the early embryo, such as faster development, higher total cell number and greater total glucose metabolism in male embryos. Expression of Y-linked genes, such as DDX3Y, EIF1AY, TSPY, USP9Y, ZFY, and ZRSR2Y, has been identified in bovine blastocysts (Hamilton et al. Theriogenology 8:1587. 2012). The ubiquitin specific peptidase 9, Y-Linked (USP9Y) gene is involved in spermatogenesis and shortening or deletions cause azoospermia or oligozoospermia, but its role in early embryo development is not known. This study examined the role of USP9Y in pre-implantation bovine embryo development by investigating the effects of USP9Y knock-down by siRNA injection on development to the blastocyst stage. In vitro embryos produced according to Ashkar et al. (Hum Reprod, 252: 334. 2010) were microinjected at the 1-cellstage at 16 h post-fertilization in 3 treatment groups: Non-injected (NI), scrambled siRNA injected (SI), and USP9Y knockdown siRNA injected (KD). USP9Y-specific siRNA was designed (Invitrogen custom primer software) assuring no homology to the X counterpart USP9X. After microinjection, lysed zygotes were removed and the remaining zygotes were cultured to the blastocyst stage. Cleavage and blastocyst rates were assessed at 48 and 168h post-fertilization, respectively. Relative transcript levels of USP9Y and the male specific gene DDX3Y were quantified in blastocysts by quantitative PCR (qPCR) with GAPDH and PPIA as reference genes. One-way ANOVA analysis showed that zygotes microinjected with either scrambled or USP9Y siRNA resulted in a significant increase in the number of lysed zygotes compared to non-injected controls (SI: 8.4% n = 538, KD: 28% n = 698 vs NI: 0% n = 487, respectively; P < 0.05). Scrambled and USP9Y siRNA microinjection significantly decreased cleavage rates (NI: 69.0% n = 487, SI: 42.8% n = 493 and KD: 43.9% n = 503; P < 0.05), while only USP9Y siRNA injection significantly decreased blastocyst rate (NI: 17.7%, SI: 12.9% and KD: 5.5%; P < 0.05) when compared to noninjected. qPCR relative quantification of DDX3Y to determine blastocyst sex in the three groups and showed only 27% of male blastocysts in KD group, compared to 44% in both NI and SI groups (P < 0.05). When the extent of the knock-down was assessed by qPCR, KD blastocysts showed approximately 25% less USP9Y expression compared to NI and SI groups. In conclusion, approximately 25% effective knock-down of USP9Y was reflected in significantly less male embryos reaching the blastocyst stage, allowing us to speculate that a baseline level of USP9Y might be necessary for development of males. Further experiments achieving a higher USP9Y knock-down efficiency will confirm our findings.

We described the clinical, cytogenetic and molecular findings of 17 clinical equine cases presented for abnormal sexual development and infertility. Six horses with an enlarged clitoris had an XX, SRY-negative genotype, which displayed male-like behavior (adult individuals). Bilateral ovotestes were noted in 2 of those cases, while another case showed increased levels of circulating testosterone. Six horses with a female phenotype, including normal external genitalia, had an XY, SRY-negative genotype. These individuals had small gonads and an underdeveloped internal reproductive tract. Four horses with normal appearing external genitalia had an XY, SRY-positive genotype, 3 of them had hypoplastic testes and male-like behavior. In addition, one young filly with enlarged clitoris and hypoplastic testes had the same genotype but did not show male-like behavior due to her age. Three of these horses were related with 2 being siblings. These findings demonstrate the diversity of disorders of sexual development seen in the horse. Furthermore, they emphasize the need for further research to identify genes involved in abnormal sex determination and differentiation in the horse.

Early pregnancy diagnosis and monitoring play an important role following embryo transfer in sheep. The aims of the current study were to investigate (i) the pattern of serum progesterone profiles in sheep carrying somatic cell nuclear transfer (SCNT)-derived (clone) pregnancies, and (ii) the frequency of pregnancy loss during development following SCNT embryo transfer. Sheep SCNT embryos were made using standard nuclear transfer techniques. Day 7 embryos were surgically transferred to oestrus-synchronized recipients (n = 27). As a control, normal fertile ewes (n = 12) were bred by natural breeding. Serum was collected from all the ewes on the day of estrus (day 0 sample), 7 days post-estrus (day 7 sample) and 19 days post-estrus (day 19 sample) and every 10 days thereafter until lambing or pregnancy loss occurred. Serum progesterone (P4) was assessed using enzyme immunoassay. Pregnancy was confirmed by ultrasound scanning on day 35 of pregnancy followed by subsequent scanning every 10 days. In control ewes, pregnancy rate on day 35 was 83.3% (10/12), whereas in the ewes that received SCNT embryos, it was 22.2% (6/27; p < 0.05). The day 45 pregnancy rate in the control ewes was 83.3%, whereas in the SCNT embryo recipients it was 11.0% (p < 0.05). Hormone analysis revealed that SCNT embryo recipients exhibited a significantly lower P4 profiles at different time points in pregnancy compared to controls (p < 0.05). This study highlights the use of serum progesterone in combination with ultrasound for the investigation of embryo loss and crucial times during development of normal and SCNT embryos in sheep. Further, the serum P4 levels directly reflect the degree of placental development in these two groups.

Wood bison (Bison bison athabascae) are currently classified as threatened in Canada. Interspecies somatic cell nuclear transfer (iSCNT) is a valuable tool for embryo production in non-domestic species in which access to gametes is limited. Unlike fertilization, SCNT allows preservation of the entire genome, thus avoiding dilution of valuable alleles, an important factor for the preservation of genetic diversity. The present study compared the developmental competence of iSCNT embryos reconstructed from adult female wood bison ear fibroblasts (bison NT) with development of embryos reconstructed from adult female cattle ear fibroblasts (cattle NT). Domestic cattle (Bos taurus) oocytes were used as recipient ooplasm for both donor cell types. In vitro fertilized (IVF) and parthenogenetic (PA) cattle embryos were used as controls. Fibroblast cultures at passages 3 to 5 confluent for 5 days were used for SCNT. Mature oocytes were enucleated, reconstructed by transfer of donor cells, and fused with an electrical stimulus of 1.5 kV cm–1 for 40 μs in 0.28 m mannitol containing 100 μm CaCl2 and MgCl2. Oocytes for parthenogenesis and following reconstruction were activated for 5 min in 5 μm ionomycin followed by 5 h in 10 μg mL–1 cycloheximide. Embryos produced by IVF, PA, and SCNT were cultured in modified synthetic oviductal fluid medium at 38.5°C in 5% CO2, 5% O2, 90% N2. Cleavage, blastocyst development to day 8, apoptosis (TUNEL assay, Roche Diagnostics, IN, USA), and total cell number were evaluated. Statistical analyses were carried out using one-way ANOVA, followed by Tukey post hoc analysis or the equivalent nonparametrical Kruskal-Wallis test. Cleavage rate was significantly (P < 0.05) higher in the IVF group than in all other groups (86.9 ± 2.9% v. 71.6 ± 4.5% to 78.1 ± 5.1%). Blastocyst rates, expressed as a percentage of cleaved embryos, were similar among all treatment groups (33.4 ± 3.3% to 39.8 ± 5.7%) except for bison NT which had significantly (P < 0.05) lower development to blastocyst (19.2 ± 5.5%). The percentages of TUNEL-positive cells among PA embryos (6.6 ± 1.5%) and bison NT embryos (6.7 ± 2.4%) were significantly (P < 0.05) higher than in IVF embryos (4.2 ± 1.0%), but similar to cattle NT embryos (5.4 ± 1.7%), which did not differ from the IVF group. Total cell number was significantly (P < 0.05) higher in the IVF group than in all other groups (133.2 ± 10.2 v. 91.2 ± 7.8 to 100.1 ± 12.9). These results confirm that in vitro-matured domestic cattle oocytes can serve as suitable recipients of wood bison somatic cells and that iSCNT may provide a possible alternative for embryo production and genetic preservation of endangered cattle species. Both the incidence of apoptotic cells and total cell number did not differ between cattle and bison NT embryos; thus other factors must play a role in the significantly decreased blastocyst development observed in bison NT embryos. This work was supported by Endangered Species Reserve Fund, Toronto Zoo, and the Canada Research Chairs program.

The effects of activation by 6-dimethylaminopurine (6-DMAP) and cycloheximicle (CHX) on the development and chromosomal complement of sheep parthenogenetic and SCNT embryos were investigated. The results revealed that the blastocyst development of parthenogenetic embryos was significantly higher (P < 0.05) in 6-DMAP activated oocytes, compared to those activated with CHX (21.0 +/- 0.9 vs. 14.9 +/- 0.5, respectively). In contrast, the blastocyst frequencies did not significantly differ (P > 0.05) between the two activation treatment groups for SCNT embryos. The 6-DMAP or CHX treatment did not result in any significant difference in the blastocyst total cell number in either parthenote or SCNT embryos. The chromosomal analysis revealed that all the parthenogenetic embryos (100.0%) derived from 6-DMAP treatment, were chromosomally abnormal whereas in CHX-treated embryos, it was significantly lowered (93.6%, P < 0.05). Conversely, the proportions of chromosomally abnormal SCNT embryos did not significantly differ (P > 0.05) among the 6-DMAP and CHX-treated embryo groups (60.0% vs. 56.2%, respectively). This study demonstrated that oocyte activation agents such as DMAP and CHX have differing effects on meiotic or mitotic nuclei. The study also highlighted the feasibility of using bovine X and Y chromosome specific painting probes in sheep embryos.

The early reprogramming events following somatic cell nuclear transfer (SCNT) determine the fate of the cloned embryo and its development to a healthy viable offspring. In the present study, we undertook a detailed immunocytochemical study of the patterns of both microtubules and chromatin during the first cell cycle of sheep nuclear transfer embryos after fusion and artificial activation using either 6-dimethylaminopurine (6-DMAP) or cycloheximede (CHX). Sheep oocytes were collected from abattoir ovaries and matured in vitro for 18-20 h and enucleated; fetal fibroblasts were transplanted using standard SCNT techniques. Reconstructed cell-cytoplast couplets were fused and activated with ionomycin, followed by culture in two separate groups containing 6-DMAP (2 mM) or CHX (10 g/mL) for 3 h. Following activation, embryos were cultured in in vitro culture (IVC) medium for blastocyst development. Embryos (n = 15, 3 replicates) were randomly removed from culture at various time points and stained using standard immunocytochemical methods to observe microtubule and nuclear configurations. Images were captured using laser scanning confocal microscopy. Results reveled that at 1 h post-fusion, 63.3% of reconstructed embryos underwent nuclear envelope breakdown (NEBD) and premature chromosome condensation (PCC) was apparent as chromosomes were situated on a non-polar spindle. The remaining embryos showed abnormal spindle and DNA configurations including chromosome outliers, congression failure, and non-NEBD. At 1 h post-activation (hpa), the embryos treated with 6-DMAP had already formed a clearly visible pronucleus (diameter 6-8 m), whereas in the CHX-treated group, none of the embryos were at pronuclear stage; instead most of the latter embryos showed two masses of chromatin. At 1 hpa, 6-DMAP- and CHX-treated embryos showed one swelled pronucleus with a mean diameter of 8.4 1.3 m and 25.8 0.8 m, respectively (P < 0.05). At 16 hpa, embryos from both treatment groups still showed one swelled pronucleus. In the 6-DMAP-treated embryos, most of the embryos showed a metaphase spindle with aligned chromosomes of the first mitotic division as early as 18-10 hpa, whereas in the CHX-treated group embryos were still at the pronuclear stage. Typical 2-cell division was seen in most of the 6-DMAP-treated embryos between 24 and 30 hpa, but it was slightly delayed in CHX-treated embryos (32-35 hpa). Blastocyst development rates in the 6-DMAP- and CHX-treated groups were 21.4 5.6% and 14.0 6.3%, respectively (P < 0.05). In summary, artificial activating agents 6-DMAP and CHX exhibited different effects on chromatin remodeling, cell cycle progression, and the degree of pronuclear swelling which may explain the poor developmental rates and abnormal chromosome complements observed for cloned embryos. This work was funded by NSERC, OMAF, and International Council for Canadian Studies.

High embryo loss occurs during the first week of in vitro bovine embryo development with a high percentage of embryo death and arrest. These early stages of development are regulated by stored maternal mRNAs that accumulate in the oocyte cytoplasm during its growth phase. In our in vitro production (IVP) system approximately 13.5% of embryos arrest at the 2–4-cell stage, displaying no characteristics of apoptosis. We hypothesized that these arrested embryos enter an oxidative stress-induced senescence-like state. We previously showed that elevated levels of reactive oxygen species and increased levels of the stress-adaptor protein p66shc were associated with this early embryonic arrest. The aim of this study was to selectively down-regulate p66shc mRNA levels in bovine oocytes, using post-transcriptional gene silencing by RNA interference (RNAi), to study the effects of p66shc mRNA “knock-down” on early arrest of IVP bovine embryos. Approximately 12,000 short hairpin (sh) RNAi molecules specific for p66shc were microinjected into bovine germinal vesicle (GV) oocytes. Experiments consisted of a control group undergoing IVF alone, and two groups microinjected with and without p66shc shRNAi molecules. Quantification of p66shc mRNA by real-time PCR was used to assure down-regulation of p66shc mRNA in 2-cell embryos collected at 35 hours post-insemination (hpi). The amount of p66shc mRNA detected in control IVF, vehicle-injected, and p66shc RNAi-injected groups was 0.92 ± 0.02 × 10-5 pg, 1.2 ± 0.20 × 10-5 pg, and 0.46 ± 0.06 × 10-5 pg, respectively. This statistically significant (P < 0.001) reduction in p66shc mRNA levels by 54% upon p66shc shRNAi microinjection was selective for p66shc mRNA, as both histone H2a and p53 mRNA levels were not altered. Percentage of 2–4-cell arrest was evaluated at Day 8 post-insemination and related to p66shc mRNA down-regulation. While there were no significant differences in the percentage of 2–4-cell arrested embryos between the control (13.3 ± 0.8%) and vehicle-microinjected (10.8 ± 0.7%) embryos, there was a significant decrease (P < 0.001) in the incidence of arrest in p66shc shRNAi-microinjected embryos (0.9 ± 0.9%). Quantification by real time PCR in blastocysts from the three groups showed no significant differences in p66shc mRNA levels (P = 0.314) among control IVF, vehicle, or p66shc hairpin RNAi microinjected at 0.59 ± 0.1 × 10-5 pg, 0.57 ± 0.2 × 10-5 pg, and 0.51 ± 0.2 × 10-5 pg, respectively. This suggests that p66shc down-regulation by RNAi might be temporary. Thus, using this novel approach of RNAi and microinjection of oocytes at the GV stage, we were able to selectively down-regulate the expression of p66shc mRNA and correlate this down-regulation with a significant decrease in 2–4 cell arrest. These data reinforce our hypothesis that p66shc is involved in a stress-induced pathway that executes a senescent-like embryonic arrest at the 2–4-cell stage, thereby postulating the possible role of p66shc as a new molecular marker for developmental competence. Statistics performed using ANOVA with Fisher LSD test for multiple comparisons. This work was funded by NSERC, CIHR, OGS, and OMAFRA.

High embryo loss occurs in the first week of bovine embryo development, with a high percentage of embryonic arrest. We hypothesized that arrested embryos enter a 'senescence-like state' and that both the cell cycle regulatory protein p53 and the stress-related protein p66(shc), which are involved in the onset of senescence in somatic cells, are responsible for this early embryonic arrest. In our in vitro production system, 13.5 +/- 0.5% of embryos arrest at the 2-4-cell stage. First cleavage occurs between 26 and 48 h post insemination (hpi), with early cleaving embryos showing only 0.6 +/- 0.3% arrest, with later cleaving embryos exhibiting up to 14.2 +/- 0.9% arrest. We compared 2-4-cell embryos collected at 28 hpi with those arrested at the 2-4-cell stage collected at day 8 post insemination. Quantification by real-time PCR and by semi-quantitative immunofluorescence showed significantly higher p66(shc) mRNA and protein levels in both arrested and late cleaving embryos versus 28 hpi embryos. By comparison, no significant changes in p53 mRNA, protein and phosphorylation levels were detected. Taken together, these results demonstrate that embryonic developmental potential is related to the time of first cleavage and that p66(shc), but not p53, is up-regulated in early arrested in vitro-produced bovine embryos.