Molecular origin of mitotic aneuploidies in preimplantation embryos

Mitotic errors are common in human preimplantation embryos. The occurrence of mitotic errors is highest during the first three cleavages after fertilization and as a result about three quarters of human preimplantation embryos show aneuploidies and are chromosomally mosaic at day three of development. The origin of these preimplantation mitotic aneuploidies and the molecular mechanisms involved are being discussed in this review.At later developmental stages the mitotic aneuploidy rate is lower. Mechanisms such as cell arrest, apoptosis, active correction of the aneuploidies and preferential allocation of the aneuploid cells to the extra-embryonic tissues could underlie this lower rate.Understanding the mechanisms that cause mitotic aneuploidies in human preimplantation embryos and the way human preimplantation embryos deal with these aneuploidies might lead to ways to limit the occurrence of aneuploidies, in order to ultimately increase the quality of embryos and with that the likelihood of a successful pregnancy in IVF/ICSI. This article is part of a Special Issue entitled: Molecular Genetics of Human Reproductive Failure.     

The Relationship between Cell Number,Division Behavior and Developmental Potential of Cleavage Stage Human Embryos: A Time-Lapse Study

Day 3 cleavage embryo transfer is routine in many assisted reproductive technology centers today. Embryos are usually selected according to cell number, cell symmetry and fragmentation for transfer. Many studies have showed the relationship between cell number and embryo developmental potential. However, there is limited understanding of embryo division behavior and their association with embryo cell number and developmental potential. A retrospective and observational study was conducted to investigate how different division behaviors affect cell number and developmental potential of day 3 embryos by time-lapse imaging. Based on cell number at day 3, the embryos (from 104 IVF/intracytoplasmic sperm injection (ICSI) treatment cycles, n = 799) were classified as follows: less than 5 cells (< 5C; n = 111); 5–6 cells (5–6C; n = 97); 7–8 cells (7–8C; n = 442), 9–10 cells (9–10C; n = 107) and more than 10 cells (>10C; n = 42). Division behavior, morphokinetic parameters and blastocyst formation rate were analyzed in 5 groups of day 3 embryos with different cell numbers. In <5C and 5–6C embryos, fragmentation (FR; 62.2% and 30.9%, respectively) was the main cause for low cell number. The majority of 7–8C embryos exhibited obvious normal behaviors (NB; 85.7%) during development. However, the incidence of DC in 9–10C and >10C embryos increased compared to 7–8C embryos (45.8%, 33.3% vs. 11.1%, respectively). In ≥5C embryos, FR and DC significantly reduced developmental potential, whereas <5C embryos showed little potential irrespective of division behaviors. In NB embryos, the blastocyst formation rate increased with cell number from 7.4% (<5C) to 89.3% (>10C). In NB embryos, the cell cycle elongation or shortening was the main cause for abnormally low or high cell number, respectively. After excluding embryos with abnormal division behaviors, the developmental potential, implantation rate and live birth rate of day 3 embryos increased with cell number.     

Postnatal alveolar development of the rabbit.

Previous studies of alveolarization have used rats or lambs; however, neither closely reflects human alveolar development. We characterized alveolar development in rabbits (n = 3-7 /group) at 28 days gestation (dg) to 9 mo to determine whether they followed the human pattern more closely. The right lung was made up of 30% alveolar and 50% duct space at 28 dg to 3 days and of 50 and 30%, respectively, at 14 days to 9 mo. Tissue fraction and alveolar wall thickness decreased by 40% 28 dg to birth. At birth, approximately 4.5% of the number of alveoli seen at 9 mo were present, with alveolar number increasing progressively well into adulthood. The rate of alveolar formation was high around birth, decreasing progressively with age. Alveolar volume increased more than twofold (28 dg to birth) and continued to increase postnatally to 16 wk. Surface fraction decreased by 17% (28 dg to 3 days), after which it remained uniform. Our findings suggest that the timing of onset of alveolarization in humans and rabbits is similar and that rabbits may be used to model postnatal influences on alveolar development.     

Reversal of postmortem degeneration of mouse oocytes during meiotic maturation in vitro

The developmental capacity of oocytes matured in vitro following isolation at the germinal vesicle stage from freshly killed mice (control) was compared with that of oocytes isolated from the carcasses of mice killed 3, 6, 9, and 12 hr earlier. The yield of intact, cumulus cell-enclosed oocytes decreased as the interval between death of the animal and removal of the ovary increased. After 15-16 hr of culture of medium containing follicle-stimulating hormone, the frequency of germinal vesicle breakdown, extrusion of a polar body, and cumulus expansion was equivalent in oocytes of all groups. The frequency of development of inseminated ova to 2-cell stage embryos in the control, 3, and 6 hr postmortem groups was the same but declined markedly in the 9 and 12 hr groups. There was also no difference in the frequency of blastocyst development from 2-cell stage embryos between the control, 3, 6, and 9 hr postmortem groups, but the 2-cell embryos in the 12 hr postmortem group did not develop to blastocysts. Thirty-six percent of the 2-cell stage embryos from the 6 hr postmortem group developed to live young after transfer to foster mothers. Follicles of 6 hr postmortem ovaries showed degeneration manifested as prominent crystalline inclusions within the oocytes and many pyknotic granulosa cells. The crystals disappeared within 1 hr of culture and the secondary oocytes appeared normal. The cultured oocyte-cumulus cell complexes, therefore, reversed degenerative changes induced by the death of the animal. This study demonstrates the feasibility of recovering developmentally competent oocytes from valuable recently deceased zoological, agricultural, and endangered mammals.     

Aberrant expression of miR-29a/29b and methylation level of mouse embryos after in vitro fertilization and vitrification at two-cell stage

Proper epigenetic modifications during preimplantation embryo development are important for a successful pregnancy. We aim to investigate the putative influence of in vitro fertilization (IVF) and vitrification on DNA methylation in mouse preimplantation embryos. The study groups consisted of blastocyst-derived vitrified two-cell embryos, nonvitrified embryos, and a control group of in vivo derived blastocysts. We assessed developmental competence, global DNA methylation, relative expression levels of miR-29a/29b, and their target genes, Dnmt3a/3b. Vitrified embryos had a lower developmental rate as compared with nonvitrified embryos. There was no significant decrease in blastocyst cell numbers among studied groups, whereas there was a steady decline in DNA methylation after IVF and vitrification. The levels of miR-29a/29b upregulated in the experimental groups as compared with the control group. IVF and vitrification caused Dnmt3a/3b downregulations in blastocysts. The results of this study have suggested that a relationship exists between IVF and embryo vitrification with methylation interruptions in the blastocysts.     

Localized electroporation: a method for targeting expression of genes in avian embryos

Avian embryos are a popular model for cell and developmental biologists. However, analysis of gene function in living embryos has been hampered by difficulties in targeting the expression of exogenous genes. We have developed a method for localized electroporation that overcomes some of the limitations of current techniques. We use a double-barreled suction electrode, backfilled with a solution containing a plasmid-encoding green fluorescent protein (GFP) and a neurophysiological stimulator to electroporate small populations of cells in living embryos. As many as 600 cells express GFP 24-48 h after electroporation. The number of cells that express GFP depends on the number of trains, the pulse frequency and the voltage. Surface epithelial cells and cells deep to the point of electroporation express GFP. No deformities result from electroporations, and neurons, neural crest, head mesenchyme, lens and otic placode cells have been transfected. This method overcomes some of the disadvantages of viral techniques, lipofection and in vivo electroporation. The method will be useful to biologists interested in tracing cell lineage or making genetic mosaic avian embryos.     

Overlapping and specific functions of Braf and Craf-1 proto-oncogenes during mouse embryogenesis

The three mammalian Raf serine/threonine protein kinases mediate the transduction of proliferative and differentiative signals from cell surface receptors to the nucleus. In vertebrates, Raf signaling has been implicated in the progression of mouse embryos through the two-cell stage and in the induction of posterior mesoderm. However, mouse embryos mutant for each of the Raf genes exhibit no developmental defects before mid-gestation. Here we describe the phenotype of mouse mutants with different combinations of mutant Craf-1 and Braf alleles. Our results show that Raf signaling is indeed indispensable for normal development beyond the blastocyst stage. However, due to a significant redundancy between Craf-1 and Braf, either gene is sufficient for normal development until mid-gestation. The molecular and developmental mechanisms for this redundancy were investigated by monitoring the expression of Raf genes throughout embryogenesis and by biochemical studies in mutant cell lines.     

Development and apoptosis of pre-implantation porcine nuclear transfer embryos activated with different combination of chemicals

Artificial activation of oocytes is a pre-requisite for successful cloning by nuclear transfer (NT). This study investigated effect of different combination of activation chemicals such as electric pulse (E), thimerosal (Thi) + dithiothreitol (DTT), 6-dimethylaminopurine (6-DMAP), or cycloheximide (CH) on the developmental ability and the frequency of apoptosis of porcine NT embryos during the culture in vitro. NT embryos activated with chemicals showed significantly higher developmental rate to blastocyst stage compared to embryos activated with E alone (21.5%-26.6% vs. 15.7%, respectively). Of chemicals, Thi + DTT supported higher development to blastocyst stage as compared to 6-DMAP or CH (26.6% vs. 21.5%-23.4%, respectively). Apoptosis of NT embryos were analyzed by using a terminal deoxynucleatidyl transferase-mediated deoxyuridine 5-triphosphate nick-end labeling (TUNEL) assay. The onset of apoptosis of embryos activated E alone was on Day 4, whereas embryos activated with chemicals showed apoptosis on Day 3 post-activation NT embryos exposed to chemicals for activation had higher frequency of apoptosis compared to that of embryos exposed to E alone from Day 3 to Day 7 during the culture. In conclusion, this study shows that chemical activation after fusion could increase not only the developmental ability of porcine NT embryos but also the mean cell number with an increased ratio of inner cell mass (ICM) to trophectoderm (TE) cells. However, the chemical activation also could increase the frequency of apoptosis and induced apoptosis earlier in porcine NT embryos.     

Growth and differentiation schedule of mouse embryos obtained from delayed matings.

We previously showed that digit formation in mouse embryos from early morning mating seemed to progress faster than those from overnight mating. In this study, to confirm this phenomenon, we examine whether the embryos from normal (0 hr from ovulation to fertilization) and delayed matings (3, 6, or 9 hr from ovulation to fertilization) respond differently to some acute teratogens when they are treated at the same time point from mating. Five mg/kg of cytosine arabinoside (Ara-C) was given to pregnant mice intraperitoneally at 246, 249, or 252 hr (day of gestation (dg) 10) after mating. The patterns of Ara-C induced digit malformations in embryos from the delayed mating groups were those of more advanced stages, when compared with normal mating groups with the same time intervals from mating to Ara-C treatment. In other words, oocytes fertilized up to 9 hr after the presumed time of ovulation could grow similarly to those of normally fertilized oocytes. This catch-up phenomenon suggests that the ovulation clock should be used for the startpoint of the time scale of the growth and differentiation of embryos rather than fertilization clock.     

Comparison of potency between histone deacetylase inhibitors trichostatin A and valproic acid on enhancing in vitro development of porcine somatic cell nuclear transfer embryos

Epigenetic modification influences reprogramming and subsequent development of somatic cell nuclear transfer (SCNT) embryos. Such modification includes an increase in histone acetylation. Histone deacetylase inhibitors (HDACi), such as trichostatin A (TSA) and valproic acid (VPA), have been known to maintain a high cellular level of histone acetylation. Hence, treatment of nuclear transfer embryos with HDACi may increase the efficiency of cloning. The present study attempted direct comparison of TSA and VPA with regard to the potency of enhancement of in vitro development in porcine SCNT embryos. Reconstructed oocytes using fetal fibroblasts were cultured in PZM-3 containing no HDACi (control), 5 mM VPA, or 50 nM TSA for 24 h, and another 5 d thereafter without HDACi. The frequency of blastocyst formation was significantly higher (P<0.05) in embryos treated with VPA than the frequencies with TSA and without HDACi (125/306, 40.8% vs. 94/313, 30.2% vs. 80/329, 23.4%). In addition, VPA treatment significantly increased (P<0.05) the number of inner cell mass (ICM) cells compared with the control (15.6 ± 1.7 vs. 10.8 ± 2.6), whereas no differences were observed between the TSA treatment and control groups (12.9 ± 3.0 vs. 10.8 ± 2.6). The present study demonstrates that VPA enhances in vitro development of porcine SCNT embryos, particularly by an increase in blastocyst formation and in the number of ICM cells, suggesting that VPA may be more potent than TSA in supporting developmental competence of cloned embryos.     

Frequency of micronuclei in 4–8 cell mouse embryos generated after maternal gamma-irradiation in the presence and in the absence of vitamin C

The objective of this investigation was to evaluate the frequency of chromosomal aberrations expressed as micronuclei (MN) in 4–8 cell embryos generated by gamma-irradiation of female mice in the absence and in the presence of vitamin C. Female NMRI mice were whole body exposed to 4 Gy gamma-irradiation after intraperitoneal (i.p.) injection of pregnant mare’s serum gonadotrophin (PMSG) followed by injection of human chorionic gonadotrophin (HCG) and mating with non-irradiated NMRI male mice. Pregnant animals were sacrificed and embryos flushed from the oviducts and fixed on slides. Cells were treated for MN observation using standard method. To investigate the protective effect of vitamin C (ascorbic acid) on the frequency of MN, 100 mg/kg vitamin C was i.p. injected 1 h before irradiation. Results show that the frequency of MN generated in the embryos of irradiated mother compared to those of control in the non-irradiated group increased dramatically (P < 0.001). Frequency of MN in embryos generated in irradiated female mice treated with vitamin C dramatically and statistically decreased relative to the frequency observed in the irradiation only group (P < 0.001). This decrease returned the combined treatment group to a level that was not statistically different from the controls (P > 0.05). Thus, irradiation of preovulatory stage oocytes leads to stable chromosome abnormalities expressed as micronuclei in successive preimplantation embryos. Vitamin C reduces these clastogenic effects of radiation in preovulatory oocytes and thus the reduced frequency of MN in embryos is probably due to its antioxidation and radical scavenging properties.     

Normal human pluripotent stem cell lines exhibit pervasive mosaic aneuploidy

Human pluripotent stem cell (hPSC) lines have been considered to be homogeneously euploid. Here we report that normal hPSC--including induced pluripotent--lines are karyotypic mosaics of euploid cells intermixed with many cells showing non-clonal aneuploidies as identified by chromosome counting, spectral karyotyping (SKY) and fluorescent in situ hybridization (FISH) of interphase/non-mitotic cells. This mosaic aneuploidy resembles that observed in progenitor cells of the developing brain and preimplantation embryos, suggesting that it is a normal, rather than pathological, feature of stem cell lines. The karyotypic heterogeneity generated by mosaic aneuploidy may contribute to the reported functional and phenotypic heterogeneity of hPSCs lines, as well as their therapeutic efficacy and safety following transplantation.     

Experimental Infection of Sheep at 45 and 60 Days of Gestation with Schmallenberg Virus Readily Led to Placental Colonization without Causing Congenital Malformations

Background

Main impact of Schmallenberg virus (SBV) on livestock consists in reproductive disorders, with teratogenic effects, abortions and stillbirths. SBV pathogenesis and viral placental crossing remain currently poorly understood. Therefore, we implemented an experimental infection of ewes, inoculated with SBV at 45 or 60 days of gestation (dg).

Methodology

“Mourerous” breed ewes were randomly separated in three groups: eight and nine ewes were subcutaneously inoculated with 1 ml of SBV infectious serum at 45 and 60 dg, respectively (G45 and G60). Six other ewes were inoculated subcutaneously with sterile phosphate buffer saline as control group. All SBV inoculated ewes showed RNAemia consistent with previously published studies, they seroconverted and no clinical sign was reported. Lambs were born at term via caesarian-section, and right after birth they were blood sampled and clinically examined. Then both lambs and ewes were euthanatized and necropsied.

Principal Findings/Significance

No lambs showed any malformation suggestive of SBV infection and none of them had RNAemia or anti-SBV antibodies prior to colostrum uptake. Positive SBV RNA detection in organs was rare in both G45 and G60 lambs (2/11 and 1/10, respectively). Nevertheless most of the lambs in G45 (9/11) and G60 (9/10) had at least one extraembryonic structure SBV positive by RTqPCR. The number of positive extraembryonic structures was significantly higher in G60 lambs. Time of inoculation (45 or 60 dg) had no impact on the placental colonization success rate but affected the frequency of detecting the virus in the offspring extraembryonic structures by the time of lambing. SBV readily colonized the placenta when ewes were infected at 45 or 60 dg but infection of the fetuses was limited and did not lead to congenital malformations.     

Evaluation of aneuploidy frequency for chromosomes 6 and 17 in eyelid tumours using the FISH technique

Although basal cell carcinoma (BCC) and squamous cell carcinoma (SCC) are very common skin tumours, the incidence of chromosome aneuploidy with regard to the eyelid has not been investigated. We aimed to find the frequency of chromosome 6 and 17 aneuploidies in eyelid tumours' interphase nuclei with fluorescence in situ hybridization (I-FISH) with chromosome specific DNA probes. I-FISH with chromosome 6 and 17 centromere specific DNA probes was used in the eyelids of 10 patients with BCC or SCC and the peripheral blood cells of 10 healthy donors as controls. The frequency of chromosome 6 and 17 aneuploidies was significantly higher in 7 out of 10 patients and 5 out of 10 patients, respectively, than in controls, indicating a higher frequency of aneuploidy in BCC than in SCC of the eyelid. Distribution of hybridization signals for chromosome 6 and 17 was wide ranging, indicating heterogeneity of cell populations with aneuploidy between patients. These findings indicate that acquisition of chromosome aneuploidies in eyelid tumours may have an important pathogenic role in both BCC and SCC of the eyelid area.     

Teratogenic effect of D-amphetamine sulphate: histodifferentiation and electrocardiogram pattern of mouse embryonic heart

Pregnant albino mice (ICR random-bred strain) received daily injections of 50 or 100 mg/kg body weight of D-amphetamine sulphate between days 9 and 11 of gestation. Parallel control animals were injected with saline solution. Treated mice were sacrificed on day 15 or 19 of gestation. The embryos were examined for gross malformations and direct embryonic ECG recordings were made; they were weighed, and their hearts were carefully dissected. Microscopic sections of the heart were stained with hematoxylin and eosin. It was found that high doses of D-amphetamine raised the incidence of mortality in the treated pregnant mice up to 40%. The resorption rate in the survivors was high (up to 58%) following the high dose of the drug. Up to 15% of the embryos from the treated groups showed gross malformations, including skeletal and eye malformations and exencephaly. The electrocardiogram (ECG) recordings in most 19-day-old embryos from both treated groups showed a pattern with prolonged Q-T interval (PQT), similar to that of control embryos in the intermediate developmental stages (days 14-16 of gestation). The ECG of control embryos (from day 17 on) resembled that of prenatal fetuses. Microscopically, the hearts of treated embryos showed a large number of undifferentiated cardiac myoblasts. It can be inferred that high doses of D-amphetamine affect embryonic development generally and delay the histodifferentiation of the myocardium, resulting in incomplete maturation of the cardiac muscles, thus leading to the immature ECG pattern, with PQT intervals.     

Excitatory and inhibitory neuron imbalance in the intrauterine growth restricted fetal guinea pig brain: Relevance to the developmental origins of schizophrenia and autism

Neurodevelopmental disorders such as schizophrenia and autism are thought to involve an imbalance of excitatory and inhibitory signaling in the brain. Intrauterine growth restriction (IUGR) is a risk factor for these disorders, with IUGR onset occurring during critical periods of neurodevelopment. The aim of this study was to determine the impact of IUGR on excitatory and inhibitory neurons of the fetal neocortex and hippocampus. Fetal brains (n = 2) were first collected from an unoperated pregnant guinea pig at mid-gestation (32 days of gestation [dg]; term ∼67 dg) to visualize excitatory (Ctip2) and inhibitory (calretinin [CR] and somatostatin [SST]) neurons via immunohistochemistry. Chronic placental insufficiency (CPI) was then induced via radial artery ablation at 30 dg in another cohort of pregnant guinea pigs (n = 8) to generate IUGR fetuses (52 dg; n = 8); control fetuses (52 dg; n = 7) were from sham surgeries with no radial artery ablation. At 32 dg, Ctip2- and CR-immunoreactive (IR) cells had populated the cerebral cortex, whereas SST-IR cells had not, suggesting these neurons were yet to complete migration. At 52 dg, in IUGR versus control fetuses, there was a reduction in SST-IR cell density in the cerebral cortex (p = .0175) and hilus of the dentate gyrus (p = .0035) but not the striatum (p > .05). There was no difference between groups in the density of Ctip2-IR (cortex) or CR-IR (cortex, hippocampus) neurons (p > 0.05). Thus, we propose that an imbalance in inhibitory (SST-IR) and excitatory (Ctip2-IR) neurons in the IUGR fetal guinea pig brain could lead to excitatory/inhibitory dysfunction commonly seen in neurodevelopmental disorders such as autism and schizophrenia.     

Birth of African Wildcat cloned kittens born from domestic cats

In the present study, we used the African Wildcat (Felis silvestris lybica) as a somatic cell donor to evaluate the in vivo developmental competence, after transfer into domestic cat recipients, of cloned embryos produced by the fusion of African Wildcat (AWC) fibroblast cell nuclei with domestic cat cytoplasts. Cloned embryos were produced by fusion of a single AWC somatic cell to in vivo or in vitro enucleated domestic cat cytoplasts. When the two sources of oocytes were compared, fusion rate was higher using in vivo-matured oocytes as recipient cytoplasts, but cleavage rate was higher after reconstruction of in vitro-matured oocytes. To determine the number of reconstructed embryos required per domestic cat recipient to consistently establish pregnancies, AWC cloned embryos were transferred within two groups: recipients (n = 24) receiving < or =25 embryos and recipients (n = 26) receiving > or =30 embryos. Twelve recipients (46.2%) receiving > or =30 embryos were diagnosed to be pregnant, while no pregnancies were established in recipients receiving < or =25 NT embryos. Also, to determine the influence of length of in vitro culture on pregnancy rate, we compared oviductal transfer on day 1 and uterine transfer on day 5, 6, or 7. Pregnancy rates were similar after transfer of embryos on day 1 (6/12; 50.0%), day 5 (4/9; 44.4%), or day 6 (2/5; 40.0%) to synchronous recipients, but the number of fetuses developing after transfer of embryos on day 1 (n = 17), versus day 5 (n = 4) or day 6 (n = 3) was significantly different. Of the 12 pregnant recipients, nine (75%) developed to term and fetal resorption or abortion occurred in the other three (25%) from day 30 to 48 of gestation. Of a total of 17 cloned kittens born, seven were stillborn, eight died within hours of delivery or up to 6 weeks of age, and two are alive and healthy. Perinatal mortality was due to lung immaturity at premature delivery, placental separation and bacterial septicemia. Subsequent DNA analysis of 12 cat-specific microsatellite loci confirmed that all 17 kittens were clones of the AWC donor male. These AWC kittens represent the first wild carnivores to be produced by nuclear transfer.     

Comparison of hybrid and purebred in vitro-derived cattle embryos during in vitro culture

Frozen-thawed spermatozoa collected from a beef bull (Japanese Black) were used for in vitro fertilization (IVF) of matured oocytes obtained from dairy (Holstein) and beef (Japanese Black) females. Embryos were examined for fertilization, cleavage rate, interval between insemination and blastocyst production (experiment I), total cell number per embryo and sex ratio during blastocyst formation (experiment II), and blastocyst production rate of zygotes that developed to 2-, 4-, and 8-cell stages at 48h post-fertilization (experiment III). Fertilized oocytes were cultured in vitro on a cumulus cell co-culture system. The fertilization and cleavage rate of oocytes groups were similar, however, the blastocyst production rate was greater (P<0.05) in hybrid than from purebred embryos (27% versus 20%). Development of blastocysts produced from hybrid embryos developed at a faster rate than blastocysts produced from the straightbred embryos. In hybrid embryos, blastocyst production was significantly greater on day 7 (56%) and gradually decreased from 20% on day 8 to 17% on day 9. In contrast, blastocyst production rate from the purebred embryos was lower on day 7 (17%), increasing on day 8 to 59% and then decreased on day 9 to 24%. The total number of cells per embryo and sex ratio of in vitro-produced blastocysts were not different between hybrid and purebred embryos. The number of blastocysts obtained from embryos at the 8-cell stage of development by 48h post-fertilization (94%) was greater (P<0.01) than the number of zygotes producing blastocysts that had developed to the 4-cell stage (4%) and the 2-cell stage (2%) during the same interval. These results show that the blastocyst production rate and developmental rate to the blastocyst stage were different between hybrid and purebred embryos, and that almost all of the in vitro-produced blastocysts were obtained from zygotes that had developed to the 8-cell stage 48h post-fertilization.     

Developmental retardation of XO mouse embryos at mid-gestation.

It is not known why XO mouse embryos, which develop more slowly than XX embryos until early mid-gestation, reach the same stage in their growth and development as their XX littermates at the mid-gestation stage. It is hypothesized that there is an effect of 'litter size' that causes an acceleration of the development of XO embryos at mid-gestation. The present study was performed to determine whether the development of XO embryos is retarded compared with that of their XX litermates at early mid-gestation (day 8 of gestation), before reduction of litter size. The percentage of pre-somite stage XO embryos was greater than the percentage of pre-somite stage XX embryos, and the mean number of somites was greater in XX embryos than it was in XO embryos. These findings indicate that the development of XO embryos was retarded when compared with that of their XX litermates at early mid-gestation. This result is discussed with respect to the compensatory development of XO embryos at mid-gestation and the reduction of litter size shortly after early mid-gestation.