Culture of human embryos for studies on the derivation of human pluripotent cells: a preliminary investigation.

Several different culture conditions were evaluated for culturing grade 4 embryos (containing 2-4 blastomeres and with >50% fragmentation) 68 h after fertilization to the blastocyst stage. Embryos were co-cultured with buffalo rat liver (BRL) cells in Menezo's B2 medium with or without 10% v/v synthetic serum substitute (SSS), co-cultured with BRL cells in KSOM with or without 10% SSS, or cultured in KSOM with 100 nM heparin binding epidermal growth factor. The most consistent development was obtained when embryos were co-cultured with BRL cells in KSOM. Rates of development to the blastocyst stage were between 27% and 40%. After reaching the blastocyst stage, continued culture of these blastocysts was only possible in a medium without serum. In a serum-deprived medium cells attached and showed initial outgrowth, but did not survive passaging. Using another approach, inner cell masses (ICMs), isolated from blastocysts with high efficiency using immunosurgery, were able to attach to a feeder layer in the presence of serum. Some ICMs differentiated whereas others could be successfully passaged up to four times. The embryonic cells were morphologically different from murine embryonic stem cells. Instead of well-defined colonies, the human colonies were characterized by individual cells and colonies without defined borders.

Cryopreservation of germ cells from bovine fetal ovaries.

Bovine fetuses at stages required for studies of female germ cells (primordial germ cells and oogonia) become available from the abattoir at unpredictable times. To alleviate this logistical problem, a procedure to cryopreserve these ovarian germ cells has been devised. Fetal ovarian cells were dispersed and suspended in 1.5 M dimethyl sulfoxide (DMSO) prepared in modified TCM 199 medium. The suspensions were aspirated into plastic semen straws, cooled, seeded to induce ice formation at -7 degrees C, and then cooled at 1 degree C min-1 to -70 degrees C before being plunged into liquid nitrogen at -196 degrees C for storage. The straws were thawed at a moderate rate of approximately 250 degrees C min-1, the DMSO was diluted 28-fold with culture medium, and then the cells were cultured for > 2 h before their viability was tested or they were used for nuclear transfer. No statistically significant difference in viability before and after cryopreservation was detected by vital staining with fluorescein diacetate (P > 0.05). When frozen-thawed germ cells were fused to cytoplasts, the cleavage rate of the resultant reconstructed embryos 44 h after fusion was 31%, although none developed into blastocysts. It is concluded that cryopreservation of bovine fetal ovarian germ cells is feasible and can play a major role in facilitating future experimentation.

Peripheral plasma prolactin and progesterone levels in pseudopregnant goats during bromocryptine treatment.

Persistence of luteal function and accumulation of fluid within the uterus (hydrometra) are characteristics of pseudopregnancy in goats. To study the luteotrophic role of prolactin in this condition, seven seudopregnant goats were treated with bromocryptine (1 mg subcutaneously, twice daily) for 6 to 10 d. Plasma progesterone (P4) and prolactin (PRL) were measured by radioimmunoassay (RIA) in samples taken twice daily by venipuncture. Ultrasound scanning took place at regular intervals to visualize the presence of fluid within the uterus. Bromocryptine treatment effectively reduced the plasma PRL concentration in six goats. In all seven goats, a gradual decrease of the plasma P4 concentration to levels < 1.8 ng/ml occured during treatment. After bromocryptine treatment, P4 concentrations reached basal levels (<0.1 ng/ml) in two animals. In four goats, P4 concentrations remained close to 1.0 ng/ml, or even temporarily rose above the 2.0 ng/ml level. Spontaneous discharge of uterine fluid took place during (two goats) or within 4 d after bromocryptine treatment (three goats). These results indicate that prolactin plays an important luteotrophic role during pseudopregnancy in goats.

Accuracy of pregnancy diagnosis and prediction of foetal numbers in sheep with linear-array real-time ultrasound scanning.

Pregnancy diagnosis was carried out in sheep by means of transabdominal linear-array real-time ultrasound scanning. Animals were restrained standing, and the transducer was placed on the hairless area of the ventral abdominal wall just in front of the udder. Of a total of 818 tests, 724 were performed between days 29 and 89 of pregnancy, 598 animals subsequently lambed and 126 were non-lambing animals. Only 8 of these tests were wrong: 3 false positive and 5 false negative diagnoses. Sensitivity, specificity, positive- and negative predictive values for these tests were 99.2%, 97.6%, 99.5%, and 96% respectively. There was evidence to indicate that the three false positive tests were caused by foetal mortality or unobserved abortions that took place after testing. Only 2 of the 5 false negative tests were carried out after day 39 of gestation. Counting of foetal numbers (1, 2 or 3) was performed in only some animals (n = 210) between days 45 and 77 of gestation. Three groups of animals (A: 89 ewes; B: 27 PMSG-treated ewes; C: 94 ewes) were analyzed separately. Overall accuracy of all predictions was 83.1%, 37.0% and 78.7% for the 3 groups respectively. Animals in group B produced only 3 or more lambs. Sensitivity of the countings of singles, twins and triplets or more were 90.4%, 90.4% and 50% respectively for the animals from group A and 91.9%, 86% and 21.4% for the animals from group C.(ABSTRACT TRUNCATED AT 250 WORDS)

The use of prostaglandins and oxytocin for transcervical recovery of bovine fetuses at days 33-58 of gestation.

The study of bovine germ cells of known developmental stage calls for alternatives to the recovery of fetuses by surgery or slaughter. Fetuses were therefore obtained during the second month of pregnancy by aborting 49 animals using a progressively modified treatment regimen of cloprostenol, prostaglandin E2 (PGE2) and oxytocin. The viability of fetuses was monitored by ultrasonography throughout treatment. Intracervical treatment with PGE2 led to cervical dilation in all treated animals. However, retrieval of the fetuses by subsequent flushing of the uterus was successful in only two of six animals. When i.m. injections of cloprostenol were given 20-40 h before PGE2 treatment, fetuses < or = 40 days of gestation were expelled spontaneously, while the majority of fetuses > or = 50 days of gestation were retained. When i.m. injections of oxytocin were given in relation to clinical signs of impending fetal expulsion after cloprostenol and PGE2 treatment, 20 of 22 fetuses were expelled 42-53 h after the cloprostenol injection. Of these 20 fetuses, 19 were expelled 0-7 h after the cessation of fetal heartbeat. The subsequent fertility of animals was not affected. Thus, the final protocol allowed bovine fetuses to be retrieved at predictable times, within a few hours of death, with little maternal trauma and without affecting subsequent fertility.

Isolation and identification of germ cells from fetal bovine ovaries.

Gonadal cell suspensions were made from bovine fetuses of 35-55-, 56-80-, and 80-130-day age groups corresponding to the periods predominated by primordial germ cells (PGCs), oogonia, and meiotic cells, respectively. Germ cells identified on morphological criteria prior to their isolation from suspensions were compared histochemically and morphologically with cells in cryosections, impression smears, and semithin sections of similar gonads. Oocytes were distinguished by their chromosomal configurations in cell spreads. In suspensions from 35-55-day fetuses, cells considered to be PGCs stood out by their size, large nucleus, intracytoplasmic vesicles, and occasional blebbing. The somatic cells were smaller and contained little cytoplasm and few vesicles. In bovine gonads, in contrast to murine gonads, alkaline phosphatase (AP) activity was not specific enough to identify germ cells once they had entered the gonad. In ovaries from the 56-80-day age group, cells similar to PGCs, but slightly larger and with more cytoplasmic vesicles, were identified as oogonia. The cytoplasmic vesicles stained positively for lipid. In ovaries of 80-130-day fetuses, oogonia, oocytes, degenerating germ cells, and multinucleate germ cells were recognized. Degenerating germ cells exhibited a variety of morphological characteristics and were consistently positive for acid-phosphatase activity. Binucleate germ cells appeared around day 85 of gestation, while multinucleate germ cells were seen from day 95. It was concluded that bovine mitotic germ cells can be isolated from gonadal cell suspensions and that the best time to recover them is between 50 and 70 days of gestation.

Status of genomic imprinting in mouse spermatids.

The advent of human round spermatid microinjection (ROSI) into oocytes as a treatment for severe male infertility raises the question of whether spermatids have undergone all of the maturation processes necessary for normal development. It is particularly important to know whether spermatids have undergone correct genomic imprinting, which results in the parent-of-origin-specific expression of only one allele of a gene. We assessed the imprinting status of three maternally and three paternally expressed genes in interspecific hybrid embryos generated by injecting Mus castaneus spermatids into Mus musculus oocytes. We used the single nucleotide primer extension (SNuPE) assay to measure the relative expression of maternal and paternal alleles on the basis of sequence polymorphisms in the transcripts. Expression of imprinted genes in mouse embryos derived by ROSI did not differ from controls, indicating that paternal genes have undergone proper imprinting by the round spermatid stage.

Transcription and translation in bovine nuclear transfer embryos.

The development of bovine embryos reconstructed by nuclear transfer (NT) is poor compared to that of embryos produced by in vitro fertilization. One reason for this could be incomplete reprogramming of the transferred nucleus. Therefore, with a view to optimizing the conditions for NT, the reprogramming of blastomere nuclei from 16- to 32-cell-stage in vitro-fertilized (IVF) embryos was investigated following NT by fusion of individual blastomeres with cytoplasts prepared from oocytes at two different stages of maturation. Heterogeneous RNA (hnRNA) production, nucleolar ultrastructure, and protein profiles of the NT embryos up to the 8-cell stage were analyzed. In all NT embryos analyzed for their hnRNA production (n = 133), [3H]uridine incorporation was higher at the 1-, 2-, and 4-cell stages than in control IVF embryos (n = 50). Ultrastructural examination of 11 NT embryos revealed evidence of transcriptional activity; fibrillar and granular components were seen in the nucleolus at the 1-cell stage. At the 2-, 4-, and 8-cell stages, fibrillar components were still evident but granular components had become scarce. The hnRNA synthesis, however, was not reflected in the one-dimensional electrophoretic patterns of protein production in the NT embryos (n = 56); these were largely similar to those of IVF embryos (n = 34) of corresponding stages. Thus, NT embryos made in this way do not behave like equivalent IVF embryos, suggesting that reprogramming of the transferred nucleus is absent or incomplete.

Nucleolar and mitochondrial morphology in bovine embryos reconstructed by nuclear transfer.

The nucleolar and mitochondrial morphology of developing reconstructed bovine nuclear transfer (NT) embryos and stage-matched in vivo-produced control embryos were examined under the electron microscope. Each reconstructed embryo at the one-cell (n = 12), two-cell (n = 5), three-cell (n = 3), four-cell (n = 5), 5-8-cell (n = 5) and blastocyst (n = 3) stages was produced by fusion of a 16-32-cell-stage blatomere with an aged enucleated bovine oocyte. The normal and reconstructed embryos showed similar mitochondrial morphology. However, NT embryos produced several pleiomorphic forms not seen in controls, and were more heterogeneous at early stages of development. Control embryos exhibited nucleolar features considered indicative of rRNA synthesis from the eight-cell stage onwards. In contrast, the NT embryos presented nucleoli with morphology consistent with rRNA synthesis in all embryos examined, except in the three-cell and in two of the five four-cell embryos. From this nucleolar morphology, it was concluded that nuclear reprogramming does not occur immediately following nuclear transfer, but occurs gradually over the first two or three cell cycles.

Development of bovine nuclear transfer embryos made with oogonia.

The pluripotency of embryonic germ cells in the mouse suggests that mitotic bovine fetal germ cells might also be a source of pluripotent cells. To investigate the pluripotency of bovine oogonia, the development in vitro of bovine embryos reconstructed by fusing oogonia with enucleated oocytes was compared with that of embryos made similarly with either blastomeres or granulosa cells. The donor cells (fresh oogonia, cryopreserved oogonia, 16- to 32-cell-stage blastomeres, or granulosa cells) were fused to the enucleated oocytes electrically. The proportions of reconstructed embryos that had cleaved at 40 h after fusion using these types of donor cells were not significantly different (37%, 33%, 56%, and 31%, respectively; p > 0.05). However, the proportions of cleaved reconstructed embryos that developed to the blastocyst stage were 9%, 13%, 36%, and 3%, respectively, significantly higher (p < or = 0.05) with blastomeres than with the other three types of donor cells. After transfer of 3 morulae and 4 blastocysts made with oogonia into three recipient heifers, embryonic and extra-embryonic tissues developed in one animal. On recovery after 43 days gestation, this conceptus was shown to be genetically identical, at 11 microsatellite loci, to the fetus that had provided the oogonia. Cytological analysis of the embryos made with oogonia at 40-44 h after fusion and at the morula and blastocyst stages revealed that aberrant cytokinesis and nucleokinesis had given rise to multinucleated, anucleate, and polyploid cells in the reconstructed embryos. It is concluded that limited pluripotency of bovine oogonia has been demonstrated, warranting further study in this area.