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Mice have been successfully cloned from somatic and embryonic stem (ES) cells using the "Honolulu method." In the present study, different donor oocytes and different culture conditions were compared to evaluate the developmental potential of nuclear transfer embryos reconstructed with an inbred ES cell line HM-1. Oocytes were recovered from two different F1 donors B6D2F1 (C57BL/6 x DBA/2) and B6CBAF1 (C57BL/6 x CBA). There was no effect of oocyte origin on development of cloned embryos to the morulae/blastocyst stage (B6D2F1 44.1% vs. B6CBAF1 45.0%), and the transferred embryos could develop to term. Two culture conditions were compared to show their ability to support development to the morulae/blastocyst stage of reconstructed embryos with B6D2F1 oocytes. The total cell number in the cloned blastocysts cultured in M16 with 20% oxygen was much higher than that observed in CZB with 20% oxygen. Low oxygen concentration during culture of nuclear transfer embryos in CZB medium showed no beneficial effect on pre-implantation development, no embryos developed to term after transfer to surrogate mothers. Our results demonstrated that not only B6D2F1, but B6CBAF1 oocytes, can be used for nuclear transfer. M16 medium is superior for culture of nuclear transfer embryos and low oxygen concentration with CZB medium during culture shows no benefit on development of cloned embryos., (Copyright 2003 Wiley-Liss, Inc.)

Mice have been successfully cloned from both somatic cells and hybrid embryonic stem (ES) cells. Heterozygosity of the donor ES cell genome has been suggested as a crucial factor for long-term survival of cloned mice. In the present study, an inbred ES cell line, HM-1 (129/Ola), and a well-tested ES cell line, R1 (129/Sv x 129/Sv-CP), were used as donor cells to evaluate the developmental potential of nuclear transfer embryos. We found that ES cell confluence dramatically affects the developmental potential of reconstructed embryos. With the ES cell line HM-1 and 80-90% confluence, 49% of reconstructed embryos developed to the morula/blastocyst stage, 9% of these embryos developed to live pups when transferred to the surrogate mothers, and 5 of 18 live pups survived to adulthood. By contrast, at 60-70% confluence, only 22% of embryos developed to the morula/blastocyst stage, and after transfer, only a single fetus reached term. Consistent with previous reports, the nuclei of R1 ES cells were also shown to direct development to term, but no live pups were derived from cells at later passages (>20). Our results show that the developmental potential of reconstructed embryos is determined by both cell confluence and cell passage. These results also demonstrate that the inbred ES cell line, HM-1, can be used to produce viable cloned mice, although less efficiently than most heterozygous ES cell lines.

Determination of embryonic age groups or stages has been based on the Carnegie Institute collection started in 1887. Improved technology has enabled the building of a new collection of embryos of < 9 weeks gestation; these were then used to compare with the original Carnegie collection. The results suggest that in providing definitive stages that are rigidly bound by developmental events, limitations are placed on categorizing the embryo. Allocation of embryos to a specific stage can assist in identifying post-ovulatory age but overlaps between stages could lead to classification into an incorrect stage.

Transforming growth factor (TGF) is known to have the ability to modify mitogenic responses of tissues to other peptide growth factors and therefore may contribute to the rapid growth rate of an embryo. Throughout the TGF superfamily there is a similar fundamental molecular architecture. Included in this superfamily are inhibin A, activin A and activin B. It has been shown that activin is a powerful mesodermal inducing factor in the early embryo. The human embryo has shown localization of inhibin in the gonads after 16 weeks gestation but it has not been previously identified in earlier embryos. The inhibin-activin protein was found in a range of tissues including the liver stages 19-21 (alpha) and stages 19-22 (beta); oesophagus stages 19-22 (alpha and beta); stomach stages 21 and 22 (alpha and beta); gut stages 16-22 (alpha) and 21 and 22 (beta); pericardium stages 12-22 (alpha and beta); gonad stages 21 and 22 (beta) stage 22 (alpha); adrenal stages 19-22 (alpha and beta); urogenital system stages 21 and 22 (alpha and beta); yolk sac stage 12 (alpha and beta); mesenchyme stages 16-22 (alpha); surface ectoderm stages 13-22 (alpha) and stages 16-22 (beta a); notocord stages 13-22 (beta) and stages 21 and 22 (alpha); nasal, trachea and bronchi stages 19-22 (alpha and beta) leading to speculation of the role of both subunits.

The developmental age of an embryo in the first trimester of pregnancy is generally determined by ultrasound scanning and/or by calculation from menstrual age. In the original studies, validation of the estimate of gestational age by ultrasound was not possible as the exact date of conception was unknown. Variation in growth rates of identically aged fetuses has previously been reported after assisted conception and with the use of ultrasound scanning. As these pregnancies were ongoing the accuracy of the scanning results could not be determined. Comparison of scanning and direct measurements after termination of pregnancy and menstrual age were carried out to determine the accuracy in fetal dating. The results suggest that the use of ultrasound scanning to determine gestational age is of less use than previously thought, and that the use of menstrual age is severely limited.

Localization of the mRNA alpha and beta a subunits of inhibin has previously been reported in the human gonads during the second trimester. Adrenal inhibin has also been reported in the second trimester for the alpha, beta a and beta b subunits. Investigations showing localization by in-situ hybridization during the first trimester have not been reported. The results have shown hybridization of the alpha and beta a subunits, throughout the period of development studied, in a variety of tissues including the dorsal and thoracic aortas and pericardium stages 13-22 (beta a subunit); liver stages 19-21 (beta a) and stages 21-22 (alpha); mesonephos stages 21 and 22 (beta a); gonad stages (alpha and beta a); adrenal stages 19-22 (alpha); surface ectoderm stages 16-22 (beta a); mesenchyme stages 16-22 (beta a); amnion stages 13-16 (beta a); yolk sac stage 12 (alpha and beta a); cartilage stages 19-22 (beta a); and nasal proliferation stages 21 and 22 (beta a). When compared with distribution of the protein subunits it was noted that more immunostaining activity was found, suggesting that that probes were not sufficiently sensitive enough to detect all levels of mRNA expressed. It can be surmised, therefore, that the lack of visual hybridization of the mRNA cannot preclude the possibility that it is not being translated within the tissue even though hybridization was not apparent.