Embryo loss during peri-implantation can approach 20% in swine following artificial insemination or natural mating and coincides with rapid conceptus elongation. The objective of the present study was to establish a comprehensive profile of the abundant proteins of the pig conceptus at the time prior to implantation and identify stage-specific changes during elongation. The abundant proteins of a homogenous population of gestational day-11 ovoid (0.7-1 cm) and gestational day-12 filamentous (15-20 cm) porcine concepti were compared by extracting proteins from three independent conceptus pools and separating the proteins by 2-DE. Proteins in 305 spots were analyzed by MALDI-TOF or additionally by LC-MS/MS and 275 were positively identified representing 174 distinct proteins. The proteins could be classified into the following functional categories: cell proliferation/differentiation, cytoskeleton, metabolism, and stress response. Based on spot density, 35 proteins associated with cell proliferation, differentiation, apoptosis, and embryo/maternal signaling, were found to be differentially expressed between ovoid and filamentous concepti. A comparison of the protein expression profile with transcriptomic data from pig concepti of the same developmental stages identified similarities and dissimilarities between protein and mRNA expression profiles. This proteomic study helps to elucidate the biological mechanisms underlying the early embryonic development of the pig.

Comparative proteomic and regulatory network analyses of the elongating pig conceptus

BACKGROUND: Retroviral-mediated gene transfer stably introduces exogenous genes into normal and neoplastic cells of the hematopoietic system. METHODS: We used two retroviral vectors [the first, FLac, expresses a chimeric protein (Sh-ble::LacZ) between the product of the phleomycin resistance gene (Sh-ble) and the bacterial beta-galactosidase encoded by the LacZ gene; the second, NuNL vector, contains a fusion sequence (LacZ::Neo) that expresses the LacZ and the neomycin resistance genes] to transduce T lymphocytes derived from the peripheral blood of healthy human donors. Two lymphocyte activation procedures were employed: a) phytohemagglutinin/interleukin-2 (PHA/IL-2) polyclonal stimulation; b) allogeneic stimulation in a mixed irradiated or non irradiated lymphocyte reaction, both supplemented with IL-2 (MLR/IL-2). Infection was achieved by co-cultivating activated T cells with the producing amphotropic cell line pretreated with mitomycin C for 96 hours. Infection and transduction efficiency were assayed by LacZ gene expression, which is detected as indigo blue staining with the chromogenic substrate 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (X..Gal). RESULTS: The highest percentage of transduced T cells was reached on the 3rd PHA/IL-2 and on 9th MLR/IL-2 activation days. In these conditions with FLac vector we obtained up to 80% X-Gal+ cells after PHA/IL-2 activation and 66% and 44%, respectively, with non irradiated and irradiated MLR/IL-2, respectively. Up to 40% X-Gal+ cells were obtained with NuNL vector after PHA/IL-2 stimulation, 40% with irradiated and 48% with non irradiated MLR/IL-2 activated cells. In term of transduction efficiency, large variability was observed among patients. There were no immunophenotypical differences between FLac or NuNL vector-transduced cells activated by either of the two techniques and the control cells. CONCLUSIONS: Our results indicate that: a) the use of FLac or NuNL vector retroviral-mediated gene transfer into T-lymphocytes derived from peripheral blood and stimulated by either PHA/IL-2 or a MLR produces a high percentage of transduced T cells; b) MLR is a good system for generating a transduced alloreactive lymphocyte population. The combination of high transduction efficiency and the capacity to obtain alloreactive transduced lymphocytes should open up the possibility of generating new in vitro and in vivo studies with selectable genes for in vivo therapeutic use.

beta-galactosidase transduced T lymphocytes: a comparison between stimulation by either PHA and IL-2 or a mixed lymphocyte reaction.

Murnane, R.D., Ahern-Rindeli A.J., and Prieur, D.J., 1991. Ovine GM 1 gangliosidosis. SmallRumin. Res. 6:109-118. Ovine GM 1 gangliosidosis is a newly described neuronal lysosomal storage disease first observed in Suffolk sheep. Affected sheep are clinically normal at birth and exhibit ataxia at 4 to 6 months of age which rapidly progresses to prostration. Affected sheep exhibit a profound deficiency oflysosomal fl-galactosidase in fibroblasts and tissues, and a partial deficiency of c¢-neuraminidase. Abnormal accumulation of GM l ganglioside, asialo-GM 1 and neutral long chain oligosaccharides is present in the cerebrum with excretion of neutral long chain oligosaccharides in the urine. Genetic studies indicate the disease is inherited as an autosomal recessive. Pathologic lesions consist of marked distension and vacuolation of the cytoplasm in virtually all neurons (central and peripheral nervous system) and of periportal hepatocytes and renal epithelial cells. Preliminary ultrastructural studies disclosed numerous membranous whorls and lameilar stacks within membrane-bound vacuoles in neurons. Golgi staining of cerebrum discloses formation of spiny meganeurities and secondary neurite formation in pyramidal neurons. Lectin histochemistry indicates stored material has terminal saccharide moieties consisting of fl-galactose, N-acetylneuraminic acid, and N-acetylgalactosamine. No consistent clinicopathologic and no musculoskeletal abnormalities have been identified. The prevalence and economic significance of ovine GM l gangliosidosis is unknown. Most neonatal deaths and abortions of small ruminants remain undiagnosed, and possibly many have an undetermined genetic basis. The described disease emphasizes the importance of clinical and pathologic evaluation of neonatal deaths as the economic and research importance of genetic diseases are unrealized.

Ovine GM-1 gangliosidosis.

Growth and development of pig fetuses is dependent on the coordinated expression of multiple genes. Between 21 and 45 days of gestation, fetuses experience increasing growth rates that can result i...

Developmental changes in the fetal pig transcriptome.

Gene transfer into bovine cells and embryos using replication‐defective retroviral vectors encapsidated with xenotropic murine leukemia virus envelopes

Knowledge regarding the timing of embryonic expression of the mammalian genome is of relevance for the development of preimplantation diagnostic methods for human genetic diseases. For development of preimplantation diagnosis of lysosomal storage diseases, it will be necessary to know at which embryonic stage the genes for lysosomal enzymes are expressed. In previous studies by other investigators, it has been shown that lysosomal alpha- and beta-galactosidase and beta-glucuronidase in murine embryos increase 50- to 100-fold in activity between the two-cell and late blastocyst stage. We describe here expression of lysosomal beta-galactosidase in preimplantation ovine (two-cell through midblastocyst) and porcine (two-cell through late blastocyst) embryos. Expression of beta-galactosidase in ovine and porcine preimplantation embryos followed a similar rate of increase as that described for murine embryos. Activity of beta-galactosidase increased over 10-fold between the two- to four-cell and midblastocyst stages in ovine embryos, and 300-fold between the two- to four-cell and late blastocyst stages in porcine embryos. Activity expressed on a per cell basis was relatively constant in ovine embryos, as has been described in murine embryos, and increased approximately 5-fold on a per cell basis in porcine embryos. Activity of beta-galactosidase in ovine and porcine embryos initially was greater than 12-fold on a per cell or per embryo basis than in murine embryos evaluated. The knowledge of beta-galactosidase embryonic expression may provide the basis for preimplantation diagnosis of genetic beta-galactosidase deficiency in these species.

Kriss A. Hoffman

Papers

Expression of β-galactosidase in preimplantation ovine and porcine embryos