Differential immunofluorescence of fertilized mouse eggs with H-2 and non-H-2 antibody.

Mouse embryos at the two-cell and blastocyst stages, as well as unfertilized eggs, have been studied by indirect immunofluorescence for the expression of H-2 and non-H-2 histocompatibility antigens on surface membranes. Serologically-specific reactivity to non-H-2 antibody (H-3 and H-6) was observed as diffuse, patchy staining over the entire surface of the blastomeres at the two-cell stage. In contrast, no reactivity of two-cell or unfertilized egg embryos of four inbred strains was observed when antisera containing only multispecific H-2 cytolytic antibody were used. Antisera containing H-2 along with non-H-2 antibody of unknown specificity showed varying degrees of reactivity, which could be shown by absorption studies to be due to the non-H-2 content of the serum. The results suggest that the initial expression of histocompatibility genes varies and support the hypothesis that the appearance of these cell components may relate to specific stages of differentiation.

Autoradiographic evidence for insulin and insulin-like growth factor binding to early mouse embryos.

Insulin binding to mouse oocytes and preimplantation embryos was assessed by light-microscopic autoradiography. Significant insulin binding was present on the cells of morulae and increased twofold at the blastocyst stage of development. Insulin binding was markedly decreased by native insulin and to a lesser extent by insulin-like growth factors (IGFs). No specific insulin binding was detected on oocytes or embryos throughout the eight-cell stage. Specific binding of IGF-I and IGF-II was also observed on the cells of blastocyst outgrowths. The findings demonstrate that specific binding of insulin and IGF is temporally expressed on the cells of pre- and peri-implantation mouse embryos. These results confirm and extend our previous immunofluorescence study.

Post-transcriptional stimulation of transforming growth factor beta 1 mRNA by TGF-beta 1 treatment of transformed human osteoblasts.

Following exogenous administration of transforming growth factor-beta 1 (TGF-beta 1) polypeptide to the human osteosarcoma cell line TE-85, we observed a 2- to 6-fold stimulation of steady-state TGF-beta 1 mRNA. The stimulation was dose- and time-dependent, as judged from Northern blot hybridization analyses. A 2- to 6-fold increase of the TGF-beta 1 polypeptide was also found in the media of these cells after TGF-beta 1 treatments. The autostimulation of TGF-beta 1 mRNA was nullified by cycloheximide treatment of the cells. The in vitro transcription rates of the TGF-beta 1 gene by isolated nuclei were not altered by TGF-beta 1 treatment. Under conditions of transcriptional inhibition, the stability of TGF-beta 1 mRNA was enhanced nearly two-fold by TGF-beta 1 treatment. Our findings indicate that TGF-beta 1 can stimulate autologous gene expression and subsequent polypeptide translation by a post-transcriptional mechanism requiring protein synthesis in human osteoblast-like cells. The recognized versatility of TGF-beta 1 autostimulation mechanisms (transcriptional and post-transcriptional) in other mesenchymal cells may apply also to skeletal cells, further underscoring the broad and potent activities of this cytokine.

Golgi study of the primate substantia nigra. I. Quantitative morphology and typology of nigral neurons.

Neuronal morphology was analyzed in the pars compacta, reticulata, and lateralis of the substantia nigra of humans and macaques. Golgi-impregnated dendritic arborizations, reconstructed from serial sections, were described by using topological, metrical, and geometrical parameters measured in three dimensions. Morphological parameters were statistically analyzed. Cell bodies and axons were also described. The primate substantia nigra comprises few local circuit microneurons. It consists mainly of large projection neurons having large cell bodies and sparsely branched dendritic arborizations. In all subdivisions, "complex endings" and "thin processes" can be found on nigral dendrites. Axons of large neurons occasionally had initial collaterals that never form profuse arborizations. Pars reticulata neurons had a cell body surface of 520 micron2, 4 dendritic stems, and 13 dendritic tips. The total dendritic length (L) was 7,100 micron, the highest dendritic length (Lm) 1,200 micron, and the mean length of dendritic segments 320 micron. Pars lateralis neurons were similar except for their larger cell bodies (650 micron2) and longer dendritic segments (440 micron). Pars compacta neurons had larger cell bodies (860 micron2), thicker and more numerous (5 stems, 19 tips), and longer dendrites (L = 10,500 micron; Lm = 1,400 micron). Large neurons of monkeys had the same topological characteristics as human neurons but shorter dendrites. The overall shape of arborizations was highly variable and not characteristic in any subdivision. A hierarchical typology of nigral neurons is proposed, which comprises two neuronal species, the compacta and reticulata species, and a lateralis subspecies. Pallidal neurons (Yelnik et al., '84) belong to the reticulata species. The position of these species in relation to higher hierarchical levels is discussed.

A Golgi analysis of the primate globus pallidus. I. Inconstant processes of large neurons, other neuronal types, and afferent axons.

The present paper is a Golgi study, with high-power lenses, of the primate globus pallidus. Two kinds of inconstant processes of large neurons are first described: complex endings and thin processes. Complex endings are thick apparatuses terminally located on dendrites having many appendages of various types. Contacts were observed not only between striatal axons and these complex endings but also between complex endings and the soma, dendritic stems, dendritic portions or complex endings of other large pallidal neurons. Thin processes were usually beaded, very thin, and arose from any part of the dendritic tree. Contacts were seen between them and soma or dendrites of other large neurons. These thin processes were very similar to initial axonal collaterals and together constitute a common pool of processes. Complex endings and thin processes were essentially observed in the lateral nucleus of the pallidum where they apparently are evenly distributed inside the nucleus but randomly distributed on individual neurons. Two neuronal types other than large pallidal neurons were isolated: the smallest were considered to be local circuit neurons, while intermediate-sized neurons might be the origin of a particular efference. Many striatal axons gave no branches over long distances and collaterals were of two types and most frequently were short (less than 50 micron). Larger axonal arborization were rarely encountered. In addition to parallel contacts, numerous very short ones were observed. All these contacts between striatal axons and dendrites of large pallidal neurons seem to be irregularly distributed.

Maternal humoral immune response to incompatibility at multiple minor histocompatibility loci in mice.

Mice of the H-2b haplotype were mated with males of the same MHC haplotype, but differing at multiple minor histocompatibility loci. Mice were bled during each pregnancy and at 2-day intervals post-partum. The sera were assayed by indirect immunofluorescence for evidence of a humoral immune response to paternal minor histocompatibility antigens. Alloantibody was first detected in the post-partum period following the third pregnancy, and was also detected during the fourth pregnancy. Thereafter, alloantibody levels dropped and by the post-partum period following the fifth pregnancy, fell to control values. Assays on a panel of cells from mice of different inbred strains revealed specificity of the alloantibody to H-3.1, H-4.1 and H-7.1 antigens. A conventional dye exclusion cytotoxicity test revealed the pregnancy-induced alloantibody did not exhibit complement-dependent cytotoxicity. These findings are discussed in relation to the regulation and functional significance of the humoral immune response in allogeneic pregnancy.

Differential expression of minor histocompatibiility antigens on the surface of the mouse oocyte and preimplantation developmental stages.

The expression of specific minor histocompatibility antigens on oocytes and preimplantation developmental stages in the mouse has been examined using immunofluorescence. Multi-specific alloantisera in conjunction with serological adsorption and target cells of the appropriate genotype have been used to how that there is differential expression of minor histocompatibility antigens during early development. Detailed analysis showed that antigen expression can be detected on oocytes and zygotes, but not on two-cell stages. From the eight-cell stage to the blastocyst, reactivity with antibody directed against a single specificity could be detected.

Pregnancy resulting from cryopreserved human embryos using a one-step in situ dilution procedure.

In vitro fertilization and embryo transfer require the use of hormonal manipulation and surgery that may reduce the receptivity of the patient's uterus during the stimulated cycle. Cryopreservation of human embryos eliminates the need for immediate transfer, permitting them to be stored until they can be transferred during subsequent unstimulated cycles. Embryo cryopreservation is an established procedure in the breeding of laboratory and domestic animals, but has only recently been applied to humans. We report on a pregnancy using a simple cryopreservation procedure that permits embryos to be diluted out of the cryoprotectant solution without removing them from the plastic straw in which they were cryopreserved.

Is chromosome 10 a primary chromosomal abnormality in endometrial adenocarcinoma?

Seven cases of endometrial adenocarcinoma (EC) are reported. Two of these cases exhibited diploid chromosome ranges and showed simple rearrangements involving a chromosomal abnormality of chromosome 10. In four cases, the chromosome number ranged between 50 and 70; rearrangements were more complex, with many abnormalities such as homogeneously stained regions, minutes, dicentrics, and ring chromosomes. In one case, two subpopulations of cells were detected, one in a diploid chromosome range with chromosome 10 altered, and the second, very pleomorphic. These abnormalities are probably due to the evolution of a destabilized genome and represent a consequence of the advanced stage of the disease. The importance of simple abnormalities as clues to the primary chromosomal change, and the possibility that chromosome 10 represents the primary chromosomal alteration event in EC, are discussed.

A histological atlas of the macaque (Macaca mulatta) substantia nigra in ventricular coordinates.

An atlas of the macaque substantia nigra was established in ventricular coordinates. Having verified that nigral contours are stable in both Macaca mulatta and Macaca speciosa, the antero-posterior sequence of four subdivisions was described on eight vertico-transverse levels after a comparison between histological and histochemical data. The pars reticulata constitutes the anterior pole containing pale, small cell-bodies scattered among the numerous endings of the striato-pallido-nigral fibres. Anteriorly, the pars compacta forms the dorsal border of the substantia nigra, but more posteriorly, it leaves the dorsal border and develops ventral fringes. Its large cell-bodies, most often pigmented, rich in Nissl bodies and in acetylcholinesterase, are closely spaced among a few axonal fascicles. The pars lateralis contains a low density of cell-bodies and corresponds to the hilum of entrance of the striato-pallido-nigral fibres and gives rise to a nigro-tectal projection. The pars mixta, dorsally located, contains a low density of neurons varying considerably in size. It represents the hilum of exit of nigral axons which project to various targets.

Gene expression in pre-implantation mammalian embryos.

The pre-implantation mammalian embryo is initially under the control of maternal informational macromolecules that are accumulated during oogenesis. Subsequently, the genetic program of development becomes dependent upon new transcription derived from activation of the embryonic genome. Several embryonic transcripts including those that encode growth factors, cell junction components and plasma membrane ion transporters are required for normal progression of the embryo to the blastocyst stage. The pattern of genes expressed and the overall program of development is subject to the influences of genomic imprinting as well as external influences encountered by the embryo within the maternal reproductive tract.

Temperature elevation in the rat fetus due to ultrasound exposure.

The temperature elevation resulting from sonically generated heat in rat fetuses was measured for various intensity levels. The temperature elevation produced inside the fetus was higher than that on the outside surface. In live fetuses, a portion of the heat generated was carried off by the circulating blood. The temperature elevation curves were used to estimate the absorption coefficient of rat fetuses. Absorption coefficient values range from 0.065 to 0.086 Np/cm at 1 MHz. The present results are consistent with a theoretical model of temperature elevation in a heated sphere.

Insulin, insulin-like growth factors and glucose transporters: temporal patterns of gene expression in early murine and bovine embryos.

mRNA phenotyping by the reverse transcription-polymerase chain reaction (RT-PCR) method was used to compare the patterns of expression of insulin and insulin-like growth factor (IGF) ligand and receptor genes in preimplantation bovine embryos with those established previously for preimplantation murine embryos. In the early bovine embryo, transcripts for IGF-I, IGF-II and mRNAs encoding receptors for insulin, IGF-I and IGF-II were all detectable at all embryo stages from the 1-cell zygote to the blastocyst. In the mouse, IGF-II ligand and receptor mRNAs were not expressed until the 2-cell stage, and the insulin and IGF-I receptor mRNAs were not detectable until the 8-cell stage. Since transcriptional activation of the embryonic genome occurs at the 8- to 16-cell stage in the bovine embryo and at the 2-cell stage in the murine embryo, it is suggested that these transcripts are products of both the maternal and embryonic genomes in the bovine embryo whereas in the mouse they are present only after activation of the embryonic genome. Transcripts for insulin were not detected in preimplantation embryos of either species. Colloidal-gold immunocytochemistry with antibodies directed against the insulin receptor, IGF-I receptor and IGF-I ligand has confirmed the presence of these molecules in bovine blastocysts. RT-PCR and indirect immunofluorescence procedures demonstrated that the glucose transporter (GLUT) isoform 1 is present in murine embryos from the oocyte to blastocyst stage whereas GLUT 2 expression begins at the 8-cell stage.

Micromachined analytical devices: microchips for semen testing.

Micromachined devices (microchips) have been designed and tested for a range of clinically important assays. In this study we compare sperm motility determined using disposable glass microchips and a conventional Makler chamber. The 17 x 14 mm glass microchips contained three etched test structures each comprising either duplicate or quadruplicate analytical microchannels. Semen samples with sperm counts ranging from 21 to 78 million sperm per ml and forward progression scores of from 1+ to 3+ were evaluated and swimming times ranging from 360 s (3.3+ progression) to 770 s (1+,2 forward progression) observed in the microchips. Motility determined by the time taken for sperm to swim to the end of a microchannel (100 microns wide x 40 microns deep x 10 mm long) in the microchip correlated with forward progression of the sperm determined by the conventional Makler chamber method. This study demonstrates the feasibility of microchips for sperm motility testing and suggests that this technique would be applicable to the study of other types of motile cells.

Comparison of two culture systems for the in-vitro growth and maturation of mouse preantral follicles.

The objective of our study was to compare to ability of collagen-treated membranes and bovine collagen gels to maintain murine preantral follicle growth and development in-vitro. To fulfill that objective, murine follicle and oocyte growth rates were followed for ten days in culture. Meiotic competence and the capacity to reach the two-cell stage after in-vitro maturation and fertilization, respectively, were then assessed. We used preantral follicles from 12 day-old CF-1 female mice that were isolated by enzymatic digestion from ovaries. Follicles were placed either on collagen-treated membranes or embedded in a bovine collagen matrix. The follicles were grown, changing the media and obtaining measurements every other day for ten days. Following culture, the granulosa-oocyte complexes were matured; the resultant metaphase II arrested oocytes were inseminated and cultured to the two-cell stage. The data was analyzed with significance considered for probability values of p < 0.05. We performed individual measurements on 650 follicles in seven separate experiments. Forty-eight hours after initial seeding and throughout the entire length of culture, both the follicles and oocytes grown in the collagen matrix were larger than follicles cultured on collagen-treated membranes (p < .0001). However, oocyte recovery rates were higher among follicles cultured on collagen-treated membranes (p < .01). Similar percentages of meiotically competent oocytes, fertilization and cleavage rates were observed in both groups. Our results show that mouse preantral follicles display a greater growth rate when grown embedded in a collagen gel matrix. This may be due to the maintenance of a normal three-dimensional organization of the follicle within the collagen matrix. However, this system does not enhance meiotic competency or fertilization rates in the mouse when compared to culture on collagen-treated membranes.