Human testis-specific lactate dehydrogenase-C promoter drives overexpression of mouse lactate dehydrogenase-1 cDNA in testes of transgenic mice.

The three isozymes of lactate dehydrogenase, each encoded by a separate gene, are developmentally regulated and differentially expressed in tissue-specific patterns. The lactate dehydrogenase-C (LDHC, mouse Ldh3) gene is temporally expressed exclusively in the germ line during spermatogenesis, whereas lactate dehydrogenase-A (LDHA, mouse Ldh1) and B (LDHB, mouse Ldh2) genes are active in somatic tissues. To determine, therefore, whether overexpression of Ldh1 would perturb spermatogenesis, we constructed a transgene in which a sequence from the promoter region of human LDHC was coupled with mouse Ldh1 cDNA. Among nine (three males, six females) founder lines that were identified as being transgenic for the construct, one male transmitted the gene through its germ line. Homo- and heterotetramers containing the LDH-A subunit were detected in homogenates of testes from transgenic animals. We conclude that the human LDHC promoter contains the necessary regulatory sequence(s) for specific expression of mouse Ldh1 as a transgene during spermatogenesis. The fertility of the founder animal was not impaired.

Testis-specific expression of a metallothionein I-driven transgene correlates with undermethylation of the locus in testicular DNA.

Mice carrying a chimeric transgene of the human testis-specific lactate dehydrogenase cDNA driven by mouse metallothionein I promoter have been reported to express the transgene in a testis-specific manner in six founder lines. To study the mechanism by which this testis-specific expression is mediated, we have examined genomic placement, expression pattern, and methylation status of the transgene. Our results indicate that transgene expression is repressed in all somatic tissues examined even when heavy metals are administered. Nuclear run-on assays indicate that failure of expression in the liver (in which the metallothionein I promoter is highly active) occurs at the transcriptional level. In contrast, the transgene mRNA is transcribed in male germ cells and is developmentally regulated during spermatogenesis. Examination of the transgene methylation status reveals that expression is inversely correlated with hypermethylation of the locus; all CpG dinucleotides examined in the promoter region were found to be fully methylated in kidney and liver but were undermethylated in testis. Since methylation of the murine metallothionein I promoter is sufficient to inhibit its activity, it is likely that suppression of the transgene in somatic tissues is mediated by methylation.

Observations on the cooling and cryopreservation of pig oocytes at the germinal vesicle stage.

This study examined the viability of pig oocytes at the germinal vesicle stage following cooling or cryopreservation. Cumulus-intact oocytes (n = 641) were collected from slaughterhouse pig ovaries and used in two experiments. In Exp. I the viability of 1) control, 2) cryoprotectant control (CC, 1.5 M glycerol/.5 M sucrose), 3) cooled (0 degrees C) and 4) cryopreserved (-196 degrees C) oocytes was assessed after no incubation or a 24-h incubation. Survivability was judged by morphological appearance, trypan blue exclusion and fluorescein diacetate staining. Survival rate of control oocytes (90%; based primarily on morphological appearance of the cumulus) incubated 0 h was greater (P less than .05) than that of all other groups, whereas survival rate of -196 degrees C oocytes (57%) was less (P less than .05) than that of all other groups. However, vital staining of 0 degrees C and -196 degrees C oocytes showed 0% survival rate as evidenced by trypan blue uptake and lack of fluorescence. The cumulus cells surrounding oocytes that were stored at 0 degrees C or -196 degrees C survived freezing as evidenced by trypan blue exclusion and intense fluorescence. Similar differences among treatment groups were found for oocytes incubated 24 h. Exp. 2 examined the temperature at which oocytes became sensitive to cooling. Oocyte death occurred when oocytes were cooled to 15 degrees C or lower. These results demonstrate that pig oocytes at the germinal vesicle stage did not survive cooling to 15 degrees C or below. When assessing the viability of cryopreserved cumulus enclosed oocytes it is important to use vital stains in conjunction with morphological appearance.

Successful rescue of microsurgically produced homozygous uniparental mouse embryos via production of aggregation chimeras.

Homozygous uniparental mouse embryos, produced by microsurgical removal of the male pronucleus from fertilized eggs and diploidization of the female pronucleus with cytochalasin, were surrounded with blastomeres from normal embryos to produce chimeric embryos. A few of these chimeras developed into viable adults, and one female has reproduced using her homozygous uniparental cells as a source of gametes. The production and use of such chimeras in breeding programs could greatly shorten the period required for producing inbred strains of mammals. The data presented demonstrate that a homozygous uniparental mammalian genome, although not lethal to all cells, is extremely deleterious to normal embryonic development. Moreover, the results support the conclusion that the genome is imprinted differently in males and females during gametogenesis so that at fertilization the genomes are not functionally equivalent, and both are required for normal development.

Preliminary characterization of molecules that increase cell free translational activity of cardiac cytoplasmic RNA.

Previous evidence indicates that cells generate molecules that alter transcription in response to stress. Accordingly, we conducted preliminary experiments to characterize these molecules. Extracts were prepared from normal and experimental canine hearts in which a 100 mmHg gradient was created across the ascending aorta for 6 h. Experimental extracts were then treated by ultrafiltration through YM 10 and YM 30 membranes, by incubation with immobilized trypsin for 1 h at 37 degrees C and by incubation in a boiling water bath for 10 min. Extracts were perfused through isolated rat hearts for 1 h. Total cytoplasmic RNA was then extracted from the perfused heart and translated in a cell free medium containing [35S]-methionine. Incorporated label into newly synthesized protein was determined by liquid scintillation counting. A 13% mean increase in translational activity was produced by the fraction of the experimental extract passing through the YM 10 membrane compared with the material retained by YM 10 and YM 30 membranes. A 14% mean decrease in translational activity was observed in hearts perfused with experimental extracts treated with immobilized trypsin compared to untreated experimental extracts. There was no significant difference in translational activity of hearts perfused with experimental extracts subjected to boiling compared with non-boiled experimental extracts. These data suggest that the active molecules may be heat stable peptides or peptide containing substances of 10000 daltons or less in molecular weight.

The in vivo and in vitro effects of clomiphene citrate on ovulation, fertilization, and development of cultured mouse oocytes.

Clinical data indicate a high rate of discrepancy between ovulation and pregnancy rates and a high abortion rate in anovulatory women treated with clomiphene citrate. An animal model was used to examine the in vivo and in vitro effects of the drug on fertilization and preimplantation development. This work indicates that mice treated with clomiphene in the early follicular phase of pregnant mare's serum-induced cycles show a dose-dependent decrease in ovulated oocytes. No effect was seen on the further development of these ova. When the drug was administered in the late follicular phase, a dose-dependent decrease was seen in the rate of successful copulation, ovulation, and embryo development. Concomitant 17 beta-estradiol treatment reversed the copulatory, ovulatory, and developmental effects of clomiphene. A short in vitro treatment of oocytes with clomiphene prior to in vitro fertilization resulted in an increased proportion of degenerated and unfertilized ova. Moreover, this treatment caused a dose-dependent decrease in blastocyst formation.

Fertilization of mammalian eggs by sperm injection.

Mammalian sperm normally fertilize eggs in the ampulla of the oviduct after a long trip through the female reproductive tract. During this trip, the sperm become capacitated to fertilize but must overcome substantial barriers in the form of egg investments before reaching the plasma membrane of the egg. Once inside the egg the head of the sperm responds to cytoplasmic factors to decondense and form a pronucleus. All the problems of sperm penetration of the egg and its investments can be circumvented by injecting the sperm into the egg with a micropipette. Such injected sperm can participate normally in the subsequent events, typical of fertilized eggs, that lead to the formation of embryos. Sperm can also be injected into normally fertilized eggs to produce a third pronucleus and a triploid embryo. To avoid the mechanical problems of capturing a swimming sperm with a micropipette, the sperm suspension can be sonicated to break off the sperm tails. The resulting sperm heads can easily be injected into unfertilized eggs to induce the normal events of fertilization. With these injection procedures it is possible to test the fertilizing capacity of foreign sperm, of defective sperm, and even of "dead" sperm. We have found that the phenotype of the sperm does not reflect the genotype in terms of fertilizing ability after microinjection. Immotile and grossly defective sperm of the mouse when injected into the egg produce the same reactions that are produced by fertilization with healthy robust sperm.(ABSTRACT TRUNCATED AT 250 WORDS)

Parthenogenesis, homozygosity, and cloning in mammals.

The numerous strategies for reproduction, sexual and asexual, that have evolved, suggest experimental designs for altering the reproduction of any particular species. The ability to culture and manipulate mammalian eggs and embryos in vitro provides the technological basis for experimentally altering the reproduction of mammals and can lead to the generation of new and useful genotypes. All of these advances, however, are restricted by our lack of understanding of the chromosomal mechanisms by which genes are regulated during the course of cell differentiation. An attractive hypothesis postulates that programs of gene regulation during development are based upon topographic interactions among chromosomes in the interphase nucleus and that these interactions in turn require an appropriate positioning of regulatory DNA throughout the genome.

Diverse forms of stress lead to new patterns of gene expression through a common and essential metabolic pathway.

Many eukaryotic organisms respond to heat shock by synthesizing new proteins. We examined the possibility that heat shock proteins represent a particular expression of a general response to stress and that, regardless of the nature of the effective stimulus, the same proteins are synthesized. Accordingly, cardiac stress was applied in the intact rat by four methods: banding the ascending aorta, increasing body temperature to 42 degrees C, reducing body temperature to 18 degrees C, and forcing the rat to swim until exhausted. The hearts were then extirpated and analyzed for new mRNA synthesis. The extracted RNA was translated in a cell-free medium containing [35S]methionine. Translation products were resolved by two-dimensional electrophoresis and visualized by autoradiography. Lactic acid concentration in heart tissue was determined enzymatically. The results showed that two new and distinct proteins of Mr 71,000 and isoelectric points of 5.8 and 6.1 were synthesized in hearts stressed by banding and by heating but not in hearts of exhausted swimmers or in animals at reduced body temperatures. There was no significant difference in cardiac lactic acid concentration between control hearts and hearts from swimmers or cold-treated animals. However, there was a 2-fold increase in lactic acid concentration in hearts of rats with banded aortas compared to controls and a 10-fold increase in heat shocked hearts. We conclude that, under conditions in which the energy requirements of the heart are not completely met by aerobic processes, the resultant lactic acidosis creates an intracellular environment that leads to the selective activation of genes, the production of new mRNA, and the synthesis of a typical group of stress proteins.

The molecules that initiate cardiac hypertrophy are not species-specific.

Extracts from hypertrophying dog hearts perfused through isolated rat hearts increase the synthesis of messenger RNA and initiate hypertrophy in the treated hearts. Total RNA extracted from experimental and control hearts was translated in vitro and hybridized with polyuridylate. Synthesis of protein and polyadenylate-containing RNA was greater in rat hearts perfused with extracts of hypertrophying dog hearts than in control hearts. The results demonstrate that molecules from hypertrophying dog hearts are not species-specific since they are effective in stimulating transcription of messenger RNA in rat hearts as well as in dog hearts.

Manufacture of diploid/tetraploid chimeric mice.

Tetraploid mouse embryos were produced by cytochalasin B treatment. These embryos usually die before completion of embryonic development and are abnormal morphologically and physiologically. The tetraploid embryos can be rescued to develop to maturity by aggregating them with normal diploid embryos to produce diploid/tetraploid chimeric mice. The diploid/tetraploid chimeric embryos are frequently abnormal: the larger the proportion of tetraploid cells, the greater the abnormality. By karyotype analysis and by the use of appropriate pigment cell markers, we have demonstrated that two of our surviving chimeras are in fact diploid/tetraploid chimeras. One surviving chimera is retarded in growth and displays neurological abnormalities. The coat color chimerism suggests that this chimera is about 50% tetraploid. Another chimera with about 10% tetraploid pigment cells in the coat is only slightly retarded in growth and is a fertile male. Tetraploid cells are distributed in many, if not all, tissues of embryos but evidently are physiologically inadequate to support completely normal development and function in the absence of substantial numbers of normal diploid cells.

Production and reproductive performance of hexaparental and octaparental mice.

The production of hexaparental and octaparental mice following embryo aggregation is reported. These mice were progeny-tested to determine which cell components were contributing to gamete formation. One chimera (No. 15) was shown to be producing eggs from all three cell strains--white, yellow, and black. The other chimeras tested were not forming germ cells from all of the cell strains involved. The use of these animals for obtaining a minimum estimate of the number of cells giving rise to the embryo proper is discussed. The suggestion is made that multi-embryo aggregates may actually be developmentally different from normal sized embryos and this may result in modified mechanisms of cell recruitment.