Targeted disruption of the mouse estrogen sulfotransferase gene reveals a role of estrogen metabolism in intracrine and paracrine estrogen regulation.

Elicitation of biological responses by estrogen in target tissues requires the presence of ER as well as receptor-active ligand in the local microenvironment. Though much attention has been devoted to the study of the receptor in estrogen target tissues, the concept is emerging that tissue estrogen sensitivity may also be regulated by ligand availability through metabolic transformation in situ. Here, we show that targeted disruption, in the mouse, of an estrogen metabolic enzyme, estrogen sulfotransferase (EST), causes structural and functional lesions in the male reproductive system. EST catalyzes the sulfoconjugation and inactivation of estrogen and is expressed abundantly in testicular Leydig cells. Although knockout males were fertile and phenotypically normal initially, they developed age-dependent Leydig cell hypertrophy/hyperplasia and seminiferous tubule damage. Development of these lesions in the testis could be recapitulated by exogenous E2 administration in younger knockout mice, suggesting that they arose in older knockout mice from chronic estrogen stimulation. Older knockout mice were also found to have reduced testis and epididymis weights but increased seminal vesicle/coagulating gland weight because of tissue swelling. Furthermore, total and forward sperm motility of older knockout mice was reduced by 60% and 80%, respectively, and these mice produced smaller litters compared with age-matched wild-type males. These findings establish a role for EST in the male reproductive system and indicate that intracrine and paracrine estrogen activity can be modulated by a ligand transformation enzyme under a physiological setting. Thus, inhibition of estrogen metabolic enzymes by environmental chemicals, as has been demonstrated recently for the human EST, may constitute a novel mechanism of endocrine disruption in vivo.

Production of transgenic mice.

A "transgenic" mouse is identified by the integration of a foreign DNA into its genome. Such animals serve as experimental systems for the study of gene expression and are often generated as models for human diseases. Direct microinjection of DNA into the male pronucleus of a mouse zygote has been the method most extensively used in the production of transgenic mice. Our method is subdivided into three sections: First, Preinjection, where the animals, the donor eggs, and the injection tools are presented. Second, Injection, where the egg handling and micromanipulation is described. Third, Postinjection, where the surgical transfer into pseudopregnant females completes the procedural sequence. Additional sections are provided to include the materials we use and to offer a series of technical tips that cover various aspects of the transgenic process.

Dialysis addition of trehalose/glycerol cryoprotectant allows recovery of cryopreserved mouse spermatozoa with satisfactory fertilizing ability as assessed by yield of live young.

Mouse sperm cryopreservation provides a means for storing the genetic information in genetically modified mice (mutants, transgenics, and "knockouts") in a cost- and space-effective manner. Sperm from this species are highly sensitive to cryodamage, which has impeded their cryopreservation in the past. The cryoprotectant used in this study was 6% glycerol (0.65 M) plus 7.5% trehalose (0.22 M), which was added to a concentrated suspension of sperm from B6SJLF1/J mice in bicarbonate-free buffer by dialysis to minimize osmotic stress on the cells. Sperm suspensions were frozen in 0.25 mL straws and stored in liquid N2. Eggs were obtained from B6SJLF1/J superovulated females. For in vitro fertilization (IVF), 15-25 microL of sperm suspension post-thaw from one straw was added directly to each of three 1.5 mL drops of fertilization medium containing 30 eggs each, for 3 replicates per experiment. The fertilized eggs were scored for blastocyst formation, after which 12 blastocysts from each drop were implanted into pseudopregnant CD-1 females. The number of live pups were then scored at birth. Ten experiments yielded 21.7 +/- 1.4 (SD) blastocysts per 30 eggs inseminated (72%) and 7.3 +/- 0.4 (SD) live pups per 12 blastocysts implanted (61%). The overall yield of live pups was 44 per 100 eggs inseminated (44%). This yield should be satisfactory for maintaining a mouse strain through sperm cryostorage, with restart of the strain through IVF and embryo transfer. The method should also provide improvement in human sperm cryopreservation, as human sperm are less sensitive to cryodamage than are mouse sperm.

Widespread distribution of cells containing human DNA in embryos derived from mouse eggs injected with human chromosome fragments.

The possibility that metaphase chromosomes can serve as a source of genetic material for making transgenic mice was suggested by our previous finding of the incorporation of human satellite DNA into mouse embryos that were injected with microdissected human centromeric fragments. In the present study, we further examined whether this chromosome transfer method can be used to generate transgenic mice containing a portion of human chromosome 4 spanning the Huntington's disease (HD) gene. For this purpose, we used an improved method of metaphase chromosome preparation that may minimize the potential for DNA damage. Using metaphase chromosomes prepared in this manner, chromosome fragments spanning the region of chromosome 4 containing the HD gene were microdissected, retrieved, and injected into fertilized mouse eggs. The injected eggs exhibited good viability and developed with a high efficiency when implanted into foster mothers. To determine whether the human DNA from the injected chromosome fragment had been incorporated into the mouse genome, embryos were harvested at 12.5 days of gestation (dg) and analyzed by in situ hybridization using a human Alu repetitive DNA probe. This analysis showed that most of the embryos contained cells with human Alu repeats. However, all of the embryos were mosaic, and the level of mosaicism was such that we were not able to determine the precise chromosomal origin of the human DNA insert. We discuss the possible basis for the mosaicism and the potential value of such mosaic animals for studying Huntington's disease.

Introduction of human DNA into mouse eggs by injection of dissected chromosome fragments.

A procedure has been developed for introducing exogenous DNA into mouse eggs by injection of chromosome fragments. Chromosome fragments were dissected from human metaphase spreads and microinjected into the pronuclei of fertilized mouse eggs. Many of the injected eggs subsequently exhibited normal pre- and postimplantation development. Embryos obtained from eggs injected with centromeric fragments retained human centromeric DNA as demonstrated by in situ hybridization analysis. From eggs injected with noncentromeric fragments, a mouse was obtained whose tail tissue exhibited the presence of human DNA. This procedure should facilitate incorporation of very large (more than 10 megabases) DNA fragments into cells and embryos without the need for cloned sequences.

Cell surface glycoproteins mediate compaction, trophoblast attachment, and endoderm formation during early mouse development.

Early mouse embryos undergo several morphogenetic processes, such as compaction, trophoblast attachment, and endoderm formation that can be studied in vitro. Several polyspecific and monospecific antisera have been used to perturb these processes in a nontoxic, reversible fashion. One of the antibody-defined molecules, cell CAM 120/80, promotes epithelial cell adhesion, embryo compaction, and endoderm formation. The results suggest the presence of another such molecule(s) involved in these same processes. Evidence is also presented that another set of antibody-defined molecules, GP 140, involved in attachment of somatic cells to the substrate, mediates trophoblast attachment of the mouse blastocyst. The possible role of these molecules in governing the processes leading to cell lineages in the mouse embryo is discussed.

Identification and purification of a cell surface glycoprotein mediating intercellular adhesion in embryonic and adult tissue.

An antiserum against material shed into serum-free medium by MCF-7 human mammary carcinoma cells (anti-SFM II) disrupts cell-cell interactions in murine mammary tumor epithelial cells (MMTE). We now report purification of an 80 kd glycoprotein (GP80) from SFM of MCF-7 mammary carcinoma cells that blocks the activity of anti-SFM II. Anti-SFM II also inhibits compaction of eight-cell mouse embryos, and purified GP80 blocks this reaction. An antiserum against purified GP80 (anti-GP80) has all adhesion-disrupting activities displayed by anti-SFM II. It recognizes one band at 80 kd in SFM and a 120 kd band in detergent extracts of epithelial but not fibroblastic cells. In immunofluorescence studies it is restricted to sites of cell-cell interaction in cultured epithelial cells. Thus a cell surface glycoprotein of 120 kd, the medium form of which is approximately 80 kd, which is neither species nor tissue specific, is expressed at early stages of mammalian development and is found on epithelia.