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Mutations in the Cu/Zn superoxide dismutase (SOD1) gene cause familial amyotrophic lateral sclerosis (FALS) by gain of an aberrant function that is not yet well understood. The role of Cu2+in mediating the toxicity of mutant SOD1 has been earnestly contested. We tested thein vivoeffects of genetically induced copper deprivation on the ALS phenotype of transgenic mice expressing G86R mutant mouse SOD1, a protein that fails to incorporate Cu2+in its active site. Genetically copper-deficient SOD1G86Rtransgenic mice were produced by mating SOD1G86Rmales to female carriers of the X-linked mottled/brindled (Mobr) mutation. We found that the Mobr allele causes a severe (∼60%) depletion of spinal cord copper levels; however, despite the burden of double genetic lesions, it lengthens the lives of SOD1G86Rtransgenic mice by 9%. These findings provide evidence supporting a role for copper in the pathogenesis of FALS linked to SOD1 mutations.

In a clinical safety trial involving an adeno-associated virus (AAV) gene therapy vector encoding human factor IX, intrahepatic administration of the vector was associated with the finding of vector DNA in semen that persisted for several weeks. Uncertainty remains as to the route by which the vector reached semen, but the finding raised the prospect that mature sperm could be exposed to the vector and sustain integration of vector DNA. To provocatively test for the ability of AAV vectors to transduce mature sperm, we exposed mouse sperm to concentrations of the same vector used in clinical studies at concentrations ranging from 840 to 3400 particles per sperm cell, performed in vitro fertilization and embryo transfer, and evaluated newborn pups by Southern analysis for the presence of vector sequences. Of 102 pups analyzed, none showed evidence of vector DNA integration. We conclude from these studies that exposure of mature sperm to AAV gene therapy vectors is highly unlikely to lead to germline transduction.

Background Human α-galactosidase A (α-Gal A) is the lysosomal enzyme that cleaves α-galactosyl residues from glycoconjugates and is the deficient enzyme in Fabry disease. To date, there have been no studies on the regulation of this “housekeeping” gene. Methods Transgenic mice were established with either 1) a 13.3-kilobase (kb) human genomic fragment that contained 246 bp of 5′- and approximately 2.8 kb of 3′- untranslated sequences, or 2) an “intronless” construct derived from the genomic sequence with the 5′ and 3′ flanking regions intact. Tissues that expressed high levels of α-Gal A activity were examined by light and electron microscopy. Results Transgenic mice were generated with 2 and 12 copies of the genomic sequence (Lines 1 and 2) or about 60 copies of the intronless construct (Lines 3 and 4). In mice hemizygous for the genomic transgene (Lines 1 and 2), tissue α-Gal A activities were 12 to 155 times higher than those in the respective wild-type tissue, depending on tissue and transgene copy number. Of note, the high overexpression did not alter the cellular or subcellular cytoarchitecture. In contrast, α-Gal A activities expressed by mice that carried the intronless construct were only two- to sixfold more than in wild-type tissues in which the genomic transgene was highly expressed. Conclusions The remarkably high levels of α-Gal A expression in transgenic mice carrying the intact genomic sequence versus the intronless construct suggested that the genomic sequence contained a strong intronic enhancer element. Identification of this regulatory element or elements may be useful in efforts to overexpress human α-Gal A for gene therapy endeavors. In addition, overexpression of human α-Gal A did not affect cellular morphology, which indicates that its overexpression in gene therapy endeavors should be safe.

The risk of insertion of adenovirus gene therapy DNA into female germ cells during the course of somatic gene therapy was stringently tested in the mouse by injecting up to 10 10 infectious particles directly into the ovary and by incubating naked oocytes in a solution of 2 × 10 8 particles/ml for 1 h prior to in vitro fertilization (IVF). The vector used was a recombinant adenovirus carrying the bacterial lacZ gene driven by the cytomegalovirus promoter (Adβ-gal). Ovaries were stained for LacZ activity, or immunochemically for LacZ, 5–7 days after injection. Although very large amounts of LacZ activity and protein were detected, all positive staining was in the thecal portion of the ovary, with no staining seen in oocytes. In another series of experiments, mice with injected ovaries were mated, and preimplantation embryos or fetuses were analyzed either for LacZ expression or by PCR for lacZ DNA. None of 202 preimplantation embryos stained positively for LacZ and none of 58 fetuses were positive for DNA by PCR analysis. Finally, more than 1400 eggs were fertilized after exposure to the vector prior to IVF and stained as morulae for LacZ activity. Fewer than 2% of the embryos stained positively for LacZ, and experiments indicated that the staining was due to incomplete washing of the eggs prior to IVF. These data provide strong evidence that adenoviruses cannot infect oocytes and that the risk of female germ-line transduction with such vectors is very low.

Mutations in the cytoplasmic Cu/Zn superoxide dismutase (SOD1) gene on human chromosome 21q22.1 cause 10-20% of familial amyotrophic lateral sclerosis (ALS) cases. The expression of the ALS phenotype in mice carrying the murine G86R SOD1 mutation is highly dependent upon the mouse genetic background. This is similar to the phenotypic variation observed in ALS patients containing identical SOD1 mutations. In the FVB/N background, mice expressing mG86R SOD1 develop an ALS phenotype at approximately 100 days. However, when these mice were bred into a mixed background of C57Bl6/129Sv, the onset of the ALS phenotype was delayed (143 days to >2 years). Using 129 polymorphic autosomal markers in a whole genome scan, we have identified a major genetic modifier locus with a maximum lod score of 5.07 on mouse chromosome 13 between D13mit36 and D13mit76. This 5- to 8-cM interval contains the spinal muscular atrophy (SMA)-associated gene Smn (survival motor neuron) and seven copies of Naip (neuronal apoptosis inhibitory protein), suggesting a potential link between SMA and ALS.

Pathologic accumulation of neurofilament protein (NF), both within spheroids of the proximal axon and within inclusions of motor neuron somata, is a hallmark of neurodegeneration in amyotrophic lateral sclerosis (ALS). Transgenic mice that express mutations in superoxide dismutase (SOD-1), which were genetically linked to familial ALS, develop symptomatology and pathology that strongly resemble ALS and therefore provide a useful model for studying the disease. Examining NF in the G86R mutant SOD-1 transgenic mice, we previously demonstrated that phosphorylated NF accumulates in motor neuron somata of symptomatic transgenic mice. In the present study, we expand these results by examining the immunocytochemical distribution of the three subunits of NF (i.e., light, medium, and heavy chains) as well as tubulin in presymptomatic and symptomatic SOD-1 transgenic mice. Although all NF subunits, but not tubulin, accumulate along with phosphorylated NF in the spinal cord inclusions of symptomatic mice, numerous inclusions containing only light chain NF are found in the spinal cord of presymptomatic SOD-1 transgenic mice. In addition to these results in the spinal cord, intensely immunoreactive aggregates of NF-L, but not the other NF subunits or tubulin, were observed in the sciatic nerve of both symptomatic and presymptomatic mutant SOD-1 transgenic mice. These results suggest that the mechanism of NF alteration in SOD-1 transgenic mice, and also perhaps in ALS patients, originates with the disruption of NF-L, only later involving the other subunits.

The potential for adenovirus gene therapy vectors to gain access to male germ cells was rigorously tested in the mouse by injecting high titers of the vector directly into the testis and epididymis, or by exposing sperm to the vector immediately prior to or during in vitro fertilization. The adenovirus vector carried the bacterial lacZ gene (Adbeta-Gal) driven by the Rous sarcoma virus (RSV) promoter, and infection was assessed by testing for lacZ expression, either with antibodies to LacZ protein or by staining for LacZ enzymatic activity. A total of 109 plaque-forming units (PFU) was inserted into the testis or epididymis, and in vitro fertilization was performed after sperm were exposed either to 10 or 100 PFU per sperm cell. lacZ expression was examined within testes for several weeks after injection, and in preimplantation embryos produced by in vitro fertilization with sperm exposed to the gene therapy vector. Direct injection of Adbeta-Gal into either the testis or epididymis resulted in lacZ expression only within the interstitium of the testis and not within seminiferous tubules. Despite direct exposure of spermatogenic cells or mature sperm to high titers of virus, lacZ expression was likewise not detected in embryos. These findings are consistent with the conclusion that the risk is minimal for germ line integration of adenovirus vectors exposed to male reproductive cells.

International audience; Molecular mechanisms promoting neuronal death in amyotrophic lateral sclerosis (ALS) were investigated using transgenic mice that overexpressed the G86R mutated form of the Cu/Zn superoxide dismutase (SOD1) gene. We observed: (i) alteration of the Bcl-x/Bax ratio and (ii) activation of the transcription factor p53, as deduced from its location within neuron nuclei. We further demonstrated that ectopic expression of the G86R mutant SOD1 in PC12 cells enhanced both p53 expression and phosphorylation, leading to transcriptional stimulation of p53-responsive genes. These findings provide evidence that the p53 signaling pathway is activated in SOD1-linked familial ALS and may play a causative role in spinal cord neuron apoptosis by modulating the Bcl-x/Bax ratio.

In the present study we analyze the molecular mechanisms underlying motor neuron degeneration in familial amyotrophic lateral sclerosis (FALS). For this, we used a transgenic mouse model expressing the Cu/Zn superoxide dismutase (SOD1) gene with a Gly(86) to Arg (G86R) mutation equivalent to that found in a subset of human FALS. Using an optimized suppression subtractive hybridization method, a cDNA specifically up-regulated during the asymptomatic phase in the lumbar spinal cord of G86R mice was identified by sequence analysis as the KIF3-associated protein (KAP3), a regulator of fast axonal transport. RT-PCR analysis revealed that KAP3 induction was an early event arising long before axonal degeneration. Immunohistochemical studies further revealed that KAP3 protein predominantly accumulates in large motor neurons of the ventral spinal cord. We further demonstrated that KAP3 up-regulation occurs independent of any change in the other components of the kinesin II complex. However, since the ubiquitous KIF1A motor is up-regulated, our results show an early and complex rearrangement of the fast axonal transport machinery in the course of FALS pathology.

In recent years, several mouse models of amyotrophic lateral sclerosis (ALS) have been developed. One, caused by a G86R mutation in the superoxide dismutase-1 (SOD-1) gene associated with familial ALS, has been subjected to extensive quantitative analyses in the spinal cord. However, the human form of ALS includes pathology elsewhere in the nervous system. In the present study, analyses were extended to three motor nuclei in the brainstem. Mutant mice and control littermates were evaluated daily, and mutants, along with their littermate controls, were killed when they were severely affected. Brains were removed after perfusion and processed for Nissl staining, the samples were randomized, and the investigators were blinded to their genetic status. Stereologic methods were used to estimate the number of neurons, mean neuronal volumes, and nuclear volume in three brainstem motor nuclei known to be differentially involved in the human form of the disease, the oculomotor, facial, and hypoglossal nuclei. In the facial nucleus, neuron number consistently declined (48%), an effect that was correlated with disease severity. The nuclear volume of the facial nucleus was smaller in the SOD-1 mutant mice (45.7% difference from control mice) and correlated significantly with neuron number. The oculomotor and hypoglossal nuclei showed less extreme involvement (

Amyotrophic lateral sclerosis (ALS) is a fatal, paralytic disorder that primarily affects motoneurons. By combining physiological and morphological approaches, we examined the effect of a murine superoxide dismutase 1 (SOD1) mutation (G86R), which induces neurological disorders resembling human familial ALS (FALS), on the arginine vasopressin (AVP) hypothalamo-neurohypophysial axis, an unmyelinated tract poor in neurofilaments. First, we observed that G86R mice progressively consumed more water than wild-type littermates. Furthermore, levels of plasma AVP and neurohypophysial AVP content were decreased in the SOD1 mutant mice, whereas the amount of hypothalamic AVP increased in an age-dependent manner. However, hypothalamic AVP mRNA levels were not significantly modified in these animals. At the ultrastructural level, we found that the neurohypophysis of G86R mice had a decreased number of neurosecretory axons. Conversely, the presence of large axon swellings was more pronounced in the SOD1 mutant mice. In addition, the size of neurosecretory granules was higher in G86R than in wild-type animals. All these findings strongly suggest that the FALS-associated SOD1 mutation injures the hypothalamo-neurohypophysial axis by provoking early, progressive disturbances in the axonal transport of neurosecretory products from neuronal perikarya to nerve terminals. This blockade could ultimately result in degeneration of the tract, as proposed for the myelinated, neurofilament-enriched motor axons affected by ALS.

Amyotrophic lateral sclerosis (ALS) is a devastating neurologic disease characterized by progressive motor dysfunction that leads to paralysis and eventually death. There are numerous hypotheses for the pathogenesis of this disease, but the mechanisms of degeneration were difficult to investigate before the development of animal models. Transgenic mice with alterations in either the superoxide dismutase (SOD-1) or neurofilament genes display motor neuron pathology and deficits in motor function and, therefore, provide animal models for the study of ALS neurodegeneration. Using these animal models, as well as several in vitro models, researchers have made rapid progress during the last several years toward understanding the cause and mechanism of ALS neurodegeneration. These studies have demonstrated that motor neuron degeneration in ALS may be secondary to a number of causes, including neurofilament disruption, mutations in SOD-1, and glutamate excitotoxicity. Although each of these mechanisms can cause motor neuron degeneration by itself, studies of transgenic mice have indicated several points at which these mechanisms may interact, suggesting that they are components of one general mechanism of neurodegeneration.

Excitotoxicity has been hypothesized to contribute to amyotrophic lateral sclerosis (ALS) neurodegeneration. The similar pattern of vulnerability in the spinal cord of mutant superoxide dismutase (SOD-1) transgenic mice and mice treated with excitotoxins supports a role for excitotoxicity in the mechanism of degeneration. The distribution of the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) class of glutamate receptors (GluRs) with different calcium permeabilities has been proposed as an explanation for this differential vulnerability. GluR2 appears to be the dominant determinant of calcium permeability for AMPA receptors; thus, it is critical for their contribution to excitotoxic mechanisms. In this study, we investigate the distribution of GluR2 immunoreactivity in the spinal cord of control and SOD-1 transgenic mice. GluR2 immunoreactivity is present equally within vulnerable neurons (i.e., motor neurons and calretinin-immunoreactive neurons) as well as nonvulnerable neurons (i.e., calbindin-immunoreactive neurons and dorsal horn neurons). In addition, postembedding immunoelectron microscopy reveals that GluR2 is present in synapses of dorsal and ventral horn neurons and that the percentage of labeled synapses and numbers of immunogold particles per synapse do not vary between these spinal cord regions. Comparing control mice with SOD-1 transgenic mice, at both the light and the electron microscopic levels, the distribution and intensity of GluR2-immunoreactivity do not appear to be altered. These results suggest that the cellular and synaptic distribution of GluR2 is not a determinant of the selective vulnerability observed in SOD-1 transgenic mice or in ALS patients.

Transgenic mice with a G86R mutation in the mouse superoxide dismutase (SOD-1) gene, which corresponds to a mutation observed in familial amyotrophic lateral sclerosis (ALS), display progressive motor dysfunction leading to paralysis and premature death. In endstage SOD-1 transgenic mice, there is marked loss of spinal motor neurons and interneurons, accumulation of phosphorylated neurofilament inclusions, and reactive astrocytosis. The present study details the time course and ultrastructural appearance of these pathologic changes and correlates the timing of these events with the behavioral symptoms. There is no significant reduction in the number of total neurons, motor neurons, or interneurons in the ventral spinal cord of presymptomatic mice, as compared to age-matched control mice. In contrast, there is a significant reduction in the number of total neurons (−23.5%), motor neurons (-28.9%), and interneurons (-23.5%) in symptomatic SOD-1 transgenic mice. This neuron loss correlates temporally with the onset of reactive astrocytosis and the appearance of phosphorylated neurofilament inclusions. The identical timing of motor neuron and interneuron degeneration in this model of ALS strongly suggests that degeneration in the spinal cord of patients with ALS is not specifically directed at motor neurons, but rather more generally at several populations of neurons in the spinal cord. In addition, the late onset and rapid progression of neuron loss suggest that a toxic property is accumulating while the SOD-1 transgenic mice are presymptomatic, and that this toxic property must reach a threshold level before the onset of neuronal degeneration. J. Comp. Neurol. 391:64–77, 1998. © 1998 Wiley-Liss, Inc.

It has been recognized for several decades that the number of cleavage divisions which precede blastocyst formation in the mammalian embryo is rigorously fixed, such that removal of cells from the embryo, or augmentation of cell number by embryo aggregation, does not affect the timing of blastulation. Instead, embryos manipulated so as to reduce cell number form small blastocysts with fewer numbers of cells, while aggregate embryos form giant blastocysts. This tight control of the number of cleavage divisions ensures that the timing of blastocyst formation corresponds to the period of uterine receptivity for implantation. As yet, no experimental manipulation has succeeded in altering control of the number of cleavage divisions prior to blastulation, and as a consequence, the biological basis for the control mechanism is entirely obscure. We report here that removal of cytoplasm from one-celled mouse embryos does not alter the rate of cleavage, but does induce precocious formation of small blastocysts. These findings suggest that the early embryo "counts" cleavage divisions by measuring the size of its blastomeres, and that experimental reduction of cell size disturbs the counting mechanism and leads to abnormally early blastulation.

The purposes of the research reported here were first to explore a murine model for human placental and umbilical cord blood transplantation and second to evaluate the engraftment ability of ex vivo cultured hematopoietic cells. Murine near-term fetal and neonatal peripheral blood (FNPB) cells, genetically marked with the human multiple drug resistance transgene (MDR1) were used for syngeneic transplants into sublethally irradiated adult mice. Donor cells were transplanted either fresh and untreated, or after ex vivo culture in the presence of the hematopoietic growth factors recombinant murine stem cell factor, recombinant human interleukin-3 (rHu IL-3), and rHu IL-6, in a liquid culture system. To evaluate, count, and characterize FNPB progenitor cell-derived colonies, neonatal mouse mononuclear cells were cultured directly in methylcellulose with growth factors. To assess their ex vivo expansion ability, FNPB mononuclear cells were first cultured in liquid medium for 3 to 8 days and then transferred to semisolid assay plates. Evaluation of the cell counts after liquid culture showed a 1.4- to 11.6-fold increase, and the numbers of colonies observed in methylcellulose were similar to those produced by fresh FNPB cells. Donor-type engraftment was demonstrated by polymerase chain reaction (PCR) amplification of the human MDR1 transgene in the peripheral blood of all surviving animals (5 of 7 recipients of the fresh, and 3 of 8 recipients of the ex vivo–cultured cells) 2 to 4 months after transplantation. The proportion of donor leukocytes in the peripheral blood of the recipients (chimerism) was evaluated using fluorescence in situ hybridization (FISH) analysis 4 to 6 months after transplantation and ranged from 2% to 26%. In addition, bone marrow cultures were obtained from two recipient animals: one had received fresh-untreated cells and was evaluated 8 months after transplant, the other had received ex vivo cultured cells and was tested 14 months after grafting. The derived hematopoietic colonies were tested by PCR and the transgene was detected, conclusively proving long-term engraftment of donor cells. These results indicate that FNPB transplants can be successfully performed in sublethally irradiated mice with and without ex vivo culture. Long-term donor-type engraftment with sustained chimerism has been demonstrated. Thus, murine neonatal blood grafts can be used as an animal model for cord blood transplantation for gene therapy studies where complete myeloablation is not desirable and partial replacement of defective marrow may be sufficient. Furthermore, the possibility of numerically expanding hematopoietic progenitor cells contained in neonatal blood without affecting their engraftment ability could facilitate use of cord blood grafts in adult recipients.

Transgenic technology allows the introduction of new and functional genetic material into the germ line. The first advanced eukaryotes to prove amenable to such genetic modifications were mice (Gordon and others 1980; Gordon and Ruddle 1981); subsequently, a variety of species including fish and fruit flies have been subject to transgenic manipulation. Germline gene transfer has enormously increased the rate of progress in understanding mammalian development. As a consequence of this improved knowledge, important insights into many disease processes have been gained, and new strategies for genetic engineering of livestock have been devised. These many advances have been comprehensively reviewed elsewhere (Palmiter and Brinster 1986; Gordon 1989). A related publication has also previously appeared in this ILAR News (Gordon 1988). In this paper transgenic technology will be discussed with the issues of laboratory animal sciences as a reference point. The extant approaches to gene transfer will first be discussed, after which some representative examples of transgenic experiments for studying gene regulation and creating models of disease will be presented. Throughout this article, issues of reproductive management, aspects of colony maintenance that require special emphasis, and IACUC review of transgenic protocols will serve as themes. However, in the last segment of the manuscript, direct focus on specific aspects of laboratory animal management will be provided.

Transgenic mice with a G86R mutation in the mouse superoxide dismutase (SOD-1) gene, which corresponds to a mutation that has been observed in familial amyotrophic lateral sclerosis (ALS), display progressive loss of motor function and provide a valuable model of ALS. The pathology in the spinal cords of these mice was evaluated to determine whether there are chemically identified populations of neurons that are either highly vulnerable or resistant to degeneration. Qualitatively, there were phosphorylated neurofilament protein (NFP)-immunoreactive inclusions and a pronounced loss of motoneurons in the ventral horn of the spinal cord without the presence of vacuoles that has been reported in other SOD-1 transgenic mice. Neuron counts from SOD-1 and control spinal cords revealed that the percentage loss of NFP-, choline acetyltransferase (ChAT)-, and calretinin (CR)-immunoreactive neurons was greater than the percentage loss of total neurons, suggesting that these neuronal groups are particularly vulnerable in SOD-1 transgenic mice. In contrast, calbindin-containing neurons did not degenerate significantly and represent a protected population of neurons. Quantitative double-labeling experiments suggested that the vulnerability of ChAT- and CR-immunoreactive neurons was due primarily to the presence of NFP within a subset of these neurons, which degenerated preferentially to ChAT- and CR-immunoreactive neurons that did not colocalize with NFP. Our findings suggest that NFP, which has been demonstrated previously to be involved mechanistically in motoneuron degeneration, may also be important in the mechanism of degeneration that is initiated by the SOD-1 mutation. © 1996 Wiley-Liss, Inc.

Each year, world wide, tens of thousands of zygotes derived from the in-vitro insemination of human oocytes undergo polyspermic fertilization. These embryos must presently be discarded because it has never been demonstrated in any mammal that polyspermic zygotes can develop normally to term after removal of the supernumerary male pronucleus. Our study was undertaken to test the developmental potential of polyspermic zygotes corrected by micromanipulation. Mouse oocytes were inseminated zona-free, and polyspermic zygotes were manipulated so as to remove one of the two male pronuclei. Surviving embryos were then observed for further development in vitro and after transfer into pseudopregnant females. Of 58 zygotes manipulated, 18 developed to the blastocyst stage and were transferred. Five animals (two male and three females) were born. The agouti coat colour marker confirmed the genotypes of the gametes. All five animals developed to normal-appearing adults, and all five produced at least 10 normal offspring. One adult founder animal was karyotyped and found to have a normal chromosome complement. These results demonstrate for the first time that a mammalian egg that has undergone polyspermic fertilization can develop normally after restoration of the diploid state by micromanipulation. Accordingly, the results provide impetus for attempting to rescue polyspermic human embryos.

Objective To determine if unprocessed human spermatozoa could capacitate in an IVF microchamber. Design Semen was loaded into an IVF microchamber with zona-free hamster oocytes, and a hamster penetration test was performed. Setting Medical School basic research laboratory. Patients Men who provided sperm for other laboratory tests. Interventions None. Main Outcome Measures Penetration of zona-free hamster oocytes by human sperm. Results In 12 of 19 sperm samples, hamster egg penetration was detected. In most negative cases other measures of sperm function suggested that the SPA might be compromised. Conclusions Positive hamster oocyte penetration after loading of unprocessed semen into an IVF microchamber indicates that the chamber supports sperm capacitation. Use of such microchambers thus might reduce or eliminate many steps in sperm processing.

To compare routine reinsemination with 2nd day micromanipulation in patients with poor day 1 fertilization.A retrospective review of patient records.The Mount Sinai Medical Center Assisted Reproductive Technologies Program.Patients undergoing IVF-ET who had poor fertilization (35%) with standard insemination and underwent second day reinsemination of oocytes (group I, n = 84) compared with patients who underwent 2nd day micromanipulation with subzonal insemination (group II, n = 12).Fertilization rate, cleavage rate, number of embryo transfers, and pregnancy rate.Fertilization rate and cleavage rate were significantly higher in group II patients. Pregnancies per transfer were similar between groups I (3/21, 14.3%) and II (0/9, 0%). Second day fertilization was possible in 9 of 12 group II patients, and fertilization rate was higher than day 1 in all nine, however, only 50% achieved cleavage, and none achieved pregnancy.Although micromanipulating oocytes that fail to fertilize may identify occult male factor infertility, may help the clinician plan future cycles, and may result in fertilization and even transfer of embryos in some cycles, there were no pregnancies in our series, and, for now, the clinical efficacy of this procedure remains in question.

Amyotrophic lateral sclerosis is a progressive neurodegenerative disorder primarily involving motoneurons. A subset of individuals with familial autosomal dominant forms of the disease have mutations of the copper/zinc superoxide dismutase (Cu/Zn SOD, SOD-1) gene, which encodes a ubiquitously expressed enzyme that plays a key role in oxygen free radical scavenging. This observation suggests that altered or reduced SOD-1 activity may play a role in the neurodegenerative process. To explore this possibility further, we have introduced a mutation into the mouse SOD-1 gene that corresponds to one of the changes found in the human gene in familial amyotrophic lateral sclerosis. Integration and expression of this mouse gene in transgenic mice was identified by the presence of a unique restriction enzyme site in the transgene coding sequence generated by introduction of the mutation. We report here that high expression of this altered gene in the central nervous systems of transgenic mice is associated with an age-related rapidly progressive decline of motor function accompanied by degenerative changes of motoneurons within the spinal cord, brain stem, and neocortex. These findings indicate a causative relationship between altered SOD activity and motoneuron degeneration. Moreover, biochemical studies indicate normal levels of total SOD activity in transgenic mouse tissues, results that indicate that the neurodegenerative disorder does not result from a diminution of activity and, as such, represents a dominant "gain of function" mutation.

The results of subzonal sperm insertion (SUZI) have been retrospectively analysed in a subset of patients with normal sperm counts who were found to require SUZI because of poor or absent fertilization of zona-intact oocytes. This patient group is of particular interest because male factor-related infertility cannot be due to insufficient numbers of spermatozoa reaching the oocytes. Thus, failed fertilization can be attributed to deficiencies in one or more steps in the fertilization process, and SUZI provides a method of distinguishing defects of zona pellucida penetration from gamete fusion. A total of 26 such patients were treated identically to and concurrently with a much larger group of SUZI candidates who typically suffered from oligozoospermia, and fertilization results were compared. Fertilization rates after SUZI were higher in patients with normal counts than in oligozoospermic patients (51 and 26% respectively), indicating that the proportion of spermatozoa capable of fusing with the oocyte is the same or higher in the group with normal counts. In addition, nearly all SUZI procedures led to fertilization (23/26), with two out of three failed fertilizations occurring in cases where two or less oocytes were manipulated, results which further indicate that failed fertilization in these patients is not due to a defect at the level of gamete fusion. These findings suggest that infertility in these patients is based upon the inability of the spermatozoa to reach the oolemma and thus, that their fertility defect resides at the step of zona penetration.

The alpha-fetoprotein (AFP) gene is normally expressed in fetal liver and transcriptionally silent in adult tissues, but can be abnormally reactivated in hepatocellular carcinoma (HCC). We linked 7.6 kb of 5'-flanking DNA from the mouse AFP gene to the herpes simplex virus (HSV) thymidine kinase gene (tk), and a line of transgenic mice was produced that expressed TK in a pattern similar to endogenous AFP. When these AFP/tk transgenic mice were crossed to another transgenic line that develops multifocal HCC due to expression of a SV40 large T-antigen transgene under regulation of the albumin promoter/enhancer complex, a significant delay of tumor progression could be achieved by administration of ganciclovir (GCV), a cytotoxic compound that is a substrate for phosphorylation by viral, but not mammalian, TK. Control animals carrying only the tk gene were unaffected by GCV treatment. These results illustrate the feasibility of prophylactic gene therapy for ablation of cancer, utilizing a strategy in which the tk gene is regulated by a promoter expected to be active only in tumor cells.

Objective To determine the developmental potential of mouse embryos that underwent cryopreservation after micromanipulation of the zona pellucida. Design Gaps were produced in the zona pellucida of mouse oocytes or two-cell embryos by zona drilling with acid Tyrode's solution. Zona-drilled oocytes were fertilized in vitro and cultured to the two-cell stage. Two-cell embryos were frozen, thawed, and cultured to the expanded blastocyst stage. Results There was no difference in the rate of embryo survival post-thaw (248/318, 77% versus 288/345, 83.4%), or in the rate of development to the expanded blastocyst stage (91/248, 36.7% versus 88/288, 30.6%), between embryos that were zona drilled as oocytes and unmanipulated controls. Similarly, there was no difference in the rate of cryosurvival (206/217, 94.9% versus 168/187, 89.8%) or development to the blastocyst stage (154/206, 74.7% versus 132/168, 78.6%) between embryos that were fertilized in vivo and zona drilled at the two-cell stage and embryos that were unmanipulated. Conclusions These findings indicate that small gaps in the zona pellucida, such as those that result from micromanipulation, do not significantly alter the ability of embryos to withstand cryopreservation.

Three different micromanipulation procedures were used to assist human fertilization in cases of severe male factor infertility. Zona drilling was performed either with acid Tyrode's solution, mechanically following zona softening with chymotrypsin, or by partial zona dissection. The fertilization rate was lowest in the zona drilling/acid Tyrode's group (7/40; 17.5%), although no differences between groups (zona drilling/chymotrypsin: 21/84, 25%; partial zona dissection: 31/143, 21.7%) were significant. The fertilization rate was significantly increased relative to untreated eggs from the same patients only in the partial zona dissection group (31/143, 21.7% versus 4/102, 3.9%). Oocyte damage occurred at a high rate as a result of zona drilling with acid Tyrode's solution (13/41, 37%). Embryonic development was compromised after zona drilling with chymotrypsin: only 7/12 (58.3%) of the fertilized oocytes cleaved, and the morphology of many of the cleaved embryos was abnormal. Although only 61% (16/26) of the diploid embryos resulting from partial zona dissection cleaved, the embryonic morphology of these embryos was comparable with controls. No pregnancies resulted from the transfer of manipulated embryos. We conclude that although zona manipulation increases the fertilization rate, losses due to oocyte trauma, low rates of diploid fertilization, low rates of cleavage, and a high frequency of abnormal cleavage reduce the number of embryos available for transfer.

Zona drilling is a powerful new tool for increasing the efficiency of IVF in animals and humans. The procedure incurs minimal harm to the oocyte and retains potentially selective prerequisites for sperm fertilizability such as motility. The methodology results in fertilization by sperm which have spontaneously undergone the AR, a situation which contrasts with normal fertilization. However, animal studies indicate that this feature does not compromise embryo development and, further, suggest that many potentially normal sperm are excluded from participating in fertilization by virtue of having undergone the AR. This observation not only reveals fundamental characteristics of normal fertilization, but offers encouragement that zona drilling can be used to increase the number of normal live births in both animal and human systems.

With the mouse as a model, we have used zona drilling to devise procedures for safe removal of the first polar body or one or more blastomeres from cleaving embryos. These methods require minimal disruption of the zona pellucida and little or no direct contact between microtools and the materials to be biopsied. Of 175 eggs subjected to the polar body biopsy procedure, 1 was killed, and 165/174 survivors were fertilized (94.8%). For blastomere biopsy, embryos from the 2- to 16-cell stage were incubated in a chelating medium containing 100 mM sucrose for at least 30 min. The zonae were then drilled, and one or more blastomeres were "pushed" out through the hole by pressure exerted against the zona at some distance from the drilling site. In all 85 embryos biopsied, one or more additional intact blastomeres were successfully removed. Moreover, 83/84 biposied embryos that were subsequently cultured developed into blastocysts (98.8%). Although acid Tyrode's solution was used in this study, mechanical methods of zona opening were also effective. The data indicate that oocyte and embryo biopsy assisted by zona drilling is safe and does not appear to affect fertilization or development, and as such, it is applicable to genetic diagnostic procedures.

Recent advances in mammalian gamete and embryo micromanipulation have stimulated the scientific and medical communities, and to some degree the public at large, to become aware that treatment of genetic disease by direct alteration of the genetic code may soon be possible. Because these micromanipulation techniques result in modification of the genotype at the earliest stages of development, such "gene therapy" affects not only the conceptus itself but also its germ cells. Thus such genetic modifications are heritable and can be transmitted indefinitely to succeeding generations of progeny. In the presentation, both narrow and broad definitions of gene therapy will be considered with respect to the techniques upon which they are based, their potential for treatment of genetic disease, and their current feasibility.

Developments in gene therapy are certain to lead to the treatment of an increasing variety of diseases, some of which will affect patients who might wish to have children following their gene therapy treatment. These circumstances raise the concern that germline integration of gene therapy vector DNA could occur. Although our current understanding of reproductive biology and of the biodistribution of gene therapy vectors administered to extragonadal sites indicate that this risk is low, animal experiments and clinical studies designed specifically to address this question are warranted; because of this risk, every gene therapy vector should be tested for its potential to integrate into germ cells and preimplantation embryos.

The application of assisted reproductive technologies (ART) to nonhuman primates has created opportunities for improving reproductive management in breeding colonies, and for creation of new animal models by genetic modification. One impediment to the application of ART in Saimiri spp. has been the lack of an effective gonadotropin preparation for ovarian stimulation. Pregnant mare serum gonadotropin (PMSG) is inexpensive and readily available, but its repeated use in rhesus monkeys has been associated with induction of a refractory state. We have compared PMSG to recombinant human follicle stimulating hormone (rhFSH) for controlled ovarian stimulation in Bolivian squirrel monkeys. Groups of mature squirrel monkeys received rhFSH (75 IU daily) or PMSG (250 IU twice daily) by subcutaneous injection for 4 d during the breeding season (November to January) or nonbreeding season (March to September). Serum estradiol (E2) was measured daily. Follicular growth was monitored by abdominal ultrasound. During the breeding season, PMSG induced a higher E2 response than did rhFSH, with mean E2 levels being significantly higher within 3 d of stimulation. Superior follicular development in PMSG animals was confirmed by abdominal ultrasonography. During the nonbreeding season, PMSG elicited a similar increase in serum E2 levels despite the fact that basal serum E2 is typically low during the nonbreeding season. Repeated use of PMSG (or = 3 cycles of administration) produced no attenuation of the E2 response. We conclude that PMSG is highly effective for repeated cycles of controlled ovulation stimulation in the squirrel monkey.

Publisher Summary This chapter discusses a procedure for production of transgenic mice by pronuclear microinjection. When one micromanipulation device is chosen over another, the choice may dictate the use of supplies that are especially compatible with the equipment available. Microneedles are pulled from appropriate microcapillary tubing and used directly without further processing. The instrument collar for the holding pipette should be connected via tubing to a microinjection device. The instrument collar for the microneedle may be connected to a 10-cc syringe. Insert the microneedle into the pronucleus and allow the flow of DNA to cause swelling of the pronucleus. Microinjection should not be considered successful unless this swelling is seen. After microinjection, immediately remove the microneedle from the zygote. Release the zygote by applying positive pressure on the holding pipette. Allow the microinjected zygote to drift down to the floor of the dish and then move the stage so as to repeat the process on the next embryo. When all embryos are microinjected, remove them to a dish of M16 for about 1 h of culture.