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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.

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.

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.

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 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.

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.

BACKGROUND: The promulgation and diversification of micromanipulation procedures which open the zona pellucida of the oocyte or early embryo is steadily increasing the chance that zygotes will encounter infectious viral agents or gene transfer vectors derived from these agents. Such interactions could lead to toxic effects on the embryo or to insertion of foreign genes into the germ line. Adenovirus is a ubiquitous human viral pathogen that is commonly used as a gene therapy vector. However, the toxicity of this virus or its vector derivatives to embryos has not been extensively investigated. METHODS: In the present study, a mouse model was used to investigate the time of appearance of embryo toxicity, the manifestations of that toxicity, and the mechanism by which adenoviruses exert toxicity. The effects of exposure to adenovirus on in-vivo embryo development was also examined. RESULTS: These vectors exerted no deleterious effects until after the 2-cell stage, where they caused developmental delay and disorganized cleavage. Toxicity was associated with expression of a lacZ reporter gene cloned into the vectors, and with the proportion of infectious particles within the virus preparations: preparations with a high proportion of ‘empty capsids’, including a preparation of wild-type virus, were less toxic than a replication-defective vector with a high proportion of functional, infectious particles. Subzonal insertion of adenovirus vector followed by embryo transfer led to a dramatic reduction in the number of embryos which developed to term—findings which establish the physiological significance of the findings from in-vitro culture. CONCLUSIONS: These results indicate that adenoviruses and their vector derivatives are not likely to insert genetic material into the germ line, but they may pose a significant threat to the viability of human embryos undergoing opening of the zona pellucida during IVF.

ALZHEIMER'S disease (AD) affects more than 30% of people over 80 years of age1,2. The aetiology and pathogenesis of this progressive dementia is poorly understood, but symptomatic disease is associated histopathologically with amyloid plaques, neurofibrillary tangles and neuronal loss primarily in the temporal lobe and neocortex of the brain. The core of the extracellular plaque is a derivative of the amyloid precursor protein (APP)3, referred to as β/A4 (refs 4–6), and contains the amino-acid residues 29–42 that are normally embedded in the membrane-spanning region of the precursor3. The cellular source of APP and the relationship of its deposition to the neuropathology of AD is unknown. To investigate the relationship between APP over-expression and amyloidogenesis, we have developed a vector to drive expression specifically in neurons of a C-terminal fragment of APP that contains the β/A4 region, and have used a transgenic mouse system7,8 to insert and express this construct. We report here that overexpression of this APP transgene in neurons is sufficient to produce extracellular dense-core amyloid plaques, neurofibrillary tangles and neuronal degeneration similar to that in the AD brain.

Male infertility, affecting as many as 10% of the adult population, is an extremely prevalent disorder. In most cases, the cause of the condition is unknown, and genetic factors that might affect male fertility, other than some sequences on the Y chromosome, have not been identified. We report here that male mice heterozygous for a targeted mutation of the apolipoprotein B (apo B) gene exhibit severely compromised fertility. Sperm from these mice failed to fertilize eggs both in vivo and in vitro. However, these sperm were able to fertilize eggs once the zona pellucida was removed but displayed persistent abnormal binding to the egg after fertilization. In vitro fertilization-related and other experiments revealed reduced sperm motility, survival time, and sperm count also contributed to the infertility phenotype. Recognition of the infertility phenotype led to the identification of apo B mRNA in the testes and epididymides of normal mice, and these transcripts were substantially reduced in the affected animals. Moreover, when the genomic sequence encoding human apo B was introduced into these animals, normal fertility was restored. These findings suggest that this genetic locus may have an important impact on male fertility and identify a previously unrecognized function for apo B.

The missense mutation Lys-296-->Glu (K296E) in the rhodopsin gene produces an opsin with no chromophore binding site and therefore is not activated by light. Nevertheless, the mutant opsin constitutively activates transducin in vitro and causes photoreceptor degeneration in vivo, possibly by continuously activating the phototransduction cascade, analogous to constant exposure to environmental light. We studied the K296E mutation in eight lines of transgenic mice. Each line developed photoreceptor degeneration with the rate of degeneration increasing monotonically as the ratio of mutant:wild-type opsin mRNA increased. At no time in the course of degeneration was there endogenous light adaptation in the retina as measured by the electroretinogram. The mutant opsin was found to be invariably phosphorylated and stably bound to arrestin. Light-independent activation of transducin was demonstrated only after the removal of arrestin and dephosphorylation of K296E opsin. Thus, K296E opsin in vivo does not activate the phototransduction cascade because it is shut off by photoreceptor inactivation mechanisms. Our data show that the K296E mutation does not cause photoreceptor degeneration by continuous activation of phototransduction.

Objective To develop an experimental model for embryos with a defect specific to hatching, with the purpose of clarifying the mammalian embryo hatching mechanism. Design A microneedle was inserted under the zona pellucida (ZP) of mouse embryos, and either 1 2 or 1 4 of the blastomeres were mechanically destroyed. The remainder of embryos that developed into blastocysts were compared for hatching to controls wherein a microneedle was inserted and withdrawn without harming the embryo. Experiments were done to increase pressure within the perivitelline space to decrease the amount of ZP material to study the hatching mechanism. Setting University-based basic research laboratory. Results When 1 2 or 1 4 of the embryo was destroyed and the remaining cells developed into healthy blastocysts within the intact zona, hatching was significantly impaired, and zona thickness was markedly increased relative to controls. When a mineral oil droplet was inserted under the zona to enhance a possible mechanical component of hatching, manipulated embryos were not restored to normal hatching, and hatching of unmanipulated blastocysts was not improved. However, when the zona was circumferentially thinned by application of acid Tyrode's solution, the hatching defect in manipulated " 3 4 embryos" was corrected. Conclusion Because it is known that embryos with cells reduced by 1 2 or 1 4 have normal developmental potential, we conclude that the inability of such embryos to hatch from an intact zona constitutes a mouse model for a defect specific to hatching. Moreover, results from mineral oil droplet insertion and circumferential thinning of the zona indicate that normal hatching is accomplished predominantly, if not entirely, by zona lysis, not by pressure exerted against the zona by the expanding blastocyst.

A line of mice has been developed which are transgenic for an 8.2-kilobase (kb) genomic clone of the rat vasopressin (VP) gene. Using a polymerase chain reaction technique, the rat VP (rVP) transgene was shown to have tissue-specific mRNA expression in the hypothalamus, temporal lobe, parietal cerebral cortex, cerebellum, and posterior pituitary, similar to the tissue distribution of endogenous mouse and rat VP expression. Expression of transgenic rVP mRNA was also found in the lung and pancreas of the transgenic mice, sites of known ectopic expression of VP. Using two methods, Northern blot analysis with species-specific cRNA probes and a quantitative polymerase chain reaction technique, the quantity of rVP transgene mRNA was shown to regulate appropriately in response to an osmotic stimulus. After 72 h of water deprivation, the quantity of transgenic rVP mRNA increased 6.8 +/- 3.0-fold. This was not significantly different than the fold increase in mouse VP mRNA quantity seen in nontransgenic mice (4.8 +/- 1.5) but was significantly different (P0.05) than the 1.2 +/- 0.03-fold increase in rat VP mRNA seen in normal rats after water deprivation. In the rat hypothalamus, VP mRNA poly(A) tail length increases with osmotic stimulation, while in the mouse it does not. The poly(A) tail of transgenic rVP mRNA expressed in mouse hypothalamus did not increase in length after osmotic stimulation.(ABSTRACT TRUNCATED AT 250 WORDS)

The procedures described in this chapter are applicable to problems of oogenesis, fertilization, and early development. Many important procedures have not been described (e.g., pronuclear exchange). However, nearly every experimental manipulation thus far published for the mouse either directly involves the techniques included here or is closely related to them. Therefore, if all of the skills outlined in this chapter are successfully mastered, it should be possible to adapt that knowledge to most or all known experimental protocols. Successful establishment of micromanipulation is certain to have a significant and lasting positive impact on nearly any research program in mammalian development.

In male mice expressing a transgenic alpha beta TCR which recognizes a male antigen (HY), T cells which do not express normal levels of CD8 escape thymic deletion and appear in the periphery. These consist of two distinct populations, one which lacks expression of both CD4 and CD8, and one with low levels of CD8. Neither population has anti-HY reactivity, consistent with the known requirement of this TCR for CD8. We now describe the consequences of expression of both the anti-HY TCR transgene and a constitutive CD8.1 transgene on T cells of male mice. Peripheral T cells in these male 'double transgenic' mice express both the anti-HY TCR and normal levels of CD8, and can proliferate to male antigen in vitro. These cells do not express the endogenous allele of CD8 (CD8.2), suggesting that the increase in CD8 levels due to the CD8.1 transgene leads to the deletion of the CD8.2low population. In contrast, the CD8.1 transgene does not lead to the deletion of the CD8.2- population. This implies that, unlike the majority of alpha beta T cells, TCR+CD4-CD8- cells in TCR transgenic mice are not subject to deletion.

Hotfoot (ho) mutation is a recessive trait in mice, characterized by motor disorder and male sterility, that maps to chromosome 6. We have identified a transgenic mouse pedigree with a similar trait. Using genetic and molecular approaches, we have demonstrated that the foreign DNA element is located in or near the ho locus. This new allele, designated hoJwg and presumably created by insertional mutagenesis, should make it possible to clone the ho gene. Male infertility in hoJwg male homozygotes was determined to be due to inability of sperm to penetrate the zona pellucida. This was demonstrated by rescuing mutant males by a new technique of gamete micromanipulation, zona pellucida drilling. These findings show that zona drilling is useful both for analysis and preservation of animals with reduced male fertility.

The value of various sperm parameters and the zona-free hamster egg sperm penetration assay (SPA) in predicting human in vitro fertilization (IVF) failure and subsequent successful fertilization with zona drilling was assessed. In 19 couples, throughout 31 IVF cycles, a total of 153 oocytes failed to be fertilized. In subsequent 12 cycles with zona drilling, 33 of 131 (25%) were fertilized. The incidence of teratospermia and asthenospermia was significantly higher in the study group than in the control, 74% versus 32% and 42% versus 5%, respectively. Although the mean values for the performance of sperm in SPA and fertilization of human eggs after zona drilling were remarkably similar (28 +/- 6 versus 28 +/- 4), there was no correlation between individual parameters (r = 0.15). Thus, whereas male factor infertility is more likely to be associated with teratoasthenospermia, neither the SPA nor other sperm parameters have any predictive value for failure in IVF. In addition, no criterion of sperm function has yet been identified that would eliminate oligoteratoasthenozoospermic males from consideration of IVF with zona drilling.

Allophenic mice have been produced by combining embryos from normal albino mice with embryos of normally pigmented mice carrying the autosomal dominent gene for sex reversed (Sxr) in order to determine the ability of XXSxr cells to differentiate either into sperm or eggs. Sixteen male and six female allophenes have thus far produced more than 1,200 offspring, none of which arose from an XXSxr primordial germ cell. Two allophenic mice were sterile hermaphrodites, and 13 others have yet to be tested. These results indicated that an XX cell is incapable of spermatogenesis even when supplied with the male determining Sxr gene, and that this gene also prevents XX cells from undergoing oogenesis. The latter result contrasts with a previous report of a functional sex reversed oocyte in the mouse.

Purkinje cell degeneration (pcd) is an autosomal recessive mutation which maps to chromosome 13 in the mouse. The pcd mutation causes loss of cerebellar Purkinje cells, retinal photoreceptor cells, and olfactory bulb mitral cells, as well as abnormalities of spermatogenesis. pcd is among a number of autosomal recessive mutations in mice and humans that affect neurologic function and male fertility. The cloning of one or more of these loci would contribute significantly to our understanding of the genetic control of development and maintenance of affected cell types. We report here identification of a transgenic mouse line with an insertional mutation that is allelic with pcd and manifests histopathologic features indistinguishable from those of the spontaneous mutation. The creation of an allele of pcd by transgene insertion should make possible the cloning of the pcd locus.

A micromanipulation apparatus was used to produce holes in the zonae pellucidae of unfertilized mouse oocytes. A microneedle loaded with acid Tyrode's solution was brought into contact with the zona surface, and positive flow was used in conjunction with mechanical pressure to cause a localized dissolution of the zona. Treated eggs were then fertilized in vitro in comparison with control cells. The zona drilling procedure decreased the sperm count required to achieve fertilization by a factor of approximately 100. The rate of polyspermy in zona-drilled oocytes was not greater than in controls, and oocytes fertilized after drilling, when implanted into pseudopregnant foster females, developed to term at the same rate as controls. The results demonstrate that zona drilling is a safe, effective method of increasing the efficiency of fertilization in vitro and may be useful both in agriculture and medicine for conferring fertility upon males with low sperm counts.

We have devised a procedure for mechanically inserting intact, acrosome reacted spermatozoa under the mouse zona pellucida, and have examined the ability of sperm so inserted to fertilize the mouse oocyte. Sperm immobilized by a variety of different methods are unable to fertilize the egg, despite the fact that electron microscopy confirms that they are acrosome reacted. Control experiments show that the oocytes are capable of being fertilized by motile sperm after the microinjection procedure, and that the immobilized sperm are able to form male pronuclei after injection directly into the ctyoplasm. These results indicate that in addition to its importance for penetration of egg investments, sperm motility is required for fusion of the gametes. Alternatively, the findings suggest that the enzymatic machinery required for sperm motility is very similar to that utilized for gamete fusion, and that destruction of one is likely to lead to inactivation of the other.

Infertile couples who had failed to achieve fertilization of oocytes in previous trials of in vitro fertilization (IVF) were treated by IVF with zona pellucida drilling. Zona drilling entails use of micromanipulation to introduce a gap in the zona pellucida either mechanically or by localized application of a zona solvent from a microneedle. Ten couples were treated, from whom 63 oocytes were recovered for manipulation. Sixteen eggs were denuded of the cumulus oophorus only, and the remaining 47 eggs were subjected to zona drilling. Of the 16 eggs denuded but not drilled, 4 (25%) were fertilized. Of the 47 oocytes drilled, 31 survived (67%) and 10 of the surviving eggs (32%) were fertilized. The polyspermy rate for drilled eggs that fertilized was high (5/10, 50%), and polyspermic eggs were often penetrated by more than two spermatozoa. The remaining five eggs fertilized after drilling were diploid fertilizations, and in three cases cleavage was followed by embryo transfer, although pregnancies were not obtained. These data indicate that zona drilling has the potential for establishing pregnancies in instances where treatment by standard IVF would fail. In addition, results indicate that the block to polyspermy in human eggs occurs at the level of the zona pellucida.

The effects of zona drilling on mouse embryo development in vitro were evaluated. Following insemination, sperm were immediately concentrated at the area of drilling, and in zona-drilled eggs, pronuclei appeared 30-50 min earlier than in zona-intact controls. Zona-drilled oocytes fertilized at significantly higher rates than undrilled controls and, consequently, a greater percentage of eggs inseminated after zona drilling reached the blastocyst stage. The attrition rates of zona-drilled embryos at each cleavage stage did not differ significantly from controls. Manipulated embryos exhibited unique cleavage patterns. Some embryos lost their zonae entirely, whereas others became partially extruded at early cleavage stages. These anomalies led to separation of blastomeres from the zygote proper, aggregation of embryos to form giant composite morulae and blastocysts, and occasionally to formation of miniature twin blastocysts. These characteristics of cleavage indicate that although zona drilling of a cohort of oocytes is likely to lead to an increased number of live births relative to controls, some developmental abnormalities can be encountered, and these may be associated with embryo loss, spontaneous chimerism, or possibly with conception of monozygotic twins.

Functional sex reversal is a well established phenomenon in many classes of organisms and demonstrates that sex determination in those organisms is influenced by the extra-embryonic environment. Experimental reversal of chromosomally male Xenopus laevis embryos to functional females has been accomplished by adding estrogens to the water in which these embryos develop (Chang and Witschi 1955; Gallien 1955). Chromosomally male embryos of the fish Oryziat latipes have been similarly reversed by estrogens (Yamamoto 1953, 1955), and female embryos of this same species have been reversed to functional males with testosterone (Yamamoto 1958). Other experiments have reversed males of Amblystoma maculatum (Foote 1940, 1941) and of the urodele, Fleurodeles waltlii (Gallien 1950, 1951).

Partial zona dissection (PZD), a zona drilling method that uses mechanical force to open the zona pellucida while the oocyte is shrunken in a sucrose solution, was applied to 121 unfertilized 1-day-old mature human oocytes prior to reinsemination. The 115 surviving oocytes were divided into three groups in which the duration between sucrose addition and reinsemination was varied: I) Less than 20 minutes, II) 21 to 45 minutes, and III) longer than 45 minutes. There was a trend toward a reduced fertilization and polyspermy rate as the time between sucrose exposure and insemination in sucrose-free medium increased. Moreover, there was a statistically significant reduction in the number of oocytes penetrated by more than four sperm in group III (0/41) versus group I (7/34), and in group III, parthenogenetic development was observed. The incidence of polyspermy was also increased in oocytes manipulated more than 25 hours after retrieval compared with those manipulated 21-24 hours after recovery, supporting the idea that aged oocytes have a reduced ability to block polyspermy. Oocyte contraction in sucrose occurred in three different patterns: spherical, pear-shaped, and crenated. Both the fertilization and polyspermy rates were significantly higher in the crenated group. These results indicate that changes resembling activation occur following sucrose exposure and that sucrose activation can be used to reduce the risk of polyspermic fertilization in zona drilling procedures. In addition, the pattern of shrinkage in sucrose can be used as an indicator of oocyte receptivity to sperm penetration.