Production of offspring after sperm chromosome screening: an experiment using the mouse model.

STUDY QUESTION: Is it possible to produce offspring after sperm chromosome screening? SUMMARY ANSWER: It is possible to produce zygotes after examining the genome of individual spermatozoa prior to embryo production. WHAT IS KNOWN ALREADY: Chromosomal aberrations in gametes are a major cause of pregnancy loss in women treated with assisted reproductive technology. However, to our knowledge, there are no reports on the successful genomic screening of spermatozoa, although some attempts have been made using the mouse as a model. STUDY DESIGN: To prevent the transmission of chromosomal aberrations from fathers to offspring, we performed sperm chromosome screening (SCS) prior to fertilization using the mouse as a model. The production of offspring after SCS consists of (i) replication of the sperm chromosomes, (ii) analysis of one copy of the replicated sperm chromosomes, (iii) construction of a zygote using another set of chromosomes and (iv) production of a transferable embryo. MATERIALS, SETTING, METHODS: A single spermatozoon of a male mouse, with or without a Robertsonian translocation, was injected into an enucleated oocyte to allow the replication of sperm chromosomes. One of the sister blastomeres of a haploid androgenic 2-cell embryo was used for chromosome analysis. The other blastomere was fused with an unfertilized oocyte, activated and allowed to develop to a blastocyst before transfer to a surrogate mother. MAIN RESULTS AND ROLE OF CHANCE: With high efficiency, we were able to analyze sperm chromosomes in a blastomere from the androgenic 2-cell embryos and culture zygotes, with and without aberrant chromosomes, to the blastocyst stage before embryo transfer. The karyotypes of the offspring faithfully reflected those of the blastomeres used for SCS. LIMITATIONS, REASONS FOR CAUTION: This study was conducted using a mouse model; whether or not the method is applicable to humans is not known. WIDER IMPLICATIONS OF THE FINDINGS: This study has shown that it is possible to produce zygotes without any paternally inherited aberrations by examining the genome of individual spermatozoa prior to embryo production.

Mouse and human spermatozoa can be freeze-dried without damaging their chromosomes.

BACKGROUND: Although mouse spermatozoa can be freeze-dried without losing their reproductive capacity, the technique needs further improvements to reduce the incidence of chromosomal damage to spermatozoa. Effects of freeze-drying on human spermatozoa are unknown. METHODS: Mouse spermatozoa were suspended in a Tris-buffered EGTA solution briefly (10 min at 37 degrees C) or for 1-7 days at 4 degrees C before freeze-drying. Freeze-dried spermatozoa were maintained for up to 1 year at 4 degrees C before injection. Sperm chromosomes were examined during the first mitosis (cleavage) of zygotes. The ability of sperm to support embryo development was assessed by examining mid-gestation fetuses (Day 14) after transfer of 2-cell embryos to surrogate mothers. Chromosome integrity of freeze-dried human spermatozoa was examined by injecting individual spermatozoa into mouse oocytes which were previously enucleated. RESULTS: When mouse spermatozoa were freeze-dried immediately after suspension in Tris-buffered EGTA solution, only c.40% had normal chromosomes. When the mouse spermatozoa were kept in the same solution for 3-7 days before freeze-drying, 85-95% had normal chromosomes and they were able to support embryo development better than those which were in the solution briefly (P < 0.05). Freeze-dried human spermatozoa well maintained their chromosomes regardless of the duration of pre-freeze-drying incubation of spermatozoa in the Tris-buffered EGTA solution. CONCLUSIONS: Prior incubation of mouse spermatozoa in Tris-buffered EGTA solution for several days makes sperm chromosomes more resistant to freeze-drying. As the consequence, spermatozoa freeze-dried this way support embryo development better than those exposed to Tris-buffered EGTA solution only briefly. Freeze-dried human spermatozoa well maintained their chromosomes without pre-freeze-drying incubation in Tris-buffered EGTA solution.

Fertilization and developmental initiation of oocytes by injection of spermatozoa and pre-spermatozoal cells.

The essence of fertilization is the union and mingling of male and female genomes. Therefore it is not surprising that microsurgical deposition of a single spermatozoon in an oocyte (ICSI) results in the development of normal offspring. Poorly motile or structurally aberrant spermatozoa, which are unable to fertilize under ordinary conditions, are not necessarily genomically abnormal. This is the reason why normal offspring are obtained after ICSI using such spermatozoa. At present, ICSI is most successful in humans and mice, but there is no reason to believe that ICSI does not work in other animal species as well. Injection of round spermatids into oocytes (ROSI) works routinely in the mouse, but it is controversial in humans. While some investigators have claimed successes, many others have reported complete failure. There must be several reasons for this, including the difficulty of distinguishing true spermatids from other small cells. Insufficient oocyte activation following ROSI and the functional immaturity of the centrosome could also be responsible for this. In mice, it is possible to obtain normal offspring by injection of primary or secondary spermatocytes into oocytes. The nucleus of a spermatocyte undergoes meiotic division(s) within the oocyte's cytoplasm before a haploid sperm pronucleus unites with an oocyte's haploid pronucleus. However, only a few of the produced zygotes have developed into fertile offspring. There are many hurdles to clear before ROSI and spermatocyte injection becomes efficient and medically safe methods for assisted fertilization.

Reproductive semi-cloning respecting biparental origin. A biologically unsound principle.

The original debate article proposed the use of "semi-cloning" as a viable method for assisted reproduction. This debate counters the proposal as being biologically unsound. Given the fundamental limitations of chromosomal segregation and genomic imprinting, the notion of using the MII oocyte to drive haploidization of a somatic cell genome and thereby obtain a substitute for authentic gametes is ill-conceived and untenable.

Adult murine neurons: their chromatin and chromosome changes and failure to support embryonic development as revealed by nuclear transfer.

Fully differentiated neurons in adult mammalian brains do not divide; consequently, their metaphase chromosomes have never been examined. Here we report metaphase chromosome constitutions of cortical neurons in adult mice visualized by a nuclear transfer technique. We found that although some reconstructed oocytes cloned from neuronal nuclei have an apparently normal karyotype, the majority do not. Regardless of chromosome morphology, nuclei of adult neurons totally lack the ability to support embryonic development. These findings support the hypothesis that fully differentiated neurons in adult mammalian brains are genomically altered.

Birth of normal calves after intracytoplasmic sperm injection of bovine oocytes: a methodological approach.

Intracytoplasmic sperm injection (ICSI) is advantageous when only very few spermatozoa are available for insemination. Bovine spermatozoa were injected individually into matured oocytes using a piezo electric actuator. Spermatozoa were "immobilized", by scoring their tails immediately before injection, or "killed", by repeated freezing and thawing. About 4 h after ICSI, the oocytes with two polar bodies (activated by sperm injection) were selected and treated 5 min with 7% ethanol before further culture. When examined 19-21 h after ICSI, nearly 90% of the oocytes were fertilized normally (two pronuclei and two polar bodies) irrespective of the sperm treatment (immobilization or killing) prior to ICSI, but subsequent preimplantation embryo development was much superior (cleavage 72%: blastocysts 20%) after ICSI with immobilized spermatozoa than by using killed spermatozoa (cleavage 28%; blastocysts 1%). Ethanol activation of bovine oocytes with two polar bodies 4 h after ICSI improved the cleavage (33% versus 72%) and blastocyst (12% versus 20%) rates markedly (P < 0.05). Five normal calves were born after transplantation of ten blastocysts to ten surrogate cows. These results show that piezo-ICSI using immobilized spermatozoa, combined with ethanol treatment of sperm-injected oocytes, is an effective method to produce bovine offspring.

Cloning: experience from the mouse and other animals.

Cloning mammals has been successful for many years by splitting an early embryo or transferring embryonic cell nuclei into enucleated oocytes. Cloning is now possible with adult somatic cells. At present, cloning efficiency--as determined by the proportion of live offspring developed from all oocytes that received donor cell nuclei--is low regardless of the cell type (including, embryonic stem (ES) cells) and animal species used. In all animals, except of Japanese black beef cattle, the vast majority (>97%) of cloned embryos perish before reaching full term. Even in the Japanese cattle, less than 20% of cloned embryos reach the adulthood. This low efficiency of cloning seems to be due largely to faulty epigenetic reprogramming of donor cell nuclei after transfer into recipient oocytes. Cloned embryos with major epigenetic errors die before or soon after implantation. Those with relatively 'minor' epigenetic errors may survive birth and reach adulthood. We found that almost all fetuses of inbred mice die at birth from respiratory problems, while those of hybrid mice do not, suggesting that genomic heterogeneity masks-to some extent-faulty epigenetic errors. Thus far, the majority of cloned mice that survived birth, had a normal life span and were fertile. However, these animals may not be totally free of health problems. Postpubertal obesity in certain strains of mice is one example. A trial and error approach may discover better cells for cloning, but it would be wiser to understand the molecular mechanisms of epigenetic nuclear programming and reprogramming to find the way to make cloning safer and more efficient. The relatively high cloning success rate in the Japanese black cattle may provide us a clue of solving the problem of high mortality of cloned offspring.

Fertilization without spermatozoa.

Mammalian spermatozoa first acquire the ability to fertilize oocytes as they pass through the epididymis to mature. Due to recent advances in microinsemination techniques, not only mature spermatozoa, but also immature sperm cells at certain stages in the testis, have been used to construct diploid zygotes, some of which subsequently develop to normal offspring. Using round spermatids, the most youngest haploid male germ cells, normal births have been reported in the mouse, rabbit, and human. Furthermore, in the mouse, secondary and primary spermatocytes also support full term development after incorporation into immature or mature homologous oocytes. Spermatogenic cells of several species can be cryopreserved easily in simple cryoprotectant solutions. Thus, the microinsemination techniques using spermatogenic cells give us a way to treat infertility, and provide valuable information on gametogenesis, including spermatogenesis, meiosis, and genomic imprinting.

Maintenance of genetic integrity in frozen and freeze-dried mouse spermatozoa.

Chromosome stability was maintained in mouse spermatozoa after freeze-drying or freezing without cryoprotection in a simple Tris.HCl buffer containing EGTA (50 mM) and NaCl (50 mM). The ability of spermatozoa to activate oocytes spontaneously was not destroyed by freeze-drying or freezing without cryoprotection in this solution. Embryos derived after injecting oocytes with sperm heads from rehydrated freeze-dried and from thawed spermatozoa developed normally. Provided the DNA integrity of the sperm nucleus is maintained, embryos can be generated by the intracytoplasmic sperm injection technique (ICSI) from severely damaged spermatozoa that are no longer capable of normal physiological activity. This procedure was effective for preserving spermatozoa from strains (C57BL/6J, 129/SvJ, and BALB/c) in which the fertility of spermatozoa frozen conventionally is extremely poor. The technique provides an effective means of storing mouse spermatozoa from many different inbred, mutant, and transgenic strains for biomedical research.

Assessment of the developmental totipotency of neural cells in the cerebral cortex of mouse embryo by nuclear transfer.

When neural cells were collected from the entire cerebral cortex of developing mouse fetuses (15.5-17.5 days postcoitum) and their nuclei were transferred into enucleated oocytes, 5.5% of the reconstructed oocytes developed into normal offspring. This success rate was the highest among all previous mouse cloning experiments that used somatic cells. Forty-four percent of live embryos at 10.5 days postcoitum were morphologically normal when premature and early-postmitotic neural cells from the ventricular side of the cortex were used. In contrast, the majority (95%) of embryos were morphologically abnormal (including structural abnormalities in the neural tube) when postmitotic-differentiated neurons from the pial side of the cortex were used for cloning. Whereas 4.3% of embryos cloned with ventricular-side cells developed into healthy offspring, only 0.5% of those cloned with differentiated neurons in the pial side did so. These facts seem to suggest that the nuclei of neural cells in advanced stages of differentiation had lost their developmental totipotency. The underlying mechanism for this developmental limitation could be somatic DNA rearrangements in differentiating neural cells.

Placentomegaly in cloned mouse concepti caused by expansion of the spongiotrophoblast layer.

Hypertrophic placenta, or placentomegaly, has been reported in cloned cattle and mouse concepti, although their placentation processes are quite different from each other. It is therefore tempting to assume that common mechanisms underlie the impact of somatic cell cloning on development of the trophoblast cell lineage that gives rise to the greater part of fetal placenta. To characterize the nature of placentomegaly in cloned mouse concepti, we histologically examined term cloned mouse placentas and assessed expression of a number of genes. A prominent morphological abnormality commonly found among all cloned mouse placentas examined was expansion of the spongiotrophoblast layer, with an increased number of glycogen cells and enlarged spongiotrophoblast cells. Enlargement of trophoblast giant cells and disorganization of the labyrinth layer were also seen. Despite the morphological abnormalities, in situ hybridization analysis of spatiotemporally regulated placenta-specific genes did not reveal any drastic disturbances. Although repression of some imprinted genes was found in Northern hybridization analysis, it was concluded that this was mostly due to the reduced proportion of the labyrinth layer in the entire placenta, not to impaired transcriptional activity. Interestingly, however, cloned mouse fetuses appeared to be smaller than those of litter size-matched controls, suggesting that cloned mouse fetuses were under a latent negative effect on their growth, probably because the placentas are not fully functional. Thus, a major cause of placentomegaly is expansion of the spongiotrophoblast layer, which consequently disturbs the architecture of the layers in the placenta and partially damages its function.

Gamete manipulation for development: new methods for conception.

Mammalian oocytes microsurgically injected with spermatozoa can develop into normal offspring. Apparently the oocyte has the ability to decompose or eliminate such sperm components as the plasma membrane and acrosomal contents, which normally do not enter its cytoplasm. Species in which normal offspring were obtained by direct sperm injection include: human, mouse, rabbit, horse, sheep, cattle, pig, and monkey. In the mouse, normal offspring can also be obtained routinely by the injection of round spermatid nuclei into oocytes. This suggests that all post-meiotic modifications of spermatozoa (spermiogenesis, sperm maturation, capacitation and the acrosome reaction) evolved as processes solely dedicated to delivering male genomes into female gametes. Birth of normal offspring after injection of spermatocytes into maturing or mature oocytes suggests that the mechanisms controlling meiosis of male and female germ cells are similar, if not the same. Spermatozoa do not need to be morphologically normal or alive in the conventional sense to participate in embryo development; as long as they have intact genomes, they are able to produce normal offspring. Chromosomes within the first and second polar bodies can be used as substitutes for female pronuclei for the production of normal offspring. The nuclei of adult somatic cells can be used for production of animals. This procedure, involving introduction of cell nuclei into enucleated oocytes (genomic cloning), is rather inefficient at present. Many obstacles must be overcome before it is accepted as a safe, novel method to reproduce scientifically, medically or economically valuable animals. For human cloning the prime consideration must be the welfare of the child.

Comparison of intracytoplasmic sperm injection for inbred and hybrid mice.

We compared the results of intracytoplasmic sperm injection (ICSI) that leads to full term development of hybrid (B6C3F1 and B6D2F1) and inbred (C57BL/6) mouse embryos. Although fertilization and pre-implantation development of C57BL/6 eggs were similar to those of F1 hybrid eggs, post-implantation development of the embryos from C57BL/6 females was significantly poorer than those of the eggs from hybrid females. Reciprocal crosses of C57BL/6 and B6C3F1 gametes revealed that the low rate of post-implantation development of C57BL/6 embryos was due to oocyte factor(s), rather than the sperm factor.

Live human germ cells in the context of their spermatogenic stages.

BACKGROUND: Various types of live, dispersed, human testicular cells in vitro were previously compared with the morphologic characteristics of human spermatogenic germ cells in situ within seminiferous tubules. The current study extends those observations by placing live human germ cells in the context of their developmental steps and stages of the spermatogenic cycle. METHODS: Live human testicular tissue was obtained from an organ-donating, brain-dead person. A cell suspension was obtained by enzymatic digestion, and dispersed cells were observed live with Nomarski optics. Testes from 10 men were obtained at autopsy within ten hours of death, fixed in glutaraldehyde, further fixed in osmium, embedded in Epon, sectioned at 20 microm, and observed unstained by Nomarski optics. RESULTS: In both live and fixed preparations, Sertoli cells have oval to pear-shaped nuclei with indented nuclear envelopes and large nucleoli, which makes their appearance distinctly different from germ cells. For germ cells, size, shape, and chromatic pattern of nuclei, the presence of meiotic metaphase figures, acrosomic vesicles/structures, tails, and/or mitochondria in the middle piece are characteristically seen in live dispersed cells and those in the fixed seminiferous tubules. These lead to identification of live germ cells in man and placement of each in the context of their developmental steps of spermatogenesis at corresponding stages of the spermatogenic cycle. CONCLUSIONS: This comparative approach allows verification of the identity of individual germ cells seen in vitro and provides a checklist of distinguishing characteristics of live human germ cells to be used in clinical procedures or by scientists interested in studying live cells at known steps in spermatogenic development characteristic of germ cells in specific stages of the spermatogenic cycle.

Epigenetic instability in ES cells and cloned mice.

Cloning by nuclear transfer (NT) is an inefficient process in which most clones die before birth and survivors often display growth abnormalities. In an effort to correlate gene expression with survival and fetal overgrowth, we have examined imprinted gene expression in both mice cloned by nuclear transfer and in the embryonic stem (ES) cell donor populations from which they were derived. The epigenetic state of the ES cell genome was found to be extremely unstable. Similarly, variation in imprinted gene expression was observed in most cloned mice, even in those derived from ES cells of the same subclone. Many of the animals survived to adulthood despite widespread gene dysregulation, indicating that mammalian development may be rather tolerant to epigenetic aberrations of the genome. These data imply that even apparently normal cloned animals may have subtle abnormalities in gene expression.

DNA methylation variation in cloned mice.

Mammalian cloning has been accomplished in several mammalian species by nuclear transfer. However, the production rate of cloned animals is quite low, and many cloned offspring die or show abnormal symptoms. A possible cause of the low success rate of cloning and abnormal symptoms in many cloned animals is the incomplete reestablishment of DNA methylation after nuclear transfer. We first analyzed tissue-specific methylation patterns in the placenta, skin, and kidney of normal B6D2F1 mice. There were seven spots/CpG islands (0.5% of the total CpG islands detected) methylated differently in the three different tissues examined. In the placenta and skin of two cloned fetuses, a total of four CpG islands were aberrantly methylated or unmethylated. Interestingly, three of these four loci corresponded to the tissue-specific loci in the normal control fetuses. The extent of aberrant methylation of genomic DNA varied between the cloned animals. In cloned animals, aberrant methylation occurred mainly at tissue-specific methylated loci. Individual cloned animals have different methylation aberrations. In other words, cloned animals are by no means perfect copies of the original animals as far as the methylation status of genomic DNA is concerned.

Effect of cytokinesis inhibitors, DMSO and the timing of oocyte activation on mouse cloning using cumulus cell nuclei.

Cloning methods are now well described and in almost routine use. However, the frequencies of production of live offspring from activated oocytes remain at < 3% and little is known about the factors that affect these frequencies. The effects of cytokinesis inhibitors, dimethylsulphoxide (DMSO) and the cell cycle of recipient cytoplasm on the cloning of mice were examined. Reconstructed oocytes, which were activated immediately after nucleus injection and cultured without cytochalasin B, developed into blastocysts at a frequency of 30--54% and into live cloned offspring at a frequency of 2--3%. Activated zygotes did not support development to full term after nuclear transfer. Reconstructed oocytes were activated 1--3 h after nuclear transfer and were exposed separately to three inhibitors of cytokinesis (cytochalasin B, cytochalasin D or nocodazole) to examine the toxicity of these inhibitors on cloning. All of the oocytes exposed to nocodazole-containing media formed many small pseudo-pronuclei, whereas with cytochalasin-containing media most of the activated oocytes formed only two pseudo-pronuclei. Despite such differences, 42--61% of reconstructed embryos developed to the morula-blastocyst stage and 1--3% developed to full term in all groups. Addition of 1% (v/v) DMSO to the activation medium significantly improved the frequency of development to the blastocyst stage and full term; however, this improvement did not lead to a higher success rate in the generation of live cloned offspring. These results show that activated mouse oocytes/zygotes are not effective cytoplasmic recipients with the methods described and that the lack of success of cloning is not due to inhibition of cytokinesis.

Hybrid vigor, fetal overgrowth, and viability of mice derived by nuclear cloning and tetraploid embryo complementation.

To assess whether heterozygosity of the donor cell genome was a general parameter crucial for long-term survival of cloned animals, we tested the ability of embryonic stem (ES) cells with either an inbred or F(1) genetic background to generate cloned mice by nuclear transfer. Most clones derived from five F(1) ES cell lines survived to adulthood. In contrast, clones from three inbred ES cell lines invariably died shortly after birth due to respiratory failure. Comparison of mice derived from nuclear cloning, in which a complete blastocyst is derived from a single ES cell, and tetraploid blastocyst complementation, in which only the inner cell mass is formed from a few injected ES cells, allows us to determine which phenotypes depend on the technique or on the characteristics of the ES cell line. Neonatal lethality also has been reported in mice entirely derived from inbred ES cells that had been injected into tetraploid blastocysts (ES cell-tetraploids). Like inbred clones, ES cell-tetraploid pups derived from inbred ES cell lines died shortly after delivery with signs of respiratory distress. In contrast, most ES cell-tetraploid neonates, derived from six F(1) ES cell lines, developed into fertile adults. Cloned pups obtained from both inbred and F(1) ES cell nuclei frequently displayed increased placental and birth weights whereas ES cell-tetraploid pups were of normal weight. The potency of F(1) ES cells to generate live, fertile adults was not lost after either long-term in vitro culture or serial gene targeting events. We conclude that genetic heterozygosity is a crucial parameter for postnatal survival of mice that are entirely derived from ES cells by either nuclear cloning or tetraploid embryo complementation. In addition, our results demonstrate that tetraploid embryo complementation using F(1) ES cells represents a simple, efficient procedure for deriving animals with complex genetic alterations without the need for a chimeric intermediate.