Production of fertile offspring from genetically infertile male mice

Quaking viable (qk v ) is a recessive neurological mouse mutation with severe dysmyelination of the CNS and spermiogenesis failure. The molecular lesion in the qk v mutant is a deletion of Ϸ1 Mb on mouse chromosome 17 that alters the expression of the qk gene in oligodendrocytes. Complementation analysis between the qk v mutation and qk mutant alleles generated through chemical mutagenesis showed that the male sterility is a distinctive feature of the qk v allele. This observation suggested that the sperm differentiation defect in qk v is due to the deletion of a gene(s) distinct from qk. Here, we demonstrate that the deletion of Pacrg is the cause of male sterility in the qk v mutant. Pacrg is the mouse homologue of the human PARKIN-coregulated gene (PACRG), which encodes for a protein whose biochemical function remains unclear. We show that Pacrg is highly expressed in the testes in both mice and humans. In addition, the expression pattern of Pacrg during spermiogenesis suggests that it plays a role in sperm differentiation. In support of this hypothesis, we show that transgenic expression of Pacrg in testes restores spermiogenesis and fertility in qk v males. This finding provides the first in vivo evidence, to our knowledge, for the function of Pacrg in a model organism. Immunolocalization experiments on isolated spermatozoa show that the Pacrg protein is present in mature sperm. Remarkably, the mammalian Pacrg protein shares significant sequence similarities with gene products from flagellated protozoans, suggesting that Pacrg may be necessary for proper flagellar formation in many organisms.

mentioning

confidence: 86%

“…Very few morphologically abnormal immotile spermatozoa are present in the semen of qk v males, because spermatids fail to complete differentiation. A recent report (3) showed that sperm from qk v semen and testes are unable to fertilize mouse oocytes in vitro. However, the intracytoplasmic injection of qk v spermatozoa resulted in normal live offspring.…”

mentioning

confidence: 99%

Deletion of the Parkin coregulated gene causes male sterility in the quaking ^viable mouse mutant

Lorenzetti

Bishop

Justice

2004

“…This is especially true for C57BL͞6 (B6) mice, most frequently used for generation of genetically engineered mice. This problem has been overcome by partial zona dissection before in vitro fertilization (3) or intracytoplasmic sperm injection of frozenthawed or freeze-dried spermatozoa (4)(5)(6)(7).…”

mentioning

confidence: 99%

“…Immature male germ cells such as spermatids and spermatocytes are currently used to produce offspring (8)(9)(10)(11) when spermatozoa cannot be obtained due to spermatogenic arrest because of genetic mutations or as the result of in vitro manipulation of germ cells (7,10,11). Therefore, cryopreservation of these immature sperm cells is necessary, but they suffer more cryodamage than mature epididymal spermatozoa.…”

mentioning

confidence: 99%

Spermatozoa and spermatids retrieved from frozen reproductive organs or frozen whole bodies of male mice can produce normal offspring

Ogonuki

Mochida

Miki

et al. 2006

Self Cite

Cryopreservation of male germ cells is a strategy to conserve animal species and strains of animals valuable to biomedical research. We tested whether mouse male germ cells could be cryopreserved without cryoprotection by simply freezing epididymides, testes, or whole bodies. The reproductive organs were isolated from killed mice and frozen for 1 week to 1 year at ؊80°C before spermatozoa and spermatids were collected and injected into mature oocytes. Normal pups were born irrespective of strains tested (ICR and C57BL͞6). Epididymides and testes frozen and transported internationally to another laboratory by air could produce pups of inbred C57BL͞6 mice. Testicular spermatozoa retrieved from the bodies of male mice (BALB͞c nude and C3H͞He strains) that had been kept frozen (؊20°C) for 15 years could also produce normal offspring by microinsemination. Thus, freezing of either male reproductive organs or whole bodies is the simplest way to preserve male germ cells. Restoration of extinct species could be possible if male individuals are found in permafrost.cryopreservation ͉ gametes ͉ intracytoplasmic sperm injection ͉ microinsemination ͉ mouse S perm cryopreservation has been widely used for both human reproduction and animal breeding. Because much of basic research of mammalian genetics and early development has been done by using laboratory mice, and the number of genetically engineered mice (transgenesis, gene targeting, and mutagenesis) is increasing exponentially, development of simple, cost-saving, and space-effective means of mouse sperm preservation is much needed (1). Mouse sperm cryopreservation using raffinose and skim milk as cryoprotectants has been successful, but defrosted spermatozoa of some strains of mice do not fertilize eggs well (2). This is especially true for C57BL͞6 (B6) mice, most frequently used for generation of genetically engineered mice. This problem has been overcome by partial zona dissection before in vitro fertilization (3) or intracytoplasmic sperm injection of frozenthawed or freeze-dried spermatozoa (4-7).Immature male germ cells such as spermatids and spermatocytes are currently used to produce offspring (8-11) when spermatozoa cannot be obtained due to spermatogenic arrest because of genetic mutations or as the result of in vitro manipulation of germ cells (7, 10, 11). Therefore, cryopreservation of these immature sperm cells is necessary, but they suffer more cryodamage than mature epididymal spermatozoa. A cryopreservation protocol we developed for mouse spermatogenic cells in the past is rather complicated, and therefore the development of simpler techniques is required (12).Thus far, the simplest method of cryopreserving mouse spermatozoa is to freeze spermatozoa in simple salt solutions without any cryoprotectants (6). Although defrosted spermatozoa are not alive in the conventional sense, they apparently maintain their genetic integrity, because they are able to produce live offspring by microinsemination. Here we report that spermatozoa or spermatids, retrieved from f...

“…Some mutant, transgenic, and gene-targeted males are infertile because of defective spermiogenesis. The infertility of such males has been overcome by injection of spermatids into normal oocytes (6)(7)(8)(9). However, if the gonads of animals of interest lack germ cells, cloning would be the only way to propagate such lines.…”

mentioning

confidence: 99%

Propagation of an infertile hermaphrodite mouse lacking germ cells by using nuclear transfer and embryonic stem cell technology

Wakayama

Kishigami

Thuan

et al. 2004

Self Cite

Animals generated by systematic mutagenesis and routine breeding are often infertile because they lack germ cells, and maintenance of such lines of animals has been impossible. We found a hermaphrodite infertile mouse in our colony, a genetic male with an abnormal Y chromosome lacking developing germ cells. We tried to clone this mouse by conventional nuclear transfer but without success. ES cells produced from blastocysts, which had been cloned by using somatic cell nuclear transfer (ntES cells) from this mouse, were also unable to produce offspring when injected into enucleated oocytes. Although we were able to produce two chimeric offspring using these ntES cells by tetraploid complementation, they were infertile, because they also lacked developing germ cells. However, when such ntES cells were injected into normal diploid blastocysts, many chimeric offspring were produced. One such male offspring transmitted hermaphrodite mouse genes to fertile daughters via X chromosome-bearing sperm. Thus, ntES cells were used to propagate offspring from infertile mice lacking germ cells.