[show abstract][hide abstract] ABSTRACT: Telomere length maintenance in the germ line from generation to generation is essential for the perpetuation of eukaryotic organisms. This task is performed by a specialized reverse transcriptase called telomerase. While this critical function of telomerase has been well established, the mechanisms that regulate telomerase in the germ line are still poorly understood. We now show, using a Pou5f1-GFP transgenic mouse model, that telomerase suppression in quiescent male primordial germ cells (PGCs) is accompanied by a decrease in expression of murine telomerase reverse transcriptase (TERT). To further assess the role of TERT in quiescent PGCs, we developed a chicken Actb gene promoter/cytomegalovirus enhancer (CAG)-Tert transgenic mouse strain that constitutively expresses murine TERT. Telomerase activity was detected in quiescent PGCs from CAG-Tert transgenic embryos, demonstrating that re-activation of TERT expression is sufficient to restore telomerase activity in these cells and implying that TERT expression is an important mechanism of telomerase regulation in PGCs. Fluorescence-activated cell-sorting (FACS) analysis of PGC frequency and cell cycle status revealed no effect of either overexpression or deficiency of TERT in CAG-Tert transgenic mice or Tert knock-out mice respectively. These results demonstrate that TERT per se does not affect proliferation or development of PGCs, in contrast with recent studies that suggest that TERT has a telomere-independent effect in certain stem cells. It is possible that the direct effect of TERT on cell behavior may be dependent on cell type.
Biology of Reproduction 12/2006; 75(5):785-91. · 4.03 Impact Factor
[show abstract][hide abstract] ABSTRACT: We have developed a novel method for mouse transgenesis. The procedure relies on a hyperactive Tn5 transposase to insert a transgene into mouse chromosomes during intracytoplasmic sperm injection. This procedure integrates foreign DNA into the mouse genome with dramatically increased effectiveness as compared to conventional methods such as pronuclear microinjection and traditional sperm injection-mediated transgenesis. Our data indicate that with this method, transgenic mice, both hybrids and inbreds, can be produced more consistently and with lower numbers of manipulated oocytes required for traditional microinjection methods. The transposase-mediated transgenesis technique is also effective with round spermatids, offering the potential for rescuing the fertility of azoospermic animals using sperm precursor cells.
Biology of Reproduction 01/2006; 73(6):1157-63. · 4.03 Impact Factor
[show abstract][hide abstract] ABSTRACT: Recent studies showed that ICSI with cauda epididymal or ejaculated sperm of infertile mice or men, respectively, was less effective in fertilization and normal embryo development than ICSI using sperm from the testes. These studies suggested that sperm nuclear quality declined after release from the testis, but the site where this loss of fertility occurs has not been localized.
We performed ICSI with testicular, caput, and cauda epididymal sperm from infertile Tnp1-/-Tnp2+/- mutant mice, which have a minimal level of transition nuclear proteins and are sterile by natural mating.
When the heads of motile sperm from the testis or caput epididymis of Tnp1-/-Tnp2+/- males were injected into enucleated mouse oocytes, sperm chromosomes showed no difference from those of wild-type mice, but the chromosomes from sperm taken from the cauda epididymis of mutant males showed increased abnormalities. Injection of testicular or caput epididymal sperm from Tnp1-/-Tnp2+/- males into intact oocytes resulted in normal embryonic and fetal development and yields of liveborn equivalent to wild-type, but cauda sperm from Tnp1-/-Tnp2-/- mice produced lower implantation rates and yields of liveborn than did those from wild-type mice.
These results demonstrate that in mice with sperm chromatin abnormalities, the decline in fertility of sperm with ICSI occurs after the caput epididymis. The advantage of using caput epididymal sperm for ICSI in certain situations may be considered as an approach to be tested in human assisted reproduction.
Human Reproduction 12/2005; 20(11):3101-8. · 4.67 Impact Factor
[show abstract][hide abstract] ABSTRACT: The low efficiency of current microinjection-based animal transgenesis techniques is largely the result of poor embryo survival. We have developed a new, bacterial recombinase-based transgenesis method. Intracytoplasmic sperm injection (ICSI) of single stranded DNA (ssDNA) complexed with E. coli recombinase RecA into mouse metaphaseII (MII) arrested oocytes resulted in RecA-dependent transgenesis. This approach offers significant advantages over pronuclear microinjection and previous ICSI-based transgenesis approaches in terms of improved embryo survival, which translates into greater transgenesis efficiency. It also opens the possibility to attempt experiments, which may affect gene targeting by homologous recombination into DNA of mammalian single celled pre-implantation embryos.
[show abstract][hide abstract] ABSTRACT: Keratin 9 (K9) is one of the components of the perinuclear ring of the manchette found in developing spermatids but is predominantly expressed in the epidermis of the footpad (palm and sole in human epidermis). As an initial step to determine the function of K9 protein in sperm development, we have generated a mutant mouse by homologous recombination of the targeting vector containing the disrupted K9 gene in which the neo(r) gene was inserted into the intron 6. This insertion resulted in the expression of two K9 mRNAs: a wild-type K9 mRNA, in which intron 6 with the neo(r) gene was completely spliced out, and a mutated form in which only a portion of the intron 6 between neo(r) gene and exon 7 was spliced out. While both heterozygous (K9(+/neo)) and homozygous (K9(neo/neo)) mutant mice expressed the wild-type form of K9 protein, the expression profile of the wild-type K9 in K9(neo/neo) mutants was modified. In addition, the open reading frame of the aberrant mRNA terminated at the exon 6/intron 6 splice site, resulting in a truncated K9 protein. Both K9(+neo) and K9(neo/neo) male mice displayed spermatids with ectopic manchette. Coiled tails were seen in maturing spermatids and epididymal sperm of mutant mice and sperm with deformed tails displayed forward motility. A predominant sperm anomaly was residual cytoplasm at the end of the mitochondria-containing middle piece tail segment. The residual cytoplasm displayed vesicles with random in situ motion, suggesting a transport impediment toward the distal end of the sperm tail. All mutant mice were fertile. Surprisingly, in oocyte nuclear injection experiments using K9(neo/neo) sperm donor, 76% of the resulting animals displayed a deletion of the neo(r) gene from the intron 6 of the mutated K9 allele. Results of this study support the view that intron 6 influences the transcriptional efficiency of the K9 gene by decreasing production of wild-type K9 and changing the expression of K9 proteins.
Molecular Reproduction and Development 11/2005; 72(2):259-71. · 2.81 Impact Factor
[show abstract][hide abstract] ABSTRACT: Reversible infertility can be induced in male mice by oral administration of the alkylated imino sugars N-butyldeoxynojirimycin (NB-DNJ) and N-butyldeoxygalactonojirimycin (NB-DGJ). Spermatozoa of these mice have grossly misshapen heads and reduced motility. Because NB-DNJ and related compounds may hold promise as nonhormonal male contraceptives, a comprehensive examination of their effects on male reproduction is necessary. To this end, we further examined reproductive properties of the dysmorphic spermatozoa that are produced after short-term imino sugar administration at the minimal dose that completely abolishes the ability of male C57BL/6 mice to produce offspring by natural mating. Here, we report that, in vitro, the abnormal spermatozoa from the NB-DNJ- and NB-DGJ-treated mice were unable to fertilize oocytes. In addition, we investigated whether the imino sugars damage the genetic integrity of spermatozoa. To test this, we microsurgically injected deformed spermatozoa from imino sugar-treated males into oocytes. The deformed spermatozoa from the testis were able to activate oocytes very efficiently, but those from the cauda epididymis often failed to do so. This problem was overcome when the sperm-injected oocytes were treated with a parthenogenetic agent, Sr(2+). Oocytes injected with the misshapen spermatozoa from NB-DNJ- and NB-DGJ-treated mice developed (with or without Sr(2+) treatment) into live offspring that grew normally and were normally fertile. This indicates that during short-term administration, alkylated imino sugars alter sperm morphology and physiology but do not diminish the genetic potential of spermatozoa.
Biology of Reproduction 05/2005; 72(4):805-13. · 4.03 Impact Factor
[show abstract][hide abstract] ABSTRACT: The histone-to-protamine transition is important in the formation of spermatozoa. In mammals this involves two steps: replacement of histones by transition nuclear proteins (TPs) and replacement of TPs by protamines. To determine the functions of the TPs and their importance for sperm development, we generated mice lacking both TPs, since mice lacking only TP1 or TP2 were fertile. Our results indicated that TP1 and TP2 had partially complemented each other. In mice lacking both TPs, nuclear shaping, transcriptional repression, histone displacement, and protamine deposition proceeded relatively normally, but chromatin condensation was irregular in all spermatids, many late spermatids showed DNA breaks, and protamine 2 was not posttranslationally processed. Nevertheless, genomic integrity was maintained in mature spermatids, since efficient fertilization and production of offspring were achieved by intracytoplasmic sperm injection. However, many mature spermatids were retained in the testis, epididymal spermatozoa were drastically reduced in number and were highly abnormal, and the mice were sterile. Most epididymal spermatozoa were incapable of fertilization even using intracytoplasmic sperm injection. Thus, in mammals TPs are required for normal chromatin condensation, for reducing the number of DNA breaks, and for preventing the formation of secondary defects in spermatozoa, eventual loss of genomic integrity, and sterility.