Male advantage observed for in vitro fertilization mouse embryos exhibiting early cleavage.

PURPOSE: Mouse embryos forming blastocoele early vs those forming late are predominantly male. We examined whether the male advantage could be recognized at an earlier stage of development. METHODS: The IVF embryos were classified into early, intermediate, and late development groups based on the time of the third cleavage, and the Zfy and Xist genes were detected to identify their sex in the classified embryos. Furthermore, embryos that were classified based on the time of the third cleavage were transferred to recipient animals and the sex ratio of the fetuses was determined at birth. RESULTS: Approximately 90% of the early-developing embryos that exhibited third cleavage as early as 47 hours after insemination were male when analyzed using PCR at the blastocyst stage. PCR analysis showed that 61% of the intermediate-developing embryos (third cleavage occurring 48-50 hours after insemination) and 45% of late-developing embryos (third cleavage occurring at 51 hours or later postinsemination) were male. After embryo transfer, the early-developing embryos produced 80% males, while intermediate- and late-developing embryos produced 56% and 45% males, respectively. CONCLUSIONS: Male embryos tend to develop faster than female embryos during early stage of preimplantation in mice.

Development of renal failure in PargParp-1 null and Timm23 hypomorphic mice.

Poly(ADP-ribose) glycohydrolase (Parg) is a central enzyme for poly(ADP-ribose) degradation. We established a Parg+/- mice strain by deletion of a part of exon 1 and around 0.4-kb upstream of sequences of the Parg gene. Parg-/- embryos obtained by intercrossing the Parg+/- mice died in utero between 4.5 and 9.5 days postcoitum. We examined whether poly(ADP-ribose) polymerase-1 (Parp-1) deficiency could rescue embryonic lethality of Parg-/- mice. Parg-/-Parp-1-/- mice were born viable at a reduced frequency from the expected mendelian ratio in the intercross progeny of Parg+/-Parp-1-/- mice. The results suggest a possibility that the presence of Parp-1 is responsible for the lethality of Parg-/- embryos, and Parg molecules or Parg activity degrading poly(ADP-ribose) might be important for embryogenesis. In Parg-/-Parp-1-/- mice, Parg protein was not detected in various tissues, and the protein level of Timm23, a 5'-upstream gene of Parg, was reduced compared with that in Parg+/+Parp-1-/- mice. Parg-/-Parp-1-/- mice showed retarded growth compared with Parg+/+Parp-1-/- mice, and died within 3 months of age accompanied with severe renal failure. Glomerular sclerosis, tubular dilatation, and hyaline casts in the kidney were observed in Parg-/-Parp-1-/- mice. An increase in blood urea nitrogen (p < 0.05), a marked increase of albumin level in urine (p < 0.01) and its concomitant decrease in serum (p < 0.05) were also detected in Parg-/-Parp-1-/- mice compared with the Parg+/+Parp-1-/- counterpart. The results imply that the combined Parg and Parp-1 loss with a hypomorphic state of Timm23 leads to the development of severe renal failure.

Effect of pressure at primary drying of freeze-drying mouse sperm reproduction ability and preservation potential.

Freeze-dried spermatozoa are capable of participating in normal embryonic development after injection into oocytes and thus useful for the maintenance of genetic materials. We recently reported that long-term preservation of freeze-dried mouse spermatozoa by conventional methods requires temperatures lower than -80 degrees C. Successful permanent preservation of mouse spermatozoa at much higher temperatures requires thorough investigation of the freeze-drying procedure. Thus, we examined the relationship between the pressure at primary drying and the preservation potential of freeze-dried mouse spermatozoa. Three different primary drying pressures were applied to evaluate the effect of pressure on freeze-dried spermatozoa under varying storage conditions and the rate of development measured. The developmental rate of embryos to the blastocyst stage from intracytoplasmic sperm injection by freeze-dried spermatozoa at pressures of 0.04, 0.37, and 1.03 mbar without storage were 59% (337/576), 71% (132/187), and 33% (99/302) respectively. When stored at 4 degrees C for 6 months, the rate was 13% (48/367), 50% (73/145), and 36% (66/182) respectively. These results show that primary drying pressure is an influential factor in the long-term preservation of freeze-dried mouse spermatozoa.

Viral envelope protein gp64 transgenic mouse facilitates the generation of monoclonal antibodies against exogenous membrane proteins displayed on baculovirus.

We have been investigating the functional display of multipass membrane protein such as transporter or G-protein coupled receptor on the budded baculovirus (BV). We tested the use of a viral envelope protein gp64 transgenic mouse for the direct immunization of these membrane proteins displayed on BVs. The gp64 transgenic mice showed only a weak response to virus compared to wild type BALB/c mice. Immunizing gp64 transgenic mice with the BV expressing peptide transporter PepT1, we obtained 47 monoclonal antibodies (mAbs). These mAbs were specific to the PepT1 on the pancreatic cancer cells AsPC-1 by fluorocytometric analysis and exhibited antibody-dependent cellular cytotoxicity or complement-dependent cytotoxicity to AsPC-1. We also generated 7 mAbs by immunizing gp64 transgenic mice on a CCR2-deficient background with the BV expressing chemokine receptor CCR2 together with partially purified CCR2. These mAbs possessed specific binding to CCR2 in CHO cells on fluorocytometric analysis, and exhibited neutralizing activities for ligand-dependent inhibition of cyclic AMP production. This method provides a powerful tool for the generation of therapeutic/diagnostic mAbs against membrane proteins.

Fertility of mice receiving vitrified adult mouse ovaries.

Cryopreservation of the ovaries is a useful technology for preservation of germ cells from experimental animals, because if the female founder is infertile or has mutated mitochondrial DNA, preservation of female germ cells is necessary. Although it is possible to cryopreserve immature mouse ovaries with a high degree of viability by vitrification with a mixture of several cryoprotectants, the viability of cryopreserved adult mouse ovaries is still unknown. Here, we investigated the viability of mouse ovaries at various ages after cryopreservation by vitrification techniques. Donor ovaries were collected from 10-day-, 4-week-, 10-week- and 7-month-old, female, nulliparous, green fluorescence protein (GFP)-transgenic mice and cryopreserved by vitrification. The vitrified-warmed ovaries were orthotopically transplanted to 4- or 10-week-old mice. GFP-positive pups were obtained in all experimental groups. In the 4-week-old recipients, the percentages of GFP-positive pups among the total pups from recipients transplanted with ovaries of 10-day-, 4-week-, 10-week- and 7-month-old donors were 44%, 9%, 12% and 4% respectively. In the 10-week-old recipients, the percentages of GFP-positive pups among the total pups from recipients transplanted with ovaries of 10-day-, 4-week-, 10-week- and 7-month-old donors were 36%, 16%, 2% and 9% respectively. Furthermore, GFP-positive pups also were obtained from recipients transplanted with ovaries of donors without normal estrous cyclicity. Our results indicate that cryopreservation of mouse ovaries by vitrification is a useful method for the preservation of female germ cells from mice of various ages.

Parp-1 deficiency does not enhance liver carcinogenesis induced by 2-amino-3-methylimidazo[4,5-f]quinoline in mice.

The susceptibility of poly(ADP-ribose) polymerase-1 (Parp-1) knockout mice to 2-amino-3-methylimidazo[4,5-f]quinoline (IQ)-induced liver carcinogenesis was analyzed. Twelve-week-old male Parp-1(+/+), Parp-1(+/-) and Parp-1(-/-) mice of the C57BL/6 congenic strain were fed a diet containing IQ at a concentration of 300 ppm or a control diet for 60 weeks. Hepatocellular carcinomas were observed only in 1/19, 2/18 and 1/17 of the Parp-1(-/-), Parp-1(+/-) and Parp-1(+/+) mice, respectively. Parp-1 deficiency did not affect the susceptibility of mice to carcinogenicity of IQ, which produces bulky DNA adducts that are repaired mainly through the nucleotide excision repair pathway. This result is in sharp contrast to the increased susceptibility of Parp-1(-/-) mice to carcinogenesis induced by alkylating agents that produce DNA damage repaired mainly through base excision repair and DNA strand break repair pathways.

Parp-1 deficiency causes an increase of deletion mutations and insertions/rearrangements in vivo after treatment with an alkylating agent.

Accumulated evidence suggests that Parp-1 is involved in DNA repair processes, including base excision repair, single-strand and double-strand break repairs. To understand the precise role of Parp-1 in genomic stability in vivo, we carried out mutation analysis using Parp-1 knockout (Parp-1-/-) mice harboring two marker genes, gpt and red/gam genes. Spontaneous mutant frequencies of both genes in the bone marrows and livers did not differ significantly between Parp-1-/- and Parp-1+/+ mice (P>0.05). After treatment with an alkylating agent, N-nitrosobis(2-hydroxypropyl)amine (BHP), the mutant frequency of the red/gam genes in the liver in Parp-1-/- mice was 1.6-fold higher than that in Parp-1+/+ mice (P<0.05). Categorization of the mutations revealed that deletions larger than 1 kb or those accompanying 1-5 bp insertions at the deletion junctions, as well as rearrangements, were more frequently observed in Parp-1-/- than in Parp-1+/+ mice (P<0.05, respectively). In contrast, mutant frequencies of the gpt gene in the livers of Parp-1(-/-) and Parp-1(+/+) mice after BHP treatment were both elevated and there was no significant difference between the genotypes. These results indicate that Parp-1 is implicated in suppressing deletion mutations in vivo, especially those accompanying small insertions or rearrangements.

Possibility of long-term preservation of freeze-dried mouse spermatozoa.

Freeze-dried mouse spermatozoa are capable of participating in normal embryonic development after injection into oocytes. When the freeze-dried spermatozoa are used as a method for storage of genetic materials, however, it is essential to assure the relevance of long-term preservation over several decades or centuries. Thus, we applied the theory of accelerated degradation kinetics to freeze-dried mouse spermatozoa. Thermal denaturation kinetics were determined based on Arrhenius plots derived from transition-state theory analysis at three elevated temperatures: 30, 40, and 50 degrees C. Accelerated degradation kinetics were calculated by extrapolation of Arrhenius plots. This theory also is being applied to the long-term stability of drugs. The estimated rate of development to the blastocyst stage at 3 and 6 mo and at 1, 10, and 100 yr of sperm storage at 4 degrees C were 21.60%, 7.91%, 1.00%, 0%, and 0%, respectively. At -80 degrees C, estimated development rates to the blastocyst stage that would be expected after 100 yr of storage did not decline significantly. In addition, after 3 or 6 mo of storage at 4 or -80 degrees C, preimplantation development of the embryos derived from intracytoplasmic sperm injection (ICSI) was examined. The actual developmental rates to the blastocyst stage from ICSI by freeze-dried sperm stored for 3 mo at 4 and -80 degrees C were 21% and 62%, respectively, and the rates for such sperm stored for 6 mo were 13% and 59%, respectively. These results indicate that the determination of accelerated degradation kinetics can be applied to the preservation of freeze-dried mouse spermatozoa. Furthermore, for long-term preservation, freeze-dried mouse spermatozoa appear to require being kept at lower than -80 degrees C.

Scavenger receptor expressed by endothelial cells I (SREC-I) mediates the uptake of acetylated low density lipoproteins by macrophages stimulated with lipopolysaccharide.

Scavenger receptor expressed by endothelial cells I (SREC-I) is a novel endocytic receptor for acetylated low density lipoprotein (LDL). Here we show that SREC-I is expressed in a wide variety of tissues, including macrophages and aortas. Lipopolysaccharide (LPS) robustly stimulated the expression of SREC-I in macrophages. In an initial attempt to clarify the role of SREC-I in the uptake of modified lipoproteins as well as in the development of atherosclerosis, we generated mice with a targeted disruption of the SREC-I gene by homologous recombination in embryonic stem cells. To exclude the overwhelming effect of the type A scavenger receptor (SR-A) on the uptake of Ac-LDL, we further generated mice lacking both SR-A and SREC-I (SR-A(-/-);SREC-I(-/-)) by cross-breeding and compared the uptake and degradation of Ac-LDL in the isolated macrophages. The contribution of SR-A and SREC-I to the overall degradation of Ac-LDL was 85 and 5%, respectively, in a non-stimulated condition. LPS increased the uptake and degradation of Ac-LDL by 1.8-fold. In this condition, the contribution of SR-A and SREC-I to the overall degradation of Ac-LDL was 90 and 6%, respectively. LPS increased the absolute contribution of SR-A and SREC-I by 1.9- and 2.3-fold, respectively. On the other hand, LPS decreased the absolute contribution of other pathways by 31%. Consistently, LPS did not increase the expression of other members of the scavenger receptor family such as CD36. In conclusion, SREC-I serves as a major endocytic receptor for Ac-LDL in LPS-stimulated macrophages lacking SR-A, suggesting that it has a key role in the development of atherosclerosis in concert with SR-A.

Comparison of fertility between intracytoplasmic sperm injection and in vitro fertilization with a partial zona pellucida incision by using a piezo-micromanipulator in cryopreserved inbred mouse spermatozoa.

Cryopreservation of mouse spermatozoa has been widely applied for maintenance of transgenic and knockout lines. However, the fertility of cryopreserved spermatozoa from some inbred strains such as C57BL/6 and BALB/c is extremely poor. We have recently reported that a partial zona-pellucida incision by piezo-micromanipulator (ZIP) significantly improves the fertilization rate and subsequent embryonic development after in vitro fertilization (IVF) using cryopreserved C57BL/6 transgenic mouse spermatozoa and that inbred C57BL/6 mice could be produced by intracytoplasmic sperm injection (ICSI). These findings prompted us to compare the efficiency of fertilization and subsequent embryonic development between ICSI and IVF with ZIP (ZIP/IVF) using cryopreserved spermatozoa. In conventional IVF, BALB/cA, C57BL/6J, and B6C3F1 cryopreserved spermatozoa fertilized 19%, 0%, and 51% of oocytes, respectively. The fertilization rates of manipulated oocytes by ICSI versus ZIP/IVF using cryopreserved BALB/cA spermatozoa were 52% versus 68%, cryopreserved C57BL/6J spermatozoa were 43% versus 63%, and cryopreserved B6C3F1 spermatozoa were 58% versus 82%, respectively. In these strains, fertilization rates for ZIP/IVF were significantly higher (P < 0.05) than for other techniques. However, embryonic development to term for oocytes fertilized by cryopreserved spermatozoa was not significantly different between ZIP/IVF and ICSI in C57BL/6J and B6C3F1. The overall efficiency of mouse production in ZIP/IVF was higher than for ICSI and conventional IVF in C57BL/6J and B6C3F1. Furthermore, ZIP/IVF required approximately 3.3 times less manipulation time than did ICSI. Our results indicate that ZIP is a useful assisted reproductive technique for IVF of ova by cryopreserved spermatozoa and improves production in some mouse strains.

Parp-1 deficiency implicated in colon and liver tumorigenesis induced by azoxymethane.

Poly(ADP-ribose) polymerase-1 (Parp-1) is activated by DNA strand breaks and functions in the maintenance of genomic integrity and cell death control. On the other hand, Parp-1 is also involved in transcriptional regulation of various genes, and the relationship between Parp-1 deficiency and susceptibility to tumorigenesis has not been fully elucidated. In the present study, Parp-1(-/-) mice, harboring exon 1 disruption in Parp-1, and Parp-1(+/+) animals were administered azoxymethane (AOM) at a dose of 10 mg/kg body weight once a week for 6 weeks. At 30 weeks after the first carcinogen treatment, mice were sacrificed. The incidence of animals bearing either adenomas or adenocarcinomas in the colon and the average number of colon tumors per mouse were significantly higher in Parp-1(-/-) mice than in Parp-1(+/+) animals. beta-Catenin accumulation was observed in 43/44 of Parp-1 (-/-) tumors and 19/21 of the Parp-1(+/+) tumors and was not statistically different between the genotypes. This suggests that most tumors developed through a pathway involving the alteration of Wnt-beta-catenin signaling in both Parp-1(-/-) and Parp-1(+/+) mice. In the liver, where AOM is primarily activated, the incidence of animals bearing nodules and the average number of nodules per section were significantly increased in Parp-1(-/-) mice compared with Parp-1(+/+) mice. Therefore, the results indicate that susceptibility to AOM-induced tumorigenesis in the colon and also in the liver is enhanced in Parp-1(-/-) mice, and Parp-1 could have a substantial role in colon and liver tumorigenesis.

Parp1-deficiency induces differentiation of ES cells into trophoblast derivatives.

Embryonic stem (ES) cells deficient in the enzyme poly(ADP-ribose) polymerase (Parp1) develop into teratocarcinoma-like tumors when injected subcutaneously into nude mice that contain cells with giant cell-like morphology. We show here that these cells express genes characteristic of trophoblast giant cells and thus belong to the trophectoderm lineage. In addition, Parp1(-/-) tumors contained other trophoblast subtypes as revealed by expression of spongiotrophoblast-specific marker genes. The extent of giant cell differentiation was enhanced, however, as compared with spongiotrophoblast. A similar shift toward trophoblast giant cell differentiation was observed in cultures of Parp1-deficient ES cells and in placentae of Parp1(-/-) embryos. Analysis of other cell lineage markers demonstrated that Parp1 acts exclusively in trophoblast to suppress differentiation. Surprisingly, trophoblast derivatives were also detected in wildtype tumors and cultured ES cells, albeit at significantly lower frequency. These data show that wildtype ES cells contain a small population of cells with trophectoderm potential and that absence of Parp1 renders ES cells more susceptible to adopting a trophoblast phenotype.

Possible expansion of "Window of Implantation" in pseudopregnant mice: time of implantation of embryos at different stages of development transferred into the same recipient.

Blastocyst implantation and successful establishment of pregnancy require delicate interactions between the embryo and maternal environment. It is believed that the growth of transferred embryos of different ages is synchronized during preimplantation development and that such embryos are implanted in the uterus at the same time. To define the time of synchronization for developing embryos of different ages, embryos at two different stages of development were transferred separately into the oviducts of the same recipient. We then examined the subsequent development of the embryos at various time intervals after transfer. Pronucleus (PN) stage eggs were transferred separately to the right or left oviduct of recipients on Day 0, while eight-cell embryos (8C) were transferred to the other oviduct. For 8C, 5%, 63%, and 74% of transferred embryos were implanted in the uterus at 42, 66, and 90 h posttransfer, respectively. In contrast, none of the transferred PN was implanted until 90 h posttransfer. At 90 h posttransfer, 59% of the PN had successfully implanted. Histological examination revealed that developmental stage of the embryos in both groups synchronized around 162 h posttransfer, even though the implantation was accelerated in 8C compared with PN. Our results indicate that embryos of advanced stage transferred to the oviduct implant in the uterus in advance of younger embryos and that the uterine development is synchronized at the neural plate, presomite stage. Our results strongly suggest that uterine receptivity for implantation is expandable in pseudopregnant mice.

Developmental expression of EphB6 in the thymus: lessons from EphB6 knockout mice.

A member of the largest family of receptor protein kinases, EphB6, lacks its intrinsic kinase activity, but it is expressed in normal human tissues. To investigate the physiological function of EphB6, we generated EphB6 deficient mice. EphB6(-/-) mice developed normally, revealed no abnormality in general appearance, and were fertile. Although a developmental increase of EphB6 in the fetal thymus was confirmed, T-cell development in various lymphoid organs of EphB6(-/-) mice was comparable to those of EphB6(+/+). Even in fetal thymus organ cultures, any developmental differences of EphB6(-/-) and EphB6(+/+) thymocytes were undetectable. The different binding characteristics to ephrin-Fc proteins between EphB6(-/-) and EphB6(+/+) thymocytes demonstrated that ephrin-B2 is the unique ligand for EphB6 among eight known ephrins. While EphB6 was a dominant receptor that binds to ephrin-B2 in adult thymocytes, fetal ones also expressed another EphB that binds to ephrin-B2. Overlapping expression of the EphB subfamily in the fetal thymus might compensate for the loss of EphB6 during the thymic development.

Role of Lkb1, the causative gene of Peutz-Jegher's syndrome, in embryogenesis and polyposis.

Peutz-Jeghers syndrome (PJS) is a dominantly inherited human disorder characterized by gastrointestinal hamartomatous polyposis and mucocutaneous melanin pigmentation. LKB1 (STK11) serine/threonine kinase is the product of the causative gene of PJS, which has been mapped to chromosome 19p13.3. However, several studies have produced results that are not consistent with a link between LKB1 gene mutation and PJS. We constructed a knockout gene mutation of Lkb1 to determine whether it is the causative gene of PJS and to examine the biological role of the Lkb1 gene. Lkb1(-/-) mice died in utero between 8.5 and 9.5 days postcoitum. At 9.0 days postcoitum, Lkb1(-/-) embryos were generally smaller than their age-matched littermates, showed developmental retardation, and did not undergo embryonic turning. Multiple gastric adenomatous polyps were observed in 10- to 14-month-old Lkb1(+/-) mice. Our results indicate that functional Lkb1 is required for normal embryogenesis and that it is related to tumor development. The Lkb1(+/-) mouse is suitable for studying molecular mechanism underlying the development of inherited gastric tumors in PJS.

Effect of partial incision of the zona pellucida by piezo-micromanipulator for in vitro fertilization using frozen-thawed mouse spermatozoa on the developmental rate of embryos transferred at the 2-cell stage.

Cryopreservation of mouse spermatozoa is widely used, although considerable strain differences in fertilization rates using frozen-thawed mouse spermatozoa have been described. The C57BL/6 mouse strain is a very widely used for establishment of transgenic mice, but the fertilization rate associated with the use of cryopreserved C57BL/6 spermatozoa is very low compared with rates for other inbred strains. We have recently solved this difficulty by in vitro fertilization (IVF) in combination with partial zona pellucida dissection (PZD). However, this technique requires culture of fertilized eggs with PZD in vitro up to morula or blastocyst stage before transfer into the uterus because blastomeres are lost after transfer into the oviduct because of the relatively large artificial slit in the zona pellucida. To overcome this problem, we performed a partial zona pellucida incision by using a piezo-micromanipulator (ZIP) for IVF with frozen-thawed mouse spermatozoa. The blunt end of the micropipette touched the surface of the zona pellucida of the oocytes, and piezo pulses were used to incise the zona pellucida while the pipette was moved along by the surface of zona pellucida. The length of the incision was pir/6 microm. When cumulus-free ZIP and PZD oocytes were inseminated with frozen-thawed genetically modified C57BL/6J spermatozoa, the fertilization rates of ZIP and PZD oocytes were 52% and 48%, respectively. After embryo transfer at the 2-cell stage, 18% and 2% of the transferred embryos with ZIP and PZD developed to term, respectively. This difference was significant (P < 0.05). When ZIP and PZD zygotes were cultured to blastocyst stage and subsequently transferred to uterine horns of recipient animals, the difference between ZIP and PZD zygotes for development rate to full term was not significant. Our results indicate that ZIP is an effective alternative technique for IVF using cryopreserved mouse spermatozoa and subsequent embryo transfer.