Germfree status of mice obtained by embryo transfer in an isolator environment

The technique of embryo transfer has been evaluated for the purpose of changing the mouse stocks to a germfree (GF) status. Our results show reproducible and quality-assured conversion of animals to those which are negative for the presence of microorganisms. Rapid and easy access to GF mice is advantageous for studies of selected microflora and their cross-talks with the host, when applying, e.g. genomic, proteomic and metabolic methodology.The study involved embryo transfer in an isolator environment, thereby allowing implantation of cleansed embryos into GF recipients under well-controlled conditions. The recipient females gave birth normally and took care of the offspring as if they were their own pups, thus enhancing the survival rate. Access to full technical resources required to maintain GF isolators are, however, a prerequisite. In this study, we used stainless steel isolators designed by Gustafsson (1959), on which a stereomicroscope was mounted to facilitate embryo transfer inside the isolator.The use of embryo transfer and isolator techniques will facilitate the availability of various mouse mutant models under different gnotobiotic conditions, GF, monoxenic or polyxenic animals, to enable comparison with conventional animals for physiological and pathophysiological studies.     

Surgical recovery and successful surgical transfer of conventionally frozen-thawed embryos in the farmed European polecat (Mustela putorius)

Surgical transfer of in vivo produced conventionally frozen-thawed embryos of farmed European polecat (Mustela putorius) was investigated as a part of an ex-situ preservation program which has the long-term aim of developing a genome resource bank for the endangered European mink (Mustela lutreola). Eighteen oestrous yearling European polecat donors were mated once daily on two consecutive days using 13 fertile males. The donors were surgically flushed for embryos 8-9 days after the first mating. The embryo recovery rate was 60% (116 embryos/193 corpora lutea). The embryos were cryopreserved with 1.5 M ethylene glycol in a programmable freezer using a conventional slow freezing protocol. The thawed embryos were surgically transferred either after dilution with 0.5 M sucrose or directly without removal of ethylene glycol. To induce ovulation, eight recipient females were mated once daily on two consecutive days with vasectomized males starting 7 or 8 days before embryo transfer. The recipients received 7-11 embryos each and three recipients delivered a total of nine pups after a gestation length of 44-46 days. The embryo survival rate was 10% (9 pups/93 frozen embryos). This report describes the first successful cryopreservation of embryos in the Mustelidae family resulting in viable offspring. The low embryo survival rate, however, indicates that the freezing-thawing protocol needs to be improved.     

Evaluation of mouse blastocyst implantation rate by morphology grading

The aim of our study is to observe the relationship between the blastocyst morphology and the implantation rate for mice. Mouse embryos obtained from the superovulated-ICR mice were cultured in vitro from 1-cell zygotes to blastocysts. Mouse blastocysts were then classified into 3 grades: grade I, small blastocysts; grade II, large blastocysts; grade III, hatching blastocysts. They were independently transferred into the uterus of recipient females mated with vasectomized male mice on 96 hours after the zygotes were cultured in vitro. The successful implantation was checked by injection of Chicago Sky Blue 6B on the second day after embryo transfer. Although there was no significant difference in the implantation rates between the grade III and grade II, grade I was significantly decreased, as compared with grade III. Grade I and grade II was also significantly decreased in both the diameter of blastocysts and cell number of inner cell mass (ICM) and trophectoderm (TE), as compared with grade III. These findings indicate that the expanded and hatching blastocyst selections for embryo transfer in in vitro fertilization were evaluated with the high implantation rate.     

Coprophagy in the germfree mouse

Coprophagy was observed in germfree (GF) ICR mice of both sexes, and the results were compared with those of conventional mice. Frequency of coprophagy per animal per day in GF mice was 5.1 in males and 5.8 in females. In conventional (CV) mice, the frequencies were 6.2 in males and 5.3 in females (data from Zoological Science 2:249-255, 1985), with no significant differences compared with GF mice. Coprophagy in CV mice was frequently observed during 6-8 hr after lighting, whereas such close time relationships tended to weaken in GF animals. In a comparison of levels of constituents per unit weight between feces and diet, fecal crude protein and crude fat exhibited lower values than those in the diet. Levels of fecal crude ash and crude fiber were higher than those in the diet, and nitrogen-free extract was almost equal to that in the diet. No essential difference in these tendencies was found compared with CV mice. Levels of fecal vitamin B1, B2, B12 and folic acid were lower than those in the diet. In CV mice, except for vitamin B1, these vitamins exhibited either almost equal or much higher levels compared with those in the diet (data from Experimental Animals 35: 381-386, 1986). From the fact that coprophagy was observed in GF mice, it is suggested that the behavior is inherent in the mouse.     

Production of identical twin and triplet mice by nuclear transplantation

Transplantation of a single nucleus from two- or four-cell embryos into one of the enucleated blastomeres of a two-cell embryo resulted in successful production of identical triplet and twin mice. The proportion of reconstituted embryos that developed in blastocysts was 71% (84/118) when four-cell embryos were used as donors of nuclei; 10 sets of quadruplet and nine sets each of triplet and twin blastocysts were obtained by this technique. After transfer to recipients, 30% (18/61) developed to term, and one set of identical triplet and four sets of identical twin mice were obtained. When two-cell embryos were used as donors of nuclei, 79 (95%) sets of twin embryos developed to blastocysts. Of 38 twin blastocysts transferred to recipients, 21 sets (55%) developed to term as identical twin mice. These results demonstrate that the enucleated two-cell embryo develops in vitro after transfer of a nucleus from a two- or four-cell embryo and the resultant blastocyst has high potential for development to term after transfer to a recipient.     

Effect of intestinal flora on megaenteron of mice.

CF#1 germfree (GF) and conventional (CV) mice as well as offspring of conventionalized GF (GF-CV) mice were orally inoculated with Escherichia coli 0115a, c: K(B), a causative agent of megaenteron in mice. Although CV and GF mice showed no clinical signs and survived, all of the GF-CV mice died with diarrhea by day 14 after inoculation. Thickened wall of the large intestine, microscopically showing proliferation of crypt type cells, was seen in GF and GF-CV mice but not in CV mice. In addition, in GF-CV mice, hemorrhage and severe erosion with marked inflammatory reactions were observed. While the inoculated E. coli could not colonize in CV mice, a level of 10⁸ cells/g feces was maintained in GF mice from day 1 after inoculation to the end of examination (on day 28) and in GF-CV mice from day 5 to the time of death. Newly prepared germfree (GF-CV-GF) mice obtained hysterectomy from GF-CV mice showed a low sensitivity as comparable to that in GF mice. On the other hand, ex-germfree mice produced by oral administration of feces of GF-CV mice showed severe infection as comparable to that seen in GF-CV mice. These results suggest that the intestinal flora may play roles both on protecting from the infection of pathogenic E. coli and on enhancing the infection.     

Commensal bacteria exacerbate intestinal inflammation but are not essential for the development of murine ileitis

The pathogenesis of Crohn's disease has been associated with a dysregulated response of the mucosal immune system against intraluminal Ags of bacterial origin. In this study, we have investigated the effects of germfree (GF) conditions in the SAMP1/YitFc murine model of Crohn's disease-like ileitis. We show that the bacterial flora is not essential for ileitis induction, because GF SAMP1/YitFc mice develop chronic ileitis. However, compared with disease in specific pathogen-free (SPF) mice, ileitis in GF mice is significantly attenuated, and is associated with delayed lymphocytic infiltration and defective mucosal expression of Th2 cytokines. In addition, we demonstrate that stimulation with purified fecal Ags from SPF, but not GF mice leads to the generation of IL-4-secreting effector lymphocytes. This result suggests that commensal bacteria drive Th2 responses characteristic of the chronic phase of SAMP1/YitFc ileitis. Finally, adoptive transfer of CD4-positive cells from GF, but not SPF mice induces severe colitis in SCID recipients. These effects were associated with a decreased frequency of CD4(+)CD25(+)Foxp3(+) T cells in the mesenteric lymph nodes of GF mice compared with SPF mice, as well as lower relative gene expression of Foxp3 in CD4(+)CD25(+) T cells in GF mice. It is therefore apparent that, in the absence of live intraluminal bacteria, the regulatory component of the mucosal immune system is compromised. All together, our results indicate that in SAMP1/YitFc mice, bacterial flora exacerbates intestinal inflammation, but is not essential for the generation of the chronic ileitis that is characteristic of these mice.     

16S rRNA gene sequence‐based analysis of clostridia related to conversion of germfree mice to the normal state

Aims: To determine phylogenetic groups of clostridia inhabiting the mouse intestine that are essential for normalization of germfree (GF) mice. Methods and Results: Using both the culture method and cloning, clostridia inhabiting the mouse intestine were isolated, and phylogenetic analysis based on 16S rRNA gene sequences was carried out. As a result, the isolates were found to have novel sequences, and no isolate was determined to be identical to previously known identified clostridia. Although the taxonomy of mouse intestinal clostridia was complex, many of them belonged to Clostridium clusters XIVa and IV in conventional (CV) and limited flora mice and ex‐germfree mice administered chloroform‐treated CV mouse faeces. The clostridia that belonged to cluster XIVa were most often present and showed the highest diversity. Conclusions: Clostridia belonging clusters XIVa and IV are dominant in the mouse intestine as in other gut ecosystems. The novel groups in these clusters are essential for normalization of GF mice. Significance and Impact of the Study: The results of this study can be applied in the strict control of mouse intestinal microbiota and will provide important information for normalization of GF mice and also for research on microbiology of the mouse intestine.     

Effects of germfree status and food restriction on longevity and growth of mice.

An investigation was undertaken to study the effects of germfree (GF) status and mild food restriction on life span in GF and specific pathogen-free (SPF) male ICR mice either full-fed (ad libitum) or on a restricted diet of 4.5 grams per day (equivalent to approximately 80% of full-fed intake) from five-week-old. The mean life span of the full-fed SPF and GF mice was 75.9 and 88.9 weeks respectively, while the mean life span of the food-restricted SPF and GF mice was 117.5 and 109.6 weeks, respectively. Mice in both GF and SPF food-restricted groups were characterized by lower body weight and increased survival. These findings suggest that the cessation of growth may be importantly and perhaps causally related to longevity. The GF mice survived longer than the SPF mice, but the combination of GF status with food restriction did not seem to extend life span more than food restriction alone.     

Immunological function of food-restricted germfree and specific pathogen-free mice.

The effects of food restriction on immune function was investigated in germfree (GF) and specific pathogen-free (SPF) mice. They were maintained from five weeks of age under either full-fed or food-restricted conditions to 4.5 grams per day (equivalent to approximately 80% of full-fed intake) of a commercial diet. Longest survival rate was attained in food-restricted SPF mice followed by food-restricted GF, full-fed GF, and full-fed SPF animals. Food-restricted GF mice showed shorter survival rate than their SPF counterparts. This result suggests that food restriction may be just as effective as GF status for extending life span. Immune function declined significantly with age in full-fed groups of GF and SPF mice. In both food-restricted GF and SPF mice, mitogenic response to concanavalin A or lipopolysaccharide and antibody response to sheep red blood cells were lower early in life and became higher later in life as compared with full-fed mice. Hence, the maintenance of effective immunological function until old age may be the reason for food-restricted groups to live slightly longer than full-fed groups.     

Utero-tubal Embryo Transfer and Vasectomy in the Mouse Model

The transfer of preimplantation embryos to a surrogate female is a required step for the production of genetically modified mice or to study the effects of epigenetic alterations originated during preimplantation development on subsequent fetal development and adult health. The use of an effective and consistent embryo transfer technique is crucial to enhance the generation of genetically modified animals and to determine the effect of different treatments on implantation rates and survival to term. Embryos at the blastocyst stage are usually transferred by uterine transfer, performing a puncture in the uterine wall to introduce the embryo manipulation pipette. The orifice performed in the uterus does not close after the pipette has been withdrawn, and the embryos can outflow to the abdominal cavity due to the positive pressure of the uterus. The puncture can also produce a hemorrhage that impairs implantation, blocks the transfer pipette and may affect embryo development, especially when embryos without zona are transferred. Consequently, this technique often results in very variable and overall low embryo survival rates. Avoiding these negative effects, utero-tubal embryo transfer take advantage of the utero-tubal junction as a natural barrier that impedes embryo outflow and avoid the puncture of the uterine wall. Vasectomized males are required for obtaining pseudopregnant recipients. A technique to perform vasectomy is described as a complement to the utero-tubal embryo transfer.     

Comparative studies of some functional and morphological parameters in the livers of germfree, conventional and ex-germfree mice.

Some parameters of hepatic function and morphology were studied to compare germfree (GF) and conventional (CV) BALB/c mice. The levels of lipid peroxide (LPO) and aniline-hydroxylase (AH) activity in the livers and the serum total cholesterol (TC), triglyceride (TG) and phospholipid (PL) were significantly lower in GF than in CV 8-week-old mice. There were no significant differences in the histology and lectin-histochemistry of the livers in the GF and CV mice. On the other hand, in ex-GF mice which were induced by housing 4-week-old GF mice together with age-matched CV mice, the levels of LPO and AH activity in the liver and the serum TC, TG and PL contents increased rapidly within the first week and then approached values almost identical to those in CV mice 4 weeks later (i.e. at 8 weeks of age). The histologic picture of the liver was similar among the GF, CV and ex-GF mice.     

三种微生物学级别的SSB小鼠生长和繁殖性能的比较

本文对无菌，清洁和普通三种微生物学级别的近交系ＳＳＢ小鼠核心群的生长，繁殖性能的观察结果进行比较分析。在哺乳期风无菌小鼠生长优于清洁级和普通级小鼠；而在离乳后育成初期体重增长又慢于者。计算机统计分析表明：三种级别种鼠的各胎次胎间隔无显著差异。清洁和无菌种鼠的产仔数和离乳率均无显著差异；而普通种鼠的繁殖性能不如清洁和无菌种鼠。     

Fate of superfluous sperm products after vasectomy and in the normal male tract of the mouse

The progression of 3H-labelled spermatozoa (thymidine or arginine) was followed through the tracts of unilaterally vasectomized, bilaterally vasectomized, oligozoospermic (t6/tw5) and normal mice; the regional lymph nodes were also investigated. The same rate of sperm production and transport was found in normal and in vasectomized tracts, down to the corpus epididymidis; there was some delay in spermatozoa entering the cauda in the vasectomized tracts. In the mouse, therefore, vasectomy does not affect the rates of sperm production or transport until just before the blockage in the swollen cauda epididymidis. Radioactivity appeared in the caudal and 'para-aortic' lymph nodes as the radioactive spermatozoa passed from the corpus, showing that this is one route of disposal of spermatozoa, or of sperm products, after vasectomy. Naturally oligozoospermic and normal mice gave similar results; again the caudal, iliac and renal lymph nodes received radioactive spermatozoa/sperm products. Some loss of (by definition) superfluous spermatozoa in the normal male tract therefore occurs naturally by this route, and we suggest that vasectomy further exploits this physiological pathway. This would account for the finding that many males do not make antisperm antibodies after vasectomy, just as normal males do not, even though their lymph nodes normally receive spermatozoa/sperm products.     

Intrabursal transfer of spermatozoa (ITS): a new route for artificial insemination of mice.

Artificial insemination (AI) by direct injection of epididymal spermatozoa into the reproductive tract of females is simpler and more convenient than in vitro fertilization (IVF) and subsequent transfer of fertilized eggs to recipient oviducts for simultaneous acquisition of a large number of pups. Introduction of epididymal spermatozoa into oviducts via the oviductal wall or via vaginal and intrauterine routes is currently the most commonly used method for AI in mice. In this study, we explored another route for AI of the mouse and found that transfer of spermatozoa into a space near the infundibulum between the ovary and ovarian bursa enables in vivo fertilization of ovulated oocytes at the ampulla. When 1 microL of a sperm suspension containing 1 x 10(4) spermatozoa freshly isolated from B6C3F1 males was intrabursally injected into superovulated B6C3F1 females on E (embryonic day) 0.4 (10:00 AM), 5 of 7 females yielded 2-cell embryos with rates of efficiency ranging from 4 to 21% (11% on average), which were much lower than those (91% on average) for embryos obtained by natural mating. All the 2-cell embryos derived from injection of sperm developed in vitro to hatched blastocysts. Similar results were obtained from injection of 1 microL of sperm suspension containing 1 x 10(3) spermatozoa, although in vivo fertilizing ability was slightly improved (28% on average). When 1 microL of sperm suspension containing 1 x 10(4) spermatozoa was injected intrabursally into superovulated females that had been mated with vasectomized males, 6 of 10 mice (60%) yielded 19 normal mid-gestational fetuses with an average litter size of 3.2, which was much lower than that (14.5) for embryos obtained by natural mating. Although the present findings appear to be preliminary, this technique, based on the intrabursal transfer of spermatozoa, will be of practical use for AI in mice, particularly for transgenic and mutant mice that are often difficult to breed.     

过继转输的父系抗原耐受T细胞诱导受体母-胎免疫耐受的机制研究

To study the effect of adoptive transfer of paternal antigen-tolerant T cells on recipient reactive T cells, CBA/JxDBA/2 mating was recruited as an abortion-prone model, and CBA/JxBALB/c mating as a successful pregnancy model. The abortion-prone CBA/J females mated with DBA/2 males were injected intraperitoneally with rat anti-mouse CD80 and CD86 mAb or rat isotype IgG at day 4 after gestation (time of implantation). The purified T cells were obtained from spleen of the pregnant CBA/J mice using magnetic beads at day 9 after gestation and labeled with CFSE in vitro. The CFSE-labeled T cells were intravenously injected into other CBA/J females mated with DBA/2 males at day 4 after gestation. The proliferation of recipient splenocytes in response to DBA/2 stimulator cells was evaluated at day 9 after gestation in vitro, and the expressions of intracellular cytokines and costimulatory molecules in CFSE +/- T cells were analyzed by flow cytometry. The results showed that adoptive transfer of either paternal antigen-tolerant T cells or T cells from BALB/c-mated CBA/J mice significantly suppressed the proliferation of recipient splenocytes in response to DBA/2 stimulator cells and resulted in lower frequency of cells positive for IL-2, IFN-gamma, CD28 and higher frequency of IL-10,CTLA-4-producing cells in both CFSE+ CD3+ population and CFSE- CD3+ population compared with adoptive transfer of T cells from isotype IgG-treated CBA/J mice, whereas the frequency of IL-4-producing cells did not appear significant change. Our findings suggest that paternal antigen-tolerant T cells transferred in recipient not only function as antigen-specific suppresser cells but also disable the recipient reactive T cells, which co-suppresses maternal rejection to the allogeneic fetus, thus resulting in the decrease of the embryo resorption rate of the abortion-prone mice to that of the normal pregnancy mice.     

Correlation of increased concentration of ovine endometrial cyclooxygenase 2 with the increase in PGE2 and PGD2 in the late luteal phase

The effect of intraoviductal embryos on endometrial receptivity was studied by intraendometrial and intrauterine embryo transfer. Five-week-old female ICR mice were mated after superovulation; a vaginal plug confirmed day 1 of pregnancy. On day 4 (90 h after hCG injection), blastocysts were collected and transferred to pseudopregnant female mice and to recipient mice in which the uterotubal junction had been ligated bilaterally on day 1 of pregnancy. Three embryos per uterine horn, a total of six embryos per recipient mouse at days 1-6, were transferred to the endometrium or uterine cavity and implantation and pregnancy rates were calculated. The implantation rate for intraendometrial embryo transfer to recipients of days 3, 5 and 6 was significantly higher for uterotubal junction-ligated mice (72.2, 20.8 and 9.7%, respectively) than for pseudopregnant mice (55.0, 8.3 and 0.0%, respectively). The implantation rate for intrauterine embryo transfer to recipients at days 2, 5 and 6 was significantly higher for uterotubal junction-ligated mice (11.1, 25.0 and 8.3%, respectively) than for pseudopregnant mice (0.0, 3.3 and 0.0%, respectively). Uterotubal junction-ligated mice achieved implantation and bore neonates by intrauterine embryo transfer on days 2 and 6, whereas no implantation was achieved in pseudopregnant mice. The difference in implantation rate could not be explained by a difference in progesterone concentration between the groups. The distribution of proliferating cells in the endometrium was also studied immunohistochemically by use of anti-proliferating cell nuclear antigen (PCNA) antibody in the recipient mice. PCNA-positive cells were more abundant in uterotubal junction-ligated mice and demonstrated a marked extension from the epithelium to the stroma over time, in contrast to those in pseudopregnant mice. These findings indicate that an intraoviductal embryo exerts a biological effect by sending a signal to the endometrial epithelium and stroma, thus facilitating endometrial receptivity to the embryo and improving the rate of implantation.     

Differences in survival among germfree mice following transfer to a conventional colony.

Sex and strain differences in survival were studied in 7-9 month old germfree mice following transfer to a conventional colony. One outbred and three inbred strains were observed. All outbred CD-1 mice survived transfer and in 4 months increased their weight by 50%. The majority of inbred mice survived 7 months after transfer. Sex differences in survival were evident throughout the experimental period and were most marked 7 months after transfer. An unexpected new finding was the viability of the male sex in germfree mice after transfer. Possible explanations are considered.     

Plasmodium berghei and Eperythrozoon coccoides: Antibody and immunoglobulin synthesis in germfree and and conventional mice simultaneously infected

The immune response to Eperythrozoon coccoides and the malaria parasite Plasmodium berghei was evaluated in germfree (GF) and conventionally reared (CV) mice infected with both parasites. Following infection, the mice showed significant changes in the levels of the immunoglobulins IgM, 7Sγin1, 7Sγ2a, and 7Sγ2b, but no detectable changes in IgA. Increases in immunoglobulin levels were first observed in GF mice, but by the twelth day both GF and CV mice had comparable levels. 7Sγ2a globulin had a bimodal distribution in both groups of mice which probably was due to heterogeneity in the allotype of this immunoglobulin. IgM levels closely paralleled the antibody responses to P. berghei suggesting that most of the antibody to this parasite was IgM. Relatively low levels of antibody to both parasites, in comparison to the large immunoglobulin response, were detected in GF and CV mice. The possible causes for the low titers are discussed.     

Participation of embryonic genotype in the pregnancy block phenomenon in mice.

Pregnancy block by male pheromones in mice differs in incidence depending on the combination of strains. Female mice of BALB/cA strain mated with BALB/cA males show a 100% pregnancy block when exposed to males of inbred strain DDK shortly after copulation (Chung et al., Biol Reprod 1997; 57:312-319). In the present study, BALB/cA females mated with the males of other strains--CBA/J, C3H/HeN, C57BL/6Cr, and IXBL--showed higher pregnancy rates (66.6-87. 5%) even when they were exposed to DDK males. In the pharmacological induction of pregnancy block with dopamine agonist (bromocriptine, 4 mg/kg BW), BALB/cA females mated with BALB/cA males showed a 100% pregnancy block. In contrast, BALB/cA females mated with CBA/J, C3H/HeN, and C57BL/6Cr males showed higher pregnancy rates (40-70%). These results suggest that the better pregnancy rate of BALB/cA females mated with alien males may be due to the stronger viability of F(1) embryos. This interpretation was confirmed by an embryo transfer experiment in which a higher implantation rate was observed when BALB/cA embryos grown in BALB/cA females exposed to BALB/cA males were transferred into recipient BALB/cA females exposed to DDK males. These results suggest that the embryonic genotype or viability of the embryo is one factor contributing to the occurrence of pregnancy block by male pheromones in mice.