Modification of survival rate of mouse embryos developing in heterozygous females for ovum mutant gene

The DDK syndrome (polar infertility) is caused by an incompatibility system due to the ovum mutant (Om) locus. For brevity, the following gene symbols are used in the present report: DDK allele, Om; C57BL/6Cr allele, +. In this investigation, we first attempted to introduce the Om allele of DDK strain into the genetic background of C57BL/6Cr strain. The attempt resulted in the production of no young at the third generation of successive backcrosses. Secondly, mating experiments were performed with heterozygous (Om/+) females having background genes of C57BL/6Cr and DDK strains in the ratios 1:1(B1D), 3:1(B3D), 7:1(B7D), and 15:1(B15D). The survival rate of the embryos as judged by the percentage number of live fetuses/number of corpora lutea at Day 12 of pregnancy was 41.3 +/- 3.2%, 27.3 +/- 3. 2%, 16.4 +/- 3.3%, and 11.3 +/- 3.2% (mean +/- SEM) in the B1D, B3D, B7D, and B15D females, respectively, when they were mated with C57BL/6Cr males. Furthermore, the increased embryonic mortality in the heterozygous (Om/+) females with more background genes of C57BL/6Cr strain was found to be due to a failure in blastocyst formation, as in the DDK syndrome. The parallelism between the proportion of C57BL/6Cr background genes and embryonic mortality has led to a hypothesis proposing the participation of a modifier gene, namely that a mechanism similar to allelic exclusion may be working in the synthesis of cytoplasmic factor of eggs and that only the Om allele is activated during oogenesis to produce DDK-type cytoplasmic factor in heterozygous (Om/+) females having a modifier gene in the homozygous state.     

Viability of frozen-thawed mouse embryos is affected by genotype

Embryos from mice of five different genotypes were evaluated for their ability to survive cryopreservation as measured by post-thaw in vitro development. In Study 1, ovulation was induced with a standardized pregnant mares' serum gonadotropin (PMSG)/human chorionic gonadotropin (hCG) regimen, after which females were mated with males of the same genotype to produce incrossed embryos. Four- to 8-cell embryos were frozen in 1.5 M dimethyl sulfoxide (DMSO) at a rate of 0.5 degrees C/min to -80 degrees C and stored in liquid nitrogen. Following thawing at room temperature, embryos were cultured and development was evaluated 24 h later. The mean (+/- SEM) number of 4- to 8-cell embryos/pregnant female by stock/strain were: N:NIH(S), 6.8 +/- 0.8; N:NIH(S)-B, 5.8 +/- 0.5; N:GP(S), 6.5 +/- 0.6; C57BL/6N, 9.7 +/- 1.0; C3H/HeN MTV-, 9.5 +/- 0.9 (P less than 0.05). Post-thaw in vitro development was related to genetic background; the proportion of embryos culturing after thawing was: N:NIH(S), 49%; N:NIH(S)-B, 61%; N:GP(S), 66%; C57BL/6N, 75%; C3H/HeN MTV-, 56% (P less than 0.05). Study 2 was conducted to evaluate the influence of mating various females to males of a genotype known to have a lower post-thaw embryo survival rate. N:NIH(S)-B, N:GP(S), C57BL/6N, and C3H/HeN MTV- female mice were mated with N:NIH(S) males to produce hybrid embryos. Post-thaw embryo survival was reduced (P less than 0.05) in three of the four hybrid groups. Fresh incrossed and hybrid embryos from each study were cultured for 24 h and yielded culture rates ranging from 95% to 99% (P greater than 0.05) among all groups.(ABSTRACT TRUNCATED AT 250 WORDS)     

BALB/c alleles at modifier loci increase the severity of the maternal effect of the "DDK syndrome"

The Om locus was first described in the DDK inbred mouse strain: DDK mice carry a mutation at Om resulting in a parental effect lethality of F(1) embryos. When DDK females are mated with males of other (non-DDK) inbred strains, e.g., BALB/c, they exhibit a low fertility, whereas the reciprocal cross, non-DDK females x DDK males, is fertile (as is the DDK intrastrain cross). The low fertility is due to the death of (DDK x non-DDK)F(1) embryos at the late-morula to blastocyst stage, which is referred to as the "DDK syndrome." The death of these F(1) embryos is caused by an incompatibility between a DDK maternal factor and the non-DDK paternal pronucleus. Previous genetic studies showed that F(1) mice have an intermediate phenotype compared to parental strains: crosses between F(1) females and non-DDK males are semisterile, as are crosses between DDK females and F(1) males. In the present studies, we have examined the properties of mice heterozygous for BALB/c and DDK Om alleles on an essentially BALB/c genetic background. Surprisingly, we found that the females are quasi-sterile when mated with BALB/c males and, thus, present a phenotype similar to DDK females. These results indicate that BALB/c alleles at modifier loci increase the severity of the DDK syndrome.     

Female mice of DDK strain are fully fertile in the intersubspecific crosses with Mus musculus molossinus and M. m. castaneus

The female mice of DDK strain are almost infertile when mated with males from other strains. This phenomenon is caused by the early death of F1 embryos owing to the incompatibility system attributed to the ovum mutant (Om) locus on Chromosome (Chr) 11 and known as DDK syndrome. In the present study, DDK females were found to be fully fertile in the intersubspecific matings with the males of two wild mouse-derived strains, MOM (originated from Japanese wild mice, Mus musculus molossinus) and Cas (originated from Philippine wild mice, M. m. castaneus), indicating that no incompatibility exists between DDK oocytes and spermatozoa of MOM and Cas strains. Furthermore, this compatibility has been confirmed by the following two findings: (1) Normal fertility was shown by the two types of backcrosses, DDK females x F(1) (DDK female x MOM male) males and DDK females x F(1) (DDK female x Cas male) males; and (2) the offspring from these backcrosses segregated equally into the homozygotes and heterozygotes as genotyped by the microsatellite markers closely linked to Om locus. MOM and Cas strains would be useful for further investigations on the Om locus. On the other hand, the litter size of F(1) [C57BL/6Cr (B6) female x Cas male] females mated with B6 males was about half that of the mating with DDK males. It would be interesting to investigate whether this reduction in fertility is related to the Om locus or not.     

Colony-stimulating factor-1 blocks early pregnancy in mice

To elucidate the mechanisms underlying the suspected immune-related pregnancy failures in humans, we established experimental systems to induce pregnancy blocking and abortion in mice. One system, based on the preimmunization of C57BL/6J females with a syngeneic regressor tumor, is described. Such females fail to develop normal gestations when mated to C57BL/6J x DBA/2 F-1 (B6D2F-1) males or DBA/2 males but sustain normal pregnancies when impregnated by CBA/J or C57BL/6 males. An investigation into the cause of these male-specific pregnancy failures led us to identify colony-stimulating factor-1 (CSF-1) as responsible for both pregnancy-block and resorption of embryos. Indeed, injection of very small amounts of CSF-1 into plugged females, for the first 5 days of pregnancy, was sufficient to block B6D2F-1-induced gestations but had no effect on CBA/J-mated females. It also induced a high rate of fetal resorptions in the sensitive mating. These results suggest a novel mechanism underlying pregnancy failures: a mechanism based on cytokines and their effect on early embryonic development in certain mating combinations.     

Improved in vitro fertilization and development by use of modified human tubal fluid and applicability of pronucleate embryos for cryopreservation by rapid freezing in inbred mice

We examined in vitro fertilizability and development of 10 inbred mouse strains (C57BL/6J, C57BL/10, C57BL/10.D2/newSn, C57BL/10-Thy1.1, C57BL/10.Br/Sn, C3H/He, RFM/Ms, STS/A, BALB/c-nu and C.B-17/Icr), and the viability of frozen-thawed in vitro fertilized (IVF) embryos after embryo transfer (ET). In seven strains, fertilizability was significantly greater in modified human tubal fluid (mHTF) compared with modified Krebs-Ringer's bicarbonate solution (TYH medium). The TYH medium supported almost no fertilization in four strains. More than 80% of IVF embryos developed to the blastocyst stage by 120 h in potassium-enhanced simplex optimization medium (KSOM). Reciprocal fertilization between C57BL/6J and BALB/c-nu gametes in TYH medium yielded poor fertilization o f BALB/c-nu due to spermatozoal deficiencies. Increased concentrations of bovine serum albumin and spermatozoa during capacitation and Percoll washing did not drastically affect fertilization. The mHTF, but not TYH medium, supported BALB/c-nu spermatozoa penetration into the zona pellucida irrespective of capacitation media. In vitro fertilized embryos frozen-thawed rapidly were transferred to surrogate mothers at the two-cell stage. Compared with that of unfrozen controls, rapid freezing had no significant effect on fetus development except in C57BL/10.D2/newSn mice. These results suggest that mHTF medium is superior with respect to IVF of inbred mice, and that KSOM adequately supports in vitro fertilized embryo development in inbred mice. The data also indicate that rapid freezing of pronucleate embryos following IVF is suitable for cryopreservation and embryo banking of inbred mice and for the production of genetically modified mice.     

Maternal transmission ratio distortion at the mouse Om locus results from meiotic drive at the second meiotic division

We have observed maternal transmission ratio distortion (TRD) in favor of DDK alleles at the Ovum mutant (Om) locus on mouse chromosome 11 among the offspring of (C57BL/6 x DDK) F(1) females and C57BL/6 males. Although significant lethality occurs in this backcross ( approximately 50%), differences in the level of TRD found in recombinant vs. nonrecombinant chromosomes among offspring argue that TRD is due to nonrandom segregation of chromatids at the second meiotic division, i.e., true meiotic drive. We tested this hypothesis directly, by determining the centromere and Om genotypes of individual chromatids in zygote stage embryos. We found similar levels of TRD in favor of DDK alleles at Om in the female pronucleus and TRD in favor of C57BL/6 alleles at Om in the second polar body. In those embryos for which complete dyads have been reconstructed, TRD was present only in those inheriting heteromorphic dyads. These results demonstrate that meiotic drive occurs at MII and that preferential death of one genotypic class of embryo does not play a large role in the TRD.     

DDK egg-foreign sperm incompatibility in mice is not between the pronuclei

A high rate of normal postimplantation development was achieved when the pronuclei of embryos from matings of DDK females with (CBA X C57BL/6J)F1 males were transplanted into enucleated embryos of non-DDK origin. This shows that the DDK egg cytoplasm, not the maternal pronucleus, is involved in the late preimplantation-lethal incompatibility.     

Rescue of the mouse DDK syndrome by parent-of-origin-dependent modifiers

When females of the DDK inbred mouse strain are mated to males of other strains, 90-100% of the resulting embryos die during early embryonic development. This DDK syndrome lethality results from incompatibility between an ooplasmic DDK factor and a non-DDK paternal gene, which map to closely linked loci on chromosome 11. It has been proposed that the expression of the gene that encodes the ooplasmic factor is subject to allelic exclusion in oocytes. Previous studies have demonstrated the existence of recessive modifiers that increase lethality in the C57BL/6 and BALB/c strains. These modifiers are thought to skew the choice of allele undergoing allelic exclusion in the oocytes of heterozygous females. In the present study, we demonstrate the presence of modifiers in three Mus musculus domesticus wild-derived strains, PERA, PERC, and RBA. These modifiers completely rescued DDK syndrome lethality. We mapped the major locus that is responsible for rescue in PERA and PERC crosses to proximal chromosome 13 and named this locus Rmod1 (Rescue Modifier of the DDK Syndrome 1). Our experiments demonstrate that PERA or PERC alleles at Rmod1 rescue lethality independently of allelic exclusion. In addition, rescue of the lethal phenotype depends on the parental origin of the Rmod1 alleles; transmission through the dam leads to rescue, while transmission through the sire has no effect.     

The locus Om,responsible for the DDK syndrome,maps close to Sigje on mouse Chromosome 11

The DDK inbred strain of mouse has a striking particularity: when DDK females are crossed to males of other strains they exhibit a reduced fertility, whereas the reciprocal crosses (non-DDK females x DDK males) are fertile (Wakasugi et al. 1967; Wakasugi 1973). The low fertility results from an early embryonic lethality, the F₁ embryos dying near the late morula-early blastocyst stage. Genetic analyses (Wakasugi 1974) and nuclear and cytoplasmic transfers (Renard and Babinet 1986; Babinet et al. 1990; Mann 1986), have shown that the failure of the embryos to develop is due to an incompatibility between a DDK maternally encoded cytoplasmic product and the non-DDK paternal genome. In order to elucidate the genetic determinism of this embryonic lethality, we have analyzed the fertility of male progeny from a backcross BALB/c females x (BALB/c x DDK)F₁ males and that of males from a set of recombinant inbred (RI) strains, established from DDK and BALB/c progenitors, when mated with DDK females. Our results indicate that a single locus, Om, is responsible for the DDK syndrome and is located on Chromosome (Chr) 11, very close to the Sigje locus.     

Effect of genotype on the efficiency of mouse embryo cryopreservation by vitrification or slow freezing methods

We examined possible genotype effects on the survival of 8- to 16-cell mouse embryos isolated from four inbred strains (C57BL/6N, BALB/cAnN, DBA/2N, and C3H/HeN), a outbred stock (ICR), and various crosses after cryopreservation by vitrification or conventional slow freezing using glycerol solutions. The rates of in vitro development of C57BL/6N, BALB/cAnN, C3H/HeN, and ICR embryos to expanded blastocysts ranged from 86% to 94% after slow freezing and 85% to 97% after vitrification. The cryopreservation method did not significantly influence in vitro embryo survival after thawing (P >0.05). Although genotype significantly influenced the in vitro survival of embryos (P = 0.008), this presumably resulted from an increased difficulty in assessing the quality grade of C3H/HeN embryos prior to cryopreservation. The rates in vivo development of C57BL/6N, BALB/cAnN, C3H/HeN, DBA/2N, and ICR embryos to normal day 18–19 fetuses ranged from 19% to 64% after slow freezing and from 18% to 63% after vitrification. The in vivo development of cryopreserved embryos was significantly influenced by cryopreservation method and genotype (P = 0.01 and P = 0.001, respectively). Vitrification yielded significantly higher rates of in vivo development than that after slow freezing (P > 0.05). In vivo development rates of DBA/2N and ICR♀ X B6D2F1 ♂ embryos after cryopreservation were significantly higher than that of embryos from BALB/cAnN and C3H/HeN mice (P < 0.05). These results indicate that parental genotype exerts little or no effect on the ability of embryos to develop in vitro after vitrification or slow freezing. Differences in the ability of cryopreserved embryos to develop normally in vivo may reflect inherent genotype related differences in their post-implantation developmental potential and not their sensitivity to cryoinjury. © 1995 Wiley-Liss, Inc.

1 This article is a US Government work and, as such, is in the public domain in the United States of America.

     

Sex and strain differences of mouse variant salivary proteins

Proteins of the mouse saliva are resolved into about 20 discrete bands by polyacrylamide gel electrophoresis. Sexual dimorphism and monomorphism were found in a subset (Msp-1) of these salivary proteins from different inbred strains. This sexual dimorphism involves a fast moving band (F-type) and a slow moving one (S-type). Mature males of seven strains (A/J, AKR, CBA/J, C3H/HeN, A/Sn, B10.A, and B10.BR) exhibit the S phenotype while mature females of these strains were typed as F. Sexually immature males and females of these strains were uniformly typed as F, but at puberty (5-6 weeks of age) the phenotype of the males switched to type S, while the phenotype of the females remained the same. This switch to type S at puberty did not take place in males of four strains (BALB/cAnn, B10.D2, C57BL/6, and C57BL/10); therefore, we conclude that these strains were sexually monomorphic with regard to Msp-1. The phenotype of mature males of C3H/HeN reverted to type F following castration, whereas castrated males and mature females switched to type S in response to testosterone administration. The testosterone treatment had no effect on the type S phenotype of males and females of the sexually monomorphic BALB/cAnn strain. The male-specific type S phenotype of Msp-1 was seen only in mice with H-2 haplotype a or k; thus an association with H-2 haplotype was suggested. All F1 males of reciprocal crosses involving the sexually dimorphic and monomorphic strains (e.g., C3H/HeN X BALB/cAnn) demonstrated the type S phenotype at puberty.     

Long-term cryopreservation of mouse sperm

The objective was to determine if mouse sperm can maintain their fertilizing ability after being frozen for >10 y and whether the offspring derived from these sperm had normal fertilizing ability and phenotype. We cryopreserved sperm from six strains of mice (C57BL/6J, DBA/2N, BALB/cA, C3H/HeJ, B6D2F1 and B6C3F1) in a solution containing 18% (w/v) raffinose and 3% (w/v) skim milk, and preserved them in liquid nitrogen for >10 y. To assess the normality and fertilizing ability of these sperms, they were thawed and used for in vitro fertilization of oocytes of the same strains. Fertilization rates for C57BL/6J, DBA/2N, BALB/cA, C3H/HeJ, B6D2F1 and B6C3F1 were 66.4, 92.3, 72.8, 32.9, 60.3 and 53.7%, respectively. Furthermore, 38.3, 15.0, 43.3, 26.1, 38.3 and 16.7% of the embryos transferred to pseudopregnant females developed and produced live offspring that had normal phenotype and fertility.     

Dispersion of the cumulus oophorus and dissolution of the zona pellucida in oocytes of DDK and other strains of mice

The time needed in vitro for granulosa cell dispersal with hyaluronidase and for lysis of the zona pellucida with α-chymotrypsin was estimated for unfertilized eggs from three inbred strains of mice, DDK, C57BL, and C3H, and one outbred strain, Q. Granulosa cells were dispersed most rapidly in the DDK and most slowly in the C3H strain. The time needed for dissolution of the zona pellucida was also shortest for DDK eggs and longest for C3H eggs. The results suggest that the high sensitivity to enzymes of the granulosa layer and the zona pellucida of DDK eggs may allow more than one spermatozoon to penetrate through these barriers before the block against polyspermy is completed. The percentage of unfertilized eggs containing spermatozoa under the zona pellucida on the day of copulation was abnormally high in matings of females from the DDK inbred strain with males from DDK and C57BL strains. This may suggest some disturbances in establishing contact between the egg plasma membrane and fertilizing spermatozoa.     

Sex and strain differences of mouse variant salivary proteins

Abstract. Proteins of the mouse saliva are resolved into about 20 discrete bands by polyacrylamide gel electrophoresis. Sexual dimorphism and monomorphism were found in a subset (Msp-1) of these salivary proteins from different inbred strains. This sexual dimorphism involves a fast moving band (F-type) and a slow moving one (S-type). Mature males of seven strains (A/J, AKR, CBA/J, C3H/HeN, A/Sn, B10.A, and B10.BR) exhibit the S phenotype while mature females of these strains were typed as F. Sexually immature males and females of these strains were uniformly typed as F, but at puberty (5–6 weeks of age) the phenotype of the males switched to types, while the phenotype of the females remained the same. This switch to type S at puberty did not take place in males of four strains (BALB/cAnn, B10.D2, C57BL/6, and C57BL/10); therefore, we conclude that these strains were sexually monomorphic with regard to Msp-1. The phenotype of mature males of C3H/HeN reverted to type F following castration, whereas castrated males and mature females switched to type S in response to testosterone administration. The testosterone treatment had no effect on the type S phenotype of males and females of the sexually monomorphic BALB/cAnn strain. The male-specific type S phenotype of Msp-1 was seen only in mice with H-2 haplotype a or k; thus an association with H-2 haplotype was suggested. All F₁ males of reciprocal crosses involving the sexually dimorphic and monomorphic strains (e.g., C3H/HeN × BALB/cAnn) demonstrated the type S phenotype at puberty.     

Pregnancy rates, prenatal and postnatal survival of offspring, and litter sizes after reciprocal embryo transfer in DBA/2JHd, C3H/HeNCrl and NMRI mice

Success of embryo transfer is often a limiting factor in transgenic procedures and rederivation efforts, and depends on the genetic background of the donor and recipient strains used. Here we show that embryo transfer to DBA/2J females is possible, and present data on pre- and postnatal success rates after reciprocal embryo transfer using the inbred DBA/2J and C3H/HeN, and outbred NMRI strains. The highest embryo yield was achieved in outbred NMRI females, but embryo yields were similar in DBA/2J and C3H/HeN mice following superovulation despite poor estrus cycle synchronization in DBA/2J females. In-strain transfer of DBA/2J blastocysts (transfer of embryos to recipients from the same strain) resulted in pregnancy rates (57.1%) similar to those obtained following in-strain transfer of C3H/HeN (60.0%) and NMRI mice (83.3%), although the prenatal survival rate of blastocysts was low. Moreover, from the pups born only half survived the postnatal period after transfer of DBA/2J and C3H/HeN blastocysts to DBA/2J recipients. These problems were not observed when transferring NMRI-blastocysts to C3H/HeN and DBA/2J mothers. The number of blastocysts transferred also had a positive effect on the success of embryo transfer. In conclusion, C3H/HeN and DBA/2J females can be used as recipients for embryo transfer procedures for certain donor strains like NMRI, as one major determinant seems to be the genetic background of the embryos transferred. We also recommend to increase the number of DBA/2J blastocysts transferred, and to foster the DBA/2J pups to other DBA/2J mothers postnatally for in-strain transfer of DBA/2J mice.     

Role of intestinal goblet cells in the expulsion of Gymnophalloides seoi from mice

Role of intestinal goblet cells (GCs) in the expulsion of Gymnophalloides seoi was studied using 4 strains of mice, ICR, C3H/ HeN, BALB/c, and C57BL/6, after infection with 200 metacercariae isolated from oysters. On day 7 postinfection (PI), significantly higher (P < 0.05) worm recovery rates (WRRs) were observed in ICR (29.5 +/- 12.0%) and C3H/HeN (14.8 +/- 8.2%) than in BALB/c (5.7 +/- 5.3%) and C57BL/6 (0.8 +/- 1.1%) mice. Alteration of the GC mucins was marked in C57BL/6 mice. On day 14 PI, 5.2 +/- 5.2% and 0.6 +/- 0.7% of worms were recovered only from ICR and C3H/HeN mice. When C57BL/6 mice were immunosuppressed with prednisolone, WRR on day 7 PI increased to 11.7 +/- 13.9%, whereas the GC hyperplasia and mucin alteration diminished significantly. The results suggest that expulsion of G. seoi from the intestine is dependent on immune responses of the host, and GCs may be an important effector.     

Mating Increases Neuronal Tyrosine Hydroxylase Expression and Selectively Gates Transmission of Male Chemosensory Information in Female Mice

Exposure to chemosensory signals from unfamiliar males can terminate pregnancy in recently mated female mice. The number of tyrosine hydroxylase-positive neurons in the main olfactory bulb has been found to increase following mating and has been implicated in preventing male-induced pregnancy block during the post-implantation period. In contrast, pre-implantation pregnancy block is mediated by the vomeronasal system, and is thought to be prevented by selective inhibition of the mate’s pregnancy blocking chemosignals, at the level of the accessory olfactory bulb. The objectives of this study were firstly to identify the level of the vomeronasal pathway at which selective inhibition of the mate’s pregnancy blocking chemosignals occurs. Secondly, to determine whether a post-mating increase in tyrosine hydroxylase-positive neurons is observed in the vomeronasal system, which could play a role in preventing pre-implantation pregnancy block. Immunohistochemical staining revealed that mating induced an increase in tyrosine-hydroxylase positive neurons in the arcuate hypothalamus of BALB/c females, and suppressed c-Fos expression in these neurons in response to mating male chemosignals. This selective suppression of c-Fos response to mating male chemosignals was not apparent at earlier levels of the pregnancy-blocking neural pathway in the accessory olfactory bulb or corticomedial amygdala. Immunohistochemical staining revealed an increase in the number of tyrosine hydroxylase-positive neurons in the accessory olfactory bulb of BALB/c female mice following mating. However, increased dopamine-mediated inhibition in the accessory olfactory bulb is unlikely to account for the prevention of pregnancy block to the mating male, as tyrosine hydroxylase expression did not increase in females of the C57BL/6 strain, which show normal mate recognition. These findings reveal an association of mating with increased dopaminergic modulation in the pregnancy block pathway and support the hypothesis that mate recognition prevents pregnancy block by suppressing the activation of arcuate dopamine release.     

The impaired pregnancy outcome in murine congenital toxoplasmosis is associated with a pro-inflammatory immune response, but not correlated with decidual inducible nitric oxide synthase expression

Congenital toxoplasmosis is associated with adverse pregnancy outcome. Despite the type 1 immune response, C57BL/6 mice are more susceptible than BALB/c mice to Toxoplasma gondii infection. Additionally, successful pregnancy appears to be correlated with type 2 T helper maternal immunity and regulatory T cells. In order to investigate the mechanisms of susceptibility/resistance to congenital toxoplasmosis in mice with different genetic backgrounds and the influence of inducible nitric oxide synthase in pregnancy outcome, groups of C57BL/6, BALB/c and C57BL/6 iNOS(-/-) females were orally infected with T. gondii ME-49 strain on day 1 of pregnancy and were sacrificed on day 8 p.i. and day 19 p.i. The uterus and placenta were evaluated for the foetal resorption rate, parasite load, immunological and histological changes. C57BL/6 mice presented inflammatory foci in the decidua (endometrium) of the uterus at a higher frequency than BALB/c mice on day 8 p.i., and a large number of pregnant C57BL/6 mice presented necrotic implantation sites. The parasite was seldom found in the uterus or placenta of either lineage of mice. Interestingly, there was no observed difference in inducible nitric oxide synthase expression in the uterus and placenta of infected mice. In addition, higher levels of TNF-α were detected in serum samples from C57BL/6 mice compared with BALB/c mice. Accordingly, C57BL/6 mice presented with levels of 90% abortion compared with 50% in BALB/c mice on day 19 p.i. C57BL/6 iNOS(-/-) mice showed low placental parasite counts and high absorption rates, similar to wild type mice. The data suggest that the impaired pregnancy outcome due to T. gondii infection in C57BL/6 mice could be associated with a higher inflammatory response leading to cell apoptosis and necrosis of implantation sites compared with BALB/c mice, and this phenomenon was not due to inducible nitric oxide synthase expression in the decidua.     

Performance of ten inbred mouse strains following assisted reproductive technologies (ARTs)

Superovulation, in vitro fertilization, embryo cryopreservation, and embryo transfer are assisted reproductive technologies (ARTs) widely used in laboratory mice. Inbred strains of mice have inherent genetic differences that cause them to respond differently to these technologies. Knowing how common inbred strains will perform when used for ARTs will ensure the most efficient use of mice, time, and resources. In this study, we characterized the ability of 10 inbred strains: 129S1/SvImJ, A/J, BALB/cJ, BALB/cByJ, C3H/HeJ, C57BL/6J, DBA/2J, FVB/NJ, NOD/LtJ, and SJL/J to superovulate, fertilize in vitro, and produce live pups subsequent to embryo transfer. Three-week-old female mice were superovulated using eCG (5.0 IU) and hCG (5.0 IU). The resulting oocytes were fertilized in vitro in human tubal fluid medium with spermatozoa of the same strain. The following day, two-cell embryos were either transferred into pseudopregnant recipient females or cryopreserved. The cryopreserved embryos were later thawed and transferred into pseudopregnant recipient females. Differences in response to superovulation, fertilization, and number of live born produced after embryo transfer were observed between strains, substantiating the influence of genetic variability on ARTs. The response to the superovulation treatment varied among strains and ranged from 5+/-1(A/J) to 40+/-3 (129S1/SvImJ) normal oocytes per female. The average proportion of oocytes that fertilized ranged among strains from 24% (129S1/SvImJ) to 93% (DBA/2J and A/J). The average proportion of two-cell embryos that were transferred into recipient females and subsequently developed into live pups varied from 5% (A/J) to 53% (C57BL/6J) for fresh embryos and from 18% (BALB/cByJ) to 45% (129S1/SvImJ) for thawed embryos.