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a Laboratory of Animal Reproduction, Graduate School of Bioagricultural Sciences, Nagoya University, Chikusa, Nagoya, Aichi, 464–8601, Japan
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The reduction in fertility of DDK females is known to be variable according to strain of stud males [2, 3, 9, 11], suggesting that the expression of DDK syndrome is variable depending on the genetic background of fertilized eggs. Therefore, we made an attempt to introduce the Om allele into the genetic background of C57BL/6Cr strain by successive backcrosses of heterozygous (Om/+) females x C57BL/6Cr males. However, this mating system failed to proceed at the third generation, because presumptive heterozygous (Om/+) females in this generation produced no young when mated with C57BL/6Cr males. This is not in agreement with the original observation that fertility of F1 heterozygous (Om/+) females mated with C57BL/6Cr (+/+) males is 50% of the fully fertile cross, F1 heterozygous (Om/+) females x DDK (Om/Om) males [4]. In the present report, we describe 1) reduction in fertility of heterozygous (Om/+) females in successive backcrosses with C57BL/6Cr males; 2) the way in which mortality of embryos increases in crosses of heterozygous (Om/+) females x C57BL/6Cr males as the genetic background of the heterozygous (Om/+) females approaches that of the C57BL/6Cr strain; and 3) the issue of whether or not the increase in embryonic death is due to failure in blastocyst formation as in the DDK syndrome. In parallel with these investigations, a preliminary survey was performed on the genetic factors responsible for the increase in embryonic death.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Strains of mice used in the present study were C57BL/6Cr and DDK. C57BL/6Cr was purchased from Shizuoka Laboratory Animal Corporation (Hamamatsu, Japan), and DDK was introduced from Laboratory of Animal Genetics, Graduate School of Bioagricultural Sciences, Nagoya University. Production colonies were made with the introduced individuals, and their descendants were used for the experiments. All mice were kept in the animal room in controlled conditions of 23.0 ± 1°C and 14L:10D (lights-on between 0500 and 1900 h). A pelleted diet (CA-1; Nippon Clea, Tokyo, Japan) and water were given continuously. All animal procedures were performed according to the Guidelines for Animal Experimentation of Nagoya University.
Introduction of Om Gene into the Genetic Background of C57BL/6Cr Strain
F1 females (C57BL/6Cr females x DDK males) were backcrossed to C57BL/6Cr males. The resulting backcross females were mated with C57BL/6Cr males and classified into the following two types according to their litter size: normal types (litter size: 7–14) and heterozygotes (litter size: 1–6). The selected heterozygous (Om/+) females were mated with C57BL/6Cr males, and the same procedure was repeated in the following generation.
Preparation of Mice to Examine Influence of Genetic Background on Reproductive Performance and Embryonic Death
Four kinds of heterozygous (Om/+) mice—B1D, B3D, B7D, and B15D, which theoretically possess background genes of C57BL/6Cr and DDK strains at 1:1, 3:1, 7:1, and 15:1 ratios, respectively—were produced as follows. B1D mice were F1 hybrids of C57BL/6Cr females x DDK males. Other heterozygous (Om/+) mice—B3D, B7D, and B15D—were obtained systematically by crosses of C57BL/6Cr females and males of the newly established BID, BIIID, and BVIID strains, which are homozygous for Om locus (Om/Om) and theoretically possess background genes of C57BL/6Cr and DDK strains at 1:1, 3:1, and 7:1 ratios, respectively. BID strain was founded on the homozygous (Om/Om) females and males selected from F2 offspring of C57BL/6Cr females x DDK males by progeny test with the following criteria: a male was mated with a DDK female and judged as a homozygote (Om/Om) when the litter size was 8 or more, while a female was mated with a C57BL/6Cr male and judged as a homozygote (Om/Om) when the litter size was 2 or less. BIIID strain was produced as follows: heterozygotes (Om/+) were selected by progeny test from the backcross offspring of C57BL/6Cr females x F1 males, and the strain was established with homozygotes (Om/Om) selected from the intercross offspring of the above heterozygous (Om/+) females and males. BVIID strain was founded on homozygotes (Om/Om) selected from the offspring produced by the intercross of heterozygous (Om/+) B7D females and males. Each strain was maintained by full sib-matings after establishment. The males of BID, BIIID, and BVIID used for production of B3D, B7D, and B15D mice were in the first to third generations (counting the foundation homozygotes as the first generation), and one of the BIIID males at the third generation was used for establishment of BVIID strain.
Mating Procedure and Examination of Reproductive Performance
The female mice were used for mating at the ages of 2–5 mo, and males were used at the ages of 2–7 mo. One female was caged with one male after 1700 h and examined daily for a vaginal plug between 0900 and 1200 h. The day when the plug was found was recorded as Day 0 of pregnancy.
Pregnancy testing was performed at Day 12 of pregnancy by increase in body weight and presence of descended blood from the uterine horns into the vagina (placental sign). Some females judged as pregnant were killed by cervical dislocation and dissected just after the diagnosis, and numbers of corpora lutea, implantation sites, and live fetuses were counted. The others were kept for examination of litter size at parturition, which included live and dead young.
Observation of Embryos at Day 3 of Pregnancy
Embryos were collected between 1200 and 1500 h by flushing uterine contents with M2 medium [13] into a watch glass, observed under a dissecting microscope (magnification: x12 to x40), and classified into normal embryos (morula, early blastocyst, and late blastocyst) and abnormal ones. Early and late blastocysts were distinguished according to the criterion of whether the blastocoele was smaller or larger than one half of the whole embryo (smaller: early blastocyst; larger: late blastocyst).
Histological Observation of Embryos at Days 4 and 5 of Pregnancy
Uterine horns were dissected out between 1200 and 1500 h together with oviducts, ovaries, and attached adipose tissue and placed on a piece of filter paper in a Petri dish. The ovaries were cut off, and the number of corpora lutea was counted under a dissecting microscope. The uterine horns were arranged straight on the filter paper and fixed with Bouin's solution. After removal of attached adipose tissue and separation of both horns, they were immersed in new Bouin's solution for 24 h, washed in 70% ethanol, dehydrated with a graded alcohol series, and embedded in paraffin. Sections were made at 8 µm for the uterine horns at Day 4 and at 10 µm for those at Day 5. Sections were stained with Mayer's hematoxylin and eosin and observed under a light microscope.
Statistical Analysis of the Data
For analysis of significance of the differences between groups, one of two methods was used: Student's unpaired t-tests or one-way ANOVA both of which were followed by post hoc analysis with Fisher's protected least significant difference (PLSD) tests. Fitness test for a segregation ratio was performed by chi-square test. A level of P > 0.05 was taken as indicating that an observed segregation ratio was in accord with the expected ratio.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In order to investigate this unexpected phenomenon, 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). Table 1 shows the results of dissection at Day 12 of pregnancy and litter size at parturition in the reciprocal crosses of heterozygous (Om/+) mice x C57BL/6Cr or DDK strain. Four kinds of control crosses—C57BL/6Cr females x B1D, B3D, B7D, and B15D males—were combined because they showed consistent results. In all four experimental crosses, mean litter size at parturition was smaller than in the control, and the difference was significant (P < 0.01). Litter size in the cross B1D (F1) females x C57BL/6Cr males was about one half that of the control, and a stepwise reduction was seen among the experimental crosses. Specifically, litter size of B3D females was about one half that of B1D females, and the difference was significant (P < 0.01). Furthermore, litter size of B7D females was about one half that of B3D females, and the difference was also significant (P < 0.05). However, no further significant reduction was seen between B7D and B15D females (P > 0.05). In addition, mean litter size in DDK females mated with B7D males was about one half that in the reciprocal cross, B7D females x DDK males.
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Number of live fetuses at Day 12 of pregnancy was similar to litter size at parturition in each cross, and number of implants in all experimental crosses was intermediate between numbers of corpora lutea and live fetuses, indicating that embryonic death occurred around the time of implantation. The same was observed in the cross of DDK females x B7D males. Figure 2 shows the survival rate of embryos estimated as the percentage number of live fetuses/number of corpora lutea in the experimental and control crosses. The survival rate in the control cross was 81.1 ± 2.0%, and that of B1D females was 41.3 ± 3.2%, which was approximately one half of the control value. Furthermore, a stepwise decrease was seen as the proportion of C57BL/6Cr background genes increased; i.e., the survival rate was 27.3 ± 3.2% in B3D females, 16.4 ± 3.3% in B7D females, and 11.3 ± 3.2% in B15D females.
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Table 2 shows the results of embryological observation at Day 3 of pregnancy. Mean numbers of normal and abnormal embryos were similar in all crosses, but the developmental stages of embryos were retarded in the experimental crosses, i.e., proportion of morulae was larger and that of late blastocysts was smaller as compared with findings in the control (P < 0.01). Table 3 shows the results of histological observation of embryos at Days 4 and 5 of pregnancy, and some micrographs of embryos are shown in Figure 3. At Day 4 of pregnancy, well-developed blastocysts (Fig. 3A) were classified as normal and blastocysts with small size (Fig. 3B) were also counted as normal, although some were quite small and seemed to be in the early stage of developmental arrest. In contrast, embryos remaining at the morula stage were counted as abnormal (Fig. 3, C and D). At Day 5 of pregnancy, embryos that had developed beyond the incipient egg cylinder stage (Fig. 3E) were regarded as normal; and degenerating morulae (Fig. 3F) or implantation sites with a few scattered embryonic cells or no embryonic cells but showing proliferation of stromal cells (decidual reaction) were counted as abnormal. Number of normal embryos was smaller in the experimental crosses than in the control (P < 0.01). In contrast, number of abnormal embryos was greater in the experimental crosses (P < 0.01). Embryonic death thus began to increase after the morula stage in the heterozygous (Om/+) females mated with C57BL/6Cr males; it was also recognized that the number of normal embryos decreased gradually as the heterozygous (Om/+) females possessed more C57BL/6Cr background genes. A common feature of abnormal embryos was a defect in blastocyst formation (Fig. 3, C, D, and F). Figure 4 shows changes in the number of normal embryos or live fetuses during pregnancy in the experimental and control crosses. It is clearly seen that embryonic death during Days 3–5 of pregnancy (the same stage as in the DDK syndrome) increased in B3D, B7D, and B15D females mated with C57BL/6Cr males. Summarizing the results described above, it is clear that litter size decreased in a stepwise manner in the heterozygous (Om/+) females mated with C57BL/6Cr males as their genetic background approached that of C57BL/6Cr strain; and this reduction in litter size is attributed to increase in embryonic death due to a defect in blastocyst formation as in the DDK syndrome.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The findings described above can be interpreted in two ways. First, according to the hypothesis of Wakasugi [4], an egg produced by heterozygous (Om/+) F1 females has two kinds of cytoplasmic substance, i.e., o-substance (incompatible with + allele) and O-substance (compatible with + allele); and the compatibility of a fertilized egg is determined depending on which of these substances interacts first with the paternal allele (+) brought in by spermatozoa. The two kinds of combinations take place equally; therefore, compatible-viable embryos and incompatible-lethal embryos appear in the ratio of 1:1 in the mating of F1 females x C57BL/6Cr males. This interaction between egg and sperm factors is distorted by some mechanism working after sperm penetration in the direction of increasing the incompatible combination as the genetic background of heterozygous (Om/+) females approaches that of C57BL/6Cr strain. A second hypothesis may be proposed, namely, that the two kinds of eggs are produced in the heterozygous (Om/+) females: the one having o-substance only and the other having O-substance only. Mechanisms similar to allelic exclusion [14] may be operating during oogenesis for production of the cytoplasmic substance of eggs. Allelic exclusion occurs equally in heterozygous (Om/+) F1 females; however, it is directed to the production of more eggs having o-substance only as the genetic background of the heterozygous (Om/+) females approaches that of C57BL/6Cr strain. In other words, the eggs produced in the heterozygous (Om/+) females with genetic background similar to C57BL/6Cr strain may be mostly DDK type, i.e., eggs possessing o-substance only.
Considering the above two hypotheses, the second one seems to be natural and more probable. The parallelism between the proportion of C57BL/6Cr background genes in the heterozygous (Om/+) females and embryonic mortality suggests participation of a modifier gene, and an attempt to hypothesize this possibility is presented in Table 4. C57BL/6Cr and DDK strains are homozygous for the modifier gene (tentatively named m for C57BL/6Cr and M for DDK). In the M/m heterozygous F1 females (C57BL/6Cr x DDK), allelic exclusion occurs equally; therefore, eggs with o-substance and those with O-substance are produced in the ratio of 1:1. In the B3D females, two genotypes (M/m and m/m) are equally included, and M/m females produce eggs with o-substance and those with O-substance in a 1:1 ratio. In m/m females, only eggs with o-substance are made. Therefore, the incompatible and compatible embryos appear in the ratio of 3:1 when B3D females are mated with C57BL/6Cr males. In B7D females, one quarter are M/m and three quarters are m/m; therefore, one eighth of embryos are compatible and seven eighths are incompatible when mated with C57BL/6Cr males. Likewise, one sixteenth are compatible and fifteen sixteenths are incompatible with regard to embryos in B15D females mated with C57BL/6Cr males. Survival rates calculated on the basis of this hypothesis are in accord with those shown in Figure 2. Both B7D and B15D females mated with C57BL/6Cr males showed 10–15% embryonic viability. On the other hand, the viability of the F1 embryos in the cross of DDK females x C57BL/6 males is also about 10% [3]. Therefore, the eggs produced by B7D and B15D females may be mostly DDK type with respect to the cytoplasmic factor.
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Figure 5 shows the frequency distribution of four kinds (B1D, B3D, B7D, and B15D) of heterozygous (Om/+) females mated with C57BL/6Cr males according to the survival rate of embryos expressed as the percentage number of live fetuses/number of corpora lutea at Day 12 of pregnancy. When the females with 25% or higher survival rate are regarded as the F1 type and those with less than 25% as the DDK type, proportions of DDK-type and F1-type females in each cross are in accord with those shown in Table 4, and fitness judged by chi-square test is as follows: B3D: 0.5 < P < 0.7; B7D: 0.5 < P < 0.7; and B15D: 0.7 < P < 0.9. These analyses suggest participation of a modifier gene in the production of cytoplasmic factor of eggs; i.e., a mechanism similar to allelic exclusion may be working and only Om allele is activated during oogenesis to produce DDK-type cytoplasmic factor in the heterozygous (Om/+) females having a modifier gene in the homozygous state. As the next step it would be interesting to investigate fertility of the heterozygous (Om/+) females having more background genes of DDK strain. Recently, Pardo-Manuel de Villena et al. [15] also presented evidence showing participation of modifier genes in the expression of fertility phenotype of females heterozygous for Om gene in matings with C57BL/6J males.
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It seems unlikely that a modifier gene exerts its effect through the male genome in view of the following data. In the cross of DDK females x B7D heterozygous (Om/+) males, mean number of live fetuses at Day 12 of pregnancy and mean litter size at birth were 3.9 ± 1.0 and 3.5 ± 0.5, respectively, which could be regarded as approximately 50% of the values in the reciprocal cross, B7D heterozygous (Om/+) females x DDK males (8.8 ± 0.4 and 8.4 ± 0.4, respectively). These values are also similar to mean number of live fetuses at Day 14 of pregnancy (5.3 ± 1.3) and mean litter size at birth (4.1 ± 1.6) in the cross of DDK females x (C57BL/6 x DDK) F1 heterozygous (Om/+) males [4, 11]. However, in order to obtain a concrete conclusion it would be necessary to investigate the fertility of the heterozygous (Om/+) males having a large proportion of DDK background genes in matings with DDK females.
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FOOTNOTES |
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1 This work was supported in part by a grant-in-aid for scientific research to N.W. from the Ministry of Education, Science, Sports and Culture of Japan (No. 05304022). 
2 Correspondence. FAX: 81 52 789 4012; w16341a{at}nuagr1.agr.nagoya-u.ac.jp
Accepted: November 8, 1999.
Received: June 22, 1999.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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