INTRODUCTION

The development of mouse preimplantation stage embryos in vivo and in vitro is affected by strain genotype (Dandekar and Glass, 1987). Genes within the mouse histo-compatibility complex (H-2), e.g. the Preimplantation-embryo-development (Ped) gene, influence the time of the first cleavage division and subsequent rate of embryonic development (Goldbard et al., 1982). The Ped gene is located in the H-2 complex and manifests itself as two functional alleles controlling fast (dominant) or slow embryonic development (Warner et al., 1987). The Ped gene is linked to the Qa-2 subregion within the H-2 complex. Expression of Qa-2 antigens by fast developing mouse embryos correlates with Ped gene pheno-type: Qa-2^a (Goldbard et al., 1982). The Ped gene product may be the Qa-2^a protein. Mouse Qa-2^a antigens have been detected on mouse oocytes and on two-cell, eight-cell and blastocyst stage embryos (Krco and Goldbard, 1977; Cozed and Warner, 1981). In addition, strains that are Qa-2^a positive have shorter gestation times, larger litters, and higher birth weights (Roderick, 1980).

Preimplantation blastocyst formation is characterized by the formation of a blastocoel cavity and the initial cell differentiation of two distinct cell populations: the inner cell mass (ICM) and the trophectoderm (TE). The initiating stage for preim-plantation cell differentiation is the morula (Johnson, 1979) and for determination is at or just prior to the 8-cell stage (Cosby et al., 1988). Many factors or conditions may regulate embryonic differentiation, including, but not limited to, nucleocytoplasmic ratios, cellular apposition, or epigenetic interactions. It is not known if the Ped gene participates in blastocyst cell differentiation during mouse preimplantation embryo development.

Therefore, the study objective was to determine the differentiation of cells to the ICM and TE in Qa-2^a positive (Qa-2^a:Ped +) and Qa-2^a negative (Qa-2^a:Ped −) mouse blastocysts.

MATERIALS AND METHODS

Cumulus complexes (CC) were recovered from the excised ovi-ducts of pregnant mare's serum gonadotropin (PMSG; 5 IU; Sigma Chem. Co., St. Louis, MO, USA) and human chorionic gonadotropin (hCG; 5 IU; Sigma) primed BALB/cJ (Qa-2^a:Ped +) and BALB/cByJ (Qa-2^a:Ped −) female mice (3-4 weeks of age). The interval between PMSG and hCG injections was 48 hr. Primed females were mated with fertile BALB/cJ or BALB/cByJ males (12+ weeks of age) immediately following the administration of hCG. CC were collected 14 hr post-hCG and cultured in modified Minimum Essential Medium-alpha modification (α-MEM; Sigma), as previously described (Roudebush et al., 1994; Roudebush and Duralia, 1996), in an atmosphere of 5% CO₂ in air, 95% relative humidity at 37°C to the hatched blastocyst stage.

Assessment of the Qa-2^a:Ped gene's influence on cell differentiation, allocation of preimplantation embryonic cells to the ICM or to TE, was determined by differential labelling with polynucleotide-specific fluorochromes as previously described (Hardy et al., 1989; Roudebush et al., 1994). Briefly, TE cells are labelled with 0.1 mg/ml propidium iodide (Sigma) during immunosurgery and 10.0 μg/ml bisbenzimide (Hoechst 33258; Sigma). ICM cells are labelled only with bisbenzimide. Fluorescence microscopy was performed on a Nikon-Diaphot inverted microscope equipped with an epifluorescence filter combination UV-1A. Under the aforementioned conditions, ICM cells fluoresce blue and TE cells fluoresce red (Fig. 1).

Fig. 1

Fluorescence imaging of a mouse hatched blastocyst. Blue, inner cell mass; Red, trophoectoderm.

Data were evaluated by Student's t test and 2 × 2 chi-squared contingency table.

RESULTS AND DISCUSSION

A total of 963 BALB/cJ and 1,106 BALB/cByJ mouse preimplantation mouse embryos were collected and cultured as described. The effect of the Qa-2^a:Ped gene on mouse preim-plantation embryo development in α-MEM is shown in Table 1. The effect of the Qa-2^a:Ped gene on mouse preimplantation formation in α-MEM is provided in Fig. 2 and Table 2.

Table 1

The effect of the Qa-2^a:Ped gene on BALB/cJ and BALB/cByJ preimplantation embryo development

Fig. 2

The effect of the Ped gene on BALB/cJ and BALB/cByJ blasto-cyst differentiation. A, Significantly different (P < 0.001); B, approaches significance (P = 0.062).

Table 2

The effect of the Ped gene on per cent cell allocation in BALB/cJ and BALB/cByJ hatched blastocysts

The Qa-2^a:Ped + BALB/cJ mouse 2-cell embryo required 120 hr to develop to the hatched blastocyst stage, whereas, the Qa-2^a:Ped − BALB/cByJ mouse 2-cell stage embryos required 134 hr to develop to the hatched blastocyst stage. A significantly (P < 0.001) higher number of BALB/cJ (57.3%) than BALB/cByJ (45.8%) 2-cell stage embryos developed to the hatched blastocyst stage (Table 1), suggesting that the Qa-2^a:Ped gene may influence not only the rate of development but also the ability to develop to the hatched blastocyst stage. The BALB/cByJ mouse hatched blastocyst had a significantly (P < 0.001) lower number of ICM (mean 29) than the BALB/cJ (mean 59) mouse hatched blastocyst (Fig. 2). There was no significant difference in the number of TE cells between the two mouse strains (Fig. 2), however, the difference did approach significance (P = 0.062). There was no significant difference in the total number of hatched blastocyst cells between the two mouse strains (Fig. 2). A proportionally higher number of preimplantation cells differentiate to the ICM than to the TE in the Qa-2^a:Ped positive BALB/cJ (42.4%) blasto-cyst than in the Qa-2^a:Ped negative (20.3%) BALB/cByJ blastocyst (Table 2). The results suggest that the Qa-2^a:Ped gene may have an influential role in preimplantation embryo cell differentiation.

Preimplantation embryo development is genetically controlled by both maternal and embryonic genes. Mouse preim-plantation stage embryos develop at different rates in vivo and in vitro; this difference in cleavage division rate between slow and fast developing mouse strains is maintained in vitro (Brownell and Warner, 1988) and is strain dependent (Streffer et al., 1980; Dandekar and Glass, 1987). This effect has been ascribed to the Preimplantation-embryo-development or Ped gene, and may be an effect of the Qa-2 locus. Fast, but not slow, developing mouse embryos express the Qa-2^a antigen (Xu et al., 1994). The embryonic Ped gene phenotype is an intrinsic property of the embryos themselves (Brownell and Warner, 1988). The Qa-2 locus is located within the mouse major histocompatability complex (H-2) on chromosome 17 (Green, 1989) and is a product of the H-2 Q9 gene (Xu et al., 1994). However, background genes, in addition to the H-2 associated Ped, play a role in the control of the rate early preimplantation embryonic cleavage (Goldbard and Warner, 1982).

In this study, it was found that the embryos of two related inbred mouse strains (BALB/cJ and BALB/cByJ) cultured under similar conditions: (1) develop at different cleavage rates; the BALB/cByJ embryos requiring additional time to reach the hatched blastocyst stage; and (2) will have a proportionally different number of cells differentiating to the blastocysts inner cell mass or trophectoderm although the total cell number remained relatively constant.

Human preimplantation stage embryos, between patients, fertilized and cultured in vivo and recovered from the uterus have been reported to be at different stages of embryonic development (Buster et al., 1984). Human in vitro fertilized embryos, between patients, also develop at different rates (Roudebush, unpublished observations). This suggests that some genetic component(s) regulate human embryonic development.

Other embryonic factors which have reported to effect cleavage (mitotic) rates include, but are not limited to: platelet-activating factor (Roberts et al., 1993) and growth factors (Paria and Dey, 1990). Additional studies are warranted to determine if the Ped gene interacts with these other factors in the control and regulation of preimplantation embryo development.