The endometrium responds differently to cloned versus fertilized embryos

Abstract

Although somatic cell nuclear transfer (SCNT) cloning is more efficient in cattle than in any other species tested so far, there is a high rate of pregnancy failure that has been linked to structural and functional abnormalities of the placenta. We tested the hypothesis that these changes may originate from disturbed embryo-maternal interactions in the peri-implantation period. Therefore, we evaluated the response of the endometrium to SCNT embryos (produced from 7 different fetal fibroblast cell lines) as compared with embryos derived from in vitro fertilization (IVF). SCNT embryos and IVF embryos were cultured under identical conditions to the blastocyst stage (day 7) and were transferred to corresponding recipients, which were slaughtered at day 18 of pregnancy. The mRNA profiles of endometrium samples were obtained using a custom cDNA microarray enriched for transcripts differentially expressed in the endometrium and/or oviduct epithelium during the estrous cycle and/or early pregnancy. Overall, the variation in mRNA profiles was greater in the SCNT group than in the IVF group. Furthermore, 58 transcripts were differentially abundant in endometria from SCNT and IVF pregnancies. Prominent examples are orphan nuclear receptor COUP-TFII and connexin 43, both known to play important roles in uterine receptivity and conceptus placentation. These findings suggest that placental failure in bovine clone pregnancies may originate from abnormal embryo-maternal communication that develops during the peri-implantation period. Endometrium transcriptome profiles may serve as a tool to evaluate SCNT embryos for their ability to establish pregnancy and develop a functional placenta.

Fig. 1.

Experimental design used to study the maternal response to SCNT vs. IVF embryos. IVF embryos were produced by fertilization of oocytes from nonrelated cows with semen from a single bull. SCNT embryos were produced from fibroblasts of different fetuses (half-sibs). IVF and SCNT embryos were cultured under identical conditions. Two IVF or SCNT embryos (day 8, grade 1) were transferred per recipient. On day 18, recipients were slaughtered, and pregnancy was verified by the presence of at least 1 normally developed conceptus. Endometrium samples (IVF: n = 10; SCNT: n = 9 from embryos of 4 different genotypes) then were processed for mRNA expression profiling using the BOE array. Differentially abundant transcripts were verified by RT-qPCR analysis in those endometrial samples and in additional samples from 8 other SCNT pregnancies with embryos derived from 3 additional fetal fibroblast donor cell cultures. FF, fetal fibroblasts.

Fig. 2.

Cluster analysis based on differentially expressed genes. Hierarchical cluster analysis based on mean centered values (log 2 value of a gene minus mean of all log 2 values of this gene) of the significant genes for samples and genes was performed (HCL support tree, Pearson correlation, MultiExperiment Viewer 4.0). The trees were calculated with 100 iterations, and support for the tree is shown by a color code. The fetal fibroblast cell line used for SCNT and the number of concepti present at the time of sample collection are indicated.

Fig. 3.

Comparison of expression profiles obtained by array analysis and qPCR. Microarray data are shown as normalized values (generalized logarithm). Data from the qPCR are shown as 35–CP for optimal visualization of array and qPCR data in the same diagram. The IDs of the BOE array cDNA clones are indicated.

Fig. 4.

In situ localization of NR2F2, MX2, and GJA1 mRNA in the bovine uterus on days 16 and 19 of pregnancy. Cross-sections of the uterine wall from pregnant (P) heifers were hybridized with radiolabeled antisense or sense bovine NR2F2, MX2, or GJA1 cRNAs and are presented under brightfield and darkfield illumination after counterstaining with hematoxylin. Car, caruncle; GE, glandular epithelium; LE, luminal epithelium; Myo, myometrium; S, stroma; V, blood vessel. All photomicrographs are displayed at the same width of field (450 μm). Original magnification: 25×.