Hepatoma-derived growth factor: from the bovine uterus to the in vitro embryo culture.

Early in cow embryo development, hepatoma-derived growth factor (HDGF) is detectable in uterine fluid. The origin of HDGF in maternal tissues is unknown, as is the effect of the induction on developing embryos. Herein, we analyze HDGF expression in day 8 endometrium exposed to embryos, as well as the effects of recombinant HDGF (rHDGF) on embryo growth. Exposure to embryos did not alter endometrial levels of HDGF mRNA or protein. HDGF protein localized to cell nuclei in the luminal epithelium and superficial glands and to the apical cytoplasm in deep glands. After uterine passage, levels of embryonic HDGF mRNA decreased and HDGF protein was detected only in the trophectoderm. In fetal fibroblast cultures, addition of rHDGF promoted cell proliferation. In experiments with group cultures of morulae in protein-free medium containing polyvinyl alcohol, adding rHDGF inhibited blastocyst development and did not affect cell counts when the morulae were early (day 5), whereas it enhanced blastocyst development and increased cell counts when the morulae were compact (day 6). In cultures of individual day 6 morulae, adding rHDGF promoted blastocyst development and increased cell counts. Our experiments with rHDGF indicate that the growth factor stimulates embryonic development and cell proliferation. HDGF is synthesized similarly by the endometrium and embryo, and it may exert embryotropic effects by autocrine and/or paracrine mechanisms.

Elements of functional genital asymmetry in the cow.

Asymmetry in the cow affects ovarian function and pregnancy. In this work we studied ovarian and uterine asymmetry. Synchronised animals, in which in vitro-produced embryos (n=30-60) had been transferred on Day 5 to the uterine horn ipsilateral to the corpus luteum (CL), were flushed on Day 8. Ovulatory follicle diameter, oestrus response and total protein flushed did not differ between sides. However, a corpus luteum in the right ovary led to plasma progesterone concentrations that were higher than when it was present in the left ovary. Fewer embryos were recovered from the left than the right horn. Among 60 uterine proteins identified by difference gel electrophoresis, relative abundance of nine (acyl-CoA dehydrogenase, very long chain; twinfilin, actin-binding protein, homologue 1; enolase 1; pyruvate kinase isozymes M1/M2 (rabbit); complement factor B Bb fragment ; albumin; fibrinogen gamma-B chain; and ezrin differed (P<0.05) between horns. Glucose concentration was higher, and fructose concentration lower, in the left horn. In a subsequent field trial, pregnancy rates after embryo transfer did not differ between horns (51.0±3.6, right vs 53.2±4.7, left). However, Day 7 blood progesterone concentrations differed (P=0.018) between pregnant and open animals in the left (15.9±1.7 vs 8.3±1.2) but not in the right horn (12.4±1.3 vs 12.4±1.2). Progesterone effects were independent of CL quality (P=0.55). Bilateral genital tract asymmetry in the cow affects progesterone, proteins and hexoses without altering pregnancy rates.

In vitro cultured bovine endometrial cells recognize embryonic sex.

Endometrial cell co-culture (ECC) with single embryo may reflect endometrium responses in vivo. Bovine Day-6 in vitro-produced morulae were cultured until Day-8 in modified synthetic oviductal fluid (mSOF), or on the epithelial side of ECC. Expression of epithelial- and stromal-cell transcripts was analyzed by RT-PCR in ECC with one male (ME) or female embryo (FE). Concentrations of ARTEMIN (ARTN) and total protein were determined in epithelial cell-conditioned medium. ECCs yielded embryos with more cells in the inner cell mass than embryos cultured in mSOF. Embryos altered transcript expression only in epithelial cells, not in stromal ones. Thus, ME induced larger reductions than FE and controls (i.e., no embryos cultured) in hexose transporter solute carrier family 2 member 1 (SLC2A1) and member 5 (SLC2A5), connective tissue growth factor (CTGF), artemin (ARTN), and interferon alpha and beta receptors subunit IFNAR1 and IFNAR2. FE reduced SLC2A1 and SLC2A5, and increased ARTN expression with respect to controls. ME tended to reduce total protein concentration (P < 0.082) in ECC-conditioned medium, while ARTN protein and gene expressions strongly correlated (R > 0.90; P < 0.05) in the group of ME or FE, but not in controls (without embryo). Isolated male and female embryos may differentially release signaling factors that induce sexually dimorphic responses in endometrial cells.

Early embryonic and endometrial regulation of tumor necrosis factor and tumor necrosis factor receptor 2 in the cattle uterus.

Tumor necrosis factor (TNF) alpha likely mediates embryomaternal communication in mammals. In bovine, we have previously found that the uterine fluid of heifers that carried early embryos shows downregulation in the TNF and nuclear factor κB system. In this work, we assessed the expression of TNF and its receptor TNFR2 in the bovine endometrium and embryos during blastocyst development. Moreover, to explore the endometrial immune response to early embryos, we analyzed the number of CD45 leukocytes in the bovine endometrium. Day 8 endometrium and blastocyst recovered from animals after transfer of Day 5 embryos showed TNF and TNFR2 mRNA transcription and protein colocalization. The presence of embryos increased endometrial TNF and TNFR2 protein, whereas endometrial leukocytes decreased. Blastocysts exposed to the uterine tract had undetectable levels of TNF and lower levels of TNFR2 mRNA. These results suggest that the endometrium might lower the TNF concentration in the blastocyst by (1) regulating TNF secretion into the uterine fluid and (2) inducing decreased TNF and TNFR2 mRNA transcription in the embryo. Thus, TNF and TNFR2 might participate in early embryomaternal communication.