No abstract

Uterine secretion from the endometrial glandular epithelium provides optimal conditions for early embryonic development. The uterine milk protein (UTMP), a member of the serine proteinase inhibitor superfamily, has been demonstrated to be a major progesterone-induced glycoprotein secreted by the endometrium during pregnancy. Previous transcriptomic analysis revealed that UTMP was highly abundant at estrus in the bovine endometrium (Bauersachs et al. 2005 J. Mol. Endocrinol. 34, 889–908). Here we describe a detailed characterization of UTMP mRNA expression at several time points during the bovine estrous cycle and the pre-implantation period. Simmental heifers were monitored with respect to serum progesterone (P4) and estradiol-17β (E2), and slaughtered at estrus or 3.5, 12, 15, or 18 days after estrus, or at Day 15 or 18 of pregnancy (n = 4 per group). The uterus was divided into corpus and caudal, middle and cranial parts of the ipsilateral uterine horn for sampling of intercaruncular endometrium. In addition, effects of steroid hormones were investigated by stimulating an endometrial cell culture obtained from Day 8 animals (n = 4) with physiological doses of P4 or E2. In all cases, UTMP mRNA was quantified by real-time RT-PCR. Pronounced changes of UTMP mRNA abundance were detected during the estrous cycle. Expression was highest at estrus, followed by a remarkable decrease at Day 3.5. There was no difference between pregnant and non-pregnant animals at Day 15. Cycling animals displaying a high P4 and low E2 content (P4 > 2 ng mL-1 and E2 < 1 pg mL-1) revealed a lower expression of UTMP at Day 18 compared to the pregnant heifers, whereas animals at Day 18 progressing toward estrus (P4 < 1.5 ng mL-1 and E2 > 3 pg mL-1) exceeded the mRNA expression of the pregnant group. Following stimulation with estradiol-17β, the in vitro UTMP transcripts increased significantly. These results indicate that two different interfering stimulatory events might take place. While estradiol-17β appeared to increase UTMP mRNA expression at estrus, a second factor, most probably embryo-derived or embryo-induced, is assumed to be responsible for the UTMP rise during early pregnancy. The distinct gradient from the cranial uterine horn to the corpus at estrus was less pronounced at Day 3.5 and absent at Days 12, 15, and 18, pointing toward functional implications regarding the passing gametes, particularly sperm. An antibody raised against bovine UTMP will further validate the observed mRNA regulations on the protein level. In conclusion, bovine UTMP seems to play a decisive role for precise cyclic regulation of the bovine uterine milieu and during early embryo-maternal communication. This work was supported by the DFG FOR 478.

A combination of subtracted cDNA libraries and cDNA array hybridization was applied to identify differentially expressed genes in the bovine intercaruncular endometrium at estrus and diestrus. Six cyclic Simmental heifers were cycle-synchronized and slaughtered the morning after standing heat occurred (n = 3) or 12 days after estrus (n = 3), respectively. The uterus was divided into seven sections: corpus, plus the caudal, middle, and cranial parts of both uterine horns. Samples were collected from the epithelial layer of the lamina propria of the intercaruncular endometrium. Two subtracted libraries were prepared and analyzed by array hybridization with probes produced from the 42 tissue samples. More than 130 differentially expressed genes were identified. Among these genes, the mRNA for the uterine milk protein (UTMP) showed a much higher expression level at estrus versus diestrus. Furthermore, the expression level differed markedly between the uterus sections. Statistical significance was tested using ANOVA and Student Newman Keuls post-hoc test (GeneSpring® version 6.1, Silicon Genetics, Redwood City, CA, USA). There was a pronounced expression gradient from both cranial uterine horns (highest) to the corpus (lowest) at estrus. UTMP mRNA level was highest in the ipsilateral cranial horn. A more accurate quantification was done by quantitative real-time RT-PCR using the same RNA samples as for array hybridization and, in addition, samples from further time points of the estrous cycle and early pregnancy. The results obtained by array hybridization were clearly confirmed. UTMPs, also known as uterine serpins (US), were previously shown to be the major products secreted by the uteri of cattle, sheep, and pigs during pregnancy. The US are glycoproteins belonging to the serpin (serine proteinase inhibitor) superfamily of proteins. However, no inhibitory activity to any serine proteinase has been found. The ovine uterine serpin (OvUS) has been shown to inhibit a wide variety of immune responses, including mixed lymphocyte reaction, T cell-dependent antibody production, and NK (natural killer)-like activity, indicating a major role in protecting the conceptus from maternal immune reactions. The four members of the US family form a clade distinct from other serpins, which have probably adopted important biological functions in the reproductive biology of Artiodactyla. Therefore, the identified expression gradient of the UTMP mRNA at estrus may be of particular importance, e.g. for sperm selection, and deserves further investigation at the protein level and in functional studies. This work was supported by the Deutsche Forschungsgemeinschaft (Research Unit “Mechanisms of Embryo-Maternal Communication”; FOR 478/1).