211 TEMPORAL AND SPATIAL EXPRESSION PROFILE OF THE UTERINE MILK PROTEIN — A MEMBER OF THE SERINE PROTEASE INHIBITOR SUPERFAMILY — IN THE BOVINE ENDOMETRIUM

Abstract

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).