The Conceptus Affects Expression of VEGF and Its Receptors Prior to Implantation in Mares.

Abstract

On Day 15 or 16 or pregnancy the mobile equine conceptus "fixates," i.e. lodges in the posterior segment of one uterine horn. Coincident with fixation, endometrium dorsal to the conceptus undergoes remarkable hypertrophic and hyperplastic changes which include increased endometrial gland size and numbers, and increased blood vessel size and numbers. We hypothesized that these localized changes, leading to endometrial encroachment onto the yolk sac area of the conceptus, are conceptus mediated, and involve alteration of Vascular Endothelial Growth Factor (VEGF) and its receptors (VEGFR-1 and VEGFR-2). To test the hypothesis, endometrial tissue samples were obtained from the endometrium dorsal to the conceptus or the contralateral horn in pregnant mares, and from equivalent sites in cycling mares. Samples were collected on Days 14, (n=5), and 21, (n=11) of pregnancy, and during estrus (n=6), and diestrus, (n=6). Samples were preserved for immunohistochemistry and for real time reverse transcription polymerase chain reaction (qPCR) analyzing expression of VEGF, VEGFR-1, and VEGFR-2. Additionally, Ki-67 was evaluated in endometrium by immunohistochemistry. Antisera were diluted as follows: anti-VEGF(147) polyclonal rabbit serum, 1:200 (sc-507; Santa Cruz Biotechnology, Inc), anti-VEGFR-1 polyclonal rabbit serum, 1:200 (Flt-1 C-17: sc-316; Santa Cruz Biotechnology, Inc.), anti-VEGFR-2 monoclonal mouse serum, 1:200 (Flk-1 A-3: sc-6251; Santa Cruz Biotechnology, Inc.), and anti-Ki-67 polyclonal rabbit serum, 1:1500 (NCL-Ki67p; Novocastra Laboratories Ltd.). Immunolocalization of VEGF and VEGFR-1 did not differ between pregnant and cyclic mares. Intense staining for VEGFR-2 was observed in all endometrial layers of pregnant mares, whereas the luminal epithelium of cycling mares exhibited only weak staining. Proliferation was evident by intense Ki-67 staining at the luminal epithelium of mares during estrus, but in diestrus, Ki-67staining was practically undetectable. During pregnancy, all endometrial layers stained positively for Ki-67, with more positive cells observed on Day 21 than on Day 14. There was no apparent difference in horn-side staining of any of the antigens, regardless of reproductive status. Expression of mRNA encoding VEGFR-1 was very low, and no differences were observed among reproductive statuses. In contrast, VEGF and VEGFR-2 exhibited significant (P < 0.03 and 0.005, respectively) differences among reproductive statuses, reflecting greater mRNA expression during pregnancy (Days 14 and 21, VEGF; and Day 21, VEGFR-2) compared with estrus or diestrus. Samples for qPCR were obtained only from endometrium ipsilateral to the conceptus in this experiment, so local versus intrauterine conceptus-mediated effects cannot be assessed. However, previous studies using color Doppler ultrasonography have shown a significant increase in endometrial blood flow primarily ipsilateral to the conceptus. Furthermore, morphometric analysis in the present study indicated significantly increased numbers and size of blood vessels, as well as endometrial glands in dorsal endometrium ipsilateral to the conceptus. These data provide strong support for the hypothesis that the equine conceptus directs the provision of its own nutrients through increased maternal endometrial vascularity and endometrial secretion, and suggests that the VEGF system plays a central role in this process. LAS is supported by a CAPES Scholarship from Brazil. (poster)