REPRODUCTIONRESEARCH Regulation of homeobox A10 expression in the primate endometrium by progesterone and embryonic stimuli G B Godbole, D N Modi and C P Puri National Institute for Research in Reproductive Health, Indian Council of Medical Research, JM Street, Parel, Mumbai 400012, India Requests for offprints should be addressed to D N Modi; Email: [email protected], [email protected] Abstract Homeobox A10 (HOXA10), a member of abdominal B subclass of homeobox genes, is responsible for uterine homeosis during development. Intriguingly, in the adult murine uterus, HOXA10 has been demonstrated to play important roles in receptivity, embryo implantation, and decidualization. However, the roles of HOXA10 in the primate endometrium are not known. To gain insights into the roles of HOXA10 in the primate endometrium, its expression was studied in the endometria of bonnet monkey (Macaca radiata) in the receptive phase and also in the endometria of monkeys treated with antiprogestin onapristone (ZK98.299) or in conception cycle where the presence of preimplantation stage blastocyst was verified. In addition, the mRNA expression of HOXA11 and insulin-like growth factor-binding protein 1 (IGFBP1) was evaluated by real-time PCR in these animals.The results revealed that HOXA10 in the luteal phase primate endometrium is differentially expressed in the functionalis and the basalis zones, which is modulated in vivo by progesterone and also by the signals from the incoming embryo suggesting the involvement of HOXA10 in the process of establishment of pregnancy in primates. In addition, the results also demonstrated that the expression of IGFBP1 but not HOXA11 is coregulated with HOXA10 in the endometria of these animals. The pattern of changes in the expression of HOXA10 in response to the two stimuli suggests that endometrial receptivity and implantation not only requires a synchrony of maternal and embryonic signaling on endometrial cells in the primates but there also exists a controlled differential response among the cells of various uterine compartments. Reproduction (2007) 134 513–523 Introduction Beyond progesterone, recent studies have also suggested that the receptive stage endometrium is further Embryo implantation is a crucial step in mammalian primed by paracrine factors secreted by blastocyst prior reproduction that involves an intricate succession of to implantation. Studies in nonhuman primate model genetic and cellular interactions, all of which must be systems have demonstrated that extensive morphological synchronized and executed within an optimal time remodeling and altered expression of cytokines and frame. The crucial events that occur in the maternal growth factors have been observed in the receptive stage compartment that culminate in a successful pregnancy endometrium of bonnet monkeys that were mated and are (a) the achievement of receptive state in the the presence of an embryo had been verified (Rosario endometrium and (b) the transformation of the stromal et al. 2005a, 2005b). Intriguingly, infusion of human cells into the decidual cells. The ovarian steroid chorionic gonadotropin and/or interleukin-1b at concen- progesterone is an essential hormone that makes the trations secreted by the blastocyst in the baboon uterus conducive (‘receptive’) to accept a blastocyst for endometrium also induces morphological changes in implantation and subsequent decidualization (Wang & the endometrium and the expression of receptivity- Dey 2006). Several progesterone-regulated genes related genes is also altered (Fazleabas et al. 1999, important for receptivity are being identified in the Strakova et al. 2005). These observations lead to the murine models (Bagchi et al. 2003, Wang & Dey 2006) speculation that along with progesterone, embryonic and a number of these have been shown to play essential factors modulate the primate endometrium to endow roles in endowing receptivity in humans (Bagchi et al. receptivity for implantation and further maintenance of 2003, Wang & Dey 2006). pregnancy. q 2007 Society for Reproduction and Fertility DOI: 10.1530/REP-07-0234 ISSN 1470–1626 (paper) 1741–7899 (online) Online version via www.reproduction-online.org Downloaded from Bioscientifica.com at 10/03/2021 12:45:00PM via free access 514 G B Godbole and others Transcription factors are elements that selectively bind monkeys is demonstrated to be closely similar to to the appropriate DNA sequences on the promoter of humans, especially in the context of menstrual cycle the genes and regulate their expression and coordinate length, endometrial receptivity, and implantation the process of cell differentiation. Since endometrial (Katkam et al. 1995). In addition, the high fecundity receptivity and decidualization are critical transfor- rates of bonnet monkeys and the ready availability of an mation processes for pregnancy to occur, it is likely assay (preimplantation factor (PIF) bioassay) to detect that transcription factors whose expression in the preimplantation conceptus encouraged us to select this endometrium is under the regulation of progesterone species for the present study. and embryonic stimuli would be critical for implantation HOXA10, under the influence of progesterone, has (Zhao et al. 2006). Identification of such transcription been shown to regulate the expression of a subset of factors would facilitate in expanding our knowledge of progesterone-responsive genes. Among these is the endometrial receptivity and initiation of pregnancy. One HOXA10 ortholog HOXA11 and insulin-like growth such transcription factor that is reported to play an factor-binding protein 1 (IGFBP1), which in murine important role in endometrial physiology is the homeo- models and in vitro have been proposed to be regulated box A10 protein (HOXA10). HOXA10 is expressed in by HOXA10 (reviewed in Daftary & Taylor 2006). murine endometrium in a cycle-dependent manner and Hence, along with HOXA10, we also investigated the is regulated by progesterone (Satokata et al. 1995, expression profiles of HOXA11 and IGFBP1 mRNA in Ma et al. 1999). Furthermore, the expression of the endometria of these animals. HOXA10 is increased at the time of implantation and is maximal during decidualization, suggesting its role in initiation of pregnancy in the mouse (Satokata et al. Materials and Methods 1995). Indeed, HOXA10 knockout mice are infertile owing to failure or abnormal implantation and inhibition The Institutional Animal Ethics committee and the of stromal cell decidualization (Benson et al. 1996, Lim Committee for the Purpose of Control and Supervision et al. 1999). These observations underscore the import- of Experiments on Animals, Ministry of Social Justice and ance of HOXA10 in the regulation of murine pregnancy. Empowerment, Government of India approved the use of However, there exist fundamental differences in the the animals for the study. mechanisms of implantation between the mouse and man, making it imperative to study the factors of interest in the primate system. But in the context of HOXA10, Animals limited information exists on its roles in the regulation of implantation and pregnancy in humans. Expressed in a Adult bonnet monkeys (Macaca radiata) were housed menstrual cycle-dependent manner in the human singly under controlled conditions at the experimental endometrium (Taylor et al. 1998, Gui et al. 1999), animal facility of the institute and were fed with a diet HOXA10 is downregulated in the endometria of women composed of semi-formulated Indian bread, fresh suffering from endometriosis (Browne & Taylor 2006), seasonal fruits, eggs, and sterile water. Animals weighing 3.5–4.5 kg, which had at least two consecutive ovulatory polycystic ovary syndrome (Cermik et al. 2003) and menstrual cycles of 28–30 days and normal steroid levels idiopathic infertility (Gui et al. 1999). In vitro, HOXA10 (peak estradiol 50–75 pg/ml and progesterone expression is reportedly altered by steroids, embryo 300–600 pg/ml), were used for the present study. The culture fluid, and hydrosalphinges (Sakkas et al. 2003, cyclicity was monitored and serum estradiol and Daftary & Taylor 2006). However, its in vivo regulation in progesterone concentrations were measured by specific the primate endometrium, particularly by progesterone RIA as described previously (Katkam et al. 1995, and embryonic factors, is hitherto unknown. We Sachdeva et al. 2001). hypothesized that if HOXA10 has a role in endometrial receptivity its expression must be modulated by Antiprogestin treatment progesterone and embryonic signals in the luteal phase endometrium around the time of implantation. To study the effects of progesterone, bonnet monkeys To gain more insights into the role of HOXA10 in were treated with antiprogestin onapristone (ZK98.299) primate uterus, particularly in the process of implan- at a dose which is known to block endometrial tation and initiation of pregnancy, we decided to receptivity without affecting the hypothalamus–pitu- investigate, in detail, the expression profiles and in vivo itary–ovarian axis (Katkam et al. 1995). Our previous regulation of both HOXA10 mRNA and protein by studies have demonstrated altered expression of a progesterone and embryonic stimuli during the window number of progesterone-regulated genes in the endo- of receptivity in primates. As it is difficult to obtain metria of these animals (Puri et al. 2000, Sachdeva et al. human samples at these stages of pregnancy because of 2001) indicating the validity of the model to study ethical limitations, we chose