There is limited information regarding hormonal regulation of mucins in endocervical mucus. summary, a robust, novel 3D endocervical culture was developed, and physiologic responses to the menstrual cycle mimic of E2 and P4 levels for a period of 28 days were identified. During the reproductive lifespan of a woman, the ovary synthesizes and secretes sex steroid hormones and peptides in a programmed manner. The 2 2 defining hormones Mouse monoclonal to GSK3B of the menstrual cycle, estrogen and progesterone (P4) rise and fall, on average, every 28 days until follicle maturation ceases to occur at menopause. At each cycle, estradiol (E2) gradually increases during the follicular (proliferative) phase with the highest serum levels at the preovulatory stage (d 14) (1). The luteal (secretory) phase of the menstrual cycle is marked by decreased levels of E2 and a gradual increase in P4 typically peaking at day 21 AZD6738 (Ceralasertib) of the cycle followed by a steep decline, if there is no pregnancy, to mark the end of a 28-day cycle (1, 2). The effects of estrogen and P4 on reproductive tissues are dramatic, because they can regulate cell, tissue, and organ function. The endocervix is the tissue that lines the cervical canal, connecting the uterine cavity to the vagina (Physique 1). The endocervix is usually lined with a AZD6738 (Ceralasertib) layer of columnar epithelium, which produces and secretes mucus, forming a protective barrier to the outer environment. Depending on the hormonal influence, cervical mucus is usually permissive or refractory to sperm transport, because the composition, viscosity, and pH of the mucus varies during the menstrual cycle (3,C5). Around the time of ovulation, cervical mucus is usually thin, alkaline, and permissive to sperm, whereas after ovulation, the mucus becomes thick, more acidic, and acts as a barrier to sperm. Moreover, high P4 levels have been linked to increased susceptibility to HIV contamination due to the composition of mucins in the endocervix, decrease in secretion of chemokines and cytokines and endogenously produced AZD6738 (Ceralasertib) antimicrobials (6,C9). Open in a separate window Physique 1. Establishment of 3D cultures of the human endocervix.The endocervix tissue was obtained postsurgery, and tissue was enzymatically digested. Cells were cultured on 2D culture plates and AZD6738 (Ceralasertib) expanded. Cells were trypsinized and seeded at 2 106 cells/well onto the polystyrene scaffold membrane. Cells were cultured for 28 days in the presence of steroid hormones. The 3D models were fixed and processed for H&E staining. In contrast to other reproductive tissues, the endocervix has not been studied in extensive detail. In vitro models of the endocervix in the form of tissue explant cultures, or 2-dimensional (2D) cell cultures, have been used to study the influence of pathogens, immunity, and hormones (10). Moreover, these cultures are usually employed for a relatively short-time period (hoursCdays) due to degradation of tissue or cellular integrity after long-term cultures. Hormone treatments usually last for 24 or 48 hours, and single doses are tested, which provide only a snapshot of the effects of the hormones. In this study, we demonstrate the establishment of a novel and strong 3-dimensional (3D) culture system of the human endocervix cultured in the presence of fluctuating levels of estrogen and P4 in order to mimic a 28-day menstrual cycle. We demonstrate that these 3D models, in response to hormones, proliferate, express estrogen receptor (ER) and P4 receptor (PR), produce mucus, and secrete cytokines and growth factors. Novel secretory factors specifically driven by P4 have been identified. This new model will allow us to study the protective and permissive functions of the endocervix as it relates to contamination and fertility in a more physiological way. Materials and Methods Tissue collection Endocervical tissue samples were.