The focus of our research program in reproductive physiology is on the hormonal regulation of embryo implantation. Investigations into this phenomenon are important because failure of the embryo to implant in the uterine endometrium is responsible for 60-70% of the pregnancy losses in cattle, horses and humans. Thus, a better understanding of the physiological mechanisms controlling embryo implantation may lead to improved domestic (farm) animal production, cures for human infertility, as well as safer and more effective forms of contraception.
Successful implantation depends upon the synchronized development of the embryo (blastocyst) to a state of implantation competency and a receptive uterus; collectively referred to as the window of implantation. In most mammals, implantation takes place in response to the sequential actions of estradiol (E2) and progesterone (P4) on the uterus and embryo. And yet, the underlying mechanisms through which these hormones promote development and activation of the embryo to the competent state for implantation remain largely unknown.
In the rodent uterus, E2 is converted to metabolites called catecholestrogens, which appear to be responsible for activating the blastocyst in preparation for implantation. Catecholestrogens are formed through the aromatic hydroxylation of E2 at the C-2 or C-4 positions. The metabolism of E2 by the enzyme cytochrome P-450 1B1 (CYP1B1) results in the production of 4-hydroxycatecholestradiol (4-OHE) while CYP1A1 converts E2 to 2-hydroxycatecholestradiol (2-OHE2). The very high metabolic clearance rate of catecholestrogens implies that they function as autocrine, paracrine and intracrine mediators of the effects of E2 and not as conventional circulating hormones. In a seminal experiment, Paria et al., (1998: Endocrinology 139: 5235-5246) showed that while E2 would not activate the dormant mouse blastocyst in culture, exogenous 4-OHE2 routinely activated the blastocyst, that when transplanted into receptive females resulted in live births.
We are currently studying the role of 4-OHE2 and 2-OHE2 in activation of the mink blastocyst. The mink is a seasonal breeder, and following fertilization of the eggs, the embryo develops to the blastocyst stage of embryogenesis and enters a state of dormancy referred to as embryonic diapause. Depending on the date of breeding, mink blastocysts may remain in the diapause state for up to 50 days or more resulting in a delay in implantation. Although both E2 and P4 are thought to be responsible for embryonic and uterine development through implantation in mink, all attempts to terminate embryonic diapause with exogenous E2 and P4, alone and in combination have failed.
We hypothesize that because the mink is a seasonal breeder, with estrus behavior and implantation being induced by increasing daylength (photoperiod), that the uterus produces catecholestrogens during the window of implantation that activate the dormant blastocyst, resulting in implantation. In other words, what determines the window of implantation in mink is, in part, the seasonally regulated production of catecholestrogens by the uterus
Using immunohistochemistry we have shown that mink uterine epithelial cells express both CYP1A1 and CYP1B1 proteins. Expression of CYP1B1 was highest during estrus and diapause, whereas CYP1A1 was only detectable during estrus. This agrees with reports in the literature that the uterus (rat) exhibits primarily CYP1B1 activity (90%) with only a minimal level (10%) of CYP1A1 activity during the window of implantation. Our qPCR analyses of the mink uterus showed that CYP1B1 mRNA was higher during diapause than estrus, with no change in CYP1A1 mRNA expression.
Collectively, these findings have caused us to begin using immortalized mink uterine epithelial cells (GMMe) to elucidate the hormonal regulation of the CYP enzyme activity. The assay we currently use measures CYP1A1 and CYP1B1 activities simultaneously, which we refer to as 2/4 Hydroxylase. Recently, we discovered that E2 stimulates 2/4 Hydroxylase activity whereas P4 inhibits the actions of E2.
We now envision a system whereby E2, which is produced before P4, during follicular development, increases uterine 4-OHE2 production, such that after ovulation and fertilization, the embryo can be made implantation competent. Subsequently, after the vacant follicle develops into a corpus luteum, the rising P4 concentrations would inhibit the actions of E2, closing the window of implantation. Thus, a delicate balance would appear to exist between E2 and P4 in regulating the production of catecholestrogens by the mink uterine epithelium.
Minimum Classes: General Biology, Anatomy and Physiology, some biochemistry
Projects: Because the use of the GMMe cells in research is relatively new, the are many questions that need to be answered, creating many opportunities for students at all levels (undergraduate and graduate). By participating in our research, students can learn: Reproductive endocrinology, Experimental design and data analysis, Cell culture, Enzyme activity assays, Quantitative Polymerase Chain Reaction assays, Immunohistochemistry, ImageJ Software (NIH), and others.
Some possible project ideas include:
1. Verfiy that GMMe cells produce polyamine molecules, and determine the hormonal regulation of polyamine synthesis. Polyamines (spermine, Spermidine and Putrescine) are detected in all mammalian cells. In mink, it has been shown that the uterus and blastocyst produce polyamines and that they are implicated in activation of the dormant mink embryo. Using the GMMe cells, the student could first carry out PCR analysis to verify the expression of genes for enzymes that are rate-limiting in polyamine synthesis. Subsequently, experiments could be conducted with the GMMe cells, to determine the effects of hormones (E2, P4 and prolactin) on expression of the genes by qPCR.
2. Similar to the project described above, the uterine epithelium produces the cytokine called Leukemia inhibitory factor (LIF). That LIF is involved in implantation can be illustrated by the observation that Lif-deficient female mice exhibit implantation failure, whereas supplementation with LIF rescues this defect. We would like to determine the hormonal regulation of LIF gene expression by the GMMe cells
Any of these projects would expose students to the process of scientific discovery and perhaps be a catalyst for the selection of science as a career path.