Binding characteristics of the ovine membrane progesterone receptor alpha and expression of the receptor during the estrous cycle

Abstract

Background: Classically, progesterone has been thought to act only through the well-known genomic pathway involving hormone binding to nuclear receptors and subsequent modulation of gene expression. However, there is increasing evidence for rapid, non-genomic effects of progesterone in a variety of mammalian tissues and it is possible that a membrane PR (mPR) is causing these events. We recently isolated and characterized an ovine mPR referred to as mPR-alpha, distinct from the nuclear PR. Based on predicted structural analysis, the ovine mPR-alpha possesses seven transmembrane domains typical of G protein-coupled receptors. Despite the homology to other reported mPRs, information pertaining to the steroid binding characteristics of the ovine mPR-alpha was lacking. Additionally, the ovine mPR-alpha transcript has been identified in the hypothalamus, pituitary, uterus, ovary and corpus luteum, yet changes in expression of the ovine mPR-alpha in these tissues were not known. Consequently, the purpose of this work was to determine the steroid binding characteristics of the ovine mPR-alpha and to investigate possible changes in expression of the ovine mPR-alpha in reproductive tissues throughout the estrous cycle.

Methods: Binding studies were performed using crude membrane fractions from CHO cells expressing the mPR-alpha. Using quantitative Real-time PCR we determined the expression pattern of mRNA for the ovine mPR-alpha during the ovine estrous cycle in tissues known to express the mPR-alpha. Jugular blood samples were also collected and analyzed for serum concentrations of P4 to ensure ewes were at the appropriate stage of their cycle.

Results: Only progesterone, 20alpha-hydroxyprogesterone and 17alpha-hydroxyprogesterone were able to displace binding of 3H-P4 (P < 0.001) to membrane fractions from CHO cells expressing ovine mPR-alpha. The average B-max and Kd values for three separate experiments were 624 +/- 119 fmol/micro gram protein and 122 +/- 50 nM, respectively. Significant changes in expression of mRNA for the mPR-alpha during the estrous cycle were noted in the corpus luteum and uterus.

Conclusion: The mPR-alpha specifically binds progestins and its expression was correlated to progesterone secretion during the ovine estrous cycle. Results from the present studies suggest that mPR-alpha may have an important physiological role during the ovine estrous cycle.

Figure 1

A) Binding of ³H-progesterone (cpm) was measured in increasing amounts of crude membranes from CHO cells and CHO cells expressing mPRα. Displacement of ³H-progesterone was measured in the presence of 1000-fold excess nonradioactive progesterone. Values are a representation of three individual experiments. B) Binding of ³H-Progesterone in crude membrane fractions from CHO cells expressing the ovine mPRα at various times of incubation. Displacement of ³H-progesterone was measured in the presence of 1000-fold excess nonradioactive progesterone. Values are means ± S.E. (n = 4).

Figure 2

Representative saturation curve in the presence of increasing concentrations of ³H-progesterone. The B_max and K_d values of ³H-progesterone binding were 624 fmol/μg protein and 122 nM, respectively. The inset shows Scatchard analysis of the same data.

Figure 3

Displacement of ³H-progesterone from crude membranes of CHO cells expressing mPRα in the presence of vehicle or 1000-fold excess of nonradioactive steroids. Steroids included progesterone (P4), 17α-hydroxyprogesterone (17-OH-P4), 20α-hydroxyprogesterone (20-OH-P4), estradiol, testosterone, cortisol, or RU486. Values are means ± S.E. (n = 3). *P < 0.001, compared to vehicle control.

Figure 4

Specificity of progestin binding sites for ovine mPRα. Binding of ³H-progesterone was measured in the absence or in the presence of increasing concentrations of nonradioactive progestins. Progestins evaluated included progesterone (P4), 20α-hydroxyprogesterone (20-OH-P4) and 17α-hydroxyprogesterone (17-OH-P4). Each point represents the average ± S.D. of progestin binding expressed as a percentage of maximum ³H-progesterone binding.

Figure 5

Serum concentrations (ng/mL) of P4 in synchronized ewes. Five ewes were assigned to each time period and synchronized with Lutalyse (7.5 mg I.M.) injections two times, 4 h apart. Time periods included 3 h and 24 h post estrus and days 4, 10, and 15 of the estrous cycle. For clarity, serum concentrations of P4 are only shown from onset of estrus (d 1) to d 15. Data are means ± S.E. (n = 5 ewes/time period).

Figure 6

Comparison of mean relative expression of mRNA encoding mPRα versus mRNA encoding actin in ovine A) hypothalamus, B) non-luteal components of the ovary, C) pituitary, D) corpus luteum, or E) uterus throughout the estrous cycle (3 h and 24 h post estrus and days 4, 10, 15). Values on the y-axis (actin Ct/mPRα Ct) represent means ± SE of a minimum of three sheep. Significant differences (P < 0.05) between groups are indicated by different letters above the bars.