Urmila Verma

In vitro binding of progesterone to receptors in the human endometrium and the myometrium.

High affinity, low capacity progesterone binding receptors have been identified in the cytosol fractions of the human endometrium and the myometrium. The endometrial and the myometrial progesterone binding proteins had sedimentation coefficients of 4.5 S and 4.1 S, respectively. Analysis of the bound steroids revealed that, along with progesterone, small amounts of its metabolites (20alpha-hydroxy-4-pregnene-3-one, 5alpha-pregnane-3,20-dione, 5alpha-pregnane-20alpha-ol-3-one) were also bound to the receptor proteins. Among the steroids studied for ligand specificity, 5alpha-pregnane-3,20-dione showed the highest competition for progesterone binding sites. Progestational steroids, like chlormadinone acetate and norgestrel, did not compete for the progesterone receptors. The endometrial and the myometrial progesterone binding receptors were thermolabile and protein in nature. The molecular weight of the endometrial progestrone binding protein was about 60,000-67,000 with a molecular (Stokes) radius of 33 A and the frictional ratio of 1.26. The myometrial progesterone binding protein had a molecular weight of 56,000-58,000 with a molecular (Stokes) radius of 31 A and a frictional ratio of 1.23. The binding of corticosterone to the myometrial cytosol was only 22-34%, whereas with progesterone it was 70-95%. A study of the immunoabsorption of the plasma proteins from the endometrial and the myometrial cytosol suggested the presence of specific progesterone binding receptors in the cytosol that were different to plasma proteins. The association constant of progesterone for the endometrial progesterone receptor was 1.9 x 10(9) M(-1) and for the myometrial progesterone receptor it was 1.4 x 10(9) M(-1), values that are higher than the association constant of progesterone for corticosteroid binding globulin, which is 7 x 10(8) M(-1). The evidence suggested that the human endometrial and the myometrial progesterone binding proteins are different to the corticosteroid-binding globulin.

Screening of anti-progestins using in vitro human uterine progesterone receptor assay system.

An in vitro receptor assay system was developed to screen new antiprogestin compounds and to estimate their relative binding affinity for uterine progesterone receptors. 62 steroidal and nonsteroidal compounds including the suspected antiprogestins were screened. Normal human uterine tissues were obtained at hysterectomy operations. Premenopausal women with normal menstrual cycles and without any hormonal treatment were selected for the study. Some synthetic progestin compounds displayed high relative binding affinity (RBA) while others (metabolites of progesterone and norethisterone) showed poor or no binding affinity for human uterine progesterone receptors. Removal of the axial 19-methyl group from progesterone as in 19-norprogesterone resulted in greater binding affinity while the presence of polar groups on the molecule decreased the binding affinity. Of the 30 suspected antiprogestins tested, R2323 showed maximum competition for progesterone binding sites. Biologically, R2323 prevents implantation in women and this is attributed to its high binding affinity for the progesterone binding sites; however, the compound is associated with bleeding irregularities in women. Other compounds with high RBA are STS557 and RMI14156. The study shows that a complexity of factors (e.g., hydrophobicity, hydrophilicity, spatial requirements, optimal contact, and steric hindrances) control the ligand interactions to human uterine cytosol receptors. Screening larger numbers of antiprogestins should be done. As some of the compounds have mixed antiprogestational, antiandrogenic, and antiestrogenic properties, a comparative study on the RBA of a particular compound for the respective receptors should be carried out to better understand the biological profile and molecular interactions of the compounds.

Characterization of progesterone receptors and metabolism of progesterone in the normal and cancerous human mammary gland

The molecular interaction of progesterone with receptor proteins was investigated in 18 cancerous and 12 normal human mammary gland tissues. The time course analysis of progesterone localization showed that it was initially concentrated in the cytosol and subsequently transferred to the nucleus. In the cancerous tissue the nuclear fraction had significantly higher amounts of the steroid than in the cytosol fraction while in the normal mammary gland tissue localization of progesterone in the nuclear fraction was only slightly higher compared with the cytosolic fraction. In both the normal and cancerous mammary tissue progesterone was bound to a 4.3S receptor but a characteristic 6.7S binding component was noted in the normal mammary tissue. The number of specific binding sites in the normal and cancerous cytosol was of the order of 2.1 and 1.5 pmol/mg protein and the dissociation constant was 0.42 x > 10 -9 10 -9 < M -1 respectively. The binding component was a thermolabile protein but different than plasma proteins. Ligand specificity studies revealed that along with progesterone other progestins compete for the binding sites. In the mammary gland progesterone was metabolized mainly to 20-hydroxy-4-pregnen-3-one 5x-pregnane-320-dione and polar compounds. The breast carcinoma tissue showed higher metabolism than the normal breast tissue. In the breast carcinoma 20beta-hydroxy-4-pregnen-3-one was the major metabolite whereas in the normal mammary gland 20x-hydroxy-4-pregnen-3-one was the major metabolite. (Authors modified)

Cellular and subcellular metabolism of progesterone by the human proliferative and secretory phase endometrium and myometrium

Abstract An investigation of the metabolism of [1,2-3H]-progesterone by human proliferative and secretory phase uterine tissue and the subcellular localization and metabolism of progesterone showed that in the endometrium and the myometrium progesterone was mainly converted to 5α-pregnane-3,20-dione and 20α-hydroxy-4-pregnen-3-one. Smaller quantities of 20α-hydroxy-5α-pregnan-3-one, 6β-hydroxy-progesterone and unidentified polar metabolites were also formed. Qualitatively this metabolism did not appear to vary significantly in the human endometrium and myometrium. However, quantitative variations between the endometrium and myometrium were apparent in both the proliferative and secretory phases of the cycle. Conversion of progesterone to 5α-pregnane-3, 20-dione was higher in the endometrium than in the myometrium, while more of 20α-hydroxy-4-pregnen-3-one was formed by the myometrium. More 20α-hydroxy-4-pregnen-3-one was formed in the secretory than in the proliferative phase. Localization of progesterone in the subcellular fractions of the endometrium and myometrium showed that progesterone and its metabolites are mainly localised in the cytosol fraction, wherein they bind specifically to receptor protein. The nuclear uptake was considerably lower than that of the cytosol fraction. The subcellular metabolism of progesterone in the individual fractions, in the presence of co-factors, revealed that the conversion of progesterone was higher in the cytosol fraction. The major metabolite formed in the nuclear fraction of endometrium and myometrium was 5α-pregnane-3,20-dione. In the mitochondrial fraction 5α-pregnane-3,20-dione, 20α-hydroxy-5α-pregnan-3-one and some highly polar compounds were formed. 6β-hydroxy-progesterone was formed to a considerable extent by the microsomal and mitochondrial fractions. In the cytosol fractions of both the endometrium and myometrium, 20α-hydroxy-4-pregnen-3-one was the major metabolite with small amounts of 6β-hydroxy-progesterone, 5α-pregnane-3, 20-dione and 20α-hydroxy-5α-pregnan-3-one.

Progesterone biotransformation by the pituitary and hypothalamus of rabbit

Abstract Progesterone and its metabolites were localized in the neural tissues of rabbit against a blood concentration gradient when [ 3 HJ-progesterone was infused at a constant rate (11 μCi/h) for 7–8 h. The concentration of [ 3 H]-progesterone and its metabolites at the end of infusion varied between 80 and 125 mμCi/g in different parts of the brain. The endometrium and myometrium localized 130 and 55 mμCi/g tissue, respectively, while skeletal muscle concentrated only 6 mμCi/g tissue which was much lower than in plasma. 5α-Pregnane-3,20-dione was the major metabolite of progesterone in the hypothalamus while it was 20α-hydroxypregn-4-en-3-one in the pituitary. The metabolism of progesterone to 20α-hydroxypregn-4-en-3-one, 5α-pregnane-3,20-dione and further reduction to 5α-pregnane-3β-o1-20-one was much higher in the myometrial tissue than in the pituitary and hypothalamus. Of the subcellular fractions of hypothalamus, the nuclear preparation metabolized about 17.9% of progesterone, 5α-pregnane-3,20-dione being the major metabolite constituting 9.3% of the substrate. The maximal metabolism occurred in the soluble fraction where 20α-hydroxypregn-4-en-3-one was the major metabolite constituting 10.6% of the substrate.