Lisette Lagacé

Purification, Cloning, Complementary DNA Structure, and Predicted Amino Acid Sequence of Human Estradiol 17β‐Dehydrogenase

Seventeen-P-hydroxysteroid dehydrogenase ( 17P-HSD) is the enzyme that catalyzes the interconversion of dehydroepiandrosterone (DHEA) and Sandrostenediol, 4-androstenedione and testosterone, and estrone and estradiol. This enzyme is present in many tissues such as the placenta,’,? testis,3 kidney,4 liver,’ skin? and ovary.’ This enzyme is likely to play a key role in the regulation of intracellular concentrations of sex hormones. The androgen testosterone and the estrogen 17Pestradiol are known to play an essential role in the development, growth, and function of all tissues responsible for reproduction and fertility in men and women. Moreover, sex steroids are involved in the regulation of hormone-sensitive cancer growth, especially prostate,* b r e a ~ t , ~ and endometrial cancer.IO Despite its purification from many tissues”-I4 and its partial biochemical chara c t e r i ~ a t i o n , ~ ~ ’ ~ little was known about the structure of this enzyme. We report the purification, cloning, and deduced amino acid sequence of estradiol 17pdehydrogenase (E2DH) from human placenta. The availability of cDNAs for EzDH offers the opportunity of studying in detail the factors controlling the expression of this crucial enzyme not only in gonadal but also in several peripheral estrogen target tissues.

SELECTIVE INHIBITORY EFFECT OF PORCINE FOLLICULAR FLUID ON FOLLICLE STIMULATING HORMONE SECRETION IN ANTERIOR PITUITARY CELLS IN CULTURE

Incubation of rat anterior pituitary cells in culture with porcine follicular fluid (treated with charcoal to remove steroids) led to a marked inhibition of spontaneous FSH release while no effect was observed on basal LH secretion. The inhibitory effect of follicular fluid was, however, less specific on LHRH‐induced gonadotropin release. Although more potent on FSH than LH release, a significant inhibition of LH release induced by the neurohormone was also observed. This inhibitory activity is mainly associated with material of MW 10000 daltons.

Mechanism of action of luteinizing hormone releasing hormone and thyrotropin releasing hormone in the anterior pituitary gland and modulation of their activity by peripheral hormones.

A new era of neuroendocrinology started with the elucidation of the structure of porcine and ovine thyrotropin releasing hormone (TRH), the neurohormone controlling the activity of thyrotropin (TSH) and possibly prolactin-secreting cells, as being (pyro)Glu-His-Pro-NH2 (1,2). This achievement was soon followed by the isolation of luteinizing hormone-releasing hormone (LHRH), the neurohormone which stimulates the release of both luteinizing hormone (LH) and follicle stimulating hormone (FSH), and the characterization of LHRH as a decapeptide having the following structure: (pyro)Glu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH2 (3,4). More recently, a tetradecapeptide, H-Ala-Gly-Cys-Lys-Asn-Phe-Phe-Trp-Lys-Thr-Phe-Thr-Ser-Cys-OH, was isolated from ovine and porcine hypothalami (5,6) on the basis of its ability to inhibit growth hormone (GH) release and is called somatostatin or GH release inhibiting hormone.

Mechanism of Action of Hypothalamic Hormones and Interactions with Sex Steroids in the Anterior Pituitary Gland

Although peripheral hormones have been shown for many years to play a major role in the control of adenohypophyseal activity in man and experimental animals, in vivo approaches could not distinguish between hypothalamic and pituitary sites of action. This area of research has been much facilitated by the development of the pituitary cell culture system(Vale et al., 1972; Labrie et al., 1973). In fact, adenohypophyseal cells in primary culture have been extremely useful not only for assessment of the biological activity of analogues of thyrotropin-releasing hormone (TRH), luteinizing hormone-releasing hormone (LH-RH), and somatostatin (La-brie et al., 1973, 1976a, b; Belanger et al., 1974), but also for determination of the characteristics of interaction between hypothalamic and peripheral hormones at the adenohypophyseal level (Drouin et al., 1976a,b; Drouin and Labrie, 1976a, b).

Interactions Between Hypothalamic and Peripheral Hormones at the Anterior Pituitary Level

Although peripheral hormones have been shown for many years to play a major role in the control of adenohypophyseal activity in man and experimental animals, in vivo approaches could not dissociate between hypothalamic and pituitary sites of action. This area of research vas much facilitated by the development of the pituitary cell culture system (Vale et al., 1972; Labrie et al., 1973). In fact, adenohypophyseal cells in primary culture have been extremely useful, not only for assessment of biological activity of analogs of TRH, LHRH and somatostatin (Labrie et al., 1973; Belanger et al., 1974; Labrie et al., 1976a, b) but also for determination of the characteristics of interaction between hypothalamic and peripheral hormones at the adenohypophyseal level (Drouin et al., 1976a, b; Drouin and Labrie, 1976a, b).

LEVELS OF CALMODULIN ARE STRICTLY REGULATED IN CELLS TRANSFECTED WITH AN EXOGENOUS CaM GENE: EFFECTS ON ENDOGENOUS CaM REGULATION AND CELL CYCLE PROGRESSION

Publisher Summary This chapter discusses the successful utilization of gene transfection and characterization of one of the stably transformed cell lines, C86. The expression of the CaM gene was studied in mouse C127 clones containing extra copies of the chicken CaM gene. The constructed vector utilized for studies included the complete structural portion of the chicken CaM gene ligated to the metallothionein promotor. This hybrid gene was cloned in a vector containing the genome of the Bovine Papilloma Virus (BPV) and transfected in to mouse C127 cells. Northern blot analysis was performed on total RNA extracted from exponentially growing C127, 6.8 and C86 cells. Clone C86 has been extremely valuable in uncovering the levels of regulation for CaM. It is apparent that total levels of CaM by themselves are insufficient to trigger a more rapid cell cycle because although clone C86 has an increase in CaM, the doubling time is not shortened. The fact that the doubling time of C86 is lengthened would still suggest that CaM plays a role in cell proliferation but that this regulation is more complex than a simple concentration effect and most likely other factors such as calmodulin binding proteins must be involved.