Kenneth Savard

Measurement of urinary testosterone by gas-liquid chromatography☆

Abstract A method has been developed which employs gas-liquid chromatography for the assay of testosterone from extracts of human urine prepared according to an earlier method described by Camacho & Migeon in 1963. The trimethyl silyl ethers of testosterone and an internal standard, cistestosterone, were separated on the nitrile silicone rubber stationary phase XE-60. This method has given results which correspond satisfactorily with those obtained with UV spectrophotometry or measurement of sulfuric acid chromogens after paper chromatography.

Function of the human corpus luteum during the puerperium: Its maintenance by exogenous human chorionic gonadotropin

Abstract The human corpus luteum is maintained and continues to function throughout pregnancy. Human chorionic gonadotropin (HCG) plays a major role in the maintenance of the corpus luteum until term. After delivery, the source of HCG disappears and the corpus luteum ceases to function. Daily administration of exogenous human chorionic gonadotropin in large doses for as long as 5 days post partum appeared to maintain the function of the corpus luteum and prevented the decline usually observed after delivery in untreated subjects. Corpus luteum function was assessed by means of two parameters: the measurement of progesterone concentrations in peripheral and ovarian venous plasma and synthesis of progesterone in vitro by surviving slices of corpus luteum tissue removed at various times post partum.

Steroid formation by the avian ovary in vitro (Gallus domesticus)

Ovarian slices of the Gallus domesticus in the presence of gonadotrophin incorporate acetate-1-14C into progesterone, androstenedione, testosterone, and estradiol-17β. Distinct profiles of radioactive steroids are seen in the tissues of the molting, laying, and broody hens. There is an intense Δ5,3β-hydroxysteroid dehydrogenase reaction and 17β-hydroxysteroid dehydrogenase reaction in the granulosa cells of the laying hen; these reactions are not present in the molting hen. An intense 17β-hydroxysteroid dehydrogenase reaction, with testosterone as the substrate, occurs in the theca and stroma cells of the molting hen with only a weak reaction in the laying hen.

In vitro steroid synthesis by follicles isolated from the rabbit ovary.

Abstract Mature ovarian follicles were mechanically isolated from the ovaries of rabbits and incubated with acetate-1-14C. The resulting 14C-labelled steroidal products were extracted and purified by solvent partition and paper and thin layer chromatography followed by recrystallization to constant specific activity. The results show that under in vitro conditions, follicles are capable of synthesizing a wide variety of steroids including estrone, estradiol 17β, progesterone, 170H progesterone, testosterone and androstenedione. Addition of luteinizing hormone to the incubation medium results in an increase in 14C incorporation into all steroids. However, the gonadotropic stimulation was found to lead to a greater accumulation of radioactivity in testosterone with relatively less estrogen synthesized. The possible basis for this phenomenon is discussed.

Steroid hormone formation in the human ovary: III. Action of gonadotropins on testosterone synthesis by normal ovarian stromal tissue

Abstract The biosynthesis of radioactive testosterone in vitro from acetate-1-14C by normal human ovarian stromal tissue is described. Testosterone formation appears to be augmented in vitro by human chorionic gonadotropin and 2 human pituitary preparations containing luteinizing hormone activity.

Steroid formation in vitro in rabbit ovary.

Abstract Biosynthesis from acetate-1- 14 C of 10 steroids has been assessed in vitro by corpora lutea and interstitium of the ovaries from rabbits obtained 6 days after mating. The following steroids were synthesized: 3 β- hydroxy -Δ 5 - pregnen-20-one , progesterone, 20α and 20 β -hydroxy- Δ 4 -pregnen-3-ones, 17-hydroxyprogesterone, dehydroepiandrosterone, Δ 4 -androstene-3, 17-dione and testosterone; no radioactive estrone or estradiol-17β was found. The most highly radioactive steroids in both tissues were progesterone, pregnenolone and 20 α -hydroxy- Δ 4 -pregnen-3-one. On a per g. basis, corpus luteal tissue incorporated approximately 20 times more radioactivity into 8 steroids than interstitium.

Steroid hormone formation in the human ovary: VII: Stability of the profile of radioactive steroids formed from acetate-1-14C by the corpus luteum in vitro

Abstract Slices of human corpora lutea of pregnancy and of the cycle have been incubated in the presence of radioactive acetate, without added cofactors, under a variety of conditions. The distinct distribution of 14 C among the steroids, 3β-hydroxy-Δ 5 -pregnen-20-one, progesterone, 17-hydroxyprogesterone, Δ 4 -3, 17-androstenedione, estradiol-17β and estrone, previously described ( Hammerstein et al., Endocr. 24:597, 1964 ), is not substantially changed in further specimens nor by incubation in the presence of added gonadotropin (placental or pituitary), 3′,5′-AMP, nor by the duration of the incubation. The stability of this distribution of 14 C is interpreted as reflecting the transformation of 14 C-labelled cholesterol formed in vitro from acetate- 14 C. It is discussed in terms of the subcellular structures associated with steroidogenesis and the likelihood of their preservation under the in vitro conditions described.

The synthesis and secretion of progesterone and 20α-hydroxy-Δ4-pregnen-3-one by the rabbit ovary at various intervals after a single injection of HCG

Abstract The effect of gonadotropic stimulation on rabbit ovarian synthesis and secretion of progesterone and 20α-hydroxy-Δ 4 -pregnen-3-one has been investigated. Ovarian venous blood samples and ovarian tissues were collected at various intervals after the intravenous administration of HCG. Ovarian progestin content was assessed directly, or tissues were sliced for incubation with acetate-1- 14 C in order to label the steroidal products. The results of these studies confirm the previously reported preovulatory rise and ovulatory fall in steroid secretion and extend the findings to include a similar pattern in the de novo synthesis of the progestins, non-polar lipids and sterols. Addition of HCG directly to incubations failed to restore the lost steroidogenic capacity of the ovaries obtained at the time of ovulation. Taken together, these results indicate that the cessation in steroidogenesis at the time of ovulation must involve more complex metabolic alterations than merely the depletion of the stores of steroidogenic cholesterol substrate as has been the suggested basis for this phenomenon.

Subcellular Structure and Synthesis of Steroids in the Testis

It is becoming increasingly evident that subcellular localization of enzymes, substrates, cofactor systems, transport systems for oxygen, electrons, etc. are factors to be considered in the process of elaboration of steroid hormones. The production of the characteristic array of steroids by specific endocrine tissues, and more important, its control by gonadotropin, cannot at the present time be explained exclusively in terms of enzymes alone. Studies of various gonadal tissues in recent years reveal that the same enzymes or families of enzymes (3s-hydroxysteroid dehydrogenase-isomerase, 17-hydroxylase, C17,20-lyase, the 19-hydroxylase-aromatase system, 17s-hydroxysteroid dehydrogenase) are present in testicular tissues, in follicular tissue, in the human corpus luteum and in ovarian interstitial tissue. Yet each of these gonadal elements elaborates its own unique steroidal product.