Really Really good article…
3α-hydroxysteroid dehydrogenase (3α-HSD) converts DHT to 3-Adiol G. This article talks about conversion directions…A MUST READ…
LINK: biolreprod.org/content/60/4/855.full
Chemicals
Radiolabeled steroids, [1,2-3H(N)]dihydrotestosterone and 5α-[9,11-3H(N)]androstane-3α,17β-diol, each with measured purities of 99%
, were purchased from DuPont New England Nuclear (Boston, MA). Nonradioactive steroids were purchased from Sigma Chemical Co. (St. Louis, MO) or Steraloids (Wilton, NH). The antibody for rat liver 3α-HSD was provided by Dr. Trevor Penning (Department of Pharmacology, University of Pennsylvania, Philadelphia, PA).
Animals
Sprague-Dawley rats (dams with litters of male pups, immature males, and adult males) were purchased from Charles River Laboratories (Wilmington, MA). The male rats were 21, 35, and 90 days of age on the day of cell isolation. The animals were killed by asphyxiation with CO2 according to an animal protocol that was approved by the Institutional Animal Care and Use Committee of the Rockefeller University (Protocol 91200).
Abstract
The enzyme 3α-hydroxysteroid dehydrogenase (3α-HSD) has an important role in androgen metabolism, catalyzing the interconversion of dihydrotestosterone (DHT) and 5α-androstane-3α,17β-diol (3α-DIOL). The net direction of this interconversion will affect the amount of biologically active ligand available for androgen receptor binding. We hypothesize that in Leydig cells, differential expression of 3α-HSD enzymes favoring one of the two directions is a mechanism by which DHT levels are controlled. In order to characterize 3α-HSD in rat Leydig cells, the following properties were analyzed: rates of oxidation (3α-DIOL to DHT) and reduction (DHT to 3α-DIOL) and preference for the cofactors NADP(H) and NAD(H) (i.e., the oxidized and reduced forms of both pyridine nucleotides) in Leydig cells isolated on Days 21, 35, and 90 postpartum. Levels of 3α-HSD protein were measured by immunoblotting using an antibody directed against the liver type of the enzyme. Levels of 3α-HSD protein and rates of reduction were highest on Day 21 and lowest on Day 90. The opposite was true for the rate of 3α-HSD oxidation, which was barely detectable on Day 21 and highest on Day 90 (59.08 ± 6.35 pmol/min per 106 cells, mean ± SE). Therefore, the level of 3α-HSD protein detectable by liver enzyme was consistent with reduction but not with oxidation. There was a clear partitioning of NADP(H)-dependent activity into the cytosolic fraction of Leydig cells, whereas on Days 35 and 90, Leydig cells also contained a microsomal NAD(H)-activated 3α-HSD. We conclude that 1) the cytosolic 3α-HSD in Leydig cells on Day 21 behaves as a unidirectional NADPH-dependent reductase; 2) by Day 35, a microsomal NAD(H)-dependent enzyme activity is present and may account for predominance of 3α-HSD oxidation over reduction and the resultant high capacity of Leydig cells on Day 90 to synthesize DHT from 3α-DIOL.
INTRODUCTION
The function of fully differentiated Leydig cells is to produce testosterone. Androgen receptors are present in Leydig cells [1–3], and it has been shown that androgen has autocrine effects on developing and mature Leydig cells [4, 5]. Leydig cells are the primary site of androgenic steroid synthesis, and multiple androgens with various potencies and intracellular concentrations are ultimately secreted. Dihydrotestosterone (DHT) is the most potent, binding to the androgen receptor with a dissociation constant (Kd) of 2.0 × 10−10 M, compared to 4.0 × 10−10 M for testosterone and 10−6 M for 5α-androstane-3α,17β-diol (3α-DIOL) [6,7]. In the Leydig cell, 5α-reductase catalyzes the irreversible conversion of testosterone to DHT. Another Leydig cell enzyme, 3α-hydroxysteroid dehydrogenase (3α-HSD), catalyzes the reductive breakdown of DHT to 3α-DIOL, but it can also catalyze, in the opposite direction, the oxidative conversion of 3α-DIOL to DHT. Therefore, the relative rates of 3α-HSD oxidation and reduction reactions will have important consequences for the steady-state concentration of DHT.
In the formation of disulfide bonds, NAD§H has to be repeatedly oxidized. During fertilization and the dissolution of disulfide bonds, the reverse occurs, and NAD§+ has to be repeatedly reduced. If chromatin decondensation utilizes the mechanism of chromatin condensation, but in reverse, then this will occur through the transfer of reducing equivalents from 3α-Diol to NAD§+. We have found that the 3α-Diol bound to the head region is consistently 3 fold that of bound 5α-DHT [85], indicating that 3α-HSD is functioning as a reductase. For the enzyme to operate in the reverse direction, it is likely that activation by an external source is required. This activation could be in the form of a female sex hormone, such as estrogen or progesterone. Progesterone is known to act on the sperm to induce capacitation [86]. It could also cause 3α-HSD to function as an oxidase. There are approximately 10,000 molecules of 3α-Diol per spermatozoan [85]. This number would produce less than 1% of the reducing equivalents needed to break all disulfide bounds. Still, enough bonds could be broken to allow reduced glutathione, contained in the cytoplasm of the ovum [18], to penetrate the chromatin and affect the dissolution of the remainder of the disulfide bonds.
HMMM, author mentions 3xHSD catalyze in the OPPOSITE direction from an external source: progesterone, estradial/estrogen…Propecia is the external source, since it elevated estrogen levels… don’t you think?
It has been suggested that glutathione is an intermediary in sperm condensation [29]. If this is indeed the case, reducing equivalents still need to be transferred, for which our proposed mechanism is applicable.
The direction of the reaction catalyzed by the putative NAD(H)-dependent microsomal 3α-HSD could not be determined directly because NADP(H)-dependent activities were also present in Leydig cells. However, the previously described NADPH-dependent 3α-HSD is exclusively reductive in intact cells (in the present paper); this could account for all of the reductive activity observed in adult Leydig cells. Since the NAD/NADH ratio is normally high in intact cells [28, 29], NADH-dependent 3α-HSD reductive activity may not convert DHT to 3α-DIOL in intact Leydig cells. We hypothesize that a microsomal NAD(H)-dependent 3α-HSD catalyzes oxidative activity in vivo, which amplifies androgen action by generating the potent androgen, DHT, from the weak androgen, 3α-DIOL. This is corroborated by other studies showing that the NAD-dependent type II 11β-hydroxysteroid dehydrogenase [17, 18] and type II 17β-hydroxysteroid dehydrogenase [19] are exclusively oxidative in intact cells but catalyze NADH-dependent reduction in homogenates and subcellular fractions.