2.3. The hypothalamus-pituitary-adrenal /gonadal axis (HPA/HPG)
In addition to gonadal differentiation, normal sexual development also requires normal development of the entire HPA/HPG axis, also referred as the reproductive axis (Vilain & McCabe 1998). HPA and HPG form a functional endocrine axis with hormonal regulation and feedback loops (Fig. 8). In contrast to the testes and adrenals, which produce hormones during fetal life, the ovaries start to release steroid hormones when they are first stimulated by gonadotropins at the onset of puberty (Miller & Strauss 1999).
Figure 8. The function of hypothalamus-pituitary-adrenal (HPA) and hypothalamus-pituitary-gonadal (HPG) axes. Gonadotropin releasing hormone (GnRH) and adrenocorticotropin releasing hormone (CRH) are discharged from hypothalamic central nervous system to stimulate the function of gonadotrophs and corticotrophs in pituitary gland. In response to gonadotropins (FSH, LH) and adrenocortical tropic hormone (ACTH) the gonads and the adrenal cortex synthesize and secrete sex steroids and corticosteroids. (Modified from Morohashi 1997.)
These two axes, HPA and HPG, show significant similarity in terms of their regulation mechanisms. The hypothalamic central nervous system discharges GnRH and adrenocorticotropin releasing hormone (CRH), which are transported to the anterior pituitary, where they stimulate the gonadotrophs and corticotrophs, respectively. In response to stimulation, these cells in turn secrete the gonadotropins FSH, LH and ACTH. Furthermore, these tropic hormones stimulate the gonads and adrenal cortex to synthesise and secrete sex steroids and corticosteroids. This stimulation take two forms: chronic stimulation, lasting for several hours or days and occurring through increased levels of P450 side chain cleavage (P450scc) protein and a consequent enhancement in steroidogenic capacity, and acute regulation, occurring within minutes and mediated by steroidogenic active regulatory protein (StAR), which facilitates the movement of cholesterol into the mitochondria for conversion to pregnenolone. The steroid hormones have an inhibitory feedback effect on secretion of the tropic peptide hormones (as reviewed in Miller 1988, Morohashi 1997, Miller & Strauss 1999, Edwards & Burnham 2001).
2.3.1. Steroidogenesis
One of the main functions of the adrenal cortex and the gonads is steroid hormone synthesis. The process in which specialized cells in specific tissues synthesize steroid hormones is generally referred to as steroidogenesis. Steroid hormones can be roughly divided into five groups according to their physiological behaviour: adrenal mineralocorticoids, which regulate the salt balance and maintain blood pressure, glucocorticoids, which regulate carbohydrate metabolism and manage stress, progestogens and estrogens, which are mainly produced by the ovaries and regulate reproductive function and secondary sex characteristics in the female, and androgens, which are mainly testicular in origin and are essential for fertility and secondary sex characteristics in the male (Fig. 9). These all share certain structural similarities and arise from a common series of pathways. Cholesterol is the precursor for all the steroid hormones, and although it can be synthesised de novo from acetate, most of the cholesterol needed is derived from plasma low density lipoproteins (LDL). Tropic hormones also stimulate the uptake of LDL cholesterol in addition to stimulating steroidogenesis.
Most of the steroidogenic enzymes belong to the cytochrome P450 oxidation group, among which five enzymes are involved in adrenal steroidogenesis: P450scc (side chain cleavage), P450c11 (11--hydroxylase), P450c17 (17-α-hydroxylase), P450c21 (21-hydroxylase) and P450aldo (aldosterone synthase). The first two of these are located in the mitochondria and the last two in the endoplasmic reticulum. In addition, P450aro mediates the aromatisation of androgens to estrogens in the gonads.
All steroid hormones are synthesised from the same precursor, cholesterol, but the end product depends on the complement of enzymes present in the tissues (Miller 1998). Adrenal glomerulosa cells mainly synthesise and secrete the mineralocorticoid aldosterone, while the adrenal fasciculata cells synthesise and secrete the glucocorticoids cortisol (in humans) and corticosterone (in rodents). The theca cells in the ovaries synthesise and secrete androgens, while the granulosa cells convert these androgens to estradiol and the corpora lutea together with the placental syncytiotrophoblasts synthesise and secrete progesterone. The Leydig cells in the testis synthesise and secrete the androgen testosterone (for a review, see Stocco 2001).
The first, rate-limiting step in the synthesis of all steroid hormones is the conversion of cholesterol to pregnenolone. In mitochondria this involves three distinct reactions, which are mediated by a single enzyme, P450scc, the activity of which is not the critical factor, but rather the supply of the substrate cholesterol from the outer mitochondrial membrane to the inner mitochondrial membrane where the P450scc enzyme is located (Black et al. 1994). Absence of P450scc activity results in an inability to synthesise any steroid hormone, and leads to death due to mineralocorticoid deficiency. Patients with a lack of P450scc can be treated with glucocorticoid and mineralocorticoid steroid hormone replacement if diagnosed early enough.
Figure 9. Steroid hormone synthesis pathways. All steroid hormones will be synthesized from cholesterol and the end products can be classified according to their principal effects; mineralocorticoids (aldosterone), glucocorticoids (cortisol in human, corticosterone in rodents), progestins, androgens and estrogens.
Pregnenolone may subsequently undergo one of two conversions. It may be converted by 3HSD to progesterone, which is the first biologically important steroid in the pathway, or else it may undergo 17α-hydroxylation mediated by P450c17 to yield 17α-hydroxypregnenolone. Progesterone can also be hydroxylated by P450c17, resulting in 17α-hydroxyprogesterone. P450c17 has two activities, that of a 17α-hydroxylase and that of a C-17,20 lyase capable of breaking up the C-17,20 carbon bond of 17α-hydroxypregnenolone or 17α-hydroxyprogesterone, yielding dehydroepiandrosterone (DHEA) or androstenedione, respectively. P450c17 is a key branching point in steroid hormone synthesis, directing pregnenolone towards the sex steroids (both hydroxylation and cleavage activities of the enzyme), the glucocorticoids (only hydroxylation) or the mineralocorticoids, if neither of the enzyme activities is participating.
After the synthesis of progesterone and 17-hydroxyprogesterone P450c21 can hydroxylate these steroids at the 21 position, resulting in deoxycorticosterone and 11-deoxycortisol, respectively.
The final step in the synthesis of adrenal mineralocorticoids and glucocorticoids is mediated by P450c11, which also mediates the final steps in the synthesis of aldosterone from deoxycorticosterone.
Conversion of androstenedione to testosterone is mediated by 17-hydroxysteroid dehydrogenase type III (Geissler et al. 1994). Androgens are precursors for estrogens in the female and the aromatisation of estrogenic steroids from them is mediated by P450aro. In peripheral target tissues, the genital tubercle, labial swellings and labioscrotal folds, testosterone can further be converted to dihydrotestosterone by 5α-reductase (non-P450 enzyme).
When the P450c21 step is impaired, cortisol synthesis decreases, leading to overproduction of ACTH. When this occurs adrenal steroid synthesis is stimulated and 17-hydroxyprogesterone is converted to androstenedione and further to testosterone, leading to severe virilisation of the female fetus. This disorder is known as CAH. Mutations in StAR cause lipoid CAH, which disrupts the synthesis of all adrenal and gonadal steroids. Affected genetic males are born with normal female external genitalia. (Miller & Strauss 1999, Stocco 2001.)
2.3.2. Mutations in biosynthetic enzymes
Some autosomal recessive mutations have been identified in biosynthetic enzymes responsible for converting cholesterol to androgens. These mutations generally lead to partial male-to-female sex reversal (reviewed in MacLean et al. 1997). Mutations in human HSD3b1 are reported to lead to either ambiguous or female external genitalia in 46XY patients, so that the conversion of DHEA to androstenedione is not possible (Rheaume et al. 1994, Russell et al. 1994a). Likewise, mutations in Cyp17 lead to a female phenotype in 46XY males, if they are unable to convert either 17α-hydroxypregnenolone to DHEA or 17α-hydroxyprogesterone to androstenedione (Kagimoto et al. 1988, Yanase et al. 1990, Yanase et al. 1991). The lack of HSD17b3 results in a 46XY phenotype with male internal genitalia but female external ones. These patients lack the enzyme that converts androstenedione to testosterone (Geissler et al. 1994). Mutations in the 5α-reductase type 2 gene lead to an inability to convert testosterone to DHT. These 46XY patients have feminised external genitalia and failed prostate development (Imperato-McGinley et al. 1974).