Endocrinological aspects of the menopause.

BnasM Mtdical BidUlui (1992) Vol. 48, No. 2, pp. 262-275 © The Bntiih Council 1992

F Al-Azzawi Leicester University School of Medicine, Leicester Royal Infirmary, Leicester, UK

On average women live for about 30 years after the menopause. About 60-70% of consultations at the general practitioner and hospital specialist levels are for people over the age of 60, the majority of who are females. Therefore it is crucial to examine the effect of oestrogen prior to the menopause and how it interacts with other systems at cellular level. It is also important to examine why cells multiply and differentiate more efficiently in an oestrogenic milieu. This will give a better understanding of the degenerative processes of the menopause and will have profound implications on the design of effective hormone replacement therapy protocols.

The menopause is a consequence of oestrogen deficiency due to the depletion, or relative absence, of primordial follicles responsive to the rising level of gonadotrophins. This deficiency results in target organ failure, e.g. failure to induce endometrial proliferation and subsequent menstrua] bleeding. In women approaching the menopause, the menstrual cycle may change in rhythm, duration, and amount of blood loss, which accounts for the variable clinical manifestations of the climacteric. The menstrual cycle may cease suddenly or gradually depending on how depleted the ovaries are of these responsive primordial follicles. Life expectancy by the turn of the century was not far beyond the menopause but in die 1990s, about one third of women's life is spent after die menopause. The average age for die menopause is approximately 50 years; events like pregnancy and ovulation suppression for contraception, do not seem to have altered the age by which the ovaries are exhausted of responsive primordial fol-

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Endocrinological aspects of the menopause

ENDOCR1NOLOGICAL ASPECTS OF THE MENOPAUSE

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ENDOCRINOLOGY OF THE MENOPAUSE Living cells do not function in isolation and an elaborate communication network has to exist in order to co-ordinate various functions. This communication network can be a direct cell-cell contact or it may be a humoral agent produced from within the cell (autocrine), neighbouring cells (paracrine) or from a distant sophisticated organ such as the endocrine glands. Most of these signalling agents (which are largely water soluble) are destined to produce a rapid change within the target cells by altering an enzyme activity or by changing the permeability of cell membranes. Other factors are destined to induce a modification in genomic expression, and therefore, are typically lipid soluble and longer acting than water soluble agents. Steroid molecules and thyroxine are lipid soluble and may influence cell growth and differentiation. They are probably the best examples of hormones that act on the cell nucleus. Steroid biosynthesis Steroid hormones are derived from free cholesterol and its esterified esters (cholesteryl esters); therefore their basic chemical skeleton is identical. It is essentially a 4-ring hydrocarbon molecule with a side chain (Fig. 1). The conversion of cholesterol into pregnenolone is achieved by the side chain cleavage of the 6carbon isocaproic acid molecule. This reaction occurs in the mitochondria, and the cytochrome P-450 is responsible for its mediation. This group of reactions is the rate limiting step in steroid biosynthesis and most of the enzymes which control the steroidogenic biotransformations belong to the family of cytochrome P-450 oxygenases (hydroxylases). Ovarian steroidogenesis is a complex process which requires the interaction of several positive and negative feedback pathways


licles. In other words, it appears that these follicles are subject to a 'programmed cell death' which is initiated periodically and probably operates, at least partially, independent of the hormonal profile. Oestrogen deficiency disorders have been recognised to affect at least 75% of postmenopausal women.1 A greater understanding of the mechanisms of oestrogen action, and deficiency is therefore vital to understand and logically target pharmacological manipulations of the hypo-oestrogenic woman.

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HORMONE REPLACEMENT THERAPY

Pregnane

Pregnenolone Fig. 1 Structure of the steroid molecule and parent compounds.

involving in addition to the ovary, the hypothalamus and the anterior pituitary. The synthesis of oestradiol, unlike other oestrogens, is unique in that almost all of its production originates in the granulosa cells of responsive primordial follicles. Oestrogen metabolism The major oestrogen in women of reproductive age is oestradiol17P, which is synthesised by the granulosa cells from androgenic


Cholesterol

ENDOCRINOLOGICAL ASPECTS OF THE MENOPAUSE

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Pregnenolone

17 a OH-Preonenolooe

17 a OH-Progeeterone

P-450cir

Dehydroepiandrosterone

Androstenedkme

Fig. 2 A Biotransformatdons of major precursors of sex steroids P-450c 17= 17ahydroxylase activity.


precursors derived from theca cells. Cholesterol transport to the mitochondria of cells manufacturing steroids is activated by trophic hormones which also activate side chain oxidation. Oestrone is the principal oestrogen produced after the menopause. It is derived mainly from the peripheral conversion of androstenedione, of adrenal gland and/or ovarian origin, and aromatization of the A-ring (Fig. 2A & 2B).2~4 This conversion takes place at many extra-glandular sites but mainly in the adipose tissue. The latter pathway is enhanced with ageing and obesity. The aromatase enzymatic activity is exerted from within a transmembraneous glycoprotein complex (P-450mrom) in the presence of a flavoprotein, P-450 cytochrome reductase.5 Aromatization of the A-ring occurs in granulosa cells, Sertoli and Leydig cells and in the hypothalamus. To a lesser extent oestrone results from the hydrolysis of oestrone sulphate and this represents a large and relatively stable pool of oestrogen in the body. One fifth of oestrone sulphate may be converted to oestrone, but only 15% of oestradiol is converted

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to oestrone, which is a negligible amount (about 10 pmol/1). It is also important to remember that secreted oestradiol is reversibly oxidised to oestrone, and both oestrogens can be metabolised into oestriol. The predominant intracellular oestrogen is oestradiol, and 17P dehydrogenase favours the formation of oestrone and oestradiol. Under the effect of progesterone in the luteal phase the 17-p dehydrogenase activity increases and more oestrone is produced which is conjugated to oestrone sulphate. Oestrone sulphate is then available to enter the serum pool where it can be selectively taken up by tissues, such as the breast. It can then be, hydrolysed intracellularly to release oestrone which is converted to oestradiol. There is evidence that oestrone sulphate may not be freely available to the uterus.6 Alteration in intracellular enzymatic activity particularly that of 17-P dehydrogenase indicates that the metabolism and interconversion of oestrogens in the circulation, which are subject to splanchnic and renal clearance as well as other factors, appear to be associated with different effects in target tissues. Due to the depletion of responsive follicles after the menopause; the range of progesterone levels is similar to that of the proliferative phase, and its presence in the plasma reflects its exclusive


Fig. 2B Biotransformation of major sex steroids.


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The oestrogen receptor: A mystery Our knowledge of the mechanism of oestrogen action is rather limited. The vast spectrum of oestrogen action in various cells is complicated by the fact that it induces different responses at different times and through, apparently, different mechanisms. The origin of the oestrogen receptor has been traced to the expression of a single gene.7 The observed spectrum of oestrogen effects must be due to events taking place at a step beyond the formation of the oestrogen-receptor complex; whether this is due to synthesis of differing nucleoproteins or to the direct binding of segments of the gene sensitive to hormone-receptor binding events is unclear.8 To ensure their distribution, steroid molecules bind to plasma proteins to render them water soluble complexes. When they reach their target cells they are dissociated from their carrier protein and they pass freely through the cell membrane. They then bind to the cytosolic steroid receptor, an event which induces a conformational change in the receptor complex which triggers its migration to the nucleus.9 In the case of sex steroids, their receptor complexes are located in the nucleus. There are no detectable cytoplasmic receptors in enucleated cell preparations. Therefore oestrogen, for example, will travel freely into the nucleus to bind its receptor complex. The oestrogen receptor complex will bind a specific segment of the DNA and modifies its transcription as well as the synthesis of specific types of mRNA. The receptor has two binding domains; one to link up with the oestrogen molecule and the other domain binds the DNA segment of the chromatin. The binding sites of these two domains are independent of each other and each will induce a conformational change in the protein structure of the receptor. In the case of the DNA binding site, which combines with its respective chromatin, it maintains its oestrogen sensitivity.10 Oestrogen responsive elements are initially stimulated and are located some 1500-2000 base pairs upstream. Consequent upon the formation of the oestrogen receptor complex the


synthesis in the adrenal glands. The use of progestogens (and progesterone) in hormone replacement therapy, is to oppose the stimulatory effect of oestrogen on the endometrium by reducing the receptor content and subsequent mitotic activity. It may also affect oestrogen dependant enzymes within the cells such as hydrogenase and sulphurylase.

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Androgens The ovarian stroma continues to respond to the rising gonadotrophin levels and therefore all androgens continue to be produced after the menopause; particularly androstenedione, testosterone, dehydroepiandrosterone and its sulphated conjugate. This pattern is probably important in the speed with which degenerative changes are taking place in women who achieve the menopause naturally as opposed to those who achieve it surgically. The plasma level of testosterone in the natural menopause is less than that following surgical menopause, which indicates that a higher proportion of testosterone compared to androstenedione is derived from the suprarenal glands. In addition, plasma levels of dehydroepiandrosterone and its sulphate decline with age, despite the fact that they originate almost entirely from the adrenal glands. Gonadotrophins One of the important observations to be made during the 5-10 years prior to the menopause is a rise in serum and urinary follicular stimulating hormones (FSH) and a fall in the peak oestrogen levels.12 This rise is initially gradual but reaches its peak level after the menopause and for the following 3-5 years. A similar rise in luteinising hormone (LH) appears later man the rise in FSH level, which together with FSH are produced at a higher rate due to increased GnRH production, but the level of both hormones returns to the premenopausal range 20 years or so later.13 The cyclical nature of gonadotrophin secretion and sex steroid production is therefore lost after the menopause. Whether this phenomenon is related to a reduction in the sensitivity of the hypophyseal negative feed back, a reduction in the sensitivity of the neuroendocrine mechanism at the hypothalamic level, or even the reduction of inhibin production (or a similar factor) by the ovary independent of oestradiol is not completely clear. Nevertheless, in postmenopausal women the negative feedback mechanism of oestrogen on FSH is preserved14 and the biphasic feedback of progesterone on luteinising hormone (LH) secretion can still be demonstrated years after the menopause.15 In a remarkable study by Ranee and colleagues a morphological event is demonstrated when the infundibular neurones in the


polymerase II enzyme becomes activated, which helps the synthesis of mRNA.11


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Site of action of oestrogen The action of oestrogen is not limited to that of a 'growth factor' of the generative tract and the breasts, and in fact is widespread. For example it affects the distribution of body fat in a feminine fashion, and can also affect an individual's behaviour, in a broad sense. Other far reaching effects include its interaction with other endocrine systems and cell signalling agents. At this juncture it is illustrative to examine vasomotor symptoms as a phenomenon indirectly related to oestrogen deficiency which are alleviated with the spontaneous increase in endogenous oestrogen production and are efficiently treated with exogenous oestrogens. It is an apparent paradox to find that they also occur in pregnancy when oestrogen levels are a few orders of magnitude greater than during the follicular phase of the cycle.17 On the other hand, the high gonadotrophin levels associated with primary ovarian failure do not explain the lack of hot flushes and sweating in these women, and may challenge the direct effect of raised gonadotrophins on the vasomotor centre. If exogenous oestrogen supplement to these patients is discontinued, hot flushes will appear. Despite the fact that these vasomotor symptoms are temporally linked to the rise in the pulsatile secretion of LH, no correlation was found between the severity of these symptoms and gonadotrophin output or with opioid peptide concentration in the blood.18 Oestrogen effect on the rate of synthesis of neurotransmitters, e.g. serotonin and noradrenaline, is manifested by mood changes following the menopause, with their subsequent reversal following oestrogen therapy. The role of these neurotransmitters in modifying hypothalamic function has been widely documented, although the precise mechanism in relation to oestrogens is not completely understood. Effects of sex steroids on the immune response The dimorphic nature of the immune response has long been recognised in clinical medicine, and pregnancy represents a pro-


hypothalamus were found to be hypertrophied in oophorectomised compared to premenopausal women. They also contained a higher density of oestrogen receptor mRNA but did not contain GnRH mRNA. Therefore, the loss of inhibitory signals usually delivered by ovarian steroids may cause this dramatic change in hypothalamic anatomy.16

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foundly impressive paradox of immunological tolerance of an allograft, in the face of a concomitant increase in antibody production for example against Escherichia coli. Higher immunoreactivity, in general, can be inferred in these circumstances yet there is no plausible and consistent mechanism which can be directly implicated for the development of this clinical phenomenon. Another particular circumstance is the 'female prevalent' diseases—autoimmune diseases, of which systemic lupus erythematosus (SLE) represents a striking example. Females with SLE demonstrate a marked increase in 16a-hydroxylation products of oestrogen metabolism.19 Other examples include the more efficient ability to deal with infections, e.g. hepatitis B, where lower incidence of the carrier state is noted; although it is widely accepted that cell mediated immunity is impaired. Premenopausal women are less likely, compared to men, to accept a graft or to develop cancer.20'21 Women's susceptibility to infection increases during the premenstruum, however. The effect of oestrogen on the immune system is variable but has to be viewed with a particular reference to documented cell biological events. Oestrogen receptors have been described on T-lymphocytes, and there appears to be a dose dependent response of lymphocytic proliferation to added oestradiol. Low concentrations act as a stimulatory agent while higher concentrations are inhibitory. Although this effect has been ascribed to stimulation of the CD8 + suppressor cells,22 other data have reported an increase in CD4 +helper cells.23 Androgens have been shown to inhibit T- and B-cell maturation as well as reduce immunoglobulin secretion in mice;24-25 the relevance to the clinical situation is not clear. The hormone progesterone, on the other hand, has been shown in a number of studies to be suppressive of T-suppressor cells.26 It is too simplistic to assume that sex steroids affect the immune system by alteration of the absolute number of certain effector cells. Probably the mechanism of action is mainly via a permissive role whereby cytokines and other growth factors take on a secondary messenger role. IL-1 is a 17kD polypeptide which mediates a number of inflammatory responses.27 There are two variants of IL-1: a and p" (pi 5.0 and 7.0, respectively), which bind to the IL-1 receptor and exhibit the same range of biological activity.28 The relationship between IL-1 production and gonadal steroids have been examined through the induction of the proliferative response and of phagocytosis, which is essentially biphasic. These


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Effects of sex steroids on oncogene expression An oncogene is basically a normal gene (proto-oncogene) which is altered or abnormally expressed due to mutation or rearrangement of the DNA. The resulting transformed structural (or functional) protein is linked to the neoplastic process. The consequence of oncogene expression is the production of five possible products: nuclear proteins, cell membrane proteins with tyrosine kinase activity, cytoplasmic proteins with serine/threonine kinases, guanine nucleotide related proteins and growth factors. If an oncogene is implicated in cell transformation, it is an indication that its ancestor in the normal cell, the proto-oncogene, has a growth regulatory function. Ovarian steroid hormones are known to activate a number of these oncogenes which result in the expression of specific proteins and in DNA synthesis.31 The oestrogen receptor gene has been sequenced and found to have a strong sequence homology with the erb-A gene of the Avian Erythroblastosis Virus (AEV), which consists essentially of v-erbA and v-erb-B genes. It is the v-erb-B gene which possesses an oncogenic potential, and is homologue to Epidermal Growth Factor (EGF) receptor. EGF receptor and EGF messenger RNA in the uterus rise following oestrogen administration.32 Nevertheless, v-erb-B does not possess an EGF binding domain.33 The function of the cellular counterpart (c-erb-B) of the viral oncogene v-erbB is not known yet. These interrelationships of sex steroids with the part of the genome responsible for cellular proliferation and differentiation is fundamental not only to the understanding of the degenerative processes caused by hypogonadism but the mechanism through which therapeutic approaches are best designed. Oestrogen metabolism and the environment Dietary habits have been implicated in the regulation of many aspects of reproductive endocrinology with controversial claims


processes as well as the production of IL-1 are stimulated by low concentration of oestradiol and progesterone, but become progressively depressed when sex steroid concentration is raised.29 Tumour necrosis factor (TNF) and IL-1 are products of macrophage activation and are intimately related to osteoclastic activity and bone turnover, especially in women with endometriosis and in idiopathic osteoporosis.30 This raises the question of their role in postmenopausal osteoporosis.

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and counterclaims. There is no definitive indication that the menopause induces any more degenerative changes in the vegetarian population compared to omnivorous women, but earlier reports suggested the induction of a lower bone mass in individuals who have a high consumption of meat.34 In a cross-sectional study of vegetarian and omnivorous women there was no difference in bone mass as measured by single photon absorptiometry.35'36 We must always remember, nevertheless, that coincident with the endocrine changes of the climacteric are multiples of other physiological adjustments in various organ systems. Most represent senescent processes that continue independent of the hormonal environment. For example, the decrease in muscle mass and in bone mass and the increase in fat mass are features of senescence in both men and women. Recent studies have shown that both growth hormone and insulin-like growth factor-1 (IGF-1) profiles are higher in premenopausal women than postmenopausal women. After correcting for age-related decrease in the levels of growth hormone and IGF-1, oestrogens appear to play a major role in maintaining the high serum level of IGF-1 in younger women either directly or more likely by effecting the synthesis of IGF-1 binding proteins (IGF-lBPs). 37 However, growth hormone and IGF-1 levels rise following orally administered oestrogen replacement therapy,38 but do not rise when the oestrogen was administered by the transdermal route.39 This phenomenon is difficult to explain, but different serum oestrone: oestradiol ratios subsequent to different type, dose, and/or route of administration of oestrogens, or their first pass effect on the liver may be responsible. Therefore, this should raise the question of the effect of chronic suppression of bioavailable IGF-1 levels on IGF-1 responsive tissue, e.g. muscle mass and in adipose tissue. A serious environmental factor which affects women's health and 'the feminine factor' is cigarette smoking. There is a strong evidence showing that cigarette smoking severely reduces the bioavailability of oestrogen to the tissue. The suggested mechanism for this is that smoking increases the rate of A-ring 2-hydroxylation metabolism which results in the synthesis of 2-hydroxyoestradiol40 which are also found to be associated with a higher level of adrenal androgens.41 Smoking also lowers serum oestradiol level in women receiving oestrogen replacement therapy by increasing hepatic conjugation.42 This subject is well reviewed by Baron and colleagues.43


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CONCLUSION

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The future impact of postmenopausal hormone replacement therapy on public health is profound, particularly when we consider the size of the population in need of such treatment and the recommended duration of its use. The effects of hormone supplement as measured by the change it produces in serum components falls short from explaining proven epidemiological data, for example the alterations in plasma lipoprotein and apoprotein profiles and the relationship with coronary artery disease protection. Therefore this gap in our knowledge must be addressed at the cell biology level and simultaneously in terms of molecular chemistry within the next decade. Such data on the mechanism of action of oestrogen, and other factors, will be of enormous value in developing future therapeutic technologies in order to achieve specific effects in susceptible population.

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