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7 Gonadotrophin receptors: Correlates with normal and pathological functions of the human ovary and testis
7 Gonadotrophin Receptors: Correlates with Normal and Pathological Functions of the Human Ovary and Testis I L P O T. H U H T A N I E M I
The ovary and testis are under trophic control of three pituitary hormones: luteinizing hormone (LH), follicle-stimulating hormone (FSH) and prolactin (PRL). LH and FSH are conventionally regarded as gonadotrophins, but also PRL, due to its known direct gonadal actions, can be included into this category. The purpose of this chapter is to discuss the characteristics, functions and regulation of the gonadal receptors for these three pituitary hormones, with special emphasis on their role in the human during normal and pathological gonadal function. CHARACTERISTICS OF GONADOTROPHIN RECEPTORS Physicochemical and biochemical features Gonadotrophin action is initiated by binding of LH, FSH and PRL to highaffinity receptors on the outer surface of the plasma membrane of their target cells in the ovary and testis. The majority of our knowledge about these receptors is still derived from animal experiments. The rat testicular and ovarian LH receptors have been found to be asymmetrical glycoprotein molecules with molecular weights of about 200000, each molecule consisting of two 90 000 molecular weight subunits (for a review see Dufau and Catt, 1978). The solubilized receptors lose their binding activity after exposure to trypsin, indicating that protein forms a major component of the binding site (Dufau and Catt, 1978). Phospholipids and carbohydrates also compose integral parts of the LH-binding site. Similar properties have been described for purified preparations of rodent FSH and PRL receptors (Shiu and Friesen, 1974; Dufau and Catt, 1978). One line of receptor research attempts to produce purified receptor protein for production of receptor antibodies. These could provide a powerful tool for further studies on the receptor structure and function, and in general on the mechanisms of gonadotrophin action. Antisera have been generated against the rat ovarian LH receptor (Luborsky and Behrman, 1979; MetsikkO and Rajaniemi, 1981) and the rabbit mammary gland PRL receptor (Shiu and Friesen, 1976). Clinics in E n d o c r i n o l o g y a n d M e t a b o l i s m - - Vol. 12, No. 1, March 1983.
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Hormone--receptor interaction The interaction of gonadotrophins with their receptors is characterized by high affinity (equilibrium association constant, K a, of 109 to I0 ~ M), low capacity (number of receptors in thousands per cell), and high specificity (Dufau and Cart, 1978). One gonadotrophin has practically no crossreactivity with the receptor of another gonadotrophin. LH and HCG bind to the same receptor molecules, and HCG can be considered as a 'superagonist' to LH due to its longer half-time in the circulation and slower dissociation from the LH receptor (Huhtaniemi and Cart, 1981). Localization of the receptors Receptors for LH and FSH are only found in the gonads, whereas specific PRL binding is ubiquitously distributed throughout the body, in organs including the gonads, mammary gland, liver, kidneys, prostate and adrenals (Shiu and Friesen, 1981). LH and FSH receptors have been detected exclusively on the plasma membrane of the target cells, using autoradiographic techniques, ferritin-labelled HCG, immunofluorescence and receptor antibodies (Cart et al, 1980; Metsikk0 and Rajaniemi, 1981). However, a small fraction of the receptors is seen inside the target ceils after hormonal stimulation (i.e., internalized), and is assumed to be a part of processing and elimination of the hormone--receptor complexes (Catt and Dufau, 1981). The intracellular localization of receptors is especially clear with PRL, and it may play a role in the mechanism of action of this hormone (Rillema, 1980). MECHANISM OF GONADOTROPHIN ACTION Functional coupling of receptor binding and cellular responses The general mechanisms of membrane-receptor-mediated hormone action are described elsewhere. In brief, transfer of the LH and FSH message inside the target cell is performed at the plasma membrane level by a mediator molecule (i.e., 'second messenger'), cyclic adenosine monophosphate (cAMP). The gonadotrophin receptor is thought to float freely in the liquid matrix of the cell membrane. Upon hormone binding it is believed to undergo some conformational change that allows it to activate the adenylate cyclase enzyme floating in the inner surface of the plasma membrane. Another plasma membrane protein, the so-called nucleotide regulatory protein, is needed for activation of adenylate cyclase. Gonadotrophin stimulation in fact stimulates binding of guanosine triphosphate (GTP) to this regulatory protein, which is thereafter capable of activating adenylate cyclase. Activation of adenylate cyclase then catalyses the conversion of intracellular ATP to cAMP, the second messenger, which binds to the regulatory subunit of the protein kinase enzyme, and a catalytic subunit of protein kinase is cleaved off. The latter, in the presence of ATP, can finally catalyse a number of protein phosphorylations (Cohen, 1982). The phosphorylations regulate the activity of a number of targe t cell enzymatic and structural proteins, and the above changes are essentially those which take place within minutes during the rapid responses of gonadal
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cell functions to LH and FSH stimulation (enhanced steroidogenesis, carbohydrate metabolism and ion transport). The other part of gonadotrophin effect is slower, and leads to late responses in RNA and protein synthesis, cell growth, differentiation and division, again through phosphorylation of different proteins. It is not known in detail how the similar cAMP molecules can activate the differential fast and slow responses in the same cells, but distribution of cAMP into different intracellular pools may be the explanation. The mechanism whereby PRL mediates its actions within the cell is still unknown. It has been suggested that the internalization of PRL would play a role in this process. Subsequently, PRL action may involve changes in Na +,K +-ATPase activity, increase in cGMP and decrease in cAMP levels, and enhanced rates of prostaglandin and polyamine synthesis (Rillema, 1980).
Quantitative aspects of hormone binding and cellular response An important feature in gonadotrophin action is that only a minute fraction of the receptors needs to be occupied to obtain maximal biological response. This is especially clear with LH action, since occupancy of less than one per cent of testicular receptors is sufficient to result in maximal testosterone production. The rest of the receptors, termed 'spare' receptors, are assumed to favour formation of hormone--receptor complexes in the face of low ligand concentrations, and to provide a receptor reserve that assures availability of free receptors for continuous gonadotrophic stimulation (Dufau and Catt, 1978; Huhtaniemi, Clayton and Catt, 1982). HORMONAL REGULATION OF GONADOTROPHIN RECEPTORS Homologous receptor regulation As will be described below, the maintenance of gonadotrophin receptors is dependent on circulating levels of the hormone itself and in many cases also on other pituitary hormones. Numerous recent studies have demonstrated another kind of gonadotrophin effect on their own (homologous) receptors: stimulation with L H / H C G leads to a rapid and severe decrease in gonadal LH receptor levels. This phenomenon is termed 'receptor down-regulation' (Catt et al, 1980). Since the effect is only produced by high hormone concentrations, its physiological role is still controversial. It may play an important role in protection of the gonads from excessive hormonal stimulation. Down-regulation could also represent amplification of mechanisms taking place in a single cell (or in a specific location of its plasma membrane) during physiological stimulation at low gonadotrophin concentrations. Clearcut down-regulation effects have not been produced during FSH and PRL stimulation, and details about the' mode of autoregulation of their receptors are not yet clear. Heterologous receptor regulation Another type of down-regulation is seen with heterologous receptor systems. LH stimulation of the ovaries and testes results, in addition to LH
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receptor loss, in concomitant loss of PRL binding. The mechanism of this response may be coaggregation and cointernalization of PRL receptors together with LH receptors upon LH receptor occupancy. In addition to down-regulatory receptor losses, gonadotrophin stimulation is accompanied by loss of target cell metabolic responses -- in gonads by decrease of cAMP production and steroidogenesis (Catt et al, 1980) -- which may be another facet in the mechanisms of protection of gonadotrophin target cells from too-high trophic stimulation. GONADOTROPHIN TARGET CELLS AND EFFECTS IN THE GONADS -- ANIMAL STUDIES The ovary
Table 1 lists the ovarian and testicular target cells for LH, FSH and PRL (i.e., cells that have receptors for these three hormones). The role of gonadotrophins in the ovarian function is, in simplified terms, as follows (Catt and Pierce, 1978; Ross and Schreiber, 1978). Granulosa cells are the target of FSH action in the ovary. Together with oestrogen, FSH induces proliferation and maturation of granulosa cells. Early follicles are practically devoid of LH and PRL receptors, but they appear as a result of FSH (and oestrogen) action on granulosa cells as the follicle matures. A reciprocal decrease of FSH binding occurs simultaneously. FSH also stimulates steroidogenesis (mainly oestrogen production) by the granulosa cells. Progesterone production appears concomitantly with the development of granulosa cell LH receptors. It seems that androgen, an obligatory intermediate in granulosa cell oestrogen formation, is produced by the theca cells in response to LH stimulation. Androgen thus produced is transferred to the granulosa cells where it is aromatized under the influence of FSH. Since oestrogen stimulates follicular growth, and androgen is inhibitory, balance between these two hormones determines the fate of the developing follicle (further growth or atresia). The FSH action seems to cease after ovulation, when LH receptor level of the luteal cells reaches a high concentration. Table 1. Presence o f LH, F S H and P R L receptors in the different cellular compartments o f the ovary and testis; the results are based on observations in experimental animals Receptor
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The midcycle surge of LH is clearly essential for ovulation, but the exact role of this hormone has not been clarified in the context of the large variety of biochemical and morphological changes taking place in the ovary around ovulation. After ovulation, LH is responsible for stimulation of luteal cell progesterone production, and, if pregnancy is initiated, HCG is the trophic stimulus for progesterone production by the corpus luteum in pregnancy. PRL receptor changes seem to accompany those of the LH receptor, and PRL is known to have synergistic actions with LH in the maintenance of LH receptors and LH-stimulated steroidogenesis. Paradoxically, PRL has been shown to have both luteotrophic and luteolytic effects. The former includes a permissive effect on steroidogenesis by maintenance of LH receptors and modulation of storage and turnover of luteal cell cholesterol. High PRL levels, in contrast, have been shown to be inhibitory to FSHstimulated oestrogen production in the granulosa cell (Dorrington and Gore-Langton, 1981) and LH-stimulated progesterone production in the luteal cell (McNatty, Sawers and McNeilly, 1974). The testis
In the testis, the Sertoli cells of the seminiferous tubules are the best-known target of FSH action (Catt and Pierce, 1978; Dufau and Catt, 1978). This hormone is known to stimulate production of numerous specific proteins, some of which may be essential for sperm maturation, in Sertoli cells. The best characterized of these substances is the secretory protein 'androgenbinding protein' (ABP), known to accumulate testosterone in the tubular compartment, which is essential for normal spermatogenesis. In addition, autoradiographic studies have shown specific binding of FSH to spermatogonia of the rat testis (Orth and Christensen, 1978), but the physiological significance of this finding is still unknown. Most testicular FSH effects have been shown only in immature or hypophysectomized animals, and the role of FSH in adult testicular function, especially in humans, is therefore still somewhat unclear (Bremner et al, 1981). LH and PRL receptors are confined to the Leydig cells (Dufau and Cart, 1978). LH receptors are needed for stimulation of Leydig cell steroidogenesis, growth and differentiation. As in the ovary, PRL seems to have a synergistic action with LH on the maintenance and induction of LH receptors (Catt et al, 1980) and has also been suggested to promote steroidogenesis by regulating the supply of intratesticular cholesterol esters (Bartke, 1980). Dependence of LH receptor induction on FSH in the testis has also been proposed (Catt et al, 1980). This kind of induction is, however, difficult to explain, since testicular LH and FSH receptors are present in two different cell types (i.e., Leydig and Sertoli cells). Thus, a direct effect at least of FSH on Leydig cells seem unlikely, but a Sertoli cell-mediated mechanism is possible. Communication between testicular compartments has been discussed recently on the basis of the finding that Sertoli cells produce a gonadotrophin-releasing hormoneqike substance which then regulates gonadotrophin actions on Leydig cells (Sharpe, 1982).
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LH receptors during the normal menstrual cycle
Human ovarian LH binding has been measured at different stages of the menstrual cycle in a number of studies (see, for example, Lee et al, 1973; Wardlaw, Lauersen and Saxena, 1975; Cole et al, 1976; Rajaniemi et al, 1981b) (Figure 1). Basically, the receptors have been found to have similar characteristics to those of rodents. The binding has high affinity, low capacity, and is highest in ovarian luteal ceils (Rajaniemi et al, 1981b). Incubation conditions, molecular weight of the receptor, and effects of enzymatic treatments on binding are similar to those in the experimental animals. A remarkable difference is the species specificity of LH receptors of the human and monkey ovary (Cole et al, 1976; Cameron and Stouffer,
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1982).. These seem to bind only human luteinizing hormones (HLH and HCG), and LH preparations of lower species (e.g., ovine and bovine) have very low affinity. Rat, ovine and bovine LH receptors, by contrast, bind all these hormone preparations. This species specificity, as well as development of neutralizing antibodies to the heterologous hormone, explains the necessity of human gonadotrophin preparations in treatment of humans. There is clear evidence that LH and HCG bind to the same receptor molecules with similar affinity (Wardlaw,~ Lauersen and Saxena, 1975; Cameron and Stouffer, 1982). Compared with that of the rat, the binding capacity of the human and primate corpus luteum (per mg protein) is clearly lower (Dufau and Catt, 1978; Rajaniemi et al, 1981b; Cameron and Stouffer, 1982)~ Some LH binding is also present in the granulosa cells (Rajaniemi et al, 1981b), but highest concentration of LH receptors is found in luteal cells during the midluteal phase (McNeilly et al, 1980; Rajaniemi et al, 1981b) (Figure]). A drop in binding occurs immediately after ovulation, possibly becaus~ of receptor occupancy or down-regulation, induced by the ovulatory LH surge (Rajaniemi et al, 1981b). The number of LH receptors during the latter part of the cycle decreases (Wardlaw, Lauersen and Saxena, 1975; McNeilly et al, 1980), but some residual LH binding is still present in old corpora lutea during the next cycle (Rajaniemi et al, 1981b). The decrease of LH receptors towards the end of luteal phase has no close correlation with the fall in progesterone levels, which suggests that LH receptor loss may not be of primary importance in the involution of the corpus luteum. McNeilly et al (1980) have suggested that, as LH stimulation and progesterone production decrease during late luteal phase, the corpus luteum becomes subject to luteolytic effects of prostaglandin F2,~. However, if high gonadotrophin levels and progesterone production are maintained, as is the case if pregnancy ensues, the corpus luteum is protected from the luteolytic effects of prostaglandin. Luteal cells of the corpus luteum of pregnancy have very low LH binding, which possibly is due to occupancy of receptors by HCG, or to receptor down-regulation (Rao et al, 1977; Halme et al, 1978; Rajaniemi et al, 1981b). The latter mechanism, together with desensitization of cellular steroidogenic responses, may be responsible for cessation of hormone production in the corpus luteum during pregnancy by about seven to eight weeks of gestation, when HCG reaches very high levels in the maternal circulation. No LH receptors have been found in the ovarian stroma, but it remains to be established whether receptors are present in the theca cells. Such is the case in the rat, where the receptors have a distinct role in regulation of theca cell androgen production. LH receptors in pathophysiological conditions Little is known about levels of gonadotrophin receptors in pathophysiological conditions. Low follicular LH receptor levels have been reported in endometriosis and in polycystic ovarian disease (Rajaniemi et al, 1980; Kauppila, Rajaniemi and R0nnberg, 1982), but whether the low binding has
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a causal relationship to the pathogenesis of these conditions, or is secondary to other hormonal changes, is not known. LH receptors have also been detected in some benign and malignant ovarian tumours (Wardlaw, Lauersen and Saxena, 1975; Rajaniemi et al, 1981a), a finding of potential clinical importance since growth and/or function of some ovarian tumours may be dependent on gonadotrophins. Accordingly, measurement of gonadotrophin receptors in ovarian tumour tissue could be used as a diagnostic tool to assess benefits of treatment to eliminate pituitary gonadotrophin secretion (hypophysectomy or gonadotrophin-releasing hormone (GnRH)-agonist analogues). FSH and PRL receptors McNeilly et al (1980) have detected specific FSH binding in human corpora lutea, a surprising finding in view of the animal data that indicate a specific role for FSH in follicular but not in luteal function (see above). Also, specific PRL binding has been detected in the human ovary (Saito and Saxena, 1975; Pointdexter et al, 1979; McNeilly et al, 1980), but studies on human FSH and PRL receptors are still too preliminary to allow conclusions about their behaviour during the normal menstrual cycle or disturbances of ovarian function. There is a report of FSH receptors in an ovarian granulosa cell tumour (Davy, Torjesen and Aakvaag, 1977), which further supports the case for gonadotrophin dependence of certain ovarian neoplasms. As for PRL, the evidence for direct ovarian actions in humans is mainly based on demonstrated inhibitory effects of PRL on ovarian steroidogenesis in vitro (McNatty, Sawers and McNatty, 1974; Demura et al, 1982). Gonadotrophin receptor antibodies An intriguing pathological condition with possible gonadotrophin receptor involvement is the 'gonadotrophin-resistant ovary' syndrome (Koninckx and Brosens, 1977; Starup and Pedersen, 1978). It is characterized by primary or secondary amenorrhoea, normal development of secondary sexual characteristics, hypergonadotrophism, presence of morphologically normal follicles in the ovary, and resistance of the ovary to high gonadotrophin stimulation. Theoretically, several pathophysiological mechanisms could lead to this condition. These include abnormal or biologically inactive circulating gonadotrophin, circulating gonadotrophin antibodies, and lack of end-organ response to gonadotrophin stimulation. The occurrence of circulating gonadotrophin receptor antibodies has been discussed for several years, but evidence for such a pathogenesis did not exist until recently. Chiauzzi et al (1982) detected FSH receptor antibodies in the serum of myasthenia gravis patients, who also had hypergonadotrophic ovarian failure. The circulating serum component was immunoglobulin by biochemical and physiochemical criteria, and it inhibited FSH binding to rat testis receptors, as well as FSH-stimulated cAMP and ABP production in rat testis tubules. This finding reveals a new category of autoimmune reproductive organ disease, analogous to conditions with detectable auto-
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antibodies against receptors of insulin (type B insulin resistance with acanthosis nigricans), TSH (Graves' disease) and acetylcholine (myasthenia gravis) (Roth and Grunfeld, 1981) (see Chapters 11, 5 and 4 respectively). Clinical aspects in gonadotrophin treatment Gonadotrophin therapy is used in the induction of ovulation in patients with gonadal failure due to hypothalamic or pituitary failure (Insler and Lunenfeld, 1978). To induce follicular maturation, the treatment consists of daily injections of human menopausal gonadotrophin (HMG), which is a combination of FSH and LH, during the follicular phase. When sufficient follicular maturation is achieved (as assessed by oestrogen measurements and ultrasound study of the follicular size) ovulation is triggered by an injection of HCG either as a single or as multiple injections. Two major complications may occur with the treatment, that is, ovarian hyperstimulation and multiple pregnancies. Although the treatment attempts to mimic physiological gonadotrophin secretion, some differences from the physiological conditions are evident and may explain difficulties encountered with it. Gonadotrophins are secreted from the pituitary in a pulsatile fashion with a burst every 60 to 90 minutes in response to pulses of the hypothalamic gonadotrophin-releasing hormone. This pulsatile secretion cannot be mimicked by the currently used methods of intramuscular hormone administration of gonadotrophins. Frequent pulses of gonadotrophin secretion are likely to be important for normal follicular maturation. Tonically elevated hormone levels could lead to ovarian overstimulation. This is not infrequent, and therefore the HMG treatment must be individually adjusted in each case. Ovulation is induced by HCG, which in the commonly used doses (3000 to 10000 IU i.m.) may result in exaggerated and prolonged LH activity in the circulation. Due to the long half-life of HCG in the circulation, biologically active levels of HCG can be found in peripheral serum for four to five days after a single injection of 5000 IU (Martikainen, Huhtaniemi and Vihko, 1980). This prolonged period of high LH stimulation may partly explain occurrence of multiple ovulations. It is even possible that the persistent high levels of HCG, if given too early, may induce luteinization of unruptured follicles or, if ovulation is achieved, down-regulation of LH receptors and steroidogenic lesions in the corpus luteum. Therefore, there is an obvious need for clinical treatments with LH preparations of shorter duration of action than HCG (possibly desialylated HCG). Treatment with HMG and HCG has undoubtedly provided a valuable method for ovulation induction under carefully controlled conditions. Our present knowledge about physiological gonadotrophin secretion and mechanisms of action can, however, explain the narrow therapeutic range of treatment and provides a rationale for avoiding some of the complications. In cases of anovulation due to hypothalamic failure, pulsatile treatment with GnRH has been shown to offer a more physiological means of treatment, with minimal risk of ovarian hyperstimulation or multiple pregnancies (see Chapter 10).
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GONADOTROPHIN
R E C E P T O R S IN T H E H U M A N TESTIS
LH receptors and L H / H C G action in the human testis L H / H C G binding has been measured in both the fetal (Huhtaniemi, Korenbrot and Jaffe, 1977) and adult (Hsu et al, 1978; Davies et al, 1979; Sharpe, Wu and Hargreave, 1980; Huhtaniemi et al, 1980, 1982) h u m a n testis (Figure 2) and, as in experimental animals, the receptors are confined to the Leydig cells. The K a of H C G binding to the receptors is similar to that observed in the rat testis (about 1 x 101° M-l), but a clear quantitative difference is seen between the two species; the number of L H receptors per Leydig cell is about 1500 in the human, and about 20000 in the rat (Huhtaniemi et al, 1982). A similar species specificity to that in the ovary is seen with testicular L H receptors; the h u m a n receptors bind effectively only primate L H preparations (Davies et al, 1979). LH
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1976), which in the adult should result in LH receptor down-regulation and desensitization of androgen production. Our recent animal studies have indicated that these negative effects of gonadotrophin treatment are not present in the fetus or neonate, but are a result of pubertal sexual development (Huhtaniemi, Katikineni and Cart, 198 l). Besides the low LH receptor level of human testes, another clear difference exists in the LH-stimulated steroidogenic response between the human and rat (Huhtaniemi et al, 1982). Gonadotrophin stimulation is known to induce two responses in the testicular androgen production, a fast one (within hours) and a slow one (several days after hormone injection). The first response is very prominent in the rat with a more than 10-fold increase of circulating testosterone level. In the human, the acute response is hardly detectable, with about a 50 per cent increase in serum testosterone (Forest et al, 1980; Martikainen, Huhtaniemi and Vihko, 1980) (Figure 3).
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Figure 3. Peripheral serum levels of testosterone, 17-hydroxyprogesteroneand oestradiol in human males after an injection of 80 IU/kg HCG. The values are expressed as percentage of the control levels, which were 19.9 nmol/1 for testosterone, 2.6 nmol/l for 17-hydroxyprogesterone, and 0.10 nmol/I for oestradiol. The data are modified from those presented by Martikainen, Huhtaniemi and Vihko (1980). At around 24 to 36 hours, an increase is seen in serum levels of 17-hydroxyprogesterone and oestradiol. The oestrogen response is a specific Leydig cell response to gonadotrophin stimulation, and is known to be involved in the refractoriness of androgen synthesis due to blockade of C21 steroid side chain cleavage. Concomitant accumulation of 17-hydroxyprogesterone with the oestrogen peak is a sign of this steroidogenic blockade. The second, two-to three-fold response occurs after two to four days when the phase of steroidogenic desensitization is over. The late androgen responses are of similar magnitude and duration in the human and rat. Although
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theoretically possible, the poor acute response of the human testis is probably not due to the low LH receptor level, since the HCG-stimulated cAMP response of the human testis is comparable to those observed in the rat testis in vitro (Huhtaniemi et al, 1982). The reason for the poor steroidogenie response has been localized to a metabolic step beyond cAMP formation but before mitochondrial formation of pregnenolone, being probably due to defective supply of cholesterol for the mitochondrial steroidogenesis. It is possible that the basal circulating LH level is already stimulating near-maximal steroidogenesis, and further exogenous stimulation by the injected HCG is without effect. Whatever the reason, gonadotrophin stimulation of the human testis is almost devoid of an immediate steroidogenic response, and marked stimulation of testosterone production does not occur until two to four days later.
FSH and PRL receptors FSH receptors have been detected in the fetal (Huhtaniemi and Jaffe, unpublished observation 1982) and adult (WahlstrOm et al, 1983) human testis, but no specific PRL binding has so far been found in it. This negative finding may be due to suboptimal analytical techniques. On the other hand, there is no unequivocal evidence that PRL in the human male would have direct testicular effects. Hyperprolactinaemia has been clinically related to azoospermia, impotence and low serum testosterone (Franks et al, 1978), but the findings may be related to direct effects of PRL on pituitary gonadotrophin secretion (Bartke, 1980). Clinical aspects in gonadotrophin treatment Chorionic gonadotrophin is used in the treatment of cryptorchidism and hypogonadotrophic hypogonadism, and diagnostically to detect presence of functional testis tissue in cases of genital ambiguity. Formerly, the treatment or diagnostic tests included daily injections of HCG for several days, after which the rise in serum testosterone was monitored. We know now that an injection of HCG (about 100 IU/kg) results in biologically effective serum HCG concentration and LH receptor occupancy for about four days, since the half-life of HCG in the circulation and at the receptor site is about 24 hours (Martikainen, Huhtaniemi and Vihko, 1980). Furthermore, since the injection results in maximal steroidogenic response in 72 to 96 hours (Figure 3), HCG injections more frequently than at three-day intervals are pointless (Forest et al, 1980; Martikainen, Huhtaniemi and Vihko, 1980). For treatment of cryptorchidism or hypogonadism, an HCG injection one or two times a week is theoretically sufficient. More frequent treatment could even be harmful, due to the possibility of LH receptor down-regulation (Sharpe, Wu and Hargreave, 1980) and inhibition of enzymes involved in androgen biosynthesis (steroidogenic desensitization) (Catt et al, 1980). Such regulatory effects may also be the reason why in male patients with HCG-secreting tumours the testosterone levels remain within normal range (Kirschner, Wider and Ross, 1970). It may also be noteworthy that long-term treatment with.high doses of HCG has been observed to result in damage of the seminiferous tubules and
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GONADOTROPHIN RECEPTORS
inhibition of spermatogenesis both in humans (Maddock and Nelson, 1952) and in experimental animals (Cusan et al, 1982).
Gonadotrophin receptors in abnormal testicular tissue Sharpe, Wu and Hargreave (1980) measured LH receptors in testicular biopsy samples from males with spermatogenic arrest. Higher HCG binding was detected in samples with severe tubular damage, and was obviously due to the increased relative proportion of interstitial tissue in the testis samples. Therefore, although gonadotrophin receptor measurements can be useful in diagnosis of testicular pathology, it is necessary to couple the quantitative measurements with studies of testicular morphology (autoradiography). By analogy with the findings on human ovarian samples, changes in gonadotrophin receptor levels can be expected in cases of hypergonadotrophic hypogonadism, due either to lack of function of the receptors or to circulating receptor-blocking agents. Tubular failure after orchitis is a potential clinical condition where antibody formation against Sertoli cell structures could be expected, and possibly also against the gonadotrophin receptors. Detection of gonadotrophin receptors in testicular tumours could form a rationale for treatment to decrease gonadotrophin secretion in these malignancies. Finally, inherited lack of gonadotrophin receptors could be the pathophysiological reason for certain cases of male pseudohermaphroditism. Several cases fitting into this category have been presented in the literature (e.g., Perez-Placios et al, 1981). Such cases are typified by a 46XY karyotype, infantile female external genitalia, lack of secondary sex characteristics, elevated serum gonadotrophin levels, and low and HCGunresponsive gonadal androgen production. Both wolffian and mtillerian duct derivatives are absent. The testes are small and cryptorchid, and show absence of spermatogenesis and only a few poorly differentiated Leydig cells. Since HCG-stimulated testicular androgen production in utero is essential for development of the male genitalia (Grumbach and Conte, 1981) absence of LH receptors in the fetal testis could explain agenesis of the male genital structures. Lack of mtillerian duct development suggests normal production of the miillerian inhibitory factor by the Sertoli cells, which are not under LH regulation (Grumbach and Conte, 1981). Although no direct evidence for lack of LH receptor is yet available, the distinct clinical characteristics of these cases indicate the likelihood of this aetiology. It is therefore possible that certain cases of disturbances in sexual differentiation are due to lack of gonadotrophin receptor development during the critical fetal period of genital differentiation. ACKNOWLEDGEMENTS 1 am grateful to Professor Markku Sepp~iD. for useful advice during preparation of this manuscript. REFERENCES Bartke, A. (1980) Role of prolactin in reproduction in male mammals. Federation Proceedings, 39, 2577-2581.
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Bremner, W. J., Matsumoto, A. M,, Sussman, A. M. & Paulsen, C. A. (1981) Folliclestimulating hormone and human spermatogenesis. Journal of Clinical Investigation, 68, 1044-1052. Cameron, J. L. & Stouffer, R. L. (1982) Gonadotrophin receptors of the primate corpus luteum. I. Characterization of "5I-labelled human luteinizing hormone and human chorionic gonadotropin binding to luteal membranes of the rhesus monkey. Endocrinology, 110, 2059-2067. Catt, K. J. & Dufau, M. L. (1981) Hormone action: control of target-cell function by peptide, thyroid, and steroid hormones. In Endocrinology and Metabolism (Ed.) Felig, P., Baxter, J. D., Broadus, A. & Frohman, L. A. pp. 61-105. New York: McGraw Hill. Catt, K. J. & Pierce, J. G. (1978) Gonadotropic hormones of the adenohypophysis (FSH, LH and prolactin). In Reproductive Endocrinology (Ed.) Yen, S. S. C. & Jaffe, R. B. pp. 34-62. Philadelphia, London, Toronto: W. B. Saunders. Catt, K. J., Harwood, J. P., Clayton, R. N. et al (1980) Regulation of peptide hormone receptors and gonadal steroidogenesis. Recent Progress in Hormone Research, 36, 577-622. Chiauzzi, V., Cigorraga, S., Escobar, M. E. et al (1982) Inhibition of follicle-stimulating hormone receptor binding by circulating immunoglobulins. Journal of Clinical Endocrinology and Metabolism, 54, 1221-12i8. Clements, J. A., Reyes, F. I., Winter, J. S. D. & Faiman, C. (1976) Studies on human sexual development. III. Fetal pituitary and serum, and amniotic fluid concentrations of LH, CG and FSH. Journal ofClinicalEndocrinology and Metabolism, 42, 9-19. Cohen, P. (1982) The role of protein phosphorylation in neural and hormonal control of cellular activity. Nature, 296,613-620. Cole, F. E., Weed, J. C., Schneider, G. T. et al (1976) The specificity of gonadotropin binding by the human corpus luteum. Fertility and Sterility, 27, 921-928. Cusan, L., Pelletier, G., Belanger, A. et al (1982) Inhibition of spermatogenesis and steroidogenesis during long-term treatment with HCG in the rat. Journal of Andrology, 3, 124-133. Davies, T. F., Walsh, P. C., Hodgen, G. D. et al (1979) Characterization of the primate luteinizing hormone receptor in testis homogenates and Leydig cells. Journal of Clinical Endocrinology and Metabolism, 48,680-685. Davy, M., Torjesen, P. A. & Aakvaag, A. (1977) Demonstration of an FSH receptor in a functioning granulosa cell tumor. Acta Endocrinologiea, 85,615-623. Demura, R., Ono, M., Demura, H. et al (1982) Prolactin directly inhibits basal as well as gonadotropin-stimulated secretion o f progesterone and 17/3-estradiol in the human ovary. Journal of Clinical Endocrinology and Metabolism, 54, 1246-1250. Dorrington, J. & Gore-Langton, R. E. (1981) Prolactin inhibits oestrogen synthesis in the ovary. Nature, 290,600-602. Dufau, M. L. & Catt, K. J. (1978) Gonadotropin receptors and regulation of steroidogenesis in the testis and ovary. Vitamins and Hormones, 36,461-592. Forest, M. G., David, M., Lecoq, A. et al (1980) Kinetics of the HCG-induced steroidogenic response of the human testis. III. Studies in children of the plasma levels of testosterone and HCG: rationale for testicular stimulation test. Pediatric Research, 14,819-824. Franks, S., Jacobs, H. S., Martin, N. & Nabarro, J. D. N. (1978) Hyperprolactinaemia and impotence. ClinicalEndocrinology, 8,277-287. Grumbach, M. M. & Conte, F. A. (1981) Disorders of sexual differentiation. In Textbook of Endocrinology, 6th Edition (Ed.) Williams, R. H. pp. 423-514. Philadelphia, London, Toronto: W. B. Saunders. Halme, J., Ikonen, M., Rutanen, E. M. & Sepp~,l~i, M. (1978) Gonadotropin receptors of human corpus luteum during menstrual cycle and pregnancy. American Journal of Obstetrics and Gynecology, 131,728-734. Harwood, J. P., Conti, M., Corm, P. M. et al (1978) Receptor regulation and target cell responses. Studies on the ovarian luteal cell. Molecular and Cellular Endocrinology, 11, 121-135. Hsu, A.-F., Stratico, D., Hosaka, M. & Troen, P. (1978) Studies of the human testis. X. Properties of human chorionic gonadotropin receptor in adult testis and relation to intratesticular testosterone concentration. Journal of Clinical Endocrinology and Metabolism, 47, 529-536.
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Huhtaniemi, I. T. & Catt, K. J. (1981) Differential binding affinities of rat testis luteinizing hormone (LH) receptors for human chorionic gonadotropin, human LH and ovine LH, Endocrinology, 108, 1931-1938. Huhtaniemi, I. T., Clayton, R. N. & Catt, K. J. (1982) Gonadotropin binding and Leydig cell activation in the rat testis in vivo. Endocrinology, 111,982-987. Huhtaniemi, I. T., Katikineni, M. & Catt, K. J. (1981) Regulation of LH receptors and steroidogenesis in the neonatal rat testis. Endocrinology, 109, 588-595. Huhtaniemi, I. T., Korenbrot, C. C. & Jaffe, R. B. (1977) HCG binding and stimulation of testosterone biosynthesis in the human fetal testis. Journal of Clinical Endocrinology and Metabolism, 44,963-967. Huhtaniemi, I., Leinonen, P., Hammond, G. L. & Vihko, R. (1980) Effect of oestrogen treatment on testicular LH/HCG receptors and endogenous steroids in prostatic cancer patients. ClinicalEndocrinology, 13, 561-568. Huhtaniemi, 1., Bolton, N., Leinonen, P. et al (1982) Testicular luteinizing hormone receptor content and in vitro stimulation of cyclic adenosine 3',5'-monophosphate and steroid production: a comparison between man and rat. Journal of Clinical Endocrinology and Metabolism, 55,882-889. Insler, V. & Lunenfeld, B. (1978) Anovulation. Contributions to Gynecology and Obstetrics, 4, 6-77. Kauppila, A., Rajaniemi, H. & R0nnberg, L. (1982) Low LH(HCG) receptor concentration in ovarian follicles in endometriosis. Acta Obstetrica et Gynecologica Scandinavica, 61, 81-83. Kirschner, M. A., Wider, J. A. & Ross, G. T. (1970) Leydig cell function in men with gonadotrophin-producing testicular tumors. Journal of Clinical Endocrinology and Metabolism, 30, 504-51 I. Koninckx, R. & Brosens, I. A. (1977) The 'gonadotropin-resistant ovary' syndrome as a cause of secondary amenorrhea and infertility. Fertility and Sterility, 28,926-931. Lee, C. Y., Coulam, C. B., Jiang, N. S. & Ryan, R. J. (1973) Receptors for human luteinizing hormone in human corpora luteal tissue. Journal of Clinical Endocrinology and Metabolism, 36, 148-152. Luborsky, J. L. & Behrman, H. R. (1979) Antiserum against rat luteinizing hormone (LH) receptors. Biochemical and Biophysical Research Communications, 90, 1407-1413. Maddcok, W. O. & Nelson, W. O. (1952) The effects of chorionic gonadotropin in adult men: increased estrogen and 17-ketosteroid excretion, gynecomastia, Leydig cell stimulation and seminiferous tubule damage. Journal of Clinical Endocrinology and Metabolism, 12, 985-1014. Martikainen, H., Huhtaniemi, I. & Vihko, R. (1980) Response of peripheral serum sex steroids and some of their precursors to a single injection of HCG in adult men. Clinical Endocrinology, 13, 157-166. McNatty, K. P., Sawers, R. S. & McNeilly, A. S. (1974) A possible role for prolactin in control of steroid secretion by the human Graafian follicle. Nature, 250, 653-655. McNeilly, A. S., Kerin, J., Swanston,. I. A. et al (1980) Changes in the binding of human chorionic gonadotrophin/luteinizing hormone, follicle-stimulating hormone and prolactin to human corpora lutea during the menstrual cycle and pregnancy. Journal of Endocrinology, 87, 315-325. Metsikk6, K. & Rajaniemi, H. (1981) Antibodies to purified luteinizing hormone receptor localize the receptor at the luteal cell surface. Endocrinology, 109, 1399-1403. Orth, J. & Christensen, A. K. (1978) Autoradiographic localization of specifically bound ~2~i_ labeled follicle stimulating hormone on spermatogonia of the rat testis. Endocrinology, 103, 1944-1951. Perez-Palacios, G., Scaglia, H. E., Kofman-Alfaro, S. et al (1981) Inherited male pseudohermaphroditism due to gonadotrophin unresponsiveness. Acta Endocrinologica, 98, 148-155. Pointdexter, A. N., Buttram, V. C., Besch, P. K & Smith, R. G. (1979) Prolactin receptors in the ovary. Fertility and Sterility, 31,273-277. Rajaniemi, H. J., R0nnberg, L., Kauppila, A. et al (1980) Luteinizing hormone receptors in ovarian follicles of patients with polycystic ovarian disease. Journal of Clinical Endocrinology and Metabolism, 51, 1054-1057.
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Rajaniemi, H., Kauppila, A., R6nnberg, L. et al (1981a) LH(HCG) receptor in benign and malignant tumors of human ovary. Acta Obstetrica et Gynecologica Scandinavica (Supplement 101), 83-86. Rajaniemi, H. J., R0nnberg, L., Kauppila, A. et al (1981b) Luteinizing hormone receptors in human ovarian follicles and corpora lutea during menstrual cycle and pregnancy. Journal of Clinical Endocrinology and Metabolism, 52,307-313. Rao, Ch. V., Griffin, L. P. & Carman, F. R. (1977) Gonadotropin receptors in human corpora lutea of the menstrual cycle and pregnancy. American Journal of Obstetrics and Gynecology, 128, 146-153. Rillema, J. A. (1980) Mechanism of prolactin action. Federation Proceedings, 39, 2593-2598. Ross, G. T. & Schreiber, J. R. (1978) The ovary. In Reproductive Endocrinology (Ed.) Yen, S. S. C. & Jaffe, R. B. pp. 63-79. Philadelphia, ~ondon, Toronto: W. B. Saunders. Roth, J. & Grunfeld, C. (1981) Endocrine systems~~mechanisms of disease, target cells and receptors. In Textbook of Endocrinology, 6th Edition (Ed.) Williams, R. H. pp. 15-72. Philadelphia, London, Toronto: W. B. Saunders~ ::~ Saito, T. & Saxena, B. B. (1975) Specific receptors for prolactin in the ovary. Acta Endocrinologica, 80, 126-137. Sharpe, R. M. (1982) Cellular aspects of the inhibitory actions of LH-RH on the ovary and testis. Journal of Reproduction and Fertility, 64, 517-527. Sharpe, R. M., Wu, F. C. W. & Hargreave, T. B. (1980) Binding of human chorionic gonadotrophin to testicular biopsy tissue from infertile men and the effect of prior treatment with human chorionic gonadotrophin. Journal of Endocrinology, 86, 117-125'.... Shiu, R. P. C. & Friesen, H. G. (1974) Solubilization and purification of a prolaql,in:yecePtor from rabbit mammary gland. Journal of Biological Chemistry, 24, 7902-7911. Shiu, R. P. C. & Fiesen, H. G. (1976) Blockade of prolactin action by an antiserum to its receptors. Science, 192,259-261. Shiu, R. P. C. & Friesen, H. G. (1981) Regulation of prolactin receptors in target cells. In Receptors and Recognition, Series B (Ed.) Lefkowitz, R. J. Volume 13, pp. 69-81. London: Chapman & Hall. Starup, J. & Pedersen, H. (1978) Hormonal and ultrastructural observations in a case of resistant ovary syndrome. Acta Endocrinologica, 89,744-752. Wahlstr0m, T., Huhtaniemi, I., Hovatta, O. & Sepp~il~, M. (1983) Localization of luteinizing hormone, follicle stimulating hormone, prolactin, and their receptors in the human and rat testis using immunohistochemistry and radioreceptorassay. Journal of Clinical Endocrinology and Metabolism (in press). Wardlaw, S., Lauersen, N. H. & Saxena, B. B. (1975) The LH-HCG receptor of human ovary at various stages of the menstrual cycle. Acta Endocrino[ogica, 79, 568-576.
The ovary and testis are under trophic control of three pituitary hormones: luteinizing hormone (LH), follicle-stimulating hormone (FSH) and prolactin (PRL). LH and FSH are conventionally regarded as gonadotrophins, but also PRL, due to its known direct gonadal actions, can be included into this category. The purpose of this chapter is to discuss the characteristics, functions and regulation of the gonadal receptors for these three pituitary hormones, with special emphasis on their role in the human during normal and pathological gonadal function. CHARACTERISTICS OF GONADOTROPHIN RECEPTORS Physicochemical and biochemical features Gonadotrophin action is initiated by binding of LH, FSH and PRL to highaffinity receptors on the outer surface of the plasma membrane of their target cells in the ovary and testis. The majority of our knowledge about these receptors is still derived from animal experiments. The rat testicular and ovarian LH receptors have been found to be asymmetrical glycoprotein molecules with molecular weights of about 200000, each molecule consisting of two 90 000 molecular weight subunits (for a review see Dufau and Catt, 1978). The solubilized receptors lose their binding activity after exposure to trypsin, indicating that protein forms a major component of the binding site (Dufau and Catt, 1978). Phospholipids and carbohydrates also compose integral parts of the LH-binding site. Similar properties have been described for purified preparations of rodent FSH and PRL receptors (Shiu and Friesen, 1974; Dufau and Catt, 1978). One line of receptor research attempts to produce purified receptor protein for production of receptor antibodies. These could provide a powerful tool for further studies on the receptor structure and function, and in general on the mechanisms of gonadotrophin action. Antisera have been generated against the rat ovarian LH receptor (Luborsky and Behrman, 1979; MetsikkO and Rajaniemi, 1981) and the rabbit mammary gland PRL receptor (Shiu and Friesen, 1976). Clinics in E n d o c r i n o l o g y a n d M e t a b o l i s m - - Vol. 12, No. 1, March 1983.
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Hormone--receptor interaction The interaction of gonadotrophins with their receptors is characterized by high affinity (equilibrium association constant, K a, of 109 to I0 ~ M), low capacity (number of receptors in thousands per cell), and high specificity (Dufau and Cart, 1978). One gonadotrophin has practically no crossreactivity with the receptor of another gonadotrophin. LH and HCG bind to the same receptor molecules, and HCG can be considered as a 'superagonist' to LH due to its longer half-time in the circulation and slower dissociation from the LH receptor (Huhtaniemi and Cart, 1981). Localization of the receptors Receptors for LH and FSH are only found in the gonads, whereas specific PRL binding is ubiquitously distributed throughout the body, in organs including the gonads, mammary gland, liver, kidneys, prostate and adrenals (Shiu and Friesen, 1981). LH and FSH receptors have been detected exclusively on the plasma membrane of the target cells, using autoradiographic techniques, ferritin-labelled HCG, immunofluorescence and receptor antibodies (Cart et al, 1980; Metsikk0 and Rajaniemi, 1981). However, a small fraction of the receptors is seen inside the target ceils after hormonal stimulation (i.e., internalized), and is assumed to be a part of processing and elimination of the hormone--receptor complexes (Catt and Dufau, 1981). The intracellular localization of receptors is especially clear with PRL, and it may play a role in the mechanism of action of this hormone (Rillema, 1980). MECHANISM OF GONADOTROPHIN ACTION Functional coupling of receptor binding and cellular responses The general mechanisms of membrane-receptor-mediated hormone action are described elsewhere. In brief, transfer of the LH and FSH message inside the target cell is performed at the plasma membrane level by a mediator molecule (i.e., 'second messenger'), cyclic adenosine monophosphate (cAMP). The gonadotrophin receptor is thought to float freely in the liquid matrix of the cell membrane. Upon hormone binding it is believed to undergo some conformational change that allows it to activate the adenylate cyclase enzyme floating in the inner surface of the plasma membrane. Another plasma membrane protein, the so-called nucleotide regulatory protein, is needed for activation of adenylate cyclase. Gonadotrophin stimulation in fact stimulates binding of guanosine triphosphate (GTP) to this regulatory protein, which is thereafter capable of activating adenylate cyclase. Activation of adenylate cyclase then catalyses the conversion of intracellular ATP to cAMP, the second messenger, which binds to the regulatory subunit of the protein kinase enzyme, and a catalytic subunit of protein kinase is cleaved off. The latter, in the presence of ATP, can finally catalyse a number of protein phosphorylations (Cohen, 1982). The phosphorylations regulate the activity of a number of targe t cell enzymatic and structural proteins, and the above changes are essentially those which take place within minutes during the rapid responses of gonadal
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cell functions to LH and FSH stimulation (enhanced steroidogenesis, carbohydrate metabolism and ion transport). The other part of gonadotrophin effect is slower, and leads to late responses in RNA and protein synthesis, cell growth, differentiation and division, again through phosphorylation of different proteins. It is not known in detail how the similar cAMP molecules can activate the differential fast and slow responses in the same cells, but distribution of cAMP into different intracellular pools may be the explanation. The mechanism whereby PRL mediates its actions within the cell is still unknown. It has been suggested that the internalization of PRL would play a role in this process. Subsequently, PRL action may involve changes in Na +,K +-ATPase activity, increase in cGMP and decrease in cAMP levels, and enhanced rates of prostaglandin and polyamine synthesis (Rillema, 1980).
Quantitative aspects of hormone binding and cellular response An important feature in gonadotrophin action is that only a minute fraction of the receptors needs to be occupied to obtain maximal biological response. This is especially clear with LH action, since occupancy of less than one per cent of testicular receptors is sufficient to result in maximal testosterone production. The rest of the receptors, termed 'spare' receptors, are assumed to favour formation of hormone--receptor complexes in the face of low ligand concentrations, and to provide a receptor reserve that assures availability of free receptors for continuous gonadotrophic stimulation (Dufau and Catt, 1978; Huhtaniemi, Clayton and Catt, 1982). HORMONAL REGULATION OF GONADOTROPHIN RECEPTORS Homologous receptor regulation As will be described below, the maintenance of gonadotrophin receptors is dependent on circulating levels of the hormone itself and in many cases also on other pituitary hormones. Numerous recent studies have demonstrated another kind of gonadotrophin effect on their own (homologous) receptors: stimulation with L H / H C G leads to a rapid and severe decrease in gonadal LH receptor levels. This phenomenon is termed 'receptor down-regulation' (Catt et al, 1980). Since the effect is only produced by high hormone concentrations, its physiological role is still controversial. It may play an important role in protection of the gonads from excessive hormonal stimulation. Down-regulation could also represent amplification of mechanisms taking place in a single cell (or in a specific location of its plasma membrane) during physiological stimulation at low gonadotrophin concentrations. Clearcut down-regulation effects have not been produced during FSH and PRL stimulation, and details about the' mode of autoregulation of their receptors are not yet clear. Heterologous receptor regulation Another type of down-regulation is seen with heterologous receptor systems. LH stimulation of the ovaries and testes results, in addition to LH
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receptor loss, in concomitant loss of PRL binding. The mechanism of this response may be coaggregation and cointernalization of PRL receptors together with LH receptors upon LH receptor occupancy. In addition to down-regulatory receptor losses, gonadotrophin stimulation is accompanied by loss of target cell metabolic responses -- in gonads by decrease of cAMP production and steroidogenesis (Catt et al, 1980) -- which may be another facet in the mechanisms of protection of gonadotrophin target cells from too-high trophic stimulation. GONADOTROPHIN TARGET CELLS AND EFFECTS IN THE GONADS -- ANIMAL STUDIES The ovary
Table 1 lists the ovarian and testicular target cells for LH, FSH and PRL (i.e., cells that have receptors for these three hormones). The role of gonadotrophins in the ovarian function is, in simplified terms, as follows (Catt and Pierce, 1978; Ross and Schreiber, 1978). Granulosa cells are the target of FSH action in the ovary. Together with oestrogen, FSH induces proliferation and maturation of granulosa cells. Early follicles are practically devoid of LH and PRL receptors, but they appear as a result of FSH (and oestrogen) action on granulosa cells as the follicle matures. A reciprocal decrease of FSH binding occurs simultaneously. FSH also stimulates steroidogenesis (mainly oestrogen production) by the granulosa cells. Progesterone production appears concomitantly with the development of granulosa cell LH receptors. It seems that androgen, an obligatory intermediate in granulosa cell oestrogen formation, is produced by the theca cells in response to LH stimulation. Androgen thus produced is transferred to the granulosa cells where it is aromatized under the influence of FSH. Since oestrogen stimulates follicular growth, and androgen is inhibitory, balance between these two hormones determines the fate of the developing follicle (further growth or atresia). The FSH action seems to cease after ovulation, when LH receptor level of the luteal cells reaches a high concentration. Table 1. Presence o f LH, F S H and P R L receptors in the different cellular compartments o f the ovary and testis; the results are based on observations in experimental animals Receptor
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The midcycle surge of LH is clearly essential for ovulation, but the exact role of this hormone has not been clarified in the context of the large variety of biochemical and morphological changes taking place in the ovary around ovulation. After ovulation, LH is responsible for stimulation of luteal cell progesterone production, and, if pregnancy is initiated, HCG is the trophic stimulus for progesterone production by the corpus luteum in pregnancy. PRL receptor changes seem to accompany those of the LH receptor, and PRL is known to have synergistic actions with LH in the maintenance of LH receptors and LH-stimulated steroidogenesis. Paradoxically, PRL has been shown to have both luteotrophic and luteolytic effects. The former includes a permissive effect on steroidogenesis by maintenance of LH receptors and modulation of storage and turnover of luteal cell cholesterol. High PRL levels, in contrast, have been shown to be inhibitory to FSHstimulated oestrogen production in the granulosa cell (Dorrington and Gore-Langton, 1981) and LH-stimulated progesterone production in the luteal cell (McNatty, Sawers and McNeilly, 1974). The testis
In the testis, the Sertoli cells of the seminiferous tubules are the best-known target of FSH action (Catt and Pierce, 1978; Dufau and Catt, 1978). This hormone is known to stimulate production of numerous specific proteins, some of which may be essential for sperm maturation, in Sertoli cells. The best characterized of these substances is the secretory protein 'androgenbinding protein' (ABP), known to accumulate testosterone in the tubular compartment, which is essential for normal spermatogenesis. In addition, autoradiographic studies have shown specific binding of FSH to spermatogonia of the rat testis (Orth and Christensen, 1978), but the physiological significance of this finding is still unknown. Most testicular FSH effects have been shown only in immature or hypophysectomized animals, and the role of FSH in adult testicular function, especially in humans, is therefore still somewhat unclear (Bremner et al, 1981). LH and PRL receptors are confined to the Leydig cells (Dufau and Cart, 1978). LH receptors are needed for stimulation of Leydig cell steroidogenesis, growth and differentiation. As in the ovary, PRL seems to have a synergistic action with LH on the maintenance and induction of LH receptors (Catt et al, 1980) and has also been suggested to promote steroidogenesis by regulating the supply of intratesticular cholesterol esters (Bartke, 1980). Dependence of LH receptor induction on FSH in the testis has also been proposed (Catt et al, 1980). This kind of induction is, however, difficult to explain, since testicular LH and FSH receptors are present in two different cell types (i.e., Leydig and Sertoli cells). Thus, a direct effect at least of FSH on Leydig cells seem unlikely, but a Sertoli cell-mediated mechanism is possible. Communication between testicular compartments has been discussed recently on the basis of the finding that Sertoli cells produce a gonadotrophin-releasing hormoneqike substance which then regulates gonadotrophin actions on Leydig cells (Sharpe, 1982).
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LH receptors during the normal menstrual cycle
Human ovarian LH binding has been measured at different stages of the menstrual cycle in a number of studies (see, for example, Lee et al, 1973; Wardlaw, Lauersen and Saxena, 1975; Cole et al, 1976; Rajaniemi et al, 1981b) (Figure 1). Basically, the receptors have been found to have similar characteristics to those of rodents. The binding has high affinity, low capacity, and is highest in ovarian luteal ceils (Rajaniemi et al, 1981b). Incubation conditions, molecular weight of the receptor, and effects of enzymatic treatments on binding are similar to those in the experimental animals. A remarkable difference is the species specificity of LH receptors of the human and monkey ovary (Cole et al, 1976; Cameron and Stouffer,
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DAY OF THE MENSTRUAL CYCLE Figure 1. Variation in the number of LH receptors in the h u m a n ovary during the menstrual cycle. The bottom panel shows LH receptor concentration in follicles and corpora lutea during one cycle. Receptors in old corpora lutea are seen in the middle panel, and those of follicles undergoing atresia in the top panel. From Rajaniemi et al (1981b) Journal of Clinical Endocrinology andMetabolisrn, 52, 307-313, with kind permission.
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1982).. These seem to bind only human luteinizing hormones (HLH and HCG), and LH preparations of lower species (e.g., ovine and bovine) have very low affinity. Rat, ovine and bovine LH receptors, by contrast, bind all these hormone preparations. This species specificity, as well as development of neutralizing antibodies to the heterologous hormone, explains the necessity of human gonadotrophin preparations in treatment of humans. There is clear evidence that LH and HCG bind to the same receptor molecules with similar affinity (Wardlaw,~ Lauersen and Saxena, 1975; Cameron and Stouffer, 1982). Compared with that of the rat, the binding capacity of the human and primate corpus luteum (per mg protein) is clearly lower (Dufau and Catt, 1978; Rajaniemi et al, 1981b; Cameron and Stouffer, 1982)~ Some LH binding is also present in the granulosa cells (Rajaniemi et al, 1981b), but highest concentration of LH receptors is found in luteal cells during the midluteal phase (McNeilly et al, 1980; Rajaniemi et al, 1981b) (Figure]). A drop in binding occurs immediately after ovulation, possibly becaus~ of receptor occupancy or down-regulation, induced by the ovulatory LH surge (Rajaniemi et al, 1981b). The number of LH receptors during the latter part of the cycle decreases (Wardlaw, Lauersen and Saxena, 1975; McNeilly et al, 1980), but some residual LH binding is still present in old corpora lutea during the next cycle (Rajaniemi et al, 1981b). The decrease of LH receptors towards the end of luteal phase has no close correlation with the fall in progesterone levels, which suggests that LH receptor loss may not be of primary importance in the involution of the corpus luteum. McNeilly et al (1980) have suggested that, as LH stimulation and progesterone production decrease during late luteal phase, the corpus luteum becomes subject to luteolytic effects of prostaglandin F2,~. However, if high gonadotrophin levels and progesterone production are maintained, as is the case if pregnancy ensues, the corpus luteum is protected from the luteolytic effects of prostaglandin. Luteal cells of the corpus luteum of pregnancy have very low LH binding, which possibly is due to occupancy of receptors by HCG, or to receptor down-regulation (Rao et al, 1977; Halme et al, 1978; Rajaniemi et al, 1981b). The latter mechanism, together with desensitization of cellular steroidogenic responses, may be responsible for cessation of hormone production in the corpus luteum during pregnancy by about seven to eight weeks of gestation, when HCG reaches very high levels in the maternal circulation. No LH receptors have been found in the ovarian stroma, but it remains to be established whether receptors are present in the theca cells. Such is the case in the rat, where the receptors have a distinct role in regulation of theca cell androgen production. LH receptors in pathophysiological conditions Little is known about levels of gonadotrophin receptors in pathophysiological conditions. Low follicular LH receptor levels have been reported in endometriosis and in polycystic ovarian disease (Rajaniemi et al, 1980; Kauppila, Rajaniemi and R0nnberg, 1982), but whether the low binding has
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a causal relationship to the pathogenesis of these conditions, or is secondary to other hormonal changes, is not known. LH receptors have also been detected in some benign and malignant ovarian tumours (Wardlaw, Lauersen and Saxena, 1975; Rajaniemi et al, 1981a), a finding of potential clinical importance since growth and/or function of some ovarian tumours may be dependent on gonadotrophins. Accordingly, measurement of gonadotrophin receptors in ovarian tumour tissue could be used as a diagnostic tool to assess benefits of treatment to eliminate pituitary gonadotrophin secretion (hypophysectomy or gonadotrophin-releasing hormone (GnRH)-agonist analogues). FSH and PRL receptors McNeilly et al (1980) have detected specific FSH binding in human corpora lutea, a surprising finding in view of the animal data that indicate a specific role for FSH in follicular but not in luteal function (see above). Also, specific PRL binding has been detected in the human ovary (Saito and Saxena, 1975; Pointdexter et al, 1979; McNeilly et al, 1980), but studies on human FSH and PRL receptors are still too preliminary to allow conclusions about their behaviour during the normal menstrual cycle or disturbances of ovarian function. There is a report of FSH receptors in an ovarian granulosa cell tumour (Davy, Torjesen and Aakvaag, 1977), which further supports the case for gonadotrophin dependence of certain ovarian neoplasms. As for PRL, the evidence for direct ovarian actions in humans is mainly based on demonstrated inhibitory effects of PRL on ovarian steroidogenesis in vitro (McNatty, Sawers and McNatty, 1974; Demura et al, 1982). Gonadotrophin receptor antibodies An intriguing pathological condition with possible gonadotrophin receptor involvement is the 'gonadotrophin-resistant ovary' syndrome (Koninckx and Brosens, 1977; Starup and Pedersen, 1978). It is characterized by primary or secondary amenorrhoea, normal development of secondary sexual characteristics, hypergonadotrophism, presence of morphologically normal follicles in the ovary, and resistance of the ovary to high gonadotrophin stimulation. Theoretically, several pathophysiological mechanisms could lead to this condition. These include abnormal or biologically inactive circulating gonadotrophin, circulating gonadotrophin antibodies, and lack of end-organ response to gonadotrophin stimulation. The occurrence of circulating gonadotrophin receptor antibodies has been discussed for several years, but evidence for such a pathogenesis did not exist until recently. Chiauzzi et al (1982) detected FSH receptor antibodies in the serum of myasthenia gravis patients, who also had hypergonadotrophic ovarian failure. The circulating serum component was immunoglobulin by biochemical and physiochemical criteria, and it inhibited FSH binding to rat testis receptors, as well as FSH-stimulated cAMP and ABP production in rat testis tubules. This finding reveals a new category of autoimmune reproductive organ disease, analogous to conditions with detectable auto-
GONADOTROPHIN RECEPTORS
125
antibodies against receptors of insulin (type B insulin resistance with acanthosis nigricans), TSH (Graves' disease) and acetylcholine (myasthenia gravis) (Roth and Grunfeld, 1981) (see Chapters 11, 5 and 4 respectively). Clinical aspects in gonadotrophin treatment Gonadotrophin therapy is used in the induction of ovulation in patients with gonadal failure due to hypothalamic or pituitary failure (Insler and Lunenfeld, 1978). To induce follicular maturation, the treatment consists of daily injections of human menopausal gonadotrophin (HMG), which is a combination of FSH and LH, during the follicular phase. When sufficient follicular maturation is achieved (as assessed by oestrogen measurements and ultrasound study of the follicular size) ovulation is triggered by an injection of HCG either as a single or as multiple injections. Two major complications may occur with the treatment, that is, ovarian hyperstimulation and multiple pregnancies. Although the treatment attempts to mimic physiological gonadotrophin secretion, some differences from the physiological conditions are evident and may explain difficulties encountered with it. Gonadotrophins are secreted from the pituitary in a pulsatile fashion with a burst every 60 to 90 minutes in response to pulses of the hypothalamic gonadotrophin-releasing hormone. This pulsatile secretion cannot be mimicked by the currently used methods of intramuscular hormone administration of gonadotrophins. Frequent pulses of gonadotrophin secretion are likely to be important for normal follicular maturation. Tonically elevated hormone levels could lead to ovarian overstimulation. This is not infrequent, and therefore the HMG treatment must be individually adjusted in each case. Ovulation is induced by HCG, which in the commonly used doses (3000 to 10000 IU i.m.) may result in exaggerated and prolonged LH activity in the circulation. Due to the long half-life of HCG in the circulation, biologically active levels of HCG can be found in peripheral serum for four to five days after a single injection of 5000 IU (Martikainen, Huhtaniemi and Vihko, 1980). This prolonged period of high LH stimulation may partly explain occurrence of multiple ovulations. It is even possible that the persistent high levels of HCG, if given too early, may induce luteinization of unruptured follicles or, if ovulation is achieved, down-regulation of LH receptors and steroidogenic lesions in the corpus luteum. Therefore, there is an obvious need for clinical treatments with LH preparations of shorter duration of action than HCG (possibly desialylated HCG). Treatment with HMG and HCG has undoubtedly provided a valuable method for ovulation induction under carefully controlled conditions. Our present knowledge about physiological gonadotrophin secretion and mechanisms of action can, however, explain the narrow therapeutic range of treatment and provides a rationale for avoiding some of the complications. In cases of anovulation due to hypothalamic failure, pulsatile treatment with GnRH has been shown to offer a more physiological means of treatment, with minimal risk of ovarian hyperstimulation or multiple pregnancies (see Chapter 10).
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GONADOTROPHIN
R E C E P T O R S IN T H E H U M A N TESTIS
LH receptors and L H / H C G action in the human testis L H / H C G binding has been measured in both the fetal (Huhtaniemi, Korenbrot and Jaffe, 1977) and adult (Hsu et al, 1978; Davies et al, 1979; Sharpe, Wu and Hargreave, 1980; Huhtaniemi et al, 1980, 1982) h u m a n testis (Figure 2) and, as in experimental animals, the receptors are confined to the Leydig cells. The K a of H C G binding to the receptors is similar to that observed in the rat testis (about 1 x 101° M-l), but a clear quantitative difference is seen between the two species; the number of L H receptors per Leydig cell is about 1500 in the human, and about 20000 in the rat (Huhtaniemi et al, 1982). A similar species specificity to that in the ovary is seen with testicular L H receptors; the h u m a n receptors bind effectively only primate L H preparations (Davies et al, 1979). LH
FSH .25
.6 k
z~ Ka_1.25x10~°M 4 A K~ - 0 . 4 lx 101°M-1.20
kk
.4 ~
k
PRL
.20 e, K,~ - 2.8 x 109 M q K~,a.0.65x 108 M "1 • K ~ - 1.5x 109M 4 K.2-0.47x10 s M 4 .15
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.15 .10
/
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.2
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.4
.6
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Figure 2. Scatchard plots of HCG (LH) (left), FSH (middle) and PRL (right) binding to homogenates prepared from the same amount of human (A) and rat (A) testis tissue. The plots highlight the low amount of LH and high-affinity FSH receptors in the human testis compared with the rat, and the absenceof measurable PRL:binding in the human testis. From WahlstrOm et al (1983) Journal of Clinical Endocrinology and Metabolism (in press), with kind permission. L H receptors appear in the testis between the first and second trimester of pregnancy, and are then responsive to stimulation by the high circulating H C G level (Huhtaniemi, Korenbrot and Jaffe, 1977). This fetal H C G stimulated testosterone production is known to be responsible for male fetal genital masculinization (Grumbach and Conte, 1981). An intriguing feature of the fetal testicular activity is that a considerable amount of L H receptors and active androgen production occur simultaneously with the peak of H C G concentrations in the maternal and fetal circulation. In fetal circulation H C G reaches levels as high as 1 to 10 nmol/1 (Clements et al,
GONADOTROPHINRECEPTORS
127
1976), which in the adult should result in LH receptor down-regulation and desensitization of androgen production. Our recent animal studies have indicated that these negative effects of gonadotrophin treatment are not present in the fetus or neonate, but are a result of pubertal sexual development (Huhtaniemi, Katikineni and Cart, 198 l). Besides the low LH receptor level of human testes, another clear difference exists in the LH-stimulated steroidogenic response between the human and rat (Huhtaniemi et al, 1982). Gonadotrophin stimulation is known to induce two responses in the testicular androgen production, a fast one (within hours) and a slow one (several days after hormone injection). The first response is very prominent in the rat with a more than 10-fold increase of circulating testosterone level. In the human, the acute response is hardly detectable, with about a 50 per cent increase in serum testosterone (Forest et al, 1980; Martikainen, Huhtaniemi and Vihko, 1980) (Figure 3).
300
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o~ 100
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G 1 7-Hydroxyprogesterone
/k Oestradiol
06241 Hours
)
;
;
;
Days Time After hCG Injection
Figure 3. Peripheral serum levels of testosterone, 17-hydroxyprogesteroneand oestradiol in human males after an injection of 80 IU/kg HCG. The values are expressed as percentage of the control levels, which were 19.9 nmol/1 for testosterone, 2.6 nmol/l for 17-hydroxyprogesterone, and 0.10 nmol/I for oestradiol. The data are modified from those presented by Martikainen, Huhtaniemi and Vihko (1980). At around 24 to 36 hours, an increase is seen in serum levels of 17-hydroxyprogesterone and oestradiol. The oestrogen response is a specific Leydig cell response to gonadotrophin stimulation, and is known to be involved in the refractoriness of androgen synthesis due to blockade of C21 steroid side chain cleavage. Concomitant accumulation of 17-hydroxyprogesterone with the oestrogen peak is a sign of this steroidogenic blockade. The second, two-to three-fold response occurs after two to four days when the phase of steroidogenic desensitization is over. The late androgen responses are of similar magnitude and duration in the human and rat. Although
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I.T.-HUHTANIEMI
theoretically possible, the poor acute response of the human testis is probably not due to the low LH receptor level, since the HCG-stimulated cAMP response of the human testis is comparable to those observed in the rat testis in vitro (Huhtaniemi et al, 1982). The reason for the poor steroidogenie response has been localized to a metabolic step beyond cAMP formation but before mitochondrial formation of pregnenolone, being probably due to defective supply of cholesterol for the mitochondrial steroidogenesis. It is possible that the basal circulating LH level is already stimulating near-maximal steroidogenesis, and further exogenous stimulation by the injected HCG is without effect. Whatever the reason, gonadotrophin stimulation of the human testis is almost devoid of an immediate steroidogenic response, and marked stimulation of testosterone production does not occur until two to four days later.
FSH and PRL receptors FSH receptors have been detected in the fetal (Huhtaniemi and Jaffe, unpublished observation 1982) and adult (WahlstrOm et al, 1983) human testis, but no specific PRL binding has so far been found in it. This negative finding may be due to suboptimal analytical techniques. On the other hand, there is no unequivocal evidence that PRL in the human male would have direct testicular effects. Hyperprolactinaemia has been clinically related to azoospermia, impotence and low serum testosterone (Franks et al, 1978), but the findings may be related to direct effects of PRL on pituitary gonadotrophin secretion (Bartke, 1980). Clinical aspects in gonadotrophin treatment Chorionic gonadotrophin is used in the treatment of cryptorchidism and hypogonadotrophic hypogonadism, and diagnostically to detect presence of functional testis tissue in cases of genital ambiguity. Formerly, the treatment or diagnostic tests included daily injections of HCG for several days, after which the rise in serum testosterone was monitored. We know now that an injection of HCG (about 100 IU/kg) results in biologically effective serum HCG concentration and LH receptor occupancy for about four days, since the half-life of HCG in the circulation and at the receptor site is about 24 hours (Martikainen, Huhtaniemi and Vihko, 1980). Furthermore, since the injection results in maximal steroidogenic response in 72 to 96 hours (Figure 3), HCG injections more frequently than at three-day intervals are pointless (Forest et al, 1980; Martikainen, Huhtaniemi and Vihko, 1980). For treatment of cryptorchidism or hypogonadism, an HCG injection one or two times a week is theoretically sufficient. More frequent treatment could even be harmful, due to the possibility of LH receptor down-regulation (Sharpe, Wu and Hargreave, 1980) and inhibition of enzymes involved in androgen biosynthesis (steroidogenic desensitization) (Catt et al, 1980). Such regulatory effects may also be the reason why in male patients with HCG-secreting tumours the testosterone levels remain within normal range (Kirschner, Wider and Ross, 1970). It may also be noteworthy that long-term treatment with.high doses of HCG has been observed to result in damage of the seminiferous tubules and
129
GONADOTROPHIN RECEPTORS
inhibition of spermatogenesis both in humans (Maddock and Nelson, 1952) and in experimental animals (Cusan et al, 1982).
Gonadotrophin receptors in abnormal testicular tissue Sharpe, Wu and Hargreave (1980) measured LH receptors in testicular biopsy samples from males with spermatogenic arrest. Higher HCG binding was detected in samples with severe tubular damage, and was obviously due to the increased relative proportion of interstitial tissue in the testis samples. Therefore, although gonadotrophin receptor measurements can be useful in diagnosis of testicular pathology, it is necessary to couple the quantitative measurements with studies of testicular morphology (autoradiography). By analogy with the findings on human ovarian samples, changes in gonadotrophin receptor levels can be expected in cases of hypergonadotrophic hypogonadism, due either to lack of function of the receptors or to circulating receptor-blocking agents. Tubular failure after orchitis is a potential clinical condition where antibody formation against Sertoli cell structures could be expected, and possibly also against the gonadotrophin receptors. Detection of gonadotrophin receptors in testicular tumours could form a rationale for treatment to decrease gonadotrophin secretion in these malignancies. Finally, inherited lack of gonadotrophin receptors could be the pathophysiological reason for certain cases of male pseudohermaphroditism. Several cases fitting into this category have been presented in the literature (e.g., Perez-Placios et al, 1981). Such cases are typified by a 46XY karyotype, infantile female external genitalia, lack of secondary sex characteristics, elevated serum gonadotrophin levels, and low and HCGunresponsive gonadal androgen production. Both wolffian and mtillerian duct derivatives are absent. The testes are small and cryptorchid, and show absence of spermatogenesis and only a few poorly differentiated Leydig cells. Since HCG-stimulated testicular androgen production in utero is essential for development of the male genitalia (Grumbach and Conte, 1981) absence of LH receptors in the fetal testis could explain agenesis of the male genital structures. Lack of mtillerian duct development suggests normal production of the miillerian inhibitory factor by the Sertoli cells, which are not under LH regulation (Grumbach and Conte, 1981). Although no direct evidence for lack of LH receptor is yet available, the distinct clinical characteristics of these cases indicate the likelihood of this aetiology. It is therefore possible that certain cases of disturbances in sexual differentiation are due to lack of gonadotrophin receptor development during the critical fetal period of genital differentiation. ACKNOWLEDGEMENTS 1 am grateful to Professor Markku Sepp~iD. for useful advice during preparation of this manuscript. REFERENCES Bartke, A. (1980) Role of prolactin in reproduction in male mammals. Federation Proceedings, 39, 2577-2581.
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Bremner, W. J., Matsumoto, A. M,, Sussman, A. M. & Paulsen, C. A. (1981) Folliclestimulating hormone and human spermatogenesis. Journal of Clinical Investigation, 68, 1044-1052. Cameron, J. L. & Stouffer, R. L. (1982) Gonadotrophin receptors of the primate corpus luteum. I. Characterization of "5I-labelled human luteinizing hormone and human chorionic gonadotropin binding to luteal membranes of the rhesus monkey. Endocrinology, 110, 2059-2067. Catt, K. J. & Dufau, M. L. (1981) Hormone action: control of target-cell function by peptide, thyroid, and steroid hormones. In Endocrinology and Metabolism (Ed.) Felig, P., Baxter, J. D., Broadus, A. & Frohman, L. A. pp. 61-105. New York: McGraw Hill. Catt, K. J. & Pierce, J. G. (1978) Gonadotropic hormones of the adenohypophysis (FSH, LH and prolactin). In Reproductive Endocrinology (Ed.) Yen, S. S. C. & Jaffe, R. B. pp. 34-62. Philadelphia, London, Toronto: W. B. Saunders. Catt, K. J., Harwood, J. P., Clayton, R. N. et al (1980) Regulation of peptide hormone receptors and gonadal steroidogenesis. Recent Progress in Hormone Research, 36, 577-622. Chiauzzi, V., Cigorraga, S., Escobar, M. E. et al (1982) Inhibition of follicle-stimulating hormone receptor binding by circulating immunoglobulins. Journal of Clinical Endocrinology and Metabolism, 54, 1221-12i8. Clements, J. A., Reyes, F. I., Winter, J. S. D. & Faiman, C. (1976) Studies on human sexual development. III. Fetal pituitary and serum, and amniotic fluid concentrations of LH, CG and FSH. Journal ofClinicalEndocrinology and Metabolism, 42, 9-19. Cohen, P. (1982) The role of protein phosphorylation in neural and hormonal control of cellular activity. Nature, 296,613-620. Cole, F. E., Weed, J. C., Schneider, G. T. et al (1976) The specificity of gonadotropin binding by the human corpus luteum. Fertility and Sterility, 27, 921-928. Cusan, L., Pelletier, G., Belanger, A. et al (1982) Inhibition of spermatogenesis and steroidogenesis during long-term treatment with HCG in the rat. Journal of Andrology, 3, 124-133. Davies, T. F., Walsh, P. C., Hodgen, G. D. et al (1979) Characterization of the primate luteinizing hormone receptor in testis homogenates and Leydig cells. Journal of Clinical Endocrinology and Metabolism, 48,680-685. Davy, M., Torjesen, P. A. & Aakvaag, A. (1977) Demonstration of an FSH receptor in a functioning granulosa cell tumor. Acta Endocrinologiea, 85,615-623. Demura, R., Ono, M., Demura, H. et al (1982) Prolactin directly inhibits basal as well as gonadotropin-stimulated secretion o f progesterone and 17/3-estradiol in the human ovary. Journal of Clinical Endocrinology and Metabolism, 54, 1246-1250. Dorrington, J. & Gore-Langton, R. E. (1981) Prolactin inhibits oestrogen synthesis in the ovary. Nature, 290,600-602. Dufau, M. L. & Catt, K. J. (1978) Gonadotropin receptors and regulation of steroidogenesis in the testis and ovary. Vitamins and Hormones, 36,461-592. Forest, M. G., David, M., Lecoq, A. et al (1980) Kinetics of the HCG-induced steroidogenic response of the human testis. III. Studies in children of the plasma levels of testosterone and HCG: rationale for testicular stimulation test. Pediatric Research, 14,819-824. Franks, S., Jacobs, H. S., Martin, N. & Nabarro, J. D. N. (1978) Hyperprolactinaemia and impotence. ClinicalEndocrinology, 8,277-287. Grumbach, M. M. & Conte, F. A. (1981) Disorders of sexual differentiation. In Textbook of Endocrinology, 6th Edition (Ed.) Williams, R. H. pp. 423-514. Philadelphia, London, Toronto: W. B. Saunders. Halme, J., Ikonen, M., Rutanen, E. M. & Sepp~,l~i, M. (1978) Gonadotropin receptors of human corpus luteum during menstrual cycle and pregnancy. American Journal of Obstetrics and Gynecology, 131,728-734. Harwood, J. P., Conti, M., Corm, P. M. et al (1978) Receptor regulation and target cell responses. Studies on the ovarian luteal cell. Molecular and Cellular Endocrinology, 11, 121-135. Hsu, A.-F., Stratico, D., Hosaka, M. & Troen, P. (1978) Studies of the human testis. X. Properties of human chorionic gonadotropin receptor in adult testis and relation to intratesticular testosterone concentration. Journal of Clinical Endocrinology and Metabolism, 47, 529-536.
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Huhtaniemi, I. T. & Catt, K. J. (1981) Differential binding affinities of rat testis luteinizing hormone (LH) receptors for human chorionic gonadotropin, human LH and ovine LH, Endocrinology, 108, 1931-1938. Huhtaniemi, I. T., Clayton, R. N. & Catt, K. J. (1982) Gonadotropin binding and Leydig cell activation in the rat testis in vivo. Endocrinology, 111,982-987. Huhtaniemi, I. T., Katikineni, M. & Catt, K. J. (1981) Regulation of LH receptors and steroidogenesis in the neonatal rat testis. Endocrinology, 109, 588-595. Huhtaniemi, I. T., Korenbrot, C. C. & Jaffe, R. B. (1977) HCG binding and stimulation of testosterone biosynthesis in the human fetal testis. Journal of Clinical Endocrinology and Metabolism, 44,963-967. Huhtaniemi, I., Leinonen, P., Hammond, G. L. & Vihko, R. (1980) Effect of oestrogen treatment on testicular LH/HCG receptors and endogenous steroids in prostatic cancer patients. ClinicalEndocrinology, 13, 561-568. Huhtaniemi, 1., Bolton, N., Leinonen, P. et al (1982) Testicular luteinizing hormone receptor content and in vitro stimulation of cyclic adenosine 3',5'-monophosphate and steroid production: a comparison between man and rat. Journal of Clinical Endocrinology and Metabolism, 55,882-889. Insler, V. & Lunenfeld, B. (1978) Anovulation. Contributions to Gynecology and Obstetrics, 4, 6-77. Kauppila, A., Rajaniemi, H. & R0nnberg, L. (1982) Low LH(HCG) receptor concentration in ovarian follicles in endometriosis. Acta Obstetrica et Gynecologica Scandinavica, 61, 81-83. Kirschner, M. A., Wider, J. A. & Ross, G. T. (1970) Leydig cell function in men with gonadotrophin-producing testicular tumors. Journal of Clinical Endocrinology and Metabolism, 30, 504-51 I. Koninckx, R. & Brosens, I. A. (1977) The 'gonadotropin-resistant ovary' syndrome as a cause of secondary amenorrhea and infertility. Fertility and Sterility, 28,926-931. Lee, C. Y., Coulam, C. B., Jiang, N. S. & Ryan, R. J. (1973) Receptors for human luteinizing hormone in human corpora luteal tissue. Journal of Clinical Endocrinology and Metabolism, 36, 148-152. Luborsky, J. L. & Behrman, H. R. (1979) Antiserum against rat luteinizing hormone (LH) receptors. Biochemical and Biophysical Research Communications, 90, 1407-1413. Maddcok, W. O. & Nelson, W. O. (1952) The effects of chorionic gonadotropin in adult men: increased estrogen and 17-ketosteroid excretion, gynecomastia, Leydig cell stimulation and seminiferous tubule damage. Journal of Clinical Endocrinology and Metabolism, 12, 985-1014. Martikainen, H., Huhtaniemi, I. & Vihko, R. (1980) Response of peripheral serum sex steroids and some of their precursors to a single injection of HCG in adult men. Clinical Endocrinology, 13, 157-166. McNatty, K. P., Sawers, R. S. & McNeilly, A. S. (1974) A possible role for prolactin in control of steroid secretion by the human Graafian follicle. Nature, 250, 653-655. McNeilly, A. S., Kerin, J., Swanston,. I. A. et al (1980) Changes in the binding of human chorionic gonadotrophin/luteinizing hormone, follicle-stimulating hormone and prolactin to human corpora lutea during the menstrual cycle and pregnancy. Journal of Endocrinology, 87, 315-325. Metsikk6, K. & Rajaniemi, H. (1981) Antibodies to purified luteinizing hormone receptor localize the receptor at the luteal cell surface. Endocrinology, 109, 1399-1403. Orth, J. & Christensen, A. K. (1978) Autoradiographic localization of specifically bound ~2~i_ labeled follicle stimulating hormone on spermatogonia of the rat testis. Endocrinology, 103, 1944-1951. Perez-Palacios, G., Scaglia, H. E., Kofman-Alfaro, S. et al (1981) Inherited male pseudohermaphroditism due to gonadotrophin unresponsiveness. Acta Endocrinologica, 98, 148-155. Pointdexter, A. N., Buttram, V. C., Besch, P. K & Smith, R. G. (1979) Prolactin receptors in the ovary. Fertility and Sterility, 31,273-277. Rajaniemi, H. J., R0nnberg, L., Kauppila, A. et al (1980) Luteinizing hormone receptors in ovarian follicles of patients with polycystic ovarian disease. Journal of Clinical Endocrinology and Metabolism, 51, 1054-1057.
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Rajaniemi, H., Kauppila, A., R6nnberg, L. et al (1981a) LH(HCG) receptor in benign and malignant tumors of human ovary. Acta Obstetrica et Gynecologica Scandinavica (Supplement 101), 83-86. Rajaniemi, H. J., R0nnberg, L., Kauppila, A. et al (1981b) Luteinizing hormone receptors in human ovarian follicles and corpora lutea during menstrual cycle and pregnancy. Journal of Clinical Endocrinology and Metabolism, 52,307-313. Rao, Ch. V., Griffin, L. P. & Carman, F. R. (1977) Gonadotropin receptors in human corpora lutea of the menstrual cycle and pregnancy. American Journal of Obstetrics and Gynecology, 128, 146-153. Rillema, J. A. (1980) Mechanism of prolactin action. Federation Proceedings, 39, 2593-2598. Ross, G. T. & Schreiber, J. R. (1978) The ovary. In Reproductive Endocrinology (Ed.) Yen, S. S. C. & Jaffe, R. B. pp. 63-79. Philadelphia, ~ondon, Toronto: W. B. Saunders. Roth, J. & Grunfeld, C. (1981) Endocrine systems~~mechanisms of disease, target cells and receptors. In Textbook of Endocrinology, 6th Edition (Ed.) Williams, R. H. pp. 15-72. Philadelphia, London, Toronto: W. B. Saunders~ ::~ Saito, T. & Saxena, B. B. (1975) Specific receptors for prolactin in the ovary. Acta Endocrinologica, 80, 126-137. Sharpe, R. M. (1982) Cellular aspects of the inhibitory actions of LH-RH on the ovary and testis. Journal of Reproduction and Fertility, 64, 517-527. Sharpe, R. M., Wu, F. C. W. & Hargreave, T. B. (1980) Binding of human chorionic gonadotrophin to testicular biopsy tissue from infertile men and the effect of prior treatment with human chorionic gonadotrophin. Journal of Endocrinology, 86, 117-125'.... Shiu, R. P. C. & Friesen, H. G. (1974) Solubilization and purification of a prolaql,in:yecePtor from rabbit mammary gland. Journal of Biological Chemistry, 24, 7902-7911. Shiu, R. P. C. & Fiesen, H. G. (1976) Blockade of prolactin action by an antiserum to its receptors. Science, 192,259-261. Shiu, R. P. C. & Friesen, H. G. (1981) Regulation of prolactin receptors in target cells. In Receptors and Recognition, Series B (Ed.) Lefkowitz, R. J. Volume 13, pp. 69-81. London: Chapman & Hall. Starup, J. & Pedersen, H. (1978) Hormonal and ultrastructural observations in a case of resistant ovary syndrome. Acta Endocrinologica, 89,744-752. Wahlstr0m, T., Huhtaniemi, I., Hovatta, O. & Sepp~il~, M. (1983) Localization of luteinizing hormone, follicle stimulating hormone, prolactin, and their receptors in the human and rat testis using immunohistochemistry and radioreceptorassay. Journal of Clinical Endocrinology and Metabolism (in press). Wardlaw, S., Lauersen, N. H. & Saxena, B. B. (1975) The LH-HCG receptor of human ovary at various stages of the menstrual cycle. Acta Endocrino[ogica, 79, 568-576.