Growth Hormone Cells as Co-gonadotropes: Partners in the Regulation of the Reproductive System

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41 Kaupmann, K. et al. (1998) GABA(B)receptor subtypes assemble into functional heteromeric complexes. Nature 396, 683–687 42 White, J.H. et al. (1998) Heterodimerization is required for the formation of a functional GABA(B) receptor. Nature 396, 679–682 43 Jones, K.A. et al. (1998) GABA(B) receptors function as a heteromeric assembly of the subunits GABA(B)R1 and GABA(B)R2. Nature 396, 674–679 44 Jordan, B.A. and Devi, L.A. (1999) G-protein-coupled receptor heterodimerization modulates receptor function. Nature 399, 697–700 45 Zeng, F.Y. and Wess, J. (1999) Identification and molecular characterization of m3 muscarinic receptor dimers. J. Biol. Chem. 274, 19487–19497 46 Ng, G.Y. et al. (1996) Dopamine D2 receptor dimers and receptor-blocking peptides. Biochem. Biophys. Res. Commun. 227, 200–204 47 Tarasova, N.I. et al. (1999) Inhibition of G-protein-coupled receptor function by disruption of transmembrane domain interactions. J. Biol. Chem. 274, 34911–34915

Growth Hormone Cells as Cogonadotropes: Partners in the Regulation of the Reproductive System Gwen V. Childs

Through unique receptors, growth hormone (GH) stimulates ovarian follicles and Leydig cells, working alone or synergistically with luteinizing hormone (LH) and follicle-stimulating hormone (FSH). The source of GH might include a unique cell type that expresses mRNA encoding gonadotropin and GH and the antigens themselves, together with gonadotropin-releasing hormone (GnRH) and GH-releasing hormone (GHRH) receptors. This multifunctional cell might provide a cocktail of hormones needed to effect optimal gonadotropic activity.

Evidence accumulating over the past two decades has shown that growth hormone (GH) might function with gonadotropins as a coregulator of many reproductive system functions1. This G.V. Childs is at the Department of Anatomy, University of Arkansas School for Medical Science, Little Rock, AR 72205, USA. Tel: 11 501 686 7020, Fax: 11 501 686 6382, email: [email protected]

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article will review this evidence, focusing on GH actions in both sexes. It will conclude with a discussion of actions at the level of the pituitary, including the cellular site of origin of GH. Crucial proof for GH as a regulator requires evidence for GH receptors in the gonads. Whereas early evidence showed that radiolabeled GH binds to ovarian membranes1, the human GH

48 Zeng, F.Y. and Wess, J. (1999) Identification and molecular characterization of m3 muscarinic receptor dimers. J. Biol. Chem. 274, 19487–19497 49 Couve, A. et al. (1998) Intracellular retention of recombinant GABAB receptors. J. Biol. Chem. 273, 26361–26367 50 Kuner, R. et al. (1999) Role of heteromer formation in GABAB receptor function. Science 283, 74–77 51 Ng, G.Y. et al. (1999) Identification of a GABAB receptor subunit, gb2, required for functional GABAB receptor activity. J. Biol. Chem. 274, 7607–7610 52 Xu, Y. et al. (1999) A bioluminescence resonance energy transfer (BRET) system: application to interacting circadian clock proteins. Proc. Natl. Acad. Sci. U. S. A. 96, 151–156 53 Angers, S. et al. (2000) Detection of beta2–adrenergic receptor dimerization in living cells using bioluminescence resonance energy transfer (BRET). Proc. Natl. Acad. Sci. U. S. A. 97, 3684–3689 54 Overton, M.C. and Blumer, K.J. (2000) G protein coupled receptors function as oligomers in vivo. Curr. Biol. 10, 341–344 55 Gouldson, P.R. et al. (1998) Domain swapping in G-protein coupled receptor dimers. Protein Eng. 11, 1181–1193

(hGH) ligand could have bound to prolactin receptors1–3. Lobie et al.4 first demonstrated immunolabeling for GH receptors in specific gonadal cells of both male and female rats. Shortly thereafter, mRNA encoding the GH receptor was also detected in gonads5 and purified Leydig cells6. Recent studies also showed that transgenic mice lacking the GH receptor exhibited reduced fertility rates and reduced testosterone secretion7. Thus, collectively, the studies showed that GH could regulate specific gonadal target cells via its own receptors. • Functions for GH in the Ovary GH-deficient female rats exhibit a delay in the onset of puberty1,8–10, and GH administration restores the time of puberty onset8,9 and increases the length of the estrous cycle11. Subsequent studies have focused on the cell types targeted by GH (Table 1). GH might work with or without follicle-stimulating hormone (FSH) to promote the early development of the follicles. GH increased FSH-stimulated granulosa cell differentiation and aromatase activity in rats12 and estradiol production by human ovaries13. GH was also effective by itself13. Studies of

1043-2760/00/$ – see front matter © 2000 Elsevier Science Ltd. All rights reserved. PII: S1043-2760(00)00252-6

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Table 1. Growth hormone (GH) as a regulator of ovarian follicular development and functions Actions: GH caused an increase in:

Species

Tissue/organ/ cell type

Additive effects seen with

Refs

Aromatase activity Estradiol production Estrogen release Progesterone secretion or accumulation Inhibin production IGF-II secretion LH receptors DNA synthesis Follicular diameter Development of intermediate follicles Follicles rescued from atresia Number of antral follicles Number of corporea lutea

Rat Human Immature rat Rat Immature rat Human Rat Immature rat Immature rat GH-deficient rats GH-deficient rats Bovine Rats

Granulosa cells Ovaries Preantral follicles Ovaries Preantral follicles Granulosa cells Ovaries Ovaries Preantral follicles Ovaries Ovaries Ovaries Ovaries

FSH (required) FSH Activin A, IGF-I hCG, FSH Activin A, IGF-I – FSH (required) – Activin A, IGF-I – – – –

19 13 16 10, 19, 21 16 22 19 14, 15 16 18 18 17 11

Abbreviations: FSH, follicle-stimulating hormone; hCG, human chorionic gonadotropin; IGF, insulin-like growth factor; LH, luteinizing hormone.

hypophysectomized rats showed that GH, luteinizing hormone (LH) and prolactin induced DNA synthesis in immature rat ovaries14,15. GH might work in tandem with activin and FSH. Liu et al.16 reported that, because FSH-knockout mice exhibited follicular growth independent of FSH up to the preantral stage, other factors might be responsible for the development to this stage. In studies of preantral follicles from immature mice, GH and activin A, but not FSH, increased follicular diameter and stimulated the secretion of estradiol and inhibin16. Furthermore, whereas these effects of GH could be augmented by insulin-like growth factor I (IGF-I) and activin A, IGF-I did not stimulate these responses by itself16. This last finding suggested that GH might act directly via its receptor population to stimulate the development of preantral follicles. GH-treated heifers had an increased number of antral follicles17. GH restored the reduced ovarian weights in GH-deficient mice by promoting the development of intermediate-sized follicles, rescuing growing follicles from atresia18 and increasing the numbers of corpora lutea11. GH also stimulates progesterone and the production of LH receptors. In vivo, GH treatment enhanced the levels of progesterone secreted and, in vitro, this was seen in response to human chorionic gonadotropin (hCG) and FSH TEM Vol. 11, No. 5, 2000

(Ref. 10). Similarly, ovine GH enhanced FSH actions on ovarian granulosa cells by increasing LH receptors and progesterone (although, in this study, GH did not induce aromatase activity)19. Another group reported that GH had no effect on the synthesis of progesterone in human granulosa cells20. Hsu and Hammond21 reported that immature porcine granulosa cells responded better to GH and FSH than to either alone. GH and FSH added, in vitro, resulted in a 33-fold increase in progesterone secretion compared with 7.4- or 2.6-fold increases following FSH or GH, respectively. GH stimulated progesterone release from luteinized follicular cells22; however, lower concentrations of GH and hCG together could stimulate the release of levels of progesterone seen only with the highest concentrations of either hormone alone. Finally, in his review1, Adashi described a study showing that forskolin (which activates adenylate cyclase) increased mRNA encoding the GH receptor in granulosa cells. Thus, the synergy between gonadotropins and GH might include increased GH receptivity. Several studies have looked at IGF-I or -II as mediators in GH responses1,2. Table 1 shows that, whereas IGF-I might add to the actions of GH, it had no effect by itself on the functions tested16. GH stimulated IGF-II release from human granulosa cells in one study23, but did not stimulate IGF-I

or -II in another20. Adashi also reviewed several clinical studies showing a function for GH in the improvement of outcomes from in vitro fertilization trials and the treatment of delayed puberty1. In many cases, GH is added to reduce the dose or increase the efficacy of gonadotropin therapy. Summary GH might work by itself, or in partnership with activin and/or IGF-I, to promote the early differentiation of the ovarian follicles that are FSH independent (preantral). Later in development, it might facilitate actions by gonadotropins, perhaps by enhancing the production of LH receptors. The nature of its actions might depend on the stage of development of the cell types involved. • GH Involvement in Testicular Function Table 2 summarizes the actions of GH on the testes. GH deficiency in males results in reduced fertility. Male rats immunized against GH-releasing factor (GHRH) experience a delay in testicular growth and differentiation of germ cells24. In men, congenital GH deficiency results in a delay in the onset of puberty25,26. In dwarf rats, GH deficiency has been associated with impaired sperm counts and motility27, and reduced testicular size27,28. GH might affect Leydig cell development. If immature hypophysectomized 169

Table 2. Growth hormone actions on the testes Actions: GH caused an increase in:

Species

Tissue/organ/ cell type

Additive effects seen with

Ref.

Number of precursor mesenchymal cells STAT5b StAR

Immature hypophysectomized male rats

Testis

–

29

Rat Rat

– –

30 6

Spermatogenesis Testosterone secretion

Human: hypogonadal/hypogonadism Human: infertile men

Immature Leydig cells Progenitor, immature and adult Leydig cell cultures Testis, in vivo Testis, in vivo

Gonadotropins Gonadotropins

31 32

Abbreviations: GH, growth hormone; StAR, steroidogenic acute regulatory protein; STAT5b, signal transducer and activator of transcription 5b.

male rats are given GH, there is an increase in the number of precursor mesenchymal cells in the testis (an effect that is also seen with LH and prolactin)29. More recently, Kanzaki and Morris30 showed that GH activated the signal transducer and activator of transcription 5b (STAT5b) in immature Leydig cell cultures. In a subsequent study, Kanzaki and Morris6 showed that Leydig cell progenitors expressed mRNA encoding the GH receptor and that GH increased levels of mRNA encoding steroidogenic acute regulatory protein (StAR) in progenitor, immature and adult rat Leydig cells. Because prolactin had no effects on the expression of mRNA encoding StAR, these data suggest that the GH actions were mediated through the somatotropic receptors. Recently, Chandrashekar et al. studied testicular function in transgenic mice lacking GH receptors7. The mice had increased circulating prolactin and undetectable IGF-I. Basal and LH-stimulated testosterone secretion from isolated testes were reduced, as was the fertility rate. GH can also enhance the efficacy of gonadotropic actions in the male. Shoham et al.31 have shown that GH can work with gonadotropins to induce spermatogenesis in patients with hypogonadotropic hypogonadism. Swerdloff and Odell32 have shown that GH can increase testicular responses to treatment with gonadotropins. Summary GH can have direct effects on the differentiation of Leydig cells, leading to testosterone secretion. It might also

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work in partnership with LH and FSH. GH could be used therapeutically in the male as it has been in the female. • GH Effects on the Pituitary GH might also affect reproductive functions by paracrine actions on the pituitary (Table 3). Harvey et al.33 reported the localization of GH-binding proteins in several types of pituitary cells, including gonadotropes. Early studies by Chandrashekar and Bartke reported that GH treatment of GH-deficient Ames mice increased plasma LH levels34, and that circulating LH is increased

significantly in male transgenic mice overexpressing the gene encoding hGH (Refs 35,36). However, GH might not always be stimulatory. Longer-term GH treatment attenuated LH and prolactin secretion in adult male rats [V. Chandrashekar and A. Bartke, Proceedings, 75th Annual Meeting of The Endocrine Society, Las Vegas, 1993, p. 280 (abstract)]37. GH also reduced the postcastration rise in LH six to eight days after surgery. In addition, testosterone-induced suppression of LH secretion in castrates was attenuated by GH treatment. However,

Table 3. Growth hormone actions on the pituitary Actions: treatment with GH resulted in

Species

Type of Treatment

Increased plasma LH

GH-deficient Ames mice In vivo

34

Increased plasma LH

Transgenic mice with excess hGH

In vivo

35

Decreased plasma LH and prolactin

Adult male rats

Longer-term treatment

35, 36, a

Decreased plasma LH

Adult

14-day treatment

37

Refs

Decreased postcastration Adult rats castrated rise in LH

6–8 days, postsurgery 37

No effects on GnRHmediated LH release

Adult

14-day treatment

37

No effects on basal or GnRH-mediated FSH release

Adult

14-day treatment

37

No effects on postcastration rise in FSH

Adult

6–8 days of treatment 37

Abbreviations: FSH, follicle-stimulating hormone; GH, growth hormone; GnRH, gonadotropin-releasing hormone; hGH, human GH; IGF, insulin-like growth factor; LH, luteinizing hormone. a V. Chandrashekar and A. Bartke, Proceedings of the 75th Annual Meeting of The Endocrine Society, Las Vegas, 1993, Abstract, p. 280.

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GH did not decrease the secretory response to GnRH and neither did it affect basal, GnRH stimulated, or postcastration levels of FSH. In rats immunized against GH, antiGH sera resulted in higher basal LH and FSH levels, correlating well with the attenuating effects of GH on gonadotropin secretion [V. Chandrashekar and A. Bartke, Proceedings, 75th Annual Meeting of The Endocrine Society, Las Vegas, 1993, p. 280 (abstract)]37. However, anti-GH sera lowered the increase in GnRH-mediated LH secretion, suggesting that GH also might facilitate GnRH actions on LH secretion. The negative feedback effects of exogenous testosterone were also attenuated in GH-immunized, castrated rats, indicating different GH actions on the castration cell response to steroid feedback. When FSH was assayed, the antibodies caused higher GnRH-stimulated FSH levels, which suggests that GH might have the opposite effects on GnRH-stimulated FSH secretion.

Finally, the testes of GH-immunized rats failed to respond to the GnRHmediated increases in LH; testosterone secretion remained at low levels. This adds to previously described evidence for GH actions at the level of the testes in promoting testosterone secretion (summarized in Table 3). The authors suggested that this was either directly caused by the absence of GH or, indirectly, by a reduction in IGF-I (also the result of neutralization of GH)37. Another recent study from this same group7 focused on pituitary functions in transgenic mice with no GH receptors. In GH receptor-knockouts, the LH response to GnRH was significantly decreased, indicating that, in the mouse, GH binding to functional receptors might be needed to enable full GnRH-mediated functions. Summary Collectively, these studies suggest that GH might play a regulatory role in the pituitary, perhaps to limit LH secretion or alter the sensitivity of the cells to

GnRH and steroids. The data also show non-parallel responses when LH and FSH are compared, indicating that GH might be acting on monohormonal gonadotropes. Alternatively, the actions of GH might alter the sensitivity of the bihormonal gonadotropes to stimulation, making them functionally monohormonal (secreting only LH or FSH) • The Cellular Partnership in the Pituitary Collectively, the above groups of studies suggest that GH might work with gonadotropins to help regulate gonadal functions. If GH is a co-regulatory hormone (a co-gonadotropin), then how is its synthesis and secretion regulated? Recent studies from our laboratory suggest that this occurs via a subset of multifunctional cells that share phenotypic characteristics with both gonadotropes and somatotropes38–43 (Table 4). Cells with GH antigens contribute to the gonadotrope population by synthesizing mRNA molecules encoding the gonadotropin b subunit38. These

Table 4. Evidence for a multifunctional pituitary cell type sharing gonadotropic and somatotropic phenotypes Combined expression of

Techniques used

Evidence

Ref.

GH antigens and GH mRNA encoding LHb or FSHb

In situ hybridization/ immunolabeling; fresh cultures from cycling female rats

Up to 40% of GH cells have mRNA encoding LHb and 60% of GH cells have mRNA encoding FSHb; peak at proestrus PM

38

mRNA encoding follistatin. GH, prolactin, LHb and FSHb antigens

In situ hybridization/ immunolabeling; fresh cultures from cycling female rats

Number of cells containing mRNA encoding FS that contain each antigen adds to over 100% of total FS cells; suggests overlap in storage

44

Binding to biotinylated GnRH and GH antigens

Avidin–biotin affinity cytochemistry/ immunolabeling; one-day cultures from cycling female rats

30–40% of GH cells bind GnRH; peak expression at proestrus AM

39

Responses to inhibin

Avidin–biotin affinity cytochemistry/ immunolabeling; one-day cultures from cycling female rats

Inhibin given to proestrous rats reduced biotinylated GnRH binding from 30% to 10–12% of GH cells

40

Responses to activin

Avidin–biotin affinity cytochemistry/ immunolabeling; one-day cultures from cycling female rats

Activin given to diestrous or proestrous rats increased binding to 40% and 60% of GH cells, respectively

41

Binding to biotinylated GHRH and gonadotropin antigens

Avidin–biotin affinity cytochemistry/ immunolabeling; one-day cultures from cycling female rats

Over 60% of gonadotropes bind biotinylated GHRH; peak expression at proestrus

42

GH mRNA and gonadotropin antigens

In situ hybridization/ immunolabeling; fresh cultures from male or cycling female rats

Over 50% of gonadotropes in female express GH mRNA; peak expression at proestrus; 55% of gonadotropes in male rat express GH mRNA

43

Abbreviations: FS, follistatin; FSH, follicle-stimulating hormone; GH, gene encoding growth hormone; GH, growth hormone; GHRH, GH-releasing hormone; GnRH, gonadotropin-releasing hormone; LH, luteinizing hormone.

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sion by gonadotropes rose to over 50% by proestrus (Figs 1, 2). Another unexpected finding was the relatively high expression of GH mRNA by 50% of gonadotropes from male rat pituitaries, suggesting that there is potential for a multihormonal/multifunctional cell type in the male as well. Collectively, our studies provide evidence for a subset of pituitary cells that shares phenotypic characteristics of gonadotropes and somatotropes. If defined by its content of GH mRNA and gonadotropin antigens, it might represent as many as 50% of gonadotropes or 30% of GH cells. The cyclic pattern of expression of GH and gonadotropin mRNAs suggests that the midcycle period is the peak expression period in the female, providing for co-secretion of GH and gonadotropins. We also reported non-parallel expression of GH mRNA by LH and FSH cells43. This suggests that the GH might be expressed differentially by monohormonal and bihormonal gonadotropes and correlates with studies of GH effects on gonadotropes37. GH might feed back

to autoregulate the secretion of LH, while at the same time facilitating actions on the gonads. The feedback loop might be exerted at the level of the multifunctional cells that share somatotrope and gonadotrope phenotypes. Stimulatory effects of GH such as those seen in the mouse36, or those seen on GnRHmediated LH secretion in the rat37, could represent a facilitatory role for GH. • Differentiation of the Multihormonal/Multifunctional Cell Type This multifunctional cell might produce a cocktail of gonadotropins and GH that provides the GH needed at the time to exert its own functions and facilitate gonadotropic functions (Tables 1, 2). The differential expression of GH mRNA during the cycle43 suggests that it might be regulated by reproductive hormones to provide the right cocktail at the appropriate time. Finally, the evidence for GHRH binding by GH, prolactin and gonadotropinbearing cells42 supports a new hypothesis suggesting that these cells belong to

70 Percentage of LH or FSH cells

multihormonal cells are detected early in diestrus, and their proportions rise to a peak by the afternoon of proestrus. They also express GnRH receptors (as gonadotropes do). Over 30% of cells with GH antigens bound biotinylated analogs of GnRH during the peak expression periods (late diestrus, early proestrus)40. GH cells were also responsive to hormones that regulated GnRH receptor expression. Inhibin reduced41 and activin increased42 the expression of GnRH receptivity in cells with gonadotropin or GH antigens. The activin-mediated enhancement during diestrus in GH cells was similar to the increase seen normally from diestrus to proestrus39. Activin enhancement of GnRH binding to GH cells during proestrus was greater than that seen in vivo39, but the proportion of GH cells bound matched the proportion of GH cells that contained mRNA encoding FSH (60%)38. The detection of GH antigens in cells with GnRH receptors and mRNA encoding gonadotropins does not prove that the multihormonal cells originate from the somatotrope population. In fact, the GH antigens could have bound the GH-binding proteins found in gonadotropes by Harvey et al.33 Therefore, we identified phenotypes unique to GH cells (GH mRNA and GHRHbinding sites) to see whether we could find multifunctional activity in gonadotropes. Over half of the gonadotropes from proestrous rats bound biotinylated GHRH (Table 4)42. GHRH binding by gonadotropes was lower in male or estrous female rat pituitary cells, indicating that the multihormonal GHRH target cells were a transient subset in the cycling rat population. There were cyclic differences in the expression of GH mRNA by the female pituitaries43. Both RNase protection assays and in situ hybridization experiments showed that the lowest levels appeared during metestrus, followed by a rise to a peak during proestrus43. When cells bearing GH mRNA were further identified by dual labeling for GH or gonadotropins, only 20–30% of gonadotropes expressed GH mRNA during metestrus. However, the expres-

60 b

b

50 40 a 30 20 10 0 Male

Metestrus

Diestrus

Proestus

Estrus

Experimental group trends in Endocrinology and Metabolism

Figure 1. Analysis of dual labeling for GH mRNA and LH (dark-grey bars) or FSHb (light-grey bars) subunit antigens. This figure shows the percentages of gonadotropes that express GH mRNA in males as well as females during different stages of the cycle. ‘a’ indicates the lowest values, significantly different from all others in the estrous cycle; ‘b’ indicates intermediate values, different from all others; p ,0.05. Abbreviations: FSH, follicle-stimulating hormone; GH, gene encoding growth hormone; LH, luteinizing hormone.

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Figure 2. Dual-labeled field showing a cell (cell x) labeled for GH mRNA (grey-black) and FSHb (orange-amber) antigens (arrows) (a). The higher magnification in (b) shows labeling of cell x and a nearby unlabeled cell. (c) shows dual label for GH mRNA and LHb antigens (arrows). The open arrows in both fields show cells labeled for GH mRNA alone. Magnification (a) 3415; (b) and (c) 3825. Abbreviations: FSH, follicle-stimulating hormone; GH, gene encoding growth hormone; LH, luteinizing hormone.

Diestrus AM

Metestrus

GH mRNA LH mRNA

1

Diestrus PM

GH mRNA LH mRNA GnRH receptors

GH

2

Proestrus AM

GH mRNA LH mRNA FSH mRNA GnRH Receptors GH

3

FSH LH

LH LH

Regulators? IGF, EGF, GH?

Estrogens GH?

GH

Estrogens Activin trends in Endocrinology and Metabolism

Figure 3. Diagram showing proposed steps in the differentiation of a multifunctional cell sharing somatotropic and gonadotropic phenotypes (a ‘somatogonadotrope’). See text for details and correlation with data. Grey arrows indicate increases in hormone concentrations. Abbreviations: EGF, epidermal growth factor; GH, gene encoding growth hormone; GnRH, gonadotropin-releasing hormone; FSH, follicle-stimulating hormone; IGF, insulin-like growth factor; LH, luteinizing hormone.

the subset of cells that produce prolactin and GH (somatomammotropes). Further tests are needed to determine how prolactin cells might be involved. Figure 3 illustrates proposed steps in the differentiation of a multifunctional TEM Vol. 11, No. 5, 2000

cell that can supply the GH and gonadotropins needed to stimulate the gonads during the cycle. It is based on the timing of appearance of each product in the multifunctional cell. Because over 80% of GH cells have GHRH

receptors42, one can assume that these cells also bind GHRH. Metestrus is the low point of expression of GH mRNA and mRNA encoding gonadotropin38,43. By the morning of diestrus, cells with LH or GH antigens contain more GH mRNA43. At the same time, cells with GH antigens begin to express more LH mRNA (Ref. 38). Expression of FSH mRNA remains low during diestrus, probably reflecting negative estradiol feedback (step 1). Putative regulators have been hypothesized, including GH itself and some known to increase LH or GH synthesis. During diestrus, cells with gonadotropin or GH antigens increase the expression of GnRH receptors (step 2) so that, by the evening, over 30% of GH cells and 80% of gonadotropes bind GnRH (Ref. 39). Much evidence indicates that this is regulated by estradiol (reviewed in Refs 39–41), although GH might also regulate the sensitivity of the cells to GnRH in this manner37. FSH mRNA is increased in cells with GH antigens38 (and GH mRNA is higher in cells with FSH antigens43) by the morning of proestrus (step 3). By this time, over 50% of LH cells contain GH mRNA. Because we know that 70–80% of cells with LH antigens produce FSH mRNA (Ref. 38), these data suggest that a high proportion of the bihormonal (LH–FSH) gonadotropes are multihormonal, producing a cocktail of LH, FSH and GH. Finally, the delayed increased expression of FSH might be regulated by activin, which might continue to regulate further differentiation of this function. By the end of the day of proestrus, 60% of GH antigen-bearing cells express FSH mRNA compared with 40% producing LH mRNA (Ref. 38). This suggests that a separate FSH gonadotrope population emerges that produces a GH–FSH cocktail. This set of hormones might serve to stimulate the development of preantral and antral follicles (Table 1). • Summary and Conclusions Collectively, the findings support the hypothesis that somatotropes and gonadotropes are partners in the regulation of the reproductive system. Their populations overlap to allow joint 173

regulation of the same subset of pituitary cells by key regulatory peptides such as GnRH, GHRH, inhibin and activin. This might allow GH to regulate early differentiation of preantral follicles or Leydig cells and, after gonadotropin receptors increase, facilitate gonadotropic actions. During the estrous cycle, there is good correlation between the appearance of the cells and the need for GH facilitation. However, how do these data correlate with findings that show GH actions on gonadotropes themselves34–37? We propose that GH might also serve as an autocrine or paracrine regulator of the development or differentiation of these multifunctional cells. For example, GH might inhibit LH secretion if levels rise too high, or change the sensitivity of the cells to GnRH. The differential effects of GH on LH and FSH secretion might reflect its action on different subsets of gonadotropes. • Acknowledgements I acknowledge the excellent assistance of Geda Unabia and Diana Rougeau as these studies developed. Also, thanks to Dr Ping Wu for training and assistance in the RNase protection assays for GH mRNA. The antisera to FSH were gifts from Dr A. F. Parlow, Hormone Distribution program, NIH, USA, and the antisera to bovine LH were a gift from Dr J.G. Pierce, USDA, USA. The studies described in this review were funded by NIH R01 HD 15472 and HD 33915, and NSF IBN 9724066. References 1 Adashi, E.Y. (1992) Growth hormone as a gonadotropin. In GnRH, GnRH Analogs, Gonadotropins and Gonadal Peptides (Bouchard, P. et al., eds), Proceedings of the Third Organon Round Table Conference, Paris, pp. 569–590, Parthenon Publishing 2 Adashi, E.Y. (1992) Intraovarian regulation: the IGF–1 paradigm. In GnRH, GnRH Analogs, Gonadotropins and Gonadal Peptides (Bouchard, P. et al., eds), Proceedings of the Third Organon Round Table Conference, Paris, pp. 559–568, Parthenon Publishing 3 Davies, T.F. et al. (1980) Lactogenic receptor regulation in hormone stimulated steroidogenic cells. Nature 283, 863–965 4 Lobie, P.E. et al. (1990) Cellular localization of the growth hormone receptors/ binding protein in the male and female reproductive systems. Endocrinology 126, 2214–2221

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5 Tiong, T.S. and Herington, A.C. (1991) Tissue distribution, characterization and regulation of messenger ribonucleic acid for growth hormone receptor and serum binding protein in the rat. Endocrinology 129, 1628–1634 6 Kanzaki, M. and Morris, P. (1999) Growth hormone regulates steroidogenic acute regulatory protein expression and steroidogenesis in Leydig cell progenitors. Endocrinology 140, 1681–1686 7 Chandrashekar, V. et al. (1999) Pituitary and testicular function in growth hormone receptor gene knockout mice. Endocrinology 140, 1082–1088 8 Ramaley, J.A. and Phares, C.K. (1980) Delay of puberty onset in females due to suppression of growth hormone. Endocrinology 106, 1989–1993 9 Bartke, A. (1964) Histology of the anterior hypophysis, thyroid and gonads of two types of dwarf mice. Anat. Rec. 149, 225–229 10 Advis, J.P. et al. (1981) Activation of growth hormone short loop negative feedback delays puberty in the female rat. Endocrinology 108, 1343–1352 11 Jorgensen, K.D. et al. (1991) Effect of human growth hormone on the reproduction of female rats. Pharmacol. Toxicol. 68, 14–20 12 Hutchinson, L.A. et al. (1988) Growth hormone and insulin-like growth factor-I accelerate PMSG-induced differentiation of granulosa cells. Mol. Cell. Endocrinol. 55, 61–69 13 Mason, H.D. et al. (1990) Direct gonadotrophic effect of growth hormone on oestradiol production by human granulosa cells. J. Endocrinol. 126, R1–R4 14 Usuki S. et al. (1989) Growth hormone elevates deoxyribonucleic acid polymerase a activity in conjunction with deoxyribonucleic acid synthesis in immature rat ovaries. Bio. Med. Res. 10, 267–273 15 Usuki, S. and Shiota, M. (1989) Growth hormone elevates DNA polymerase-a activity related to DNA synthesis in ovaries of hypophysectomized immature rat. Horm. Metab. Res. 21, 455–456 16 Liu, X. et al. (1998) Effects of growth hormone, activin, and follistatin on the development of preantral follicles from immature male mice. Endocrinology 139, 2342–2347 17 Gong, J. et al. (1991) The effect of recombinant bovine somatotropin on ovarian function in heifers: follicular populations and peripheral hormones. Biol. Reprod. 45, 941–949 18 Ozawa K. et al. (1996) Recombinant human growth hormone acts on intermediate-sized follicles and rescues growing follicles from atresia. Endocr. J. 43, 87–92 19 Jia, X.C. et al. (1986) Growth hormone enhances follicle-stimulating hormone induced differentiation of cultured rat granulosa cells. Endocrinology 118, 1401–1409 20 Voutilainen, R. and Miller, W.L. (1987) Coordinate tropic hormone regulation of mRNAs for insulin-like growth factor II and the cholesterol side chain cleavage enzyme, P450ssc, in human steroidogenic tissues. Proc. Natl. Acad. Sci. U. S. A. 84, 1590–1594

21 Hsu, C.J. and Hammond, J.M. (1987) Concomitant effects of growth hormone on secretion of insulin-like growth factor I and progesterone by cultured porcine granulosa cells. Endocrinology 121, 1343–1348 22 Ramasharma, K. and Li, C.H. (1987) Human pituitary and placental hormones control human insulin-like growth factor II secretion in human granulosa cells. Proc. Natl. Acad. Sci. U. S. A. 84, 2643–2647 23 Lanzone, A. et al. (1992) Human growth hormone enhances progesterone production by human luteal cells in vitro: evidence of a synergistic effect with human chorionic gonadotropin. Fertil. Steril. 57, 92–96 24 Arsenijevic Y. et al. (1989) Growth hormone (GH) depreviation induced by passive immunization against rat GH releasing factor delays sexual maturation in the male rat. Endocrinology 124, 3050–3059 25 Laron, Z. (1993) Laron syndrome: from description to therapy. Endocrinologist 3, 21–28 26 Strobl, J.S. and Thomas, M.J. (1994) Human growth hormone. Pharmacol. Rev. 46, 1–34 27 Gravance, C.G. et al. (1997) Impaired sperm characteristics in postpubertal growth hormone deficient dwarf (dw/dw) rats. Anim. Reprod. Sci. 49, 71–76 28 Bartlett, J.M. et al. (1990) Pubertal development and testicular function in the male growth hormone deficient rat. J. Endocrinol. 126, 193–201 29 Zipf, W.B. et al. (1978) Prolactin, growth hormone, and luteinizing hormone in the maintenance of testicular luteinizing hormone receptors. Endocrinology 103, 595–600 30 Kanzaki, M. and Morris, P.L. (1998) Lactogenic hormone-inducible phosphorylation and gamma-activated site binding activities of Stat5b in primary rat Leydig cells and MA-10 mouse Leydig tumor cells. Endocrinology 139, 1872–1882 31 Shoham, Z. et al. (1992) Cotreatment with growth hormone for induction of spermatogenesis in patients with hypogonadotropic hypogonadism. Fertil. Steril. 57, 1044–1051 32 Swerdloff, R.S. and Odell, W.D. (1977) Modulating influence of FSH, GH and prolactin on LH-stimulated testosterone secretion. In The Testis in Normal and Infertile Men (Troen, P. and Nankin, H.R., eds), pp. 395–401, Raven Press 33 Harvey, S. et al. (1993) Ultrastructural colocalization of growth hormone binding protein and pituitary hormones in adenohypophyseal cells of the rat. Endocrinology 133, 1125–1130 34 Chandrashekar, V. and Bartke, A. (1993) Induction of endogenous insulin like growth factor-I secretion alters the hypothalamic–pituitary–testicular function in growth hormone-deficient adult dwarf mice. Biol. Reprod. 48, 544–551 35 Chandrashekar, V. et al. (1988) Endogenous human growth hormone (GH) modulates the effect of gonadotropin-releasing hormone on pituitary function and the gonadotropin response to the negative feedback effect of testosterone in adult male transgenic mice bearing human GH gene. Endocrinology 123, 2717–2722

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36 Chandrashekar, V. and Bartke, A. (1993) Effects of age and endogenously secreted human GH on the regulation of gonadotropin secretion in female and male transgenic mice expressing the human growth hormone gene. Endocrinology 132, 1482–1488 37 Chandrashekar, V. and Bartke, A. (1998) The role of growth hormone in the control of gonadotropin secretion in adult male rats. Endocrinology 139, 1067–1074 38 Childs, G.V. et al. (1994) Cells that express luteinizing hormone (LH) and follicle stimulating hormone (FSH) beta (b) subunit mRNAs during the estrous cycle: the major contributors contain LHb, FSHb and/or

growth hormone. Endocrinology, 134, 990–997 39 Childs, G.V. et al. (1994) Cytochemical detection of GnRH binding sites on rat pituitary cells with LH, FSH and GH antigens during diestrous upregulation. Endocrinology 134, 1943–1951 40 Childs, G.V. et al. (1997) Differential effects of inhibin on gonadotropin stores and gonadotropin releasing hormone binding to pituitary cells from cycling female rats. Endocrinology 138, 1577–1584 41 Childs, G.V. and Unabia, G. (1997) Cytochemical studies of the effects of activin on gonadotropin releasing hormone (GnRH) binding by pituitary gonadotropes and

The Mammalian Fatty Acid-binding Protein Multigene Family: Molecular and Genetic Insights into Function Ann Vogel Hertzel and David A. Bernlohr

Intracellular fatty acid-binding proteins associate with fatty acids and other hydrophobic biomolecules in an internal cavity, providing for solubilization and metabolic trafficking. Analyses of their in vivo function by molecular and genetic techniques reveal specific function(s) that fatty acid-binding proteins perform with respect to fatty acid uptake, oxidation and overall metabolic homeostasis. Intracellular fatty acid-binding proteins (FABPs) are members of a multigene family encoding ~15-kDa proteins, which bind a hydrophobic ligand in a non-covalent, reversible manner (reviewed in Refs 1–3). The nine family members have between 20% and 70% identity in their amino acid sequence. Despite the wide variance in primary sequence, numerous X-ray crystal structures have shown a common tertiary fold forming a b-barrel (Fig. 1). The barrel comprises ten antiparallel bstrands, linked by hydrogen bonds, which are organized into two nearly orthogonal b-sheets. Importantly, the A. Vogel Hertzel and D.A. Bernlohr are at the Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, 1479 Gortner Avenue, St Paul, MN 55108, USA. Tel: 11 612 624 2712, Fax: 11 612 625 5780, email: [email protected]. umn.edu

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b-sheets create an internal, water-filled cavity, lined with ~50% polar amino acids. Although the cavity is significantly larger (two-three times) than the

growth hormone cells. J. Histochem. Cytochem. 45, 1603–1610 42 Childs, G.V. et al. (1999) Differential expression of gonadotropin and prolactin antigens by GHRH target cells from male and female rats. J. Endocrinol. 162, 177–187 43 Childs, G.V. et al. (2000) Differential expression of growth hormone mRNA by somatotropes and gonadotropes in male and cycling female rats. Endocrinology 141, 1560–1570 44 Lee, B.L. et al. (1993) Expression of follistatin mRNA in somatotropes and mammotropes early in the estrous cycle. J. Histochem. Cytochem. 41, 955–960

volume of a fatty acid, typically only a single fatty acid is bound in the cavity, with the carboxylate group oriented inwards, coordinated by a tyrosine and two arginine residues. At the N-terminus of FABPs, a helix–loop–helix motif forms a cap-like structure on the ‘top’ of the barrel. Because there is no obvious opening for the fatty acids to enter or exit the cavity, the portal hypothesis was proposed. This involves a transient conformational change around the helix–loop–helix area and adjacent loops connecting b-strands, thereby allowing the fatty acid to enter or exit the cavity4 (Fig. 1). The function(s) of these proteins within cells has remained elusive. Although the in vitro binding of a fatty acid has been analyzed extensively, the in vivo function is less well defined. It has been hypothesized that owing to the low solubility of fatty acids in an

Figure 1. Ribbon diagram of the X-ray crystal structure of fatty acid-binding protein 4 (FABP4). (a) Oleate ligand (yellow) bound in the cavity of FABP4 with the helix–loop–helix motif in top part of the figure. b-Strands A–E are in front and b-strands F–J are behind. (b) ‘Top’ view of FABP4, looking down on the portal region. The portal is surrounded by the second a-helix, and loops (noted with arrows) between b-strands C and D, and b-strands E and F. This figure was created with the use of RasMol v2.6 with data from the Brookhaven Protein DataBank, Id# 1LID.

1043-2760/00/$ – see front matter © 2000 Elsevier Science Ltd. All rights reserved. PII: S1043-2760(00)00257-5

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