Developmental Endocrinology Branch, NICHD, NIH, Bethesda, MD 20892, USA
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Abstract |
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Key words: folliculogenesis/granulosa cells/PCOS/testosterone/theca
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Introduction |
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The mechanism(s) whereby androgens promote ovarian growth are unclear. Insulin-like growth factors (IGF) have been strongly implicated in ovarian functions (Adashi, 1998; Giudice et al., 1996
). Insulin-like growth factor-I (IGF-I) expression predominates in murine species (Hernandez et al., 1989
; Zhou et al., 1991
; Adashi et al., 1997
), where its expression is significantly correlated with granulosa proliferation and follicular growth (Zhou et al., 1995
). Moreover IGF-I gene deletion results in murine infertility due to impaired follicular development (Baker et al., 1996
; Zhou et al., 1997
). In ovaries of larger species, IGF-I expression tends to be less and IGF-II expression more prominent (Zhou et al., 1996
; Perks et al., 1995
). In the human ovary, IGF-I expression is minimal and IGF-II is highly abundant (Geisthovel et al., 1989
; Hernandez et al., 1992
; Zhou and Bondy, 1993
; El-Roeiy et al., 1994
). Despite variability in IGF expression, expression of the IGF-I receptor, which subserves both IGF-I and IGF-II (Willis et al., 1998
), is highly conserved across species (Bondy et al., 1994
). It has recently been shown that androgen treatment increased the recruitment of primordial follicles into the growth pool, and that this effect was correlated with increased IGF-I and IGF-I receptor gene expression by primordial follicle oocytes (Vendola et al., 1999
). In the present study, we compared IGF-I, IGF-II, IGF-I receptor and insulin receptor concentrations in granulosal, thecal and interstitial compartments of androgen-treated monkeys and follicular phase normal cycling controls.
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Materials and methods |
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RNA probes for detection of IGF-I, IGF-II, IGF-I receptor and insulin receptor mRNA were synthesized as previously described (Zhou and Bondy, 1993), and used for in-situ hybridization as previously reported in detail (Bondy et al., 1993
). Non-specific signal was determined from sense probes hybridized to parallel sections in the same incubations. All sections for each probe were hybridized and exposed in a single batch. Slides were air-dried and exposed to Hyperfilm-beta Max (Amersham, Arlington Heights, IL, USA) for 5 (IGF-II and IGF-I receptor) or 10 days (IGF-I and insulin receptor), then dipped in Kodak NTB2 nuclear emulsion, stored with desiccant at 4°C for 14 days (IGF-I receptor) or 21 days (IGF-I and insulin receptor). Slides were developed and stained with Mayer's haematoxylin and eosin for microscopic evaluation.
Quantification of mRNA
To quantify hybrid signal in the ovary interstitium, measurements were taken on randomly chosen 500 µm2 areas of tissue between follicles in the ovarian cortex, excluding thecal cells. To quantify hybrid signal in granulosa and thecal cells, 500 µm2 areas overlying dense, homogeneous populations of granulosa or thecal cells were scored in follicles of two size ranges determined by largest cross-section diameter: 100380 and 620-1000 µm (class B and D, as described in Vendola et al., 1998). The former were pre-antral with 36 layers of granulosa cells and an early thecal layer. The latter were small antral follicles with well-developed theca (Vendola et al., 1998
). There were no significant differences between values for class B and D follicles in any of the groups. Therefore, data for the two follicle populations were pooled and are presented simply as granulosa and theca cell values. These values represent the vast majority of follicles beyond the primary stage. There are too few large antral follicles (02 per animal) to obtain meaningful statistical data, and intermediate size follicles (class C, 380620 µm) are encompassed by the flanking classes. Hybrid signal was quantified by computerized silver grain counting using NIH Image, 1.57 and a Leica Laborlux microscope at a magnification of x400 under oil. Slide identifications were masked prior to analysis. Non-specific signal was determined as the number of grains over the relevant cell population in sense probe-hybridized sections; this count was subtracted from raw antisense counts prior to further analysis. The mean was taken from eight to ten measurements for each cell compartment for each animal. Group means for control, testosterone and DHT animals were compared using analysis of variance (ANOVA), followed by Fisher's least significant difference test if appropriate. P < 0.05 was taken as significant.
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Results |
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Discussion |
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The possibility that androgen-induced proliferation of granulosa and theca cells is mediated by local IGF-I action has a precedent in the uterus, where IGF-I is thought to be involved in oestrogen's proliferative effects (`oestromedin hypothesis'). Oestradiol's dramatic effects on uterine growth are spatiotemporally correlated with increased local IGF-I production (Murphy et al., 1987; Norstedt et al., 1989
; Adesanya et al., 1996
). It has recently been shown that oestradiol treatment fails to induce uterine growth in the IGF-I/ targeted gene deletion mouse (Adesanya et al., 1999
), supporting the view that IGF-I is critical to oestrogen's growth-promoting effects in the uterus. Androgens also stimulate IGF-I production and growth in the murine uterus (Sahlin et al., 1994
) and dihydroepiandrosterone, an androgen precursor, increases IGF-I production in cultured murine granulosa cells (Yan et al., 1997
). These data are consistent with the present findings of androgen-induced up-regulation of IGF-I expression in the primate ovary. To our knowledge, there have been no previous studies evaluating androgen effect on IGF-I receptor expression. The present data suggest that androgen-induced augmentation of local IGF-I and IGF-I receptor expression is instrumental in the stimulation of ovarian follicular and thecalinterstitial growth. However, available evidence from ovaries from women with polycystic ovarian syndrome (PCOS) has not yielded any consistent evidence for abnormal IGF-I concentrations (El-Roeiy et al., 1994
; Voutilainin et al., 1996
; Yap et al., 1997
). This could be due to sampling limitations inherent in working with surgical specimens, which make it difficult to obtain sufficient numbers of size-matched follicles to allow meaningful statistical comparisons. Alternatively, the acute augmentation of IGF-I and IGF receptor expression observed in our short-term studies may be a transient response to hyperandrogenism, no longer apparent in chronic conditions.
Since thecalinterstitial cells are androgenic, the fact that androgens promote ovarian thecalinterstitial growth (Vendola et al., 1998) may create the substrate for self-perpetuating hyperandrogenism. It is possible that even transient extra-ovarian hyperandrogenism stimulates excessive follicle and associated thecalinterstitial growth, which then becomes the source of ovarian hyperandrogenism. This sequence of events might account for the development of PCOS in girls with congenital adrenal hyperplasia (Lobo, 1984
). These observations may also help to explain why androgen receptor blockade is associated with reduction in follicle number and ovarian size in young women with PCOS (de Leo et al., 1998
).
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Acknowledgments |
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Notes |
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References |
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Submitted on April 19, 1999; accepted on June 17, 1999.