Androgens are essential for the normal function of mature antral follicles but also
have a role in the early stages of follicle development. Polycystic ovary syndrome
(PCOS), the most common cause of anovulatory infertility, is characterized by
androgen excess and aberrant follicle development that includes accelerated early
follicle growth. We have examined the effects of testosterone and dihydrotestosterone
(DHT) on development of isolated mouse preantral follicles in culture with the
specific aim of investigating interaction with follicle-stimulating hormone (FSH),
the steroidogenic pathway, and growth factors of the TGFβ
superfamily that are known to have a role in early follicle development. Both
testosterone and DHT stimulated follicle growth and augmented FSH-induced growth and
increased the incidence of antrum formation among the granulosa cell layers of these
preantral follicles after 72 hours in culture. Effects of both androgens were
reversed by the androgen receptor antagonist flutamide. FSH receptor expression was
increased in response to both testosterone and DHT, as was that of
Star, whereas Cyp11a1 was down-regulated. The
key androgen-induced changes in the TGFβ signaling pathway
were down-regulation of Amh, Bmp15, and their
receptors. Inhibition of Alk6 (Bmpr1b), a putative
partner for Amhr2 and Bmpr2, by dorsomorphin
resulted in augmentation of androgen-stimulated growth and modification of
androgen-induced gene expression. Our findings point to varied effects of androgen on
preantral follicle growth and function, including interaction with FSH-activated
growth and steroidogenesis, and, importantly, implicate the intrafollicular
TGFβ system as a key mediator of androgen action. These
findings provide insight into abnormal early follicle development in PCOS.
The obligatory role of follicle-stimulating hormone (FSH) in normal development and function of ovarian antral follicles is well recognized, but its function in preantral growth is less clear. The specific objective of this study was to investigate the response, in culture, to FSH of mouse preantral follicles of increasing size, focusing particularly on growth rate and gene expression. Preantral follicles were mechanically isolated from ovaries of C57BL/6 mice, 12 to 16 days postpartum, and single follicles cultured for up to 96 hours in medium alone (n = 511) or with recombinant human FSH 10 ng/mL (n = 546). Data were grouped according to initial follicle diameter in 6 strata ranging from <100 to >140 μm. Follicles of all sizes grew in the absence of FSH (P < 0.01, paired t test). All follicles grew at a faster rate (P < 0.0001) in the presence of 10 ng/mL FSH but larger follicles showed the greatest change in response to FSH. Even the smallest follicles expressed FSH receptor messenger RNA (mRNA). FSH-induced growth was inhibited by KT5720, an inhibitor of protein kinase A (PKA), implicating the PKA pathway in FSH-induced follicle growth. In response to FSH in vitro, FSH receptor mRNA (measured by quantitative polymerase chain reaction) was reduced (P < 0.01), as was Amh (P < 0.01), whereas expression of StAR (P < 0.0001) and the steroidogenic enzymes Cyp11a1 (P < 0.01) and Cyp19 (P < 0.0001) was increased. These results show heterogeneous responses to FSH according to initial follicle size, smaller follicles being less FSH dependent than larger preantral follicles. These findings strongly suggest that FSH has a physiological role in preantral follicle growth and function.
SummaryPreterm birth occurs in 10% of pregnancies and is a major cause of neonatal morbidity and mortality. The majority of cases of early preterm labour are associated with infection/inflammation, which places the fetal central nervous system at risk. Targeting immune activation is therefore an appealing therapeutic strategy for the prevention of preterm labour and neonatal brain injury. The expression of many labour-associated and inflammatory-response genes is controlled by the transcription factors nuclear factor-jB (NF-jB) and activator protein-1 (AP-1), which makes them therapeutic targets of interest. Sulfasalazine (SASP) has been shown to inhibit NF-jB and reduce lipopolysaccharide-induced cytokine concentrations in fetal membrane explants and reduce the rate of Escherichia coli-induced preterm labour in mice. Its effects upon AP-1 in the context of pregnancy are unknown. In this study the effect of SASP on interleukin-1b (IL-1b) -induced NF-jB and AP-1 activity, cytokine production and cyclo-oxygenase-2 (COX-2) expression was examined in amniocytes and myocytes. A supra-therapeutic concentration (5 mM) was required to inhibit IL-1b-induced NF-jB (P < 0Á0001) in amniocytes and IL-1b-induced NF-jB (P < 0Á01), AP-1 (P < 0Á01) and COX-2 (P < 0Á05) in myocytes. Despite inhibiting IL-1b-induced cytokines, a basal increase in IL-6 (P < 0Á01), IL-8 (P < 0Á0001) and tumour necrosis factor-a (TNF-a) (P < 0Á001) was seen with 5 mM SASP in amniocytes, and significant cytotoxic effects were seen in myocytes. The therapeutic concentration of 0Á015 mM had no inhibitory effects on pro-inflammatory mediators, but led to an augmented response to IL-1b-induced IL-6 (P < 0Á01), IL-8 (P < 0Á05) and TNF-a (P < 0Á05) in amniocytes and IL-8 (P < 0Á05) in myocytes. SASP is therefore an unlikely therapeutic candidate for the prevention of inflammation-induced preterm labour.
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