Figure 3. FSH increases FSH mRNA in adherent human monocytes.

There was variation in quantitative response between assays and between FSH preparations, but all cell preparations and all FSH sources gave qualitatively similar effects.

A. In 7 day cultures of adherent monocytes and osteoclasts, production of TNFα mRNA by real-time PCR was increased by addition of 25 ng/ml FSH. ACTH, 100 nM, is included as a control. Medium was Dubelco’s MEM with 10% fetal calf serum and 10 ng/ml CSF-1; RANKL for osteoclast differentiation was 40 ng/ml. N=2., mean ± range.

B. Different sources of FSH increase TNFα production; admixture with LH had no effect. TNFα mRNA transcription in response to three preparations of human FSH are shown. Highly pure and partially pure FSH caused significant increases in TNFα mRNA relative to controls without FSH (left bar) A mixture of pure FSH with an equal amount LH (third bar), did not change response relative to FSH alone. N=2, mean ± range.

C. Endotoxin does not contribute to the FSH effect. The Limulus amebocyte assay was used; endotoxin controls at 0.01 and 0.1 EU/ml are shown (right two bars). Osteoclast medium and each FSH sample gave results corresponding to endotoxin levels of 0.001 EU/ml or less. N=2, mean ± range.

D. Cyclic AMP is not produced in response to FSH, 25 ng/ml, 15 minutes, added to mononuclear cell cultures. A standard curve was used to calculate cAMP. Control untreated and FSH treated mononuclear cells and osteoclasts are shown, in addition to a forskolin (1 μM, 15 minutes) positive control cells (right bar). Note the logarithmic scale.