Previous studies have reported the roles of Ca²⁺ in steroidogenesis. The present study has investigated an inhibitory effect of Ca²⁺ influx through L-type Ca²⁺ channels on gene expression of steroidogenic acute regulatory (STAR) protein that regulates the transfer of substrate cholesterol to the inner mitochondrial membrane for steroidogenesis. Blocking Ca²⁺ influx through L-type Ca²⁺ channels using the selective Ca²⁺ channel blocker, nifedipine, markedly enhanced cAMP-induced STAR protein expression and progesterone production in MA-10 mouse Leydig cells. This was confirmed by utilization of different L-type Ca²⁺ channel blockers. Reverse transcription-PCR analyses of Star mRNA and luciferase assays of Star promoter activity indicated that blocking Ca²⁺ influx through L-type Ca²⁺ channels acted at the level of Star gene transcription. Further studies showed that blocking the Ca²⁺ channel enhanced Star gene transcription by depressing the expression of DAX-1 (NR0B1 as listed in the MGI Database) protein, a transcriptional repressor of Star gene expression. It was also observed that there is a synergistic interaction between nifedipine and cAMP. Normally, sub-threshold levels of cAMP are unable to induce steroidogenesis, but in the presence of the L-type Ca²⁺ channel blocker, they increased STAR protein and steroid hormone to the maximal levels. However, in the absence of minimal levels of cAMP, none of the L-type Ca²⁺ channel blockers are able to induce Star gene expression. These observations indicate that Ca²⁺ influx through L-type Ca²⁺ channels is involved in an inhibitory effect on Star gene expression. Blocking L-type Ca²⁺ channel attenuated the inhibition and reduced the threshold of cAMP-induced Star gene expression in Leydig cells.

During the aging process of males, testosterone biosynthesis declines in testicular Leydig cells resulting in decreases in various physiological functions. To explore the possibility of delaying the decline using food supplements, we have studied steroidogenic effects of a natural flavonoid, chrysin, in mouse Leydig cells. Chrysin dramatically increased cyclic AMP (cAMP)-induced steroidogenesis in MA-10 mouse Leydig tumor cells. This result was confirmed using Leydig cells isolated from mouse testes. The steroidogenic effect of chrysin is not associated with an increase in expression of the P450 side-chain cleavage enzyme, required for the conversion of cholesterol to pregnenolone. In addition, when 22(R)hydroxylcholesterol was used as a substrate, chrysin induced a non-significant increase in steroid hormone, suggesting that the majority of the observed increase in steroidogenesis was due to the increased supply of substrate cholesterol. These observations were corroborated by showing that chrysin induced a marked increase in the expression of steroidogenic acute regulatory (StAR) protein, the factor that controls mitochondrial cholesterol transfer. Also, chrysin significantly increased StAR promoter activity and StAR mRNA level. Further studies indicated that this compound depressed expression of DAX-1, a repressor in StAR gene transcription. In the absence of cAMP, chrysin did not increase steroidogenesis. However, when a sub-threshold level of cAMP was used, StAR protein and steroid hormone were increased by chrysin to the levels seen with maximal stimulation of cAMP. These results suggest that while chrysin itself is unable to induce StAR gene expression and steroidogenesis, it appears to function by increasing the sensitivity of Leydig cells to cAMP stimulation.

The essential role of arachidonic acid (AA) in steroidogenesis has been previously demonstrated. The present study continues the investigation into how AA regulates steroidogenesis by examining the effects of epoxygenase-derived AA metabolites on cAMP-stimulated steroidogenic acute regulatory (StAR) gene expression and steroid hormone production in MA-10 mouse Leydig cells. The HPLC analysis of cell extracts from MA-10 cells treated with the cAMP analog dibutyryl cAMP (dbcAMP) demonstrated an increase in three epoxygenase-generated AA metabolites: 5,6-epoxyeicosatrienoic acid (EET), 8,9-EET, and 11,12-EET. Incubating MA-10 cells with each of the EETs induced a dose–dependent increase in StAR protein expression and steroid hormone production in the presence of dbcAMP. These metabolites also significantly enhanced StAR gene transcription as determined by luciferase assays of StAR promoter activity and reverse transcriptase-PCR analysis of StAR mRNA levels. While the EETs enhanced steroidogenesis, inhibiting the activity of protein kinase A (PKA) abolished the stimulatory effects of these AA metabolites on StAR expression and steroid hormone production. This study suggests that cAMP stimulation of MA-10 cells increases epoxygenase-generated AA metabolites and the co-action of these metabolites with PKA significantly increases StAR gene expression and steroid hormone production.