Testosterone Synthesis Pathways
by Benjamin Bunting BA(Hons) PGCert
Written by Ben Bunting: BA, PGCert. (Sport & Exercise Nutrition) // British Army Physical Training Instructor // S&C Coach.
In men, the development of the genitalia and establishment of secondary male characteristics are reliant on the testosterone synthesis pathway. Testosterone is made by Leydig cells in the testis, which also produce other essential steroids for male development, including estradiol and DHT. Several other pathways are involved, but the biosynthesis of testosterone is an essential component of these processes. To understand these pathways, the following information should be helpful.
Leydig cell steroidogenesis
We have recently discovered that the mitochondria in Leydig cells play a role in testosterone synthesis. We were able to evaluate the function of mitochondria by flow cytometry using JC-1 staining. We also examined the levels of intracellular ROS using H2DCFDA staining. We found that the amount of oxLDL significantly decreased testosterone synthesis. This suggests that the mitochondria in Leydig cells may be a key regulator of male fertility.
The steroidogenic machinery in Leydig cells depends on the structure of mitochondria. The mitochondrial membrane contains the acute regulatory protein for steroid hormone synthesis. It is responsible for the conversion of cholesterol into pregnenolone and testosterone. CYP17A and 17b-HSD act in the smooth endoplasmic reticulum. These proteins are critical in Leydig cell function and can be damaged by reactive oxygen species (ROS) and a disruption of the mitochondrial membrane permeability.
LH stimulates the steroidogenic pathway in Leydig cells by triggering the synthesis of cAMP from ATP. This in turn induces the synthesis of protein kinase A (PKA), which is required for cholesterol transport to mitochondria. This in turn activates the enzymes in the endoplasmic reticulum (SER).
Hypoxia reduced NRF1 protein and mRNA expression in the testis of male mice. Similarly, it also decreased serum testosterone. This suggests that NRF1 is involved in testicular steroidogenesis. Hypoxia may cause diseases in other organs and the reproductive system. Hypoxia also decreased NRF1, which promotes testicular steroidogenesis. We believe this mechanism may play a role in male fertility disorders.
The ERK1/2 and testosterone synthesis pathways are critical for female reproduction, but what are their exact roles? One study showed that ERK1/2 inhibits the expression of Raf, a steroid hormone receptor. The study also showed that ERK1/2 phosphorylates Raf, suggesting that the two pathways are negatively regulated. However, a recent study suggests that DHEA acts directly on the ERK1/2 pathway and induces androgen excess.
After pre-incubation, p-ERK1/2 levels were reduced in pGCs. The level of total CREB protein did not change when the testosterone synthesis pathway inhibitor was used in the experiments. In the control group, levels of p-CREB proteins were significantly increased in response to DHEA treatment, while the total CREB protein did not change. Thus, p-ERK1/2 is a marker for the synthesis of testosterone.
A key role for steroid hormone synthesis in Leydig cells is the presence of a steroid hormone acute regulatory protein, or CRSP. Cholesterol transported into the mitochondrial matrix is converted to pregnenolone by the cytochrome P450scc. The latter, meanwhile, is converted to testosterone by CYP17A and 17b-HSD in the smooth endoplasmic reticulum. However, low levels of testosterone are associated with male infertility, and disorders of the male reproductive system. Although the mechanism is unclear, the results suggest that a p38 inhibitor may be a key steroid hormone regulator in the Leydig cell.
DHEA activates p-ERK1/2 and modulates 3b-HSD and 17b-HSD proteins. In addition, it has an effect on the expression of aromatase and 17b-HSD. Inhibition of p-ERK1/2 can reverse the changes in these genes, and a decrease in testosterone is seen in the DHEA-treated group. In addition, DHEA has a positive impact on the expression of these three proteins in primary Leydig cells.
The effect of CBS overexpression on testis spermatogenesis was reversed by H89, a specific inhibitor of PKA. CBS increased the expression of p-PKA, reduced cAMP level, and improved inflammation status of testis. Although CBS had no impact on PKA, it promoted the phosphorylation of the enzyme. This finding supports the hypothesis that CBS overexpression can help restore testosterone synthesis by inhibiting PDE4A and 8A.
Constitutive activation of PKA leads to accelerated cortical renewal. It also promotes the differentiation of zF cells and their conversion to reticularis-like zone at the corticomedullary junction. Both of these changes correlate with the recruitment of subcapsular and capsular progenitors. However, PKA signaling has been shown to antagonize androgen action through mevalonate pathway.
In the mouse, the hormones LH and hCG stimulate steroid synthesis through different mechanisms. Both hCG and LH activate the common receptor (LHCGR) and adenylyl cyclase through Gas protein. PkA activates cAMP-response element binding protein (ERK1/2) and increases intracellular cAMP. It also induces ERK1/2 phosphorylation. Both hormones result in a rise in intracellular cAMP. The downstream Stard1 gene expression is increased. Both hormones promote steroid synthesis within physiological limits.
LH/LHR/cAMP/PKA are the classical molecular signaling pathways for testosterone synthesis. These pathways control steroid hormone production in the testes. Hence, inhibiting LH/LHR/cAMP/PKA is important for treating conditions associated with testosterone secretion. Inhibition of PDE may also promote the secretion of testosterone. However, the significance of H2S in male reproduction is not fully understood.
cAMP-dependent protein kinase A
The cAMP-dependent protein kinases (AMPKs) control several fundamental physiological processes, including the steroidogenesis process. The activation of AMPKs leads to an increase in cAMP, which in turn activates the cAMP-dependent protein kinase (cAMP-PKA). Inhibiting PDE4 and PDE8 increases cAMP levels, increasing activity of cAMP-dependent protein kinases and testosterone synthesis.
The cAMP-dependent protein kinases A and B and the steroid hormone testosterone regulate different intracellular pathways. Interestingly, testosterone increases activity of MEF2C in cardiac myocytes, a pathway that regulates ventricular myocytes' differentiation. Moreover, testosterone induces the induction of the cAMP-dependent protein kinase A (CaMKII) enzyme.
PKA is a tetramer enzyme with two catalytic and two regulatory subunits. It is composed of two types of regulatory subunits, RIa and RIIa. The regulatory subunits activate the catalytic subunits, which in turn phosphorylate the substrate proteins. The two types of subunits are broadly distributed in tissues, with the RIa and RIIa being more abundant in the brain. RIb is found in the fat and some endocrine tissues.
In prostate cancer, PKA and CREB are important for tumor reversion, and ERK2 activation promotes cellular differentiation and apoptosis. Moreover, these two molecules are essential for testosterone synthesis. Although cAMP-dependent protein kinase A and testosterone synthesis pathways play a crucial role in cancer, there are some controversial areas of research.
CAMKs play a crucial role in the development of acute myeloid leukemia. In a study published in J Hematol Oncol, Insel PA and colleagues investigated the role of cAMP in S49 lymphoma cells. The cells were stimulated with testosterone or were not. To determine the exact role of CAMKs in cancer progression, the authors looked at the interaction between cAMP and mitochondria.
The synthesis of testosterone is required for sex development and male characteristics. Testosterone is primarily produced in Leydig cells. Autophagy is a highly active process. A recent study showed that steroidogenic cells with impaired autophagy exhibited marked changes in sexual behavior. Furthermore, autophagy-deficient Leydig cells had impaired testosterone synthesis. However, the study does not suggest that autophagy is the sole cause of testosterone synthesis.
Activation of LH and hCG by pErk1/2 and adenylyl cyclase results in a sustained increase in intracellular cAMP. This induces the activation of protein kinase A (PKA), which mediates ERK1/2 phosphorylation. These events in turn regulate the expression of target genes. This study also suggests that LH and hCG might be involved in the same pathway.
A second study used 10,000 IU hCG as an injection to measure the steroidogenic activity of a single hormone. Testosterone concentrations increased up to 2 to ten times higher than control levels at 60 minutes. Interestingly, testosterone levels peak at two to three days after hCG administration, and testosterone levels return to baseline eight to 10 days later. These findings suggest that these hormones are important for male sexual development.
In a study of rFSH and hCG in male androgen biosynthesis, researchers examined the role of gonadotropins in the synthesis of testosterone. The serum steroid hormone profiles of 25 CHH males were analysed using liquid chromatography-tandem mass spectrometry. The results were compared to those of healthy control males. The controls were matched for age, BMI, and serum testosterone levels.