Studies conducted in rats have indicated that their degree of sexual arousal is sensitive to reductions in testosterone. Testosterone levels follow a circadian rhythm that peaks early each day, regardless of sexual activity. Serious side effects may include liver toxicity, heart disease (though a randomized trial found no evidence of major adverse cardiac events compared to placebo in men with low testosterone), and behavioral changes. It is unclear if the use of testosterone for low levels due to aging is beneficial or harmful. It is bound 65% to sex hormone-binding globulin (SHBG) and 33% bound weakly to albumin. The amount of testosterone synthesized is regulated by the hypothalamic–pituitary–testicular axis (Figure 2). In addition, the 3β-hydroxyl group is oxidized by 3β-hydroxysteroid dehydrogenase to produce androstenedione. The development in 1981 by Mario Ascoli of the hormone-dependent steroid producing mouse tumor Leydig cell line, MA-10, was an important step that, over the years, has allowed the undertaking of numerous studies designed to understand the mechanism regulating Leydig cell steroidogenesis . These studies indicated that LH regulates not only Leydig cell steroidogenesis but also membrane biogenesis and/or turnover within the Leydig cells. Later, Hall and his colleagues reported that testosterone and androstenedione were produced when interstitial cells, but not seminiferous tubules, were incubated with cholesterol. At about the same time, Hall and Eik-Nes and Ewing and Eik-Nes showed that pituitary gonadotropic hormones stimulate androgen formation by testes in vitro and ex vivo. About 60 years after that, Baillie demonstrated that these cells contain the enzyme 3β-hydroxysteroid dehydrogenase . Fifty years later, Bouin and Ancel first suggested that androgens are produced by the interstitial Leydig cells . Studies conducted have found direct correlation between testosterone and dominance, especially among the most violent criminals in prison who had the highest testosterone. It is therefore the challenge of competition among males that facilitates aggression and violence. The first is the challenge hypothesis which states that testosterone would increase during puberty, thus facilitating reproductive and competitive behavior which would include aggression. There are two theories on the role of testosterone in aggression and competition. In addition, a continuous increase in vaginal sexual arousal may result in higher genital sensations and sexual appetitive behaviors. There is a time lag effect when testosterone is administered, on genital arousal in women. Women's level of testosterone is higher when measured pre-intercourse vs. pre-cuddling, as well as post-intercourse vs. post-cuddling. Men who watch sexually explicit films also report increased motivation and competitiveness, and decreased exhaustion. have been undertaken on the relationship between more general aggressive behavior, and feelings, and testosterone.|Administering exogenous testosterone, known as testosterone replacement therapy, reverses many of the symptoms of low testosterone. In hypogonadal men in whom there are deficiencies in central stimulation (hypogonadotropic hypogonadism, Kallman's syndrome), serum testosterone can be elevated directly by administering LH or hCG, or indirectly with clomiphene or aromatase inhibitors. In some men, reduced serum testosterone results from reduced serum LH (hypogonadotropic hypogonadism) . There are many other men who also present with what is referred to as "low T," including men with sickle cell disease and spinal cord injury .|Testosterone may prove to be an effective treatment in female sexual arousal disorders, and is available as a dermal patch. Androgens may modulate the physiology of vaginal tissue and contribute to female genital sexual arousal. Men who watch a sexually explicit movie have an average increase of 35% in testosterone, peaking at 60–90 minutes after the end of the film, but no increase is seen in men who watch sexually neutral films. This reaction engages penile reflexes (such as erection and ejaculation) that aid in sperm competition when more than one male is present in mating encounters, allowing for more production of successful sperm and a higher chance of reproduction. Every mammalian species examined demonstrated a marked increase in a male's testosterone level upon encountering a novel female.|Once targeted to the outer mitochondrial membrane, cholesterol must be translocated to the inner mitochondrial membrane, and there converted to pregnenolone by CYP11A1. The protein was found to be localized to mitochondria and later was shown to result from the cleavage of a 37-kDa precursor protein. Testosterone biosynthesis begins with the conversion of cholesterol to pregnenolone by the mitochondrial enzyme CYP11A1. This leads to the initiation of cholesterol release from lipid droplets or from the plasma membrane. Cholesterol can be synthesized de novo from acetate 54, 55 and stored in lipid droplets, or it can come from Leydig cell membranes including the plasma membrane . The proximity of 3β-HSD to CYP17A1 and 17β-HSD3 in the smooth endoplasmic reticulum makes it possible for each intermediate involved in testosterone synthesis to move easily and directly to the next enzyme. Species-specific pathways for the biosynthesis of testosterone from pregnenolone were identified by measuring the secretion products of in vitro perfused testes infused with specific intermediates and inhibitors .}