1. Testosterone backgrounder
Module 1
Testosterone backgrounder
Approval Number: G.MKT.GM.MH

2. Testosterone biosynthesis and its regulation
Module 1: Testosterone backgrounder
Testosterone biosynthesis and its regulation
Approval Number: G.MKT.GM.MH

3. Testosterone
Testosterone is the most important steroid made by the testis1,2
A healthy adult man produces ~6–7 mg of testosterone per day from the Leydig cells in the testes1,2
Testosterone mainly acts as an androgen, but can also be aromatized into estrogens
This provides ~25% of total daily 17β-estradiol production2
The half-life of free testosterone in the blood is only ~12 min1
Seminiferous tubules
Testosterone-producing Leydig cells
1. Nieschlag E et al. Andrology: Male reproductive health and dysfunction, 3rd edition, Springer, O’Donnell L et al. Endocrinology of the male reproductive system and spermatogenesis. Endotext. South Dartmouth (MA); 2017.

4. Testosterone biosynthesis
Testosterone is synthesized from free cholesterol in a process involving 5 enzymatic stages
Leydig cell LH
ATP
cAMP AC
Mitochondria
Smooth ER
Free cholesterol CE
StAR, PBR
Pregnenolone
Progesterone
17-Hydroxy- progesterone
Androstene- dione
Testosterone
17-Hydroxy- pregnenolone
Dehydroepi- androsterone
Androstenediol
3β-HSD
17β-HSD
17,20 Lyase
17α-Hydroxylase
P450scc
The most significant and time-critical stage in the enzymatic cascade is the conversion of cholesterol to pregnenolone. This takes place on the inner side of the membrane of the mitochondria, where the membrane’s own enzyme cytochrome P450scc (side-chain cleavage) catalyzes three consecutive stages: first hydroxylation on atom C20, then on atom C22 and then cleavage between C20 and C22, giving rise to pregnenolone and isocaproic acid. Cytochrome P450ssc, also known as 20,22-desmolase, is the crucial enzyme in all the steroid-producing tissues (adrenal gland, ovary) and is encoded by a gene on chromosome 15. Pregnenolone is the parent substance of all biologically active steroid hormones (corticosteroids, mineralocorticoids, gestagens, estrogens, androgens) and leaves the mitochondria by diffusion. It undergoes further processing in the endoplasmic reticulum. There are two pathways available: these are called the 4- or 5-synthesis pathways, depending on whether the double bond is located in ring A or ring B. The 5-synthesis pathway is preferred in man.
AC, adenylyl cyclase; ATP, adenosine triphosphate; cAMP, cyclic adenosine monophosphate; CE, cholesteryl ester; ER, endoplasmic reticulum; HSD, hydroxysteroid dehydrogenase; LH, luteinizing hormone; P450scc, cholesterol side-chain cleavage enzyme; PBR, peripheral-type benzodiazepine receptor; StAR, steroidogenic acute regulatory protein
Miller WL & Auchus RJ. Endocr Rev. 2011;32(1):81–151; Nieschlag E et al. Andrology: Male reproductive health and dysfunction, 3rd edition, Springer, 2010.

5. Cholesterol is the key substrate for testosterone biosynthesis
Conversion of cholesterol to pregnenolone is the first and rate-limiting step in testosterone biosynthesis1
The cholesterol utilized for steroidogenesis is derived from multiple sources, including:2
De novo synthesis in the ER
Mobilization of CEs stored in lipid droplets via the action of CE hydrolase, which is mediated by LH-induced formation of cAMP and stimulation of protein kinase A
Plasma lipoprotein-derived CEs obtained by either LDLR-mediated endocytosis and/or SR-BI-mediated selective uptake
cAMP, cyclic adenosine monophosphate; CE, cholesteryl ester; ER, endoplasmic reticulum; LDLR, low-density lipoprotein receptor; LH, luteinizing hormone; SR-BI, scavenger receptor class B type 1
1. Haider SG. Endocrinology. 2007;148(6):2581–2. 2. Hu J et al. Nutr Metab (Lond). 2010;7:47.

6. The hypothalamus regulates testicular function and testosterone production
Regular, intermittent GnRH secretion from hypothalamic neurons stimulates biosynthesis of LH and FSH by the pituitary gland1,2
LH and FSH travel via the bloodstream to the testes, where they initiate testosterone production and spermatogenesis, as well as systemic testosterone secretion and virilization3
This episodic GnRH release results in pulsatile LH secretion every ~60–120 min on average, although considerable variability in LH pulse patterns can exist2,3
GnRH pulse amplitude and frequency influence the amount of LH and FSH secreted by the pituitary gland4
Hypothalamic–pituitary regulation
The hypothalamus controls the function of the testis by means of the pituitary hormones, LH and FSH. In the hypothalamus, GnRH is produced under the stimulating and inhibiting influence of neurotransmitters. GnRH promotes the production and release of the gonadotropins LH and FSH in the pituitary gland.
GnRH is secreted in episodically, leading to peaks of LH secretion every ~60–120 min. This basic rhythm is modulated by numerous neurotransmitters. Endorphins display cycle-dependent effects in women but have a predominantly inhibitory action in men. Testosterone and progesterone slow down this pulse rate, presumably mediated by -endorphins. The adverse effect of stress on reproductive function is well known. Several factors are involved: corticotropin releasing hormone (CRH) inhibits GnRH secretion through direct neuronal contact between the paraventricular nucleus and preoptic region. The level of prolactin, which is often raised in stress, further reduces the GnRH pulse rate.
FSH, follicle-stimulating hormone; GnRH, gonadotropin-releasing hormone; LH, luteinizing hormone
1. O’Donnell L et al. Endocrinology of the male reproductive system and spermatogenesis. Endotext. South Dartmouth (MA); Handelsman DJ. Androgen physiology, pharmacology and abuse. Endotext. South Dartmouth (MA); Hayes F et al. Hypogonadotropic hypogonadism (Hh) and gonadotropin therapy. Endotext. South Dartmouth (MA); Nieschlag E et al. Andrology: Male reproductive health and dysfunction, 3rd edition, Springer, 2010.

7. Role of LH in testosterone biosynthesis
LH promotes Leydig cell steroidogenesis by increasing cholesterol availability and activating rate-limiting steroidogenic enzyme and cholesterol-transport proteins1
LH thus regulates testosterone biosynthesis in two ways:
Acutely at the level of StAR2
Chronically by enhancing the transcription of genes that encode a variety of enzymes in the steroidogenic pathway3
The pulsatile pattern of trophic hormone exposure maintains Leydig cell sensitivity to LH, sustaining mature male patterns of testicular testosterone secretion1
cAMP, cyclic adenosine monophosphate; LH, luteinizing hormone; StAR, steroidogenic acute regulatory protein
1. Handelsman DJ. Androgen physiology, pharmacology and abuse. Endotext. South Dartmouth (MA); Haider SG. Endocrinology. 2007;148(6):2581–2. 3. O’Donnell L et al. Endocrinology of the male reproductive system and spermatogenesis. Endotext. South Dartmouth (MA); 2017.

8. Control of testosterone biosynthesis in the hypothalamic-pituitary-testis axis
Testosterone is key to the testicular negative feedback cycle on the hypothalamus and pituitary gland1
It acts directly on androgen receptors and indirectly, by aromatization (oxidation) in the hypothalamus to estradiol1
This leads to reductions in GnRH pulse frequency and decreases in the amplitude of LH pulses, caused by reduced GnRH secretion and gonadotropin response to GnRH stimulation1
Pituitary LH and FSH secretion is also suppressed by feedback inhibition via testosterone and estradiol2
Feedback control of LH production in man occurs via testosterone and its metabolite estradiol. Testosterone has an inhibitory effect on the neurons producing GnRH, and exerts only a slight suppressant effect on pituitary LH production. In contrast, estradiol has an inhibitory action on the pituitary gland and hypothalamus.
Testosterone and estrogens also exert a negative feedback effect on FSH via their effect on GnRH.
FSH, follicle-stimulating hormone; GnRH, gonadotropin-releasing hormone; LH, luteinizing hormone
1. Handelsman DJ. Androgen physiology, pharmacology and abuse. Endotext. South Dartmouth (MA); O’Donnell L et al. Endocrinology of the male reproductive system and spermatogenesis. Endotext. South Dartmouth (MA); 2017.

9. Control of testosterone biosynthesis in the hypothalamic-pituitary-testis axis (2)
Other regulatory molecules include:
Inhibins, hormone inhibitors of FSH production secreted by Sertoli cells in the testes
Follistatin, a pituitary autocrine glycoprotein, which inhibits FSH secretion by binding to and blocking the action of activins, paracrine hormones that enhance FSH biosynthesis and secretion
Kisspeptins, a family of neuropeptides localized to the arcuate nucleus in the brain, which are upstream stimulators of GnRH secretion
GnIH, which acts upstream of GnRH and may also operate at the level of the testes as an autocrine/paracrine regulator of steroidogenesis
In addition to a number of other proteins, the hormones inhibin and activin are formed in Sertoli cells under the influence of FSH. Inhibin is an important component in the feedback system controlling FSH secretion. In isolated functional disturbances of the Sertoli cells (e.g. dysplastic gametes or following radiotherapy or chemotherapy), inhibin deficiency is marked by a sharp rise in FSH, while LH remains at normal levels. The physiological significance of FSH-stimulating activins has not been conclusively explained; however, they appear to be less important.
FSH, follicle-stimulating hormone; GnIH, gonadotropin-inhibitory hormone; GnRH, gonadotropin-releasing hormone
O’Donnell L et al. Endocrinology of the male reproductive system and spermatogenesis. Endotext. South Dartmouth (MA); 2017.

10. Regulation of testosterone secretion
GnRH
Inhibins
Gonadotropins
(LH, FSH)
Testosterone
(Estradiol)
Pituitary gland
Hypothalamus
Testis + –
Activins
Follistatin
Kisspeptins
GnIH
FSH, follicle-stimulating hormone; GnIH, gonadotropin-inhibitory hormone; GnRH, gonadotropin-releasing hormone; LH, luteinizing hormone
Adapted from Nieschlag E et al. Andrology: Male reproductive health and dysfunction, 3rd edition, Springer, 2010.

11. Testosterone binding
Approximately 98% of testosterone is bound to transport proteins and only 2% is free or ‘biologically active’
SHBG is the carrier protein for testosterone and estradiol, and has a 3-fold higher affinity for the former
SHBG synthesis occurs mainly in the liver and is regulated by the opposing actions of sex steroids
Androgen inhibits SHBG synthesis
Estrogen stimulates SHBG synthesis
An increase in SHBG plasma concentrations leads to an acute decrease in free testosterone and simultaneous stimulation of testosterone biosynthesis
Free 2%
Bound with higher affinity to SHBG
54%
Bound with lower affinity to albumin
44%
SHBG, sex hormone-binding globulin
Nieschlag E et al. Andrology: Male reproductive health and dysfunction, 3rd edition, Springer, 2010.

12. Biological effects of testosterone and its metabolites
Module 1: Testosterone backgrounder
Biological effects of testosterone and its metabolites

13. Testosterone is important in every phase of male life
Testosterone is the most important endogenous sex hormone and androgen in the male
It is vital for the proper development and functioning of the male phenotype
Testosterone and its main metabolites, DHT and estradiol, exert a number of anabolic and metabolic effects that can influence many physical and mental functions in men
Stage of male life
Effect of testosterone
In utero (embryo)
Sex differentiation
Puberty
Virilization
Adulthood
Maintenance of the male phenotype
Sexual function
Anabolic effects
DHT, dihydrotestosterone
Nieschlag E et al. Andrology: Male reproductive health and dysfunction, 3rd edition, Springer, 2010.

14. Testosterone acts on diverse target tissues to cause a variety of biological effects
Biological effect of testosterone and its metabolites
Reproductive organs1,2
Prenatal sex differentiation; pubertal virilization and development of testes, penis, epididymis, seminal vesicles and prostate; in adults, maintenance of reproductive organs, potency and sexual function, and initiation/maintenance of spermatogenesis
Muscle1
Increased volume, strength and lean body mass
Skin and hair1
Increased sebum production, hair growth and male hair pattern
Liver1,3
Increased synthesis of clotting factors, hepatic triglyceride lipase, sialic acid, α1-antitrypsin, haptoglobin; decreased production of SHBG and other hormone-binding proteins, transferrin, fibrinogen
Lipids1,2
Improvements in dyslipidemia; decreased plasma total cholesterol, LDL-C and triglycerides, and increased plasma HDL-C concentrations
Blood glucose2
Improvements in hyperglycemia; decreased fasting blood glucose and HbA1c
Bone1
Accelerated linear growth; closure of epiphyses; increased bone mass
Brain1
Improvements in cognitive function, socialization, concentration, self-confidence, mood, libido; in adults, possible neuroprotective effect
Hematopoietic system1,2
Stimulation of erythropoietin biosynthesis in the kidney; direct effects on hematopoietic stem cells, leading to increased hemoglobin production
Immune cells4
Suppression of humoral and cellular immune responses; anti-inflammatory effect
HbA1c, glycated hemoglobin; HDL-C, high-density lipoprotein cholesterol; LDL-C, low-density lipoprotein cholesterol; SHBG, sex hormone-binding globulin
1. Nieschlag E et al. Andrology: Male reproductive health and dysfunction, 3rd edition, Springer, Traish AM. Sex Med Rev. 2018;6(1):86– Nebido® Product Monograph, Bayer AG, Cutolo M et al. Ann N Y Acad Sci. 2002;966:131–42.

15. Conversion of testosterone to DHT and estradiol is a necessary process for its biological action
Testosterone is metabolized to DHT and estradiol by 5α-reductase and aromatase, respectively1
Testosterone and DHT bind to the same androgen receptor, while estradiol binds to the estrogen receptor, to regulate gene expression in target tissues1,2
Androgen and estrogen receptors are members of the steroid nuclear receptor superfamily and have a highly homologous structure, differing mostly in the binding domain2
Compared with testosterone, DHT is a more potent androgen:
Higher binding affinity for the androgen receptor2
3–10 times greater molar potency in transactivation2
Slower dissociation from the androgen receptor3
Greater ability to slow natural degradation of the androgen receptor3
The complete spectrum of action of testosterone therefore incorporates effects that are indirectly induced by conversion to DHT and estradiol
DHT, dihydrotestosterone
1. Nieschlag E et al. Andrology: Male reproductive health and dysfunction, 3rd edition, Springer, Handelsman DJ. Androgen physiology, pharmacology and abuse. Endotext. South Dartmouth (MA); Zhou ZX et al. Mol Endocrinol. 1995;9(2):208–18.

16. Androgen receptor effects Estrogen receptor effects
Direct and indirect biological effects of testosterone, DHT and estradiol
Testosterone
DHT
Estradiol
5α-reductase
Aromatase
Sex differentiation
Musculature
Secondary hair growth
Sebum production
Prostate development
Spermatogenesis
Bone mass
Erythropoiesis
Psychotropic action
Potency/libido
Lipid metabolism
Epiphyseal closure
Androgen receptor effects
Estrogen receptor effects
DHT, dihydrotestosterone
Nieschlag E et al. Andrology: Male reproductive health and dysfunction, 3rd edition, Springer, 2010; Rochira V et al. Estrogens and male reproduction. Endotext. South Dartmouth (MA); 2016.

17. Summary
Testosterone is synthesized from cholesterol in the Leydig cells of the testis in 5 enzymatic stages
As a necessary process for its biological action, testosterone is also converted to DHT by 5α-reductase and estradiol by aromatase
Testosterone biosynthesis and secretion is regulated by the hypothalamic-pituitary-testis axis
This involves pulsatile GnRH secretion from the hypothalamus, LH and FSH biosynthesis by the pituitary gland, negative feedback from testosterone and estradiol, and the action of other regulatory molecules, including inhibins, follistatin, kisspeptins and GnIH
Approximately 98% of testosterone is bound to the transport proteins, SHBG and albumin, and only 2% is free or ‘biologically active’
Testosterone is the most important endogenous sex hormone and androgen in men, and is vital for proper male development and physical and mental functioning
DHT, dihydrotestosterone; FSH, follicle-stimulating hormone; GnIH, gonadotropin-inhibitory hormone; GnRH, gonadotropin-releasing hormone; LH, luteinizing hormone; SHBG, sex hormone-binding globulin