After synthesis, testosterone diffuses fron the interstial space bathes Leydig cells into the venous system via the pampiniform plexus, at which point it is avaitable to affect target tissues.
More precisely, the biologicaally available testosterone is available to
affect target tissues. Of the total testosterone in circulation, 2% remains free, or unbound, and 98% is associated with plasma proteins.
This includes the 54% bound to albumin and 44% bound to sex hormone binding globulin (SHBG).
According to the free hormone hypothesis, it is only the unbound, free testosterone that is able to passively difusse across cell membranes and cause downstream effects.
Therefore in order for bound testosterone to contribute an appreciable effect, it must first dissociate from its binding protein. In the case of testosterone bound to SHBG, this quantity is negligible as the binding affinity is too high to dissociate a significant amount of hormone .
Conversely in the case of albumin, which has a dissociaton constant many orders of magnitude lowe, this does impact the total amount of testosterone available to cells.
Thus, bioavailable testosterone is typically defined as unbound hormone plus albumin-bound hormone.
From a laboratory perspective, total testosterone can be easily assayed and is the initial step for the evaluation of andeogen insuffiency.
Assessment of free testosterone is more espensive and time consuming, generally requiring the use of equilibrium dialysis or precipitation assays.
Despite the well-established hypotheses regarding bioavailable testosterone, there is now some evidence suggesting the existence
of endocytotic cellular uptake pathways for carrierbound steroids, however, these are likely to play a role only in limited tissues under specific physiologic conditions.
The total level of testosterone is dependent upon the relative rates of its production and metabolism. In terms of metabolism, testosterone may follow one of three enzymatic pathways.
In the first, cytochrome P450 ( CYP19) aromatizes testosterone to create the C18 estrogen estradiol.
While this conversion primarily takes place in adipose tissues. CYP19 is expressed in a number of tissues , including Leyding cells.
5 α Reductase CYP19
DHT ←←←← Testosterone →→→→ Estradiol
↓
↓
↓
↓
Hepatic
Metabolism
– Testosterone conversion and metabolism
Alternatively, testosterone may be reduced to 5ἀ-dihydrotestosterone (DHT) by 5alpha reductase.
Both testosterone and dihydrotestosterone bind to the same androgen receptor; however, DHT, as the most potent natural androgen, binds with testosterone responsible for maturation of the Wolffian ducts and DHT responsible for external virilization both in utero and during puberty.
Thus far, two forms of 5ἀ reductase have been inentified: isionzymes I and II. Type I 5ἀ reductase is predominantly expressed in the liver and somatic tissue.
Whereas type II 5alpha reductase is predominantly found in the prostate, epididymis, seminal vesicles, and genital skin.
Pharmacologic inhibitors of 5ἀ reductase have been used to relieve prostatic obstruction and consequently alleviate lower urinari tract symptoms in some patiens.
Two examples of such medications are finasteride, which is selective for the, type II isoenzyme, and dutasteride ,which is nonselective and inhibits both forms of 5alpha reductase.
Lastly, both testosterone and DHT can be degraded in the liver by a number of enzymes that will not be elaborated upon here.
The end result of this process is to generate steroids conjugated with a glucuronide or sulfate group that are ultimately excreted by the skin or the kidneys.
Hormonal Regulation of Testosterone Production
The regulation of testosterone syntesis by the Leydig cells of the testis falls into two broad categories. The first includes endocrine signaling by the gonadotropins, primarily luteinizing hormone, wich are secreted by the anterior pituitary gñand and are the key mediators of the HPG axis.
Thes second includes more discrete paracrine signaling by locally produced factors within the interstitial compartment of the testis.
Endocrine Signaling
The gonadotropins luteinizing hormone ( LH) and follicle stimulating hormone (FSH) belong to a larger family of heterodimeric proteins know as glycoprotein hormones.
As heterodimers , these molecules are composed of two different subunits.
The α subunit is common to every member of the glycoprotein family; however , depending upon the β subunit that is chosen for dimerization, a variety of related hormones may by created:
LH,FSH,chorionic gonadotropin (HCG), and tyroid stimulating hormone ( TSH).
Functionally, LH exerts its effects on testosterone production by binding to the G protein-coupled receptors on Leydig cells.
FSH exerts its effects on spermatogenesis by binding to the G protein-coupled receptors on Sertoli cells. This entire axis is tightly regulated by a negative feedback system in wich testosterone plays a pivotal role.
Gonadotropin-releasing hormone (GnRH), produced by the neurons of the hypothalamus, empties into the hypothalamic hypophyseal portal system to make its way to the anterior pituitary, where it acts to stimulatte gonadotropin release.
This process occurs in a pulsatile, coupled manner: GnRH pulses are followed by shorter-lived pulses of LH.
This episodic secretion is essential to the male reproductive axis, and continuous GnRH administration fails to achieve the same effect.
Yet, the pulsatility of GnRH secretion is not constant throughout life. Recall that during the neonatal period, Leydig cell numbers increase and testosterone levels peak in response to GnRH and LH release at 2-3 months of life.
Thereafter, LH and GnRH levels drop and the neuronal GnRH generator goes dormant for the rest of prepuberty.
With the onset of puberty, pulsatile GnRH secretions resume, and LH levels begin to rise as well, initially only at nigth but eventually throughout the day , as well
After is pulsatile scretion, LH travels to the testes where in induces steoid syntesis in both an acute and a chronic manner.
Binding of LH to the LH receptor, a G protein coupled receptor (GPCR) ,results in activation of adenylyl cyclasse with a subse-quent rise in cAMP an concomitant activation of the protein kinase A(PKA) pathway.
There is evidence that multiple other pathways may be upregulated by this GPCR:
however, in Leydig cells most investigator agree that these steroidogenic effects are primarily regulated by the GPCR/adenylyl cyclase/cAMP/PKA patway.
Activated PKA in turn results in two major downstream effects. The first consists of increased translocation of cholesterol to the mitochondrial inner membrane secondary to upregulation of the acute regulatory protein ( StAR).
The second includes increased activation of the cholesterol side-chain cleavage system, mediated both by CYP1A and CYP17.
While LH is accepted as being primarily responsible for the endocrine control of testosterone production by Leydig cells, ther is some evidence FSH may play a role as well, albeit on a more limited scale.
This likely occurs via the direct effect of Sertoli cells (triggered by FSH) on Leydig cells.
-Physiology of Testosterone Production
T
Hipotalamus _________________
| Pulsatile
GnRH
|
T→E Anterior
__________________ Pituitary _________T__________
|
| LH
|
Leydig Cells Steroidogenic
Pathway
– The hypotalamic-pituitary-testis axis
Paracrine Signaling
Several non-pituitary, locally produced factors are identified as further regulators of Leydig cell function.
Insulin-like growth factor 1( IGF) and tumor growth factor beta
(TGFẞ) have well-established influences in this regard.
Both in vitro and in vivo studies demonstrate the important role IGF-1 plays in Leydig cell development and function.
Furthermore, while the in vivo is not as cogent evidence, animal studies suggest that TGFẞ modulates both steroidogenesis and Leydig cell proliferation .
A myriad of other potential local regulators are identified in vitro in rat models, however, studies on these molecules are often difficult to definitively translate to human Leydig cells.
More recently , additional regulator are generating increased attention including insulin-like factor 3, ghrelin, and leptin.
INSL3 is a peptide that belongs to the insulin-like growth factor (IGF) and relaxin family of hormones. It previously had an established role role in testicular descent with uncertain biological significance in
adults.
However, more recent human studies have shown a correlation between
INSL3 levels and Leydig cell functional status.
While the autocrine and paracrine effects of this peptide remain to be more fully elucidated, in data collected from adult men it appears that INSL 3 operates outside of the HPG axis, reflecting both differentiation status and absolute number of Leydig cells.
Leptin and ghrelin have more firmly established roles in testosterone production and physiology than does INSL3.
While the primary function of leptin and ghrelin is to operate as a coordinated system regulating energy homeostasis, there is increasing evidence that they play a combined role in modulating testosterone levels as well.
In one model, rats fed a restricted diet with repeated administration of ghrelin showed decreased LH and testosterone with reduced testis weight.
Conversely, ablation of grhelin in leptin-deficient ob/ob mice resulted in increased steroidogenesis and reduction of testicular apoptosis.
Thus, it appears that unopposed ghrelin may exert an inhibitore effect on testosterone production. This occurs centrally, as demonstrated by decreased LH levels in rats given daily doses of ghrelin.
Peripherally at the level of the Leydig cell, as shown by in vitro models showing a ghrelin mediated inhibition of testosterone secretion in a dose dependent fashion.
Further substantiating this hypothesis are data collected from human testicular samples showing an inverse correlation between ghrelin expression by Leydig cells and peripheral testosterone levels.
Negative Feedback Control
LH levels are maintained within a narrow physiologic range. Therefore, the pulsatile, stymulatory effects of GnRH must be balanced by a refined set of negative feedback mechanism.
These mechanisme act at both the level of the anterior pituitary and the
hypothalamus, and they primarily are the result of circulating testosterone and estrogen.
Note that this discussion regarding negative feedback control only pertains to LH physiology.
Control of FSH and Sertloy cells occurs in an analogous manner and, however, involves additional factors, as primarily activin and inhibin B, which do not primarily affect LH secretion and wich do not primarily affect LH secretion.
One well-designed human study illustrates the interplay between testosterone, estrogen, and the aromatization of testosterone to estrogen in the negative feedback of LH secretion.
Prior to completion of this study, the respective contributions of these
sex steroids to negative feedback control of LH were unclear.
Furthermore, the precise sites oh inhibition via testosterone and estrogen were uncertain.
Thirteen men with idiophatic hypogonadotropic hypogonadism (IHH), who do not produce GnRH at baseline but who may be normalized with exogenous pulsatile GnRH administration, allowed the study authors to better answer these remaining questions.
By measuring the peripheral LH levels of these individuals in response to exogenous sex steroids and then comparing these values to healthy volunteers, a site of action as well as the relative contributions of testosterone and estrogen could be inferred.
Several conclusions can be made:
– Testosterone and estrogen independently inhibit LH secretion.
– Testosterone does require aromatization to estrogen to inhibit LH secretion at the pituitary but not the hypotalamus.
– While estrogen can act either location in predominantly functions at the
level of the hypotalamus.
While these discoveries do not hold inmediate clinical aplpications, they elucidate the contributions of testosterone and estrogen to negative feedback control.
Altered Testosterone Physiology
Previous discussion has focused on the physiology of testosterone production in the normal male.
There are two instances of altered physiology that deserve particular attention because of their increasing prevalence: the metabolic syndrome and the aging male.
-Metabolic Syndrome
Changes in diet and reduction in physical activity have resulted in a rising wave of global obesity, not only within developed countries but all around the world.
Not only does one´s total body fat predict important comorbidities such as coronary artery disease, stroke, and diabetes.
The distribution of body fat also makes a difference as individuals with a greater percentage of visceral fat appear to have an increased risk of metabolic consequences, as in the metabolic syndrome.
Hypogonadism is often seen in this picture, and it has been suggested that testosterone replacement may improve lipid profiles and insulin resitance in men with the metabolic syndrome.
The hypogonadal-obesity cycle attempts to explain this relationship:
increased adipose tissues lead to greater testosterone deficiency througt increased conversion of testosterone to estradiol by aromatase.
This relative defieciency of testosterone and excess of estradiol, in turn, leads to even greater fat deposition and subsequent further declines in testosterone.
In addition, overall abdominal obesity may lead to increased glucocorticoid turnover and production with disruption of the HPG/ adrenal axis, thereby leading to mild hypogonadism.
Of interest, two of the paracrine signals discussed earlier: leptin and grhelin, have been studied in some detail with regard to the metabolic syndrome.
Leptin has been demonstrated to increase in obese individuals with an attendant fall in serum testosterone.
An dministration of exogenous testosterone appears to suppress leptin
levels, but this effect is short-lived and leptin levels return to the pre-therapy range after cessation of testosterone.
The data regarding ghrelin is more limited; however, there is evidence that suggests that testosterone replacement raise ghrelin levels
back to their normal range.
-The Aging Male
After the age of 40, serum testosterone declines at a rate 0´4-2,6% per year whit an associated decrease in muscle mass, strength, sexual function, and bone mass.
This decline in testosterone independently predicts disturbances in insulin and glucose metabolism, potentially leading to the metabolic syndrome.
However, not all men will exhibit clinically significant symptoms associated
with this decline in tesosterone levels.
Using data from the Boston Area Community Healt Survey, Araujo et al, found 24% of their 1,475 subjects aged 30-79 to have low total testosterone(<300ng/mL) and 5,6% of the 1.475 patients presented symptoms.
Prevalence of low testosterone increases with age. This is
reflected by estimates obtained from the Massachussetts Male Agin Study ( MMAS).
An observational cohort of 1.709 subjects aged 40-70 and enrolled between 1987 and 1989 with two separate follow-up phases.
While initial crude prevalence of androgen deficiency at baseline was 6.0%, this increased to 12,3% during the first follow-up phases of the study between 1995 and 1997.
Why there is a range in testosterone decline and it associated symptomatology remains an area of interest.
For example, in contradiction to the typical aging male with a diminishing testosterone and minimally elevated LH, there is a population of men who mount a large enough rise in their LH to maintain a normal serum testosterone.
An increased amount of attention is being paid to these men and to the androgen receptor itself , with particular research
being devoted to the number of CAG trinucletoide repeats present in the transactivation exon of this gene.
There is a demonstrated inverse correlation between the number of CAG repeats and both the transcriptional activity of the androgen-dependent
genes and their downstream effects.
Furthermore, men with normal total testosterone concentrations and longer CAG repeats run a grater risk of developing andropausal type symptoms.
Summary
Testosterone production is a finely balanced process with many points of potential regulation starting with the translocation of cholesterol cross the mitochondrial membrane and ending with the ultimate negative feedback of testosterone in the HPG axis.
Two scenarios in wich this physiology is altered are becoming increasingly, clinically relevant: metabolic syndrome and the aging male.
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