Testosterone Synthesis and Signalling

      

Introduction:

Testosterone is an important and main androgen i.e. male sex steroid hormone which plays an essential role in the development, differentiation and functioning of the male reproductive system. Mostly the genomic effects of testosterone are described which require the mediation of androgen receptors. Non-genomic effects of testosterone have been described recently. It is having a basic structure as shown below in Fig.1-

Fig.1 Structure of testosterone (cited in steroid chemistry, 2009)

As testosterone is the principal androgen which supports spermatogenesis therefore, its synthesis is quite important as it influences the development and behaviour patterns in male (Johnson &Everitt 1980). Any deviations from normal testosterone synthesis and signalling that has been described may lead to some serious diseases and problems as such, therefore it is very important to understand how testosterone is synthesized and signalled in the body.

Synthesis of Testosterone:

Testosterone can be synthesized directly within the testis by the leydig cells and also via the inter-conversion from adrenal androgens. (Johnson&Everitt 1980)

Synthesis within the testis:

Like other steroid hormones, testosterone is also derived from common precursor cholesterol. The first step in the synthesis of testosterone is the conversion of cholesterol to pregnenolone. This is the slowest step that requires a large amount of energy for initiation. The conversion of cholesterol to pregnenolone takes place in the inner mitochondria and NADPH, oxygen and cytochrome P-450 are required along with the side chain cleavage reaction of cholesterol (Johnson &Everitt). After cholesterol is converted to pregnenolone, inter conversion takes place in smooth endoplasmic reticulum by a ‘biosynthetic unit’ which consists of a series of enzymes which are arranged together. For testosterone synthesis, this pregnenolone is converted to progesterone in the presence of 3β-dehydrogenase.The formed progesterone is then converted to 17-hydroxyprogesterone by 17-hydroxylase. Further 17-hydroxyprogesterone is converted into androstenedione by 17-hydroxylase. Now androstenedione is converted to testosterone by the action of 17β-hydroxysteroid dehydrogenase (Fig.2) (Hills 2010).

Fig.2 Testicular biosynthetic pathway of testosterone (cited in endotext.org 2009)

Synthesis via inter-conversion from adrenals:

Adrenal cortex is an important place where the production of androgen occurs. Functionally and histologically it can be divided into 3 parts viz. zonaglomerulosa (outer part), zonafasciculata (intermediate part) & the zonareticularis (central part) as shown in Fig.3. (Sanderson 2006)

Fig.3 Cross sections of Adrenal gland (cited in mcg.edu 2008)

Zonareticularis is the principle site where androgen synthesis takes place. The production of testosterone in the adrenals is under the control of Adrenocorticotropic hormone (ACTH). The production of testosterone is increased via the cAMP PK A (cyclic adenosine mono phosphate protein kinase A) pathway. After the conversion of cholesterol to pregnenolone by the action of 17α- hydroxylase, progesterone is hydyroxylated to 17-hydroxypregnenolone by 17α-hydroxylase. This 17-hydroxypregnenolone is further converted to dehydroepiandrosterone(DHEA)  by the action of 17, 20 desmolase. Now, in the zonareticularis this DHEA is converted to androstenedione in the presence of 3β-dehydrogenase. This androstenedione gets converted to testosterone by the action of 17β-hydroxysteroiddehydrogenase.Dehydroepiandrosterone (DHEA) and androstenedione are the adrenal androgens.Testosterone is further converted to dihydrotestosterone (DHT) by 5α-reductase in the testis (Fig.4). In a normal adult man about 6mg of testosterone is produced daily, out of which only 25 µg approximately is stored in the testis, which indicates that testosterone is continuously synthesised and released in the circulation. (Kaplan &Pesce 1984)

Fig.4 Testosterone synthesis pathway (cited in AACR 2010)

Regulation of testosterone expression:

Testosterone synthesis is under the control of hypothalamic-pituitary-gonadal axis. Gonadotropin releasing hormone (GnRH) is produced in the hypothalamus in a pulsatile mode and binds to the specific receptors present on the plasma membrane. At anterior pituitary GnRH stimulation leads to the pulsatile secretions of  luteinizing hormone (LH) and Follicle stimulating hormone (FSH). Testosterone is synthesised by the interstitial cells of leydig of testis by the action of LH. GnRH stimulates LH and it binds to the LH receptors and conversion of cholesterol to testosterone takes place in the interstitial cells of Leydig. LH controls the rate of testosterone synthesis and FSH is required for maintaining spermatogenesis and it acts on sertoli cells. Under the influence of FSH sertoli cells secrete androgen binding protein (ABP) as shown in Fig.5. Testosterone binds with ABP which carries it to seminiferous tubules to facilitate spermatogenesis. As sertoli cells have 5α-reductase, testosterone is converted to a more potent form dihydrotestosterone (DHT) there. (Johnson &Everitt 1980; Kaplan &Pesce 1984)

Fig.5 Regulation of gonadal steroid expression (cited in Hills 2010)

Negative feedback:

The aim of negative feedback is to maintain Homeostasis. The synthesis of testosterone depends on the action of LH and the secretion of LH is itself controlled through the negative feedback of testosterone to hypothalamus. When the amount of testosterone produced is increased, it leads to the suppression of GnRHwhich in turn effects the secretion of LH by pituitary and thus testosterone synthesis is kept under control (Fig.6). For this negative feedback to occur there are some factors which either stop GnRH secretion or inhibit testosterone receptor synthesis and in their absence, the production of testosterone will stop automatically. Thus testosterone regulates the secretions of LH by reducing LH peaks and LH frequency. Certain hormones like inhibin which is produced in sertoli cells, also has a negative feedback on pituitary FSH secretion though the effect is not much. Testosterone can cause the inhibition of both LH and FSH whereas inhibin inhibits the secretions of FSH mainly. (Elder & Dale 2000)

Fig.6 Negative feedback due to increased testosterone (cited in Hills 2010)

Testosterone signalling:

Classical Pathway:

Testosterone is an androgen and to mediate its actions or in order to execute its biological effects, it binds to the intracellular androgen receptor (AR). Androgen receptors are basically proteins which consist of a binding site for a specific androgen. Androgen receptor belongs to the nuclear receptor superfamily and consists of 4 functional domains viz. ligand binding domain (LBD), a DNA binding domain (DBD), a hinge region and an N-terminal domain (NTD). Through the action of LBD, testosterone is able to control the AR activity (Fig.7). DBD recognizes a specific sequence of DNA and the hinge region connects the LBD with DBD (Claessens et. al 2008)

Fig.7 Various domains of androgen receptor (cited in Hills 2010)

Synthesis of testosterone takes place by the classical action of AR. Androgen receptor (AR) has 2 native ligands- testosterone and 5α-DHT. The cytoplasm and nucleus consists of specific AR’s. Testosterone has a great affinity for the AR, therefore, as soon as it comes in contact with the AR; it readily binds to the ARwhich further leads to the conformational changes and this is termed as the activation of AR (Fig.8).

Fig.8 The classical testosterone signalling pathway (Walker 2010)

For the expression of genes, the activated AR binds to the androgen response elements (ARE’s) which are specific DNA sequences. For androgen the consensus DNA (Deoxyribonucleic acid) sequence reads 5'-TGTTCT-3'. In androgen responsive genes, these types of binding sequences have been described. The protein expression is controlled by the promoter of a gene and this is the region where these Androgen response elements (ARE’s) are located. When testosterone is converted to dihydrotestosterone (DHT) by 5α-reductase, the formed DHT binds with the AR and dimerization of AR takes place and this is termed as the activation of AR. This activated receptor has great affinity for the promoter region of DNA which codes for the gene expression. It now enters the nucleus and binds with the ARE’s on the DNA and thus gene expression is promoted (Fig.9). Along with ARE’s there are some other co-activator proteins and transcription factors which bring about the gene expression. Only leydig cells, peritubular cells and sertoli cells in the testis are able to express the AR. Germ cells of the mature testis are not able to express AR. Mature leydig cells have a very good expression for AR. (Walker & Cheng 2005)

Fig.9 Activation of AR and expression of genes (Hills 2010)

Therefore, AR plays a very crucial role in the classical pathway of testosterone signalling. Apart from its role in testosterone signalling, AR performs some other functions as well. From the result of AR knock out and knock-in mouse modelsit was found that activity of AR plays a crucial role in spermatogenesis as follows-:  (Kerkhofs et.al 2009)

1.      AR is necessary for progression through Meiosis I.

2.      During spermatogenesis the round spermatids are converted into elongated ones, which also require the activity of AR.

3.      During the terminal stages of spermatogenesis, AR plays a crucial role.

Non-Classical Pathway:

Apart from the classical mechanism of testosterone signalling, non-classical pathway of testosterone signalling also exists. Various studies of the past prove that testosterone can directly lead to the gene expression without AR binding to DNA. It was first indicated by Fix et al. For these studies, sertoli cells from rat testis were isolated and then cultured. Then they were stimulated with the testosterone levels less than or similar to that found in testis. This led to the increased phosphorylation of the ERK/MAP (extracellular signal-regulated kinase/mitogen activated protein) kinase and the CREB (cAMPresponse element-binding) transcription factor. As the phosphorylation of the CREB and ERK occurred more rapidly than 30-45 minutes.Testosterone also causes activation of kinase p90^RSK (p90 ribosomal S6 kinase) which induces CREB-mediated transcription. All this indicates that some other pathway does exist other than the classical one as this phosphorylation doesn’t take place by the classical mechanism. Apart from this it was found that testosterone plays an important role on the effect on the concentration of intracellular Ca⁺² concentrations. For spermatogenesis testosterone level in sertoli cells should be more than 70nM but, the binding of testosterone to the AR and the gene expression are saturated at 1nM which point towards some other signalling pathway present apart from the classical one. (Walker 2009)

Furthermore, as soon as testosterone stimulation takes place, the Ca⁺² levels in freshly isolated cultures of sertoli cells are elevated, which suggests that testosterone signalling is not fully dependent on the AR and its interactions with the DNA leading to gene expression. If it would have been so then Ca⁺² levels should not be elevated within seconds of elevation of testosterone.In sertoli cells testosterone does not increase the levels of cAMP (cyclic adenosine mono phosphate) but in the activation of MAP (Mitogen activated protein) kinase pathway, CREB (cAMP response element-binding) phosphorylation and CREB mediated transcription, it plays an important role (Fig.10). Two pathways were described in support of this – (Walker & Cheng 2005)

1^st pathway –

Testosterone binds to AR which causes activation of Src (family of protein kinases) and as soon as this activation occurs it leads to the activation of MAP kinase pathway by a series of events by Ras (Rat sarcoma) G-protein (it is a signal transducing guanine nucleotide binding protein).

2^nd pathway –

When testosterone binds to AR there is an increase in the Ca+2 levels. Once this occurs it causes activation of all the intermediates that have the capability of RasorRas like G-protein stimulation which further activates MAP kinase pathway.

Moreover it was found that testosterone can directly activate non-genomic signalling pathways irrespective of AR and DNA interaction. AR mediates testosterone induced MAP kinase and CREB (cAMP response element-binding) phosphorylation. Following evidence supports it – (Walker & Cheng 2005)

1^st – Flutamide which is an AR antagonist leads to the inhibition of testosterone mediated CREB phosphorylation.

2^nd – Sertoli cells which did not have the activity of AR, CREB phosphorylation did not take place in them.

3^rd – Sertoli cells which had the testicular feminization (tfm) AR mutant, testosterone could not increase the CREB phosphorylation in them.

Fig.10 Non-classical testosterone signalling pathways (Walker 2010)

All of these series of evidences (Walker & Cheng 2005; Walker 2009; Walker 2010) suggest that apart from the classical mechanism, testosterone acts through other pathways also in the sertoli cells. Both classical and non-classical pathways (Fig.11) play an important role in spermatogenesis and maintainingfertility.In the future, may be it will be proved that there is some co-relation between these two pathways or they may be interdependent. It may give a breakthrough in determining the male fertility as well. As most of the studies are being conducted on ratsand mice therefore, their relevance to human beings still remains a question.But, as the reproductive system of humans and rats is very much identical therefore, studies on rats remain a good source of research and information. In vivo studies will be more useful in future.

Fig.11 Classical & non-classical testosterone pathways (Walker 2009)

Conclusion:

A lot of research work is done on understanding the role of testosterone in body, its functioning, synthesis, signalling and its role in spermatogenesis. Apart from this the action of AR, AR antagonists, hormonal control of testosterone, and negative feedback by testosterone, gene expression and Ca⁺² uptakes by sertolicells is well understood today. Some years back testosterone was thought to act only through the classical mechanism but, recent research and evidences prove that apart from the classical pathway, a non-classical pathway which includes CREB and MAP kinase pathway also exists. Non-classical pathway of testosterone signalling is not yet understood fully; still the role of hormones and co-regulators remains to be fully described and most of the study has been conducted in vitro, therefore, in vivo study can provide some useful information. So, may be in coming years there will be more research in this area which will provide more information about testosterone signalling and also about the co-factors which are yet to be identified.

Dr.Bharati Sood