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It puts the T’s in fertility: testosterone and spermatogenesis (2022)
Luke Witherspoon and Ryan Flannigan
INTRODUCTION
The role of testosterone in successful spermatogenesis has been a well-documented physiologic process for several decades. Like any process in the human body, there are multiple hormonal pathways implicated in spermatogenesis, but testosterone appears as the only essential hormone that must be present to drive normal development of testis and ultimately spermatogenesis. In this paper, we review the cellular control mechanisms by which testosterone drives spermatogenesis.
*Spermatogenesis and spermiogenesis
Spermatogenesis begins with the mitotic division of spermatogonial stem cells (SSCs) into two daughter cells, one serving the role of self-renewal, while the other initiates differentiation. This occurs within the specialized niche environment found within the seminiferous tubules of the testis. This environment is comprised of several support-type cells helping to create this physical and hormonally dense environment. Current research efforts suggest that three main cell populations help to develop germ cells from their initial SSC stage into spermatozoa [1]. Within the seminiferous tubules peritubular myoid cells (PTM) line the walls of the tubules and contract to help push sperm forward in the tubules. PTM cells help form the basement membrane of the seminiferous tubules by working alongside Sertoli cells and are involved in cell signaling with SSCs [2].
Sertoli cells extend from this basement membrane of the tubules to surround and help nurture the developing germ cells by acting as a signaling interface to the extra tubular environment, while also producing and releasing several factors that help promote germ cell production and differentiation. Unlike the Leydig cells whose main stimulus is Luteinizing Hormone (LH), Sertoli cell's main regulatory hormone is Follicle Stimulating Hormone (FSH), although these cells do require testosterone stimulation for normal function [3]. Although PTM cells aid in basement membrane production, it is the Sertoli cells that create specialized cell-cell junctions to create the blood-testis barrier (BTB), partitioning the interior of the seminiferous tubules from the rest of the bodies circulatory system [4]. This barrier, and the Sertoli cells themselves, may in fact play a role in immune response and protection from infection, as Sertoli cells have been shown to upregulate innate immunity-related genes (e.g. defensin class of peptides) [5].
Leydig cells exist outside the seminiferous tubules in the interstitial space between tubules and it is here where they produce testosterone, allowing close proximity production of testosterone to the developing germ cells. Based on recent single-cell sequencing analysis of Leydig cells they appear to likely stem from a common progenitor that is shared with PTM cells [5]. This production of testosterone within the testes translates into the testes having a much higher concentration of testosterone locally than what is found anywhere else in the human body (25–125 fold higher) [6–8].
*Testosterone production
Testosterone produced by interstitial Leydig cells is driven by LH released from the anterior pituitary. LH binds the LH receptor (LHR), which in turn couples to G protein and stimulates cyclic adenosine 3’, 5’-monophosphate (cAMP). cAMP is then able to stimulate cholesterol translocation into the mitochondria. It is this step that is ultimately the rate-limiting step in steroid production [9]. Cholesterol then undergoes several enzymatic induced changes with the mitochondria to ultimately be converted into testosterone [9]. The elevation of testosterone within the testicle as compared to serum levels has not been fully elucidated, although most appear to be bioavailable within the testis with only a third of the testosterone tightly bound to sex hormone-binding globulin (SHBG), and the rest being either free or loosely bound to albumin [10, 11]. This high level of bioavailable testosterone appears to be a requirement for spermatogenesis, as sperm production appears to drop significantly when levels deviate from this high concentration [12].
*TESTOSTERONE SIGNALING
Classical pathway
Testosterone produced by Leydig cells travels into the interior of the seminiferous tubules and enacts its actions predominately via binding the androgen receptor (AR) protein. The AR is typically located within the cytoplasm and translocates to the nucleus upon activation. Interestingly germ cells do not appear to possess androgen receptors, rather it is the support cells (PTMs, Sertoli cells) where the binding of testosterone to AR’s leads to these cells performing their roles in creating a niche environment conducive to spermatogenesis [13]. In addition to these specialized cell populations possessing AR, surrounding vasculature appears sensitive to testosterone with arteriole smooth muscle cells and vascular endothelial cells expressing this receptor class [14].
Androgen receptors can potentiate testosterone signaling in several ways, and these have been historically grouped into two pathways: the classical and non-classical pathways of testosterone signaling.
In the classical pathway testosterone produced by Leydig cells diffuses into the seminiferous tubules binding to the AR receptors found within PTM and Sertoli cells. A subsequent conformational change in the AR releases this protein receptor from a sequestering heat shock protein (HSP) within the cytoplasm. Following release from the HSP, the AR is free to translocate to the nucleus where it is able to bind to androgen-responsive elements (ARE) which are regulatory DNA sequences, leading to either activation or repression of these regions through recruitment of activating or repressing regulatory proteins (Fig. 1) [15]. This entire process takes ~30-45 min, from testosterone stimulation to transcription [16]. AR modulation of gene expression appears to result in an equal split between upregulation and downregulation of gene expression. This is a somewhat surprising finding, as logically it would be assumed that AR activation would lead to gene expression [17]. Thus, it is thought more likely that some of the gene products induced by AR go on to cause downregulation of alternate genes creating this apparent milieu of gene expression [17].
Non-classical pathway
In the non-classical pathway of testosterone production alterations in cellular function occur within seconds to minutes, and may help augment some of the pathways found within the classical pathway [18].
The most commonly reported non-classical mechanisms of testosterones action reported within the literature focus on pathways contained within Sertoli cell regulation. Testosterone stimulation has been shown to induce Ca2+ influx into the Sertoli cells, although the significance of this is not yet clear in terms of its impact on spermatogenesis [19]. Testosterone stimulation has been shown to lead to a sub-population of AR migrating transiently to the plasma membrane where they interact with and activate a Src tyrosine kinase [20]. Activation of this kinase leads to stimulation of endothelial growth factor receptor (EGFR) which goes on to activate the MAP kinase cascade that results in p90RSK mediated phosphorylation of the CREB transcription factor [21]. Activation of these different kinase groups appears to be critical to spermatogenesis and possibly linked to Sertoli-germ cell attachment, as evidenced by studies with defective AR Sertoli cells showing dysfunctional binding that can be rescued with the introduction of wild type AR or AR mutants specifically activating the non-classical pathway [21]. More recently, the non-classical pathway has been shown to be required not only for Sertoli-germ cell-binding but further appears critical to normal BTB production [22].
*TESTOSTERONE REGULATION OF SPERMATOGENESIS
Testosterone in puberty
In infant males, testis tissue is primarily comprised of seminiferous cords containing immature Sertoli cells and undifferentiated state 0 spermatogonia and lined with PTMs [5]. The expression of the AR first appears in the human testis around 12 months of age in Sertoli cells [23]. As the child progresses towards puberty the number of AR-positive cells increases, until the initiation of puberty when all Sertoli cells express this protein [24]. This pre-pubertal phase appears critically important to future Sertoli cell function as it is in this phase where many of the germ-cell support features and the ability to form the BTB are developed. This has been characterized via enlarging cytoplasmic volume and adhesion to the basal lamina of the seminiferous tubules as children progress towards puberty [25, 26]. With the onset of puberty, and increased production of testosterone Sertoli cells fully mature completing the formation of the BTB, and germ cells initiate meiosis to begin sperm production [24]. Recent single-cell sequencing analysis has confirmed the importance of testosterone in Sertoli development, with testosterone-suppressed Sertoli cells exhibiting an expression profile in keeping with a pre-pubescent genotype [5].
Leydig cells show a transient period of activation in the neonatal period until approximately 6 months of age when the hypothalamic-pituitary-gonadal (HPG) axis becomes quiescent leading to a similar period of Leydig cell inactivity. At the time of puberty with the onset of testosterone signaling, the Leydig cell population resumes testosterone production [27]. Interestingly Leydig cells appear to express a constant AR level, rather than their Sertoli counterparts who as described increase AR expression towards puberty [23]. With the puberty-induced localized testosterone production, the AR located within the Sertoli cells begins their final maturation process.
*Blood-Testis-Barrier
The BTB evolves over the course of development towards its final state at/during puberty. Testosterone appears critical in this transition. The BTB reaches its adult state at puberty and this is characterized by a reduction in the number of gap junctions, with a corresponding rise in tight junctions leading to the separation of the inner tubule from the rest of the body's circulation [28]. During normal spermatogenesis, the developing spermatocyte moves off the basement membrane (stages VI-VII) and transitions through the BTB, with the old BTB dissolving and with a new BTB being deposited behind the transitioning spermatocyte [29]. This process seems at least partially testosterone sensitive with a reduction in specific tight junction proteins (claudin 3, 11, occludin) and a more permeable BTB when androgen signaling is impaired [30, 31].
*Meiosis
Impaired testosterone signaling leads to the halting of spermatogenesis during meiosis, with few cells developing to a haploid stage and abnormal morphology (no elongated spermatids) are noted [32]. The full genetic pathways through which testosterone causes this halt to remain elusive. A proteomic study of rats who were exposed to various levels of testosterone reduction indicated that the loss of testosterone in cells undergoing meiosis appeared to develop cellular stress, apoptosis, and dysfunction of posttranslational processing and DNA repair [33].
*Aromatisation of testosterone
Estrogen production within the testis is mainly located within Sertoli cells during the pre-pubescent phase, utilizing the P450 aromatase enzyme to synthesize estrogen from androgens [26]. This transitions in the mature testis to Leydig cells as the main site of aromatization [27]. Although germ cells have been shown to have no AR, they do appear to express estrogen receptors (ER) and have the ability to convert androgens to estrogens themselves [28]. Interestingly, this aromatization of androgens into estrogens appears crucial to normal spermatogenesis. In aromatase knockout mice (ArKO), although initially fertile these mice begin to show decreased fertility at 5 months of age and eventually become infertile. Evaluation of spermatogenesis in this mouse model shows a decrease in elongated spermatids and blockage of germ cell maturation in the spermatid phase [34]. A delicate balance exists, however, as overexpression of aromatase or exposure to estrogens similarly leads to disrupted spermatogenesis and increased germ cell apoptosis [35]. The pathway by which estrogen interferes with testosterone production has yet to be fully determined. Similar to testosterone levels, estrogen levels are elevated within the male reproductive tract in comparison to serum concentrations [36]. Leydig cells appear to be regulated somewhat by estrogen, with some evidence that estrogen can inhibit LH signaling, providing one possible mechanism by which estrogen could affect Leydig function [37]. Sertoli cells similarly experience some level of regulation via estrogen with the observation that this hormone is required for the expression of N-cadherin – the protein that is critical to the tight junctions that Sertoli cells form [38].
CONCLUSION
The role of testosterone in male fertility has been investigated for the better part of 80 years, although the intricate nature of how this hormone affects spermatogenesis has largely stymied researchers from fully determining the complex pathways involved.
With improved investigational tools allowing for a better understanding of how testosterone, and its by-products, affect gene and protein expression across several unique cell populations we are beginning to understand and identify new targets for directed therapies for male infertility conditions.
Luke Witherspoon and Ryan Flannigan
INTRODUCTION
The role of testosterone in successful spermatogenesis has been a well-documented physiologic process for several decades. Like any process in the human body, there are multiple hormonal pathways implicated in spermatogenesis, but testosterone appears as the only essential hormone that must be present to drive normal development of testis and ultimately spermatogenesis. In this paper, we review the cellular control mechanisms by which testosterone drives spermatogenesis.
*Spermatogenesis and spermiogenesis
Spermatogenesis begins with the mitotic division of spermatogonial stem cells (SSCs) into two daughter cells, one serving the role of self-renewal, while the other initiates differentiation. This occurs within the specialized niche environment found within the seminiferous tubules of the testis. This environment is comprised of several support-type cells helping to create this physical and hormonally dense environment. Current research efforts suggest that three main cell populations help to develop germ cells from their initial SSC stage into spermatozoa [1]. Within the seminiferous tubules peritubular myoid cells (PTM) line the walls of the tubules and contract to help push sperm forward in the tubules. PTM cells help form the basement membrane of the seminiferous tubules by working alongside Sertoli cells and are involved in cell signaling with SSCs [2].
Sertoli cells extend from this basement membrane of the tubules to surround and help nurture the developing germ cells by acting as a signaling interface to the extra tubular environment, while also producing and releasing several factors that help promote germ cell production and differentiation. Unlike the Leydig cells whose main stimulus is Luteinizing Hormone (LH), Sertoli cell's main regulatory hormone is Follicle Stimulating Hormone (FSH), although these cells do require testosterone stimulation for normal function [3]. Although PTM cells aid in basement membrane production, it is the Sertoli cells that create specialized cell-cell junctions to create the blood-testis barrier (BTB), partitioning the interior of the seminiferous tubules from the rest of the bodies circulatory system [4]. This barrier, and the Sertoli cells themselves, may in fact play a role in immune response and protection from infection, as Sertoli cells have been shown to upregulate innate immunity-related genes (e.g. defensin class of peptides) [5].
Leydig cells exist outside the seminiferous tubules in the interstitial space between tubules and it is here where they produce testosterone, allowing close proximity production of testosterone to the developing germ cells. Based on recent single-cell sequencing analysis of Leydig cells they appear to likely stem from a common progenitor that is shared with PTM cells [5]. This production of testosterone within the testes translates into the testes having a much higher concentration of testosterone locally than what is found anywhere else in the human body (25–125 fold higher) [6–8].
*Testosterone production
Testosterone produced by interstitial Leydig cells is driven by LH released from the anterior pituitary. LH binds the LH receptor (LHR), which in turn couples to G protein and stimulates cyclic adenosine 3’, 5’-monophosphate (cAMP). cAMP is then able to stimulate cholesterol translocation into the mitochondria. It is this step that is ultimately the rate-limiting step in steroid production [9]. Cholesterol then undergoes several enzymatic induced changes with the mitochondria to ultimately be converted into testosterone [9]. The elevation of testosterone within the testicle as compared to serum levels has not been fully elucidated, although most appear to be bioavailable within the testis with only a third of the testosterone tightly bound to sex hormone-binding globulin (SHBG), and the rest being either free or loosely bound to albumin [10, 11]. This high level of bioavailable testosterone appears to be a requirement for spermatogenesis, as sperm production appears to drop significantly when levels deviate from this high concentration [12].
*TESTOSTERONE SIGNALING
Classical pathway
Testosterone produced by Leydig cells travels into the interior of the seminiferous tubules and enacts its actions predominately via binding the androgen receptor (AR) protein. The AR is typically located within the cytoplasm and translocates to the nucleus upon activation. Interestingly germ cells do not appear to possess androgen receptors, rather it is the support cells (PTMs, Sertoli cells) where the binding of testosterone to AR’s leads to these cells performing their roles in creating a niche environment conducive to spermatogenesis [13]. In addition to these specialized cell populations possessing AR, surrounding vasculature appears sensitive to testosterone with arteriole smooth muscle cells and vascular endothelial cells expressing this receptor class [14].
Androgen receptors can potentiate testosterone signaling in several ways, and these have been historically grouped into two pathways: the classical and non-classical pathways of testosterone signaling.
In the classical pathway testosterone produced by Leydig cells diffuses into the seminiferous tubules binding to the AR receptors found within PTM and Sertoli cells. A subsequent conformational change in the AR releases this protein receptor from a sequestering heat shock protein (HSP) within the cytoplasm. Following release from the HSP, the AR is free to translocate to the nucleus where it is able to bind to androgen-responsive elements (ARE) which are regulatory DNA sequences, leading to either activation or repression of these regions through recruitment of activating or repressing regulatory proteins (Fig. 1) [15]. This entire process takes ~30-45 min, from testosterone stimulation to transcription [16]. AR modulation of gene expression appears to result in an equal split between upregulation and downregulation of gene expression. This is a somewhat surprising finding, as logically it would be assumed that AR activation would lead to gene expression [17]. Thus, it is thought more likely that some of the gene products induced by AR go on to cause downregulation of alternate genes creating this apparent milieu of gene expression [17].
Non-classical pathway
In the non-classical pathway of testosterone production alterations in cellular function occur within seconds to minutes, and may help augment some of the pathways found within the classical pathway [18].
The most commonly reported non-classical mechanisms of testosterones action reported within the literature focus on pathways contained within Sertoli cell regulation. Testosterone stimulation has been shown to induce Ca2+ influx into the Sertoli cells, although the significance of this is not yet clear in terms of its impact on spermatogenesis [19]. Testosterone stimulation has been shown to lead to a sub-population of AR migrating transiently to the plasma membrane where they interact with and activate a Src tyrosine kinase [20]. Activation of this kinase leads to stimulation of endothelial growth factor receptor (EGFR) which goes on to activate the MAP kinase cascade that results in p90RSK mediated phosphorylation of the CREB transcription factor [21]. Activation of these different kinase groups appears to be critical to spermatogenesis and possibly linked to Sertoli-germ cell attachment, as evidenced by studies with defective AR Sertoli cells showing dysfunctional binding that can be rescued with the introduction of wild type AR or AR mutants specifically activating the non-classical pathway [21]. More recently, the non-classical pathway has been shown to be required not only for Sertoli-germ cell-binding but further appears critical to normal BTB production [22].
*TESTOSTERONE REGULATION OF SPERMATOGENESIS
Testosterone in puberty
In infant males, testis tissue is primarily comprised of seminiferous cords containing immature Sertoli cells and undifferentiated state 0 spermatogonia and lined with PTMs [5]. The expression of the AR first appears in the human testis around 12 months of age in Sertoli cells [23]. As the child progresses towards puberty the number of AR-positive cells increases, until the initiation of puberty when all Sertoli cells express this protein [24]. This pre-pubertal phase appears critically important to future Sertoli cell function as it is in this phase where many of the germ-cell support features and the ability to form the BTB are developed. This has been characterized via enlarging cytoplasmic volume and adhesion to the basal lamina of the seminiferous tubules as children progress towards puberty [25, 26]. With the onset of puberty, and increased production of testosterone Sertoli cells fully mature completing the formation of the BTB, and germ cells initiate meiosis to begin sperm production [24]. Recent single-cell sequencing analysis has confirmed the importance of testosterone in Sertoli development, with testosterone-suppressed Sertoli cells exhibiting an expression profile in keeping with a pre-pubescent genotype [5].
Leydig cells show a transient period of activation in the neonatal period until approximately 6 months of age when the hypothalamic-pituitary-gonadal (HPG) axis becomes quiescent leading to a similar period of Leydig cell inactivity. At the time of puberty with the onset of testosterone signaling, the Leydig cell population resumes testosterone production [27]. Interestingly Leydig cells appear to express a constant AR level, rather than their Sertoli counterparts who as described increase AR expression towards puberty [23]. With the puberty-induced localized testosterone production, the AR located within the Sertoli cells begins their final maturation process.
*Blood-Testis-Barrier
The BTB evolves over the course of development towards its final state at/during puberty. Testosterone appears critical in this transition. The BTB reaches its adult state at puberty and this is characterized by a reduction in the number of gap junctions, with a corresponding rise in tight junctions leading to the separation of the inner tubule from the rest of the body's circulation [28]. During normal spermatogenesis, the developing spermatocyte moves off the basement membrane (stages VI-VII) and transitions through the BTB, with the old BTB dissolving and with a new BTB being deposited behind the transitioning spermatocyte [29]. This process seems at least partially testosterone sensitive with a reduction in specific tight junction proteins (claudin 3, 11, occludin) and a more permeable BTB when androgen signaling is impaired [30, 31].
*Meiosis
Impaired testosterone signaling leads to the halting of spermatogenesis during meiosis, with few cells developing to a haploid stage and abnormal morphology (no elongated spermatids) are noted [32]. The full genetic pathways through which testosterone causes this halt to remain elusive. A proteomic study of rats who were exposed to various levels of testosterone reduction indicated that the loss of testosterone in cells undergoing meiosis appeared to develop cellular stress, apoptosis, and dysfunction of posttranslational processing and DNA repair [33].
*Aromatisation of testosterone
Estrogen production within the testis is mainly located within Sertoli cells during the pre-pubescent phase, utilizing the P450 aromatase enzyme to synthesize estrogen from androgens [26]. This transitions in the mature testis to Leydig cells as the main site of aromatization [27]. Although germ cells have been shown to have no AR, they do appear to express estrogen receptors (ER) and have the ability to convert androgens to estrogens themselves [28]. Interestingly, this aromatization of androgens into estrogens appears crucial to normal spermatogenesis. In aromatase knockout mice (ArKO), although initially fertile these mice begin to show decreased fertility at 5 months of age and eventually become infertile. Evaluation of spermatogenesis in this mouse model shows a decrease in elongated spermatids and blockage of germ cell maturation in the spermatid phase [34]. A delicate balance exists, however, as overexpression of aromatase or exposure to estrogens similarly leads to disrupted spermatogenesis and increased germ cell apoptosis [35]. The pathway by which estrogen interferes with testosterone production has yet to be fully determined. Similar to testosterone levels, estrogen levels are elevated within the male reproductive tract in comparison to serum concentrations [36]. Leydig cells appear to be regulated somewhat by estrogen, with some evidence that estrogen can inhibit LH signaling, providing one possible mechanism by which estrogen could affect Leydig function [37]. Sertoli cells similarly experience some level of regulation via estrogen with the observation that this hormone is required for the expression of N-cadherin – the protein that is critical to the tight junctions that Sertoli cells form [38].
CONCLUSION
The role of testosterone in male fertility has been investigated for the better part of 80 years, although the intricate nature of how this hormone affects spermatogenesis has largely stymied researchers from fully determining the complex pathways involved.
With improved investigational tools allowing for a better understanding of how testosterone, and its by-products, affect gene and protein expression across several unique cell populations we are beginning to understand and identify new targets for directed therapies for male infertility conditions.
