As infertility rates across nations become a growing concern, the interest in the development of treatments, such as in vitro gametogenesis (IVG), increases. This is especially the case for male infertility. For instance, the average sperm count continues to decline across nations, while more adult and pediatric patients survive cancer only to be left with little to no options for fertility restorative therapies. Understanding the male reproductive system and the process of spermatogenesis, however, has proven to be a difficult task. Progress occurs slowly and inconsistencies remain in the literature while reports attempt to better understand spermatogonial stem cells (SSCs) in conjunction with spermatogenesis. Interestingly, stem cell behavior, the decision to self-renewal or commit to differentiation, has shown to be closely linked to the stem cell’s microenvironment (i.e. niche). Perhaps the missing pieces required to better understanding spermatogenesis are found in the re-defined perspective of SSC niche dynamics.
- spermatogonial stem cells
- stem cell therapy
- in vitro gametogenesis
- organoid engineering
The Center for Disease Control (CDC)’s Division of Vital Statistics released a recent report titled
The production of germ cells (i.e. gametogenesis), is a process that begins in embryos with the formation of primordial germ cells (PGCs) that continues differently in male and female reproductive systems. Only recently did studies show that mouse embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) can differentiate into PGC-like cells (PGCLCs) that upon transplantation gave rise to both functional sperm or oocytes [5, 6]. Importantly, the culture conditions and differentiation protocols of murine PGCLCs still require further optimization, as these cells differentiate inefficiently and lack well-defined long-term culture conditions . This is especially the case with SSCs that constitute the male testis. Understanding how these stem cells initiate spermatogenesis within the seminiferous tubules of the testis is vital for the future of IVG applications, as such knowledge would lead to optimized differentiation protocols and long-term SSC culture conditions that could be implemented for the treatment of infertility .
The focus of this chapter is on the significance of mammalian spermatogenesis as it pertains to both the successes of IVG, and to the push for innovation in the general field of reproductive medicine. We will see how SSC fate decisions establish spermatogenesis through the multifaceted interaction of the stem cell and its niche. Furthermore, we discuss how novel technologies, which allow for SSC niche mapping and in vitro preservation of the seminiferous tubules, hold the key to therapeutic and diagnostic breakthroughs—and the challenges and ethical implications that follow.
2. The spermatogonial stem cell niche establish and regulate spermatogenesis
The variation in stem cell behavior, whether it is the decision to self-renew or to commit to differentiation, is strictly linked to the stem cell’s niche . The niche is highly dynamic and has shown to direct such fate decisions in a variety of adult stem cells such as hematopoietic stem cells (HSCs) , intestinal , and epidermal stem cells . The concept of the niche extends beyond the direct cell–cell contact and is comprised of other key components such as secreted factors (i.e. chemokines, hormones, growth factor receptors), inflammation (i.e. macrophages, T cells), physical factors (i.e. shear forces, topography, elasticity/stiffness), hypoxia (i.e. glycolysis-optimizing conditions), and cellular metabolism (i.e. glucose, lipids, calcium, calcium receptors) . Importantly, the stem cell-niche communication not only occurs over short and long range distances, but is also reciprocal and significant in tissue homeostasis [13, 14, 15]. For instance, in mouse skin, the removal of the stem cell population (i.e. hair follicle stem cells) can result in niche cells dedifferentiating to replace them. In this case, the repopulation of epithelial cells (cells that do not contribute to hair growth), replace the stem cells and sustain hair regeneration . Interestingly, with respect to the field of spermatogenesis, the key components of the SSC niche also appear to be as multifaceted as the examples provided above.
2.1. Direct and indirect cell contact: SSCs, Sertoli cells, and other key players
One key player in regulating the accessibility of SSCs to other components in the niche is the Sertoli cell. These cells extend from the basal compartment of the seminiferous tubule to the adluminal region. Sertoli cells take on a constantly evolving and irregular shape that is in a continuous three-dimensional relationship with not only the SSCs, but also the differentiating spermatogonia throughout spermatogenesis . Due to the complexity of such a dynamic relationship, traditional experiments involving two-dimensional or stage-specific cell analysis may have portrayed an incomplete depiction of spermatogenesis and may have attributed to the large discrepancy found in today’s literature . Regardless, it is still worth discussing such findings because it provides a glimpse into the three-dimensional relationship between the SSC and Sertoli cells.
To begin, Sertoli cells have a large surface area that allows them to support germ cell development at a higher ratio of germ cells to Sertoli cells [17, 18]. Such a characteristic is critical for providing structural support to the germ cells, but also to germ cell movements throughout the tubule. Furthermore, the unique structural and signaling flexibility of Sertoli cells create two distinct environments within the tubules that are otherwise referred to as the blood-testis barrier (BTB). This is where the basal compartment, the region in close contact with lymph and blood, is speculated to maintain earlier staged cells of spermatogenesis, such as SSCs and early progenitors. Later stages that are committed and differentiating spermatogonia, however, appear to occur in the adluminal compartment, isolated from lymph and blood . Importantly, the BTB is created by tight or gap junctions, and desmosomes that are present between Sertoli cells. The BTB does not exist between Sertoli cells and germs cells or between germ cells [19, 20, 21, 22]. Additionally, the specialization of the BTB was first depicted in early studies of testicular transferrin. Cells located in the later staged adluminal region of the tubule (i.e. cells that do not have access to serum iron), gained access to testicular transferrin through Sertoli cells [17, 23, 24].
In addition to the role of the Sertoli cells, Leydig cells within the interstitial space of the seminiferous tubules produce and signal testosterone. Without the presence of testosterone, spermatogenesis does not proceed completely and results in male infertility. Therefore, if Leydig cells were to be removed, the germ cells that have initiated meiosis and completed differentiation begin to improperly detach from the Sertoli cells and die . Furthermore, mature sperm near the lumen of the tubule (i.e. the adluminal compartment) cannot properly release from Sertoli cells without testosterone signaling. Another major hormone, follicle-stimulating hormone (FSH) has shown to act synergistically with testosterone to increase and regulate spermatogenesis. It does this by binding to the FSH receptor (FSHR) on the surface membrane of Sertoli cells . Peritubular myoid cells (PMCs) are also located in the interstitial space of the tubules, and express the androgen receptor for testosterone. Within the seminiferous tubules, however, only Sertoli cells express the androgen receptor. Therefore, Sertoli cells, in communication with other niche cells (i.e. Leydig cells and PMCs) regulate spermatogenesis indirectly and thus impact the initiation, development, and survival of germs cells [25, 26]. Furthermore, the surface of some seminiferous tubules associates with vascular endothelium and perivascular cells . The role of such vasculature-associated cells may be involved in regulation of SSC niche dynamics. For instance, some studies found that in the prepubescent and adult testis, macrophages are closely associated with Leydig cells and play a role in the signaling and production of testosterone . Another study found populations of macrophages near the surface of the basal compartment where enriched undifferentiated spermatogonia were found. Such testicular macrophages expressed SSC proliferative and differentiating factors such as enzymes involved in retinoic acid (RA) synthesis and colony stimulating factor 1 (CSF1) signaling .
2.2. The extracellular matrix, secreted factors, and their respective receptors
The extracellular matrix (ECM) and its role in the stem cell niche vary substantially in almost every tissue . In some cases, the ECM is also involved in the maintenance of local concentrations of growth factors that direct stem cell fate or target niche cells involved in the regulation of those SC fate decisions [29, 30]. In the testis, the ECM located at the basal compartment, is made up of the basement membrane that is composed of proteins like laminin, type IV collagen, and entactin. Importantly, the basement membrane (a modified ECM) is speculated to not only interact with Sertoli cells, but also regulate SSC fate decisions. Sertoli cells even secrete components of the ECM (i.e. laminin) that is not only useful in short-term SSC culture conditions, but is also involved in Sertoli cell tight junctions, and in turn, the formation of the BTB [8, 17, 31, 32, 33]. It is the formation of tight junctions across Sertoli cells that is said to create a semipermeable barrier that restricts molecule movement based on either weight or chemical structure . The exact involvement, however, of the basement membrane with key components like laminin and the mechanisms of junction formation still require further investigation with their role in SSC niche dynamics. Nevertheless, specialized junctions have been found throughout the seminiferous epithelium and include junctions such as adherens, desmosome-like, hemidesmosome, and gap junctions that are located throughout the tubule from the basal to adluminal compartment. These junctions not only appear to control germ cell movement, but are also involved in the regulation or perhaps local concentrations of secreted factors in SSC fate decisions .
Glial cell line-derived neurotrophic factor (GDNF) is a secreted factor produced by Sertoli cells and PMCs that is linked to SSC fate determination, Sertoli cell proliferation and short-term SSC
In terms of respective receptors, in the seminiferous epithelium, some major ECM-receptors are integrins. In fact, studies have claimed that integrin-α6 and integrin-β1 are key surface markers involved in the regulation of spermatogenesis. There is still much discrepancy, however, on whether such markers are exclusively expressed on SSCs or germ cell progenitors [8, 17]. It has been shown, however, that the deletion of integrin-β1 on Sertoli cells not only reduced SSC homing (i.e. the repopulation of SCs after the removal of endogenous SCs), but also that the adhesion receptor’s association with laminin is critical for the several steps involved in SSC homing . Again, though the integrins are significant in niche dynamics, it appears that integrins cannot be used to distinguish the sub-populations of early staged germ cells (i.e. SSCs and their progenitors). Importantly, such studies use marker-based techniques (i.e. Fluorescence-activated cell sorting (FACS)) to isolate cells positive for markers such as integrin-α6, integrin-β1 or GFRα1, to then transplant back into germ cell-depleted testes for further analysis [8, 42, 43]. Though the transplantation assay  is a great tool to gauge stem cell competency, the SSC niche dynamics must also be clearly defined since germ cells have shown to behave inconsistently from
2.3. Hypoxia, metabolism and the role of inflammation
Tissue specific cell populations such as HSCs, and cardiac progenitors are found to be in low oxygen (i.e. hypoxic) microenvironments that contribute to cell survivability and maintenance [9, 47]. During hypoxic conditions, cells favor glycolysis rather than mitochondrial oxidative phosphorylation. In terms of the SSC niche, one recent report found that the reduction in O2 tension during
The general understanding regarding immune privilege (i.e. the capacity to tolerate the introduction of new antigens without the trigger of an inflammatory response) is the evolutionary adaptation to protect tissues from loss of functions due to their limited capacity for regeneration [55, 56]. In the testis, however, this protection against loss of function is for the tissue’s reproductive capacity. The production and differentiation of male germ cells are unique in that sperm matures at puberty, which is long after the maturation of the immune system and systemic self-tolerance . Due to these phenomena, the BTB plays a role in protecting the maturation of sperm from an autoimmune reaction. For instance, the various junctions and desmosomes between Sertoli cells have created such a limited access in the passage of other molecules that the composition of the basal, adluminal, and interstitial spaces differ significantly. Additionally, while the BTB’s ability to isolate meiotic and postmeiotic germ cells from lymph and blood is significant in testicular immune privilege, there are also physical and immunological components required for the immunotolerance of the testis . The expression of anti-inflammatory cytokines by immune and somatic cells and the role of androgens also play a role in the immunoprivileged niche.
Meiotic and postmeiotic germ cells express a large variety of neoantigens that emerge during puberty and long after self-tolerance is already established. Furthermore, once spermatogenesis begins, the BTB is established and immediately isolates post pubertal germ cells from the immune system . Interestingly, germ cell neoantigens are also present on SSCs and progenitors that are located in the basal compartment, and not isolated from lymph and blood, unlike the adluminal compartment of the BTB [55, 57, 58]. This suggests that other components of the testes also play a role in the immunoprivileged niche. For instance, accumulating evidence suggests that PMCs secrete cytokines like transforming growth factor-β (TGF-β), leukemia inhibitory factor (LIF), and macrophage chemoattractant protein 1 (MCP-1) that directly affect leukocytes in the interstitial space of the testis [59, 60]. Furthermore, the local high concentrations of testosterone appear to play an important role in the immunoprivileged niche within the testis. For example, when testosterone was incubated with stimulated human macrophages, monocytes and non-immune cells, the suppression of cytokines and adhesion molecules occurred while the production of anti-inflammatory cytokines increased [61, 62]. In transplantation studies, rats that were treated with estrogen to suppress Leydig cell production of testosterone immediately rejected allotransplanted cells within the seminiferous tubules. This directly contrasted the untreated control group where no rejection occurred in allotransplanted cells . Though this provides evidence that the local high concentrations of testosterone plays an important role in the immunoprivileged niche, the exact mechanism testosterone and its anti-inflammatory function on testicular leukocytes, still remains unknown. There may be an indirect regulatory mechanism involved in the balance between the expression of pro and anti-inflammatory cytokines in Sertoli cells, Leydig cells, and PMCs . Such a balance between pro and anti-inflammatory cytokines may also play a significant role in the protection and maintenance of SSCs that are not isolated from lymph and blood. For instance,
2.4. Physical factors
The physical surroundings such as the three-dimensional physical shape, shear forces, and topography (i.e. the physical arrangement of cells) all contribute to the stem cell and its niche . For example, shear forces such blood flow, have shown to play a role in either the acceleration or reduction of
Furthermore, the biophysical cues involved in the
2.5. Niche mapping and its significance in the seminiferous tubule microenvironment
Modulating the SSC niche requires well-defined and reproducible studies. Since SSCs appear to be highly interconnected with their niche, the
A recently published report characterized a three-dimensional multilayer model (termed the Three-Layer Gradient System (3-LGS)) that allowed for the reorganization of dissociated rat testicular cells into testicular organoids with the formation of a functional BTB and germ cell maintenance. This system used three concentric layers of Matrigel to not only increase the area for factor exchange but also for testicular cell reorganization into organoids to take place . Furthermore, the cellular organization of this
in vivospermatogenesis through in vitropreservation
Together, the controlled monitoring of a fluidic
4. Initiating innovation: promises, challenges, and the ethical implications for the push toward IVG
Human embryogenesis and gametogenesis is crucial to our understanding of reproduction, development, disease and evolution [87, 88]. The recent successes in the generation of human PGCLCs from human ESCs and human iPSCs solidified the prospects that the reconstitution of human IVG may be near . In the future, IVG combined with IVF can allow infertile couples trying to conceive to generate their own gametes through iPSC technology [7, 89]. Interestingly, IVG has the potential for even broader implications in reproductive medicine. With the increasing cultural shift in family planning, such as the delay in marriage and parenthood from young adults, is a shift that will most likely impact the GFR and the replacement fertility rate in any given country. Countries that have a low GFR have a larger aging population that is not only positioned to shrink, but also has fewer populations in the working age to support the older dependents. Therefore, the replacement fertility rate, highly dependent on the GFR, is the rate in which women give birth to enough babies to sustain population levels in any given country . With the availability of contraceptives, young adults can now easily delay parenthood to ages where fertility begins to decline and conceiving may become more difficult and at times no longer possible. IVG/IVF therapy combined with iPSC technology can potentially provide a solution to the significant implications in the new cultural shifts that will affect the GFR and the replacement fertility rate across countries. Until such a promising therapy can come into fruition, there are many scientific and ethical challenges that we must undertake. The need for robust and reproducible studies in both spermatogenesis and oogenesis are crucial for the future in IVG practices. It is imperative to evaluate claims of IVG and germ cell meiosis through the use of standardized benchmarks and the demonstration of the ‘gold standards’ for meiosis . Furthermore, the advancement in both knowledge (i.e. understanding SSC niche dynamics) and technology (i.e. creating comprehensive fluidic devices and viable
In terms of the ethical implications of IVG, it is imperative for us to revisit the relevant regulatory measures. For instance, 10 countries permit human embryo research under the 14 day culture rule . Of these countries, not including the United States, only seven include regulations to human IVG for medical or scientific applications. Interestingly, though the United States has no federal laws or regulations to prohibit IVG for research, the Dickey-Wicker amendment (signed in 1995) forbids federal funding for human embryo research [87, 92]. Even though the National Institutes of Health (NIH) recognized the value of it, and the CDC continues to report the GFR at record lows. Combine this with the 2017 provisional CDC report that birth rates are declining for nearly all age groups of women under 40, but rising in women aged 40–44 by 2% from 2016 . Furthermore, just as the development of IVF was initially highly controversial, it has now become a widely accepted treatment for infertility, and more commonly used among women with declining fertility. Therefore, if the scientific and societal value of human IVG research is agreed to be significant, IVG research should then be conducted under balanced regulations with careful ethics review and close oversight . Scientists, appropriate policymakers and the public should all be included for all future discussions regarding human IVG research.