Diagnosing Spermatogonial Stemness

Introduction

Familial health largely reflects the quality of traits transmitted by ancestral oocytes and spermatozoa. This fact of life endows gamete-producing germ cells with an intrinsic accountability for the well-being of heritable genomic architecture [1]. Consequently, it is fitting that pressure to preserve genomic integrity is reflected by lower frequencies of DNA mutations detected in germ cells than in somatic cells [2–4]. In mammals, self-renewing germline stem cells are considered unique to male gonads during reproductive life. This is because mitotically dividing female germ cells enter meiosis to differentiate into oocytes shortly after sex determination in mammals, which occurs midway through embryogenesis [5]. Oogenic arrests, together with selective oocyte degeneration, are hypothesized to provide additional safeguards that defend the germline from genomic abnormalities, highlighting the “female-protective model” [6, 7]. In contrast, male germline stem cells, termed “spermatogonial stem cells,” sustain spermatozoan production in testes by mitotically self-renewing over relatively long periods that span both embryonic and adult life (>50 years in humans) [8]. Accordingly, whole-genome sequencing is providing evidence that sex-dependent increases in time given for a germline to replicate its DNA correlate strongly with longstanding observations of “male mutation bias” [9–11]. In most species, our ability to unequivocally identify spermatogonia that replicate to function as germline stem cells has yet to be firmly established, but such a hypothesis appears fundamental to understanding cellular mechanisms that buffer the accumulation of transmittable DNA mutations by germlines [12, 13]. Scientists are rapidly annotating stem and progenitor spermatogonia in rodents [14–17], and these advances are being translated to other mammalian species, including primates [18–23]. Clinically, the ability to diagnose genomic stability in spermatogonial stem cells seems paramount in order to safely make the connection to their enormous prospective benefits for family planning and genetic medicine [24].

Ever-Expanding Models of Spermatogonial Development

Existence of spermatogonial stem cells was postulated near the end of the 19th Century by radiologists studying the regenerative capacity of testicular germ cells in model organisms receiving “gonotoxic” doses of ionizing radiation [25]. At that time, spermatogonial developmental hierarchy was simply assigned to “type A” spermatogonia that selectively survived radiation exposure and, presumably, restored populations of heterochromatic “type B” spermatogonia representing differentiating spermatogenic progenitors destined for meiosis. A review of publications since then reveals consistent advances from decade to decade that have led to ever-evolving theories of an expanding developmental hierarchy of >10 distinct stem, progenitor, and differentiating spermatogonial types currently classified in mammals [15, 26], which mirror a diversity of proposed mechanisms by which stem spermatogonia maintain spermatogenesis [15, 27, 28]. Presently, a prevailing view in mammals is that spermatogonial stem cells reside within a population of type “A-single” (A_s) spermatogonia that maintain spermatogenesis by their dual capacity to either self-renew as A_s spermatogonia or to form syncytia of mitotically dividing progenitor cells termed type A-paired (A_pr) and A-aligned spermatogonia (A_al) (Fig. 1) [29–31]. Disrupting self-renewal of A_s spermatogonia can drive development fully toward a progenitor fate [32–37]. In rodents, progenitor spermatogonia differentiate into mitotically dividing types A1, A2, A3, and A4 and intermediate (Int) and B spermatogonia [38–40], during which time their cell numbers or syncytium often increase >100-fold prior to entering meiosis to form spermatocytes (Fig. 1).

FIG. 1.

Spermatogonial stem cell predetermination hypothesis. A recent study by Hermann et al. [41] highlights large gaps of knowledge on the specification of spermatogonial stem cells. Based on that study and the group's previous findings [2, 44], their current study formulates a hypothesis that spermatogonial stem cells may actually derive developmentally from a subpopulation of prospermatogonia that are selectively protected genetically by unknown factors. The illustration also demonstrates independent questions (?) that will likely be raised on how their concepts integrate with presumptive germline stem cell niches in the seminiferous epithelium postnatally, and with intrinsic mechanisms determining germ cell fate. Prosgn, prospermagonia; A_s, A-single spermatogonia; A_pr, A-paired spermatogonia; A_al, A-aligned spermatogonial; A1-B, types A1 to B spermatogonia; PL-D, preleptotene to diplotene spermatocytes.

Dissecting Spermatogonial Heterogeneity

In a recent issue of Biology of Reproduction, a study by Hermann et al. [41] reported a strategy to dissect the diversity of mouse spermatogonial types during a pivotal time in gametogenesis, immediately following the transition from “prespermatogenesis” to “spermatogenesis” (reviewed by J.R. McCarrey [26]) (Fig. 1). Transition from prespermatogenesis to spermatogenesis occurs in mice between Postnatal Days 3 and 6 (P3–P6) when pools of “prospermatogonia” give rise to functionally distinct populations of stem and progenitor spermatogonia [42–44]. The group hypothesized that subpopulations of spermatogonia with discrete mRNA signatures could be identified in neonatal mice, which potentially could unveil previously unrealized heterogeneity of germ cells at the “start” of spermatogenesis, shortly after their derivation from prospermatogonia. To test their hypothesis, Hermann et al. [41] performed single-cell qPCR using a Fluidigm system (San Francisco, CA) [45] to study gene expression in spermatogonia isolated from mice at P6). The approach previously proved successful to help classify even earlier pregonadal and prespermatogenic steps in primordial germ cell development, from embryonic precursors in vivo and in vitro [46–48]. It allowed Hermann et al. to analyze gene expression in arrays of hundreds of individual testis cells comparing three distinct isolation methods. A panel of 172 qPCR primer sets were designed to analyze the relative abundance of target transcripts with reported functions in undifferentiated spermatogonia, somatic testis cells, differentiating male and female germ cells, and pluripotent stem cells. Indeed, a battery of statistical analyses consistently predicted ∼6 conspicuous clusters of amplified spermatogonial transcripts marking distinct germ cell populations at that pivotal juncture in gametogenesis. Principal component analyses were then used to distill this heterogeneity into three prominent spermatogonial signatures, which directionally, would be consistent with subtypes of stem, progenitor, and differentiating spermatogonia present in P6 mice. Thus, their study strongly supported the concept that the P6 spermatogonial pool consists of multiple subpopulations with discrete gene expression signatures, quite possibly reflecting divergence in spermatogonial fate.

Spermatogonial Stem Cell Predetermination Hypothesis

The repertoire of molecular signatures in spermatogonia unearthed by Hermann et al. [41] begs new questions surrounding the actual diversity of spermatogonia developmentally (Fig. 1). Answers to these questions are needed to fill key gaps of knowledge centered on understanding the long-term and short-term replicative potential of stem spermatogonia, and the fidelity at which prospermatogonia give rise to stem, progenitor and differentiating spermatogonia in mammals. Their study was motivated in part by earlier observations [2, 3] that ignited the “disposable soma hypothesis” posed in 1977 by Kirkwood [1]. Kirkwood's premise suggested that, because germ cells give rise to the entire subsequent generation of individuals, it would be evolutionarily advantageous for these cells to use mechanisms to maintain an enhanced level of genetic integrity [1]. This thesis was reinforced by higher spontaneous mutational frequencies in the soma versus the germline of female and male mice harboring a LacI mutation-reporter transgene [2, 3]. Interestingly, these same studies made seminal discoveries in that spermatogonia at P6 accumulated more mutations than primary spermatocytes at P18 [2, 3]. This paradox raised questions as to why the germline harbored 5 to 10 times more DNA mutations in spermatogonia at the start of spermatogenesis than in the germ cell population it presumably gave rise to shortly after the start of spermatogenesis. To place this oxymoron in context with their current studies plus accumulating examples of molecular and functional heterogeneity within populations of A_s, A_pr, and A_al spermatogonia [28, 29, 37, 49–54], the group formulated the following hypothesis: “Spermatogonial stem cell specification, either through a mechanism of predetermination or selection, may occur as a result of molecular divergence that first emerges among prospermatogonia.”

Conclusions and Future Directions

The above-described hypothesis proposes that the founding population of actual spermatogonial stem cells derives from a predetermined pool of prospermatogonia, which by some unknown mechanism(s), selectively maintains their genetic integrity at more pristine levels than other prospermatogonia (Fig. 1). Identifying prospermatogonia predestined to become either stem or progenitor spermatogonia will, in essence, break the current dogma that spermatogenesis initiates after prospermatogonia migrate from the lumen of seminiferous cords to colonize the basal lamina [26]. This theory raises questions on how “prospermatogonial stem cell” genomes could selectively be protected. Extrinsic factors that promote protection of the germline would imply existence of specialized “prospermatogonial niches” to mediate this function in the embryonic gonad. For example, does a similar version of the A_s > A_pr > A_al model occur earlier in development than generally thought, consistent with findings by Kluin and de-Rooij [42]? As presented, the spermatogonial stem cell predetermination hypothesis remains open to germline intrinsic mechanisms that, if not properly engaged, promote terminal differentiation and/or elimination of prospermatogonia. It would lead to question if DNA damage actually is a consequence of differentiation during a vulnerable developmental window or if it actually functions as a differentiation signal. In the latter case, how would mutational load “blind” prospermatogonia-derived progenitors to self-renewal factors (Fig. 1)? Conceivably, mutations above a relative threshold could facilitate the increased rates of apoptosis consistently observed in cohorts of first-generation spermatocytes derived more directly from prospermatogonia [55, 56]. In an analogous example, DNA double-strand breaks and chromatin abnormalities accumulate in MIWI2-deficient prospermatogonia due to persistent retrotransposon activation, but effects on germ cell fate are not manifested until induction of apoptosis post-mitotically at the zygotene-pachytene checkpoint [57]. Moreover, the above-described models of prospermatogonial heterogeneity remain compatible with an “inductive” influence of germline stem niches postnatally as one way for predetermined stem spermatogonia to grasp their true potential (Fig. 1). A combination of these mechanisms would increase spermatogonial heterogeneity due to greater divergence in fate, which in turn, would allow a greater diversity in spermatogonial types to be captured by single-cell qPCR.

Centered on their hypothesis, the study by Hermann et al. [42] highlights the potential to classify new spermatogonial types with respect to developmental origin and biological function. It will be interesting to see how such findings impact the course of biomedical research, to ultimately apply germline stem cells to human health, a field that prospectively will benefit by the ability to propagate and/or differentiate an individual's germline [18, 24, 58]. If “spermatogonial stemness” is largely predetermined by mechanisms linked to protecting genomic integrity (i.e., disposable soma hypothesis), how should this impact standards for therapeutic application of donor cells produced by somatic cell reprogramming? This is an especially broad question as it relates regenerative medicine. As a consequence, practical application of spermatogonial stem cells holds enormous potential to transform numerous areas of science, medicine, industry, and conservation [58–63] with the probability of donor haplotypes possessing a more ancestral background [9, 10]. However, this untapped potential is being held at bay by limited capacity to experimentally manipulate spermatogonial proliferation and differentiation from so many species outside Rodentia. Current absence of in vitro and in vivo bioassays to unequivocally measure the sperm-forming potential of donor spermatogonia in most mammalian species means that genome-wide analytical approaches similar to those applied by Hermann et al. [41] may well prove critical to safely diagnose spermatogonial stemness, as imputed by genetic integrity.