Spermatogonial stem cells in higher primates: are there differences to those in rodents?

Abstract

Spermatogonial stem cells (SSCs) maintain spermatogenesis throughout the reproductive life of mammals. While A_single spermatogonia comprise the rodent SSC pool, the identity of the stem cell pool in the primate spermatogenic lineage is not well established. The prevailing model is that primate spermatogenesis arises from A_dark and A_pale spermatogonia, which are considered to represent reserve and active stem cells, respectively. However, there is limited information about how the A_dark and A_pale descriptions of nuclear morphology correlate with the clonal (A_single, A_paired A_aligned), molecular (e.g., GFRα1, PLZF) and functional (SSC transplantation) descriptions of rodent SSCs. Thus, there is a need to investigate primate SSCs using criteria, tools, and approaches that have been used to investigate rodent SSCs over the past two decades. SSCs have potential clinical application for treating some cases of male infertility, providing impetus for characterizing and learning to manipulate these adult tissue stem cells in primates (nonhuman and human). This review recounts the development of a xenotransplant assay for functional identification of primate SSCs and progress dissecting the molecular and clonal characteristics of the primate spermatogenic lineage. These observations highlight similarities and potential differences between rodents and primates regarding the SSC pool and the kinetics of spermatogonial self-renewal and clonal expansion. With new tools and reagents for studying primate spermatogonia, the field is poised to develop and test new hypotheses about the biology and regenerative capacity of primate SSCs.

Progress characterizing spermatogonial stem cells (SSCs) and development of the spermatogenic lineage in rodents may provide insights about the identity and characteristics of nonhuman primate and human SSCs. Here we provide a review of tools and strategies used to characterize rodent SSCs and summarize classical and contemporary approaches for studying primate SSCs. We will conclude with comments on the evolutionary conservation of SSC phenotype and function from rodents to primates and future studies that may help elucidate the mode of stem cell renewal and differentiation in primates.

I. Spermatogonial stem cells in rodents

Spermatogonial stem cells (SSCs) are undifferentiated germ cells that balance self-renewing and differentiating divisions to maintain spermatogenesis throughout adult life. This is a productive stem cell system that produces millions of sperm each day while also maintaining rigorous quality control to safeguard germline integrity. Investigating the biological properties of SSCs that achieve this delicate balance in vivo will expand the understanding of stem cell/niche interactions in a variety of adult tissues and may also have implications for treating male infertility. Despite their critical importance to spermatogenesis and male fertility, the cellular and molecular characteristics of SSCs remain largely undefined. Experimental determination of the basic characteristics of SSCs requires a standardized biological assay that detects the capacity to initiate and maintain spermatogenesis. A SSC transplantation technique, developed for mice in 1994, measures this endpoint, and thus, functionally evaluates stem cell activity in any mouse testis cell preparation (Brinster & Avarbock 1994; Brinster & Zimmermann 1994). Briefly, germ cells are isolated from the testes of donor animals and transplanted into the seminiferous tubules of infertile recipients where they produce colonies of spermatogenesis and functional sperm. Only a stem cell can produce and maintain a colony of spermatogenesis and each colony in recipient testes arises from the clonogenic proliferation and differentiation of a single SSC (Dobrinski et al. 1999b; Nagano et al. 1999; Zhang et al. 2003; Kanatsu-Shinohara et al. 2006). Application of this technique in rodents revealed that SSCs from donors of all ages (newborn to adult) are competent to produce complete spermatogenesis in the testes of infertile males (Brinster & Avarbock 1994; Ohta et al. 2000; Ogawa et al. 2000; Shinohara et al. 2001; Nagano et al. 2001a; Brinster et al. 2003; Ryu et al. 2003). In addition to rodents, SSC transplantation has successfully generated complete spermatogenesis in other higher species, including goats (Honaramooz et al. 2003), pigs (Mikkola et al. 2006) and dogs (Kim et al. 2008). These results may have future implications for treating some cases of human male infertility [reviewed by (Orwig & Schlatt 2005; Brinster 2007; Schlatt et al. 2009)]. In addition, fluorescence-activated cell sorting (FACS) combined with SSC transplantation has also enabled systematic characterization of mouse SSCs as a subpopulation of mouse testis cells defined by the phenotype α6-INTEGRIN⁺, β1-INTEGRIN⁺, THY-1⁺, CD9⁺, Hoechst side population⁺, Rho123^low, αv- INTEGRIN ⁻, c-KIT⁻, major histocompatibility complex class I (MHC-I)⁻, CD45⁻(Shinohara et al. 1999; Shinohara et al. 2000; Kubota et al. 2003; Falciatori et al. 2004; Kanatsu-Shinohara et al. 2004; Lassalle et al. 2004; Fujita et al. 2005; Lo et al. 2005). Rodent SSCs can also be identified in whole mount preparations of testicular seminiferous tubules [initially described by (Clermont & Bustos-Obregon 1968)] as isolated A_single spermatogonia and probably some A_paired spermatogonia. These A_single SSCs can be distinguished in whole mount from committed progenitor spermatogonia (some A_paired and A_aligned chains of 4–16 cells) on the basement membrane of seminiferous tubules because committed cells exist as clonal chains connected by intercellular cytoplasmic bridges. Here we define progenitors as undifferentiated spermatogonia that are committed to differentiate and can undergo a finite number of self-renewing divisions. Although no SSC-specific marker has been identified, whole mount analyses indicate that GFRα1, PLZF, CDH1, NGN3 and OCT3/4 are expressed by undifferentiated stem and progenitor spermatogonia, including A_single, A_paired and A_{aligned 4–16} (Yoshida et al. 2004; Nakagawa et al. 2007; Buaas et al. 2004; Greenbaum et al. 2006; Tokuda et al. 2007; Schlesser et al. 2008). In contrast, the cKIT receptor tyrosine kinase is absent from A_single, A_paired and most A_aligned spermatogonia, but initiates expression in larger A_aligned clones (8 and 16 cells) and continues in differentiating types A1-A4, intermediate and B spermatogonia (Manova et al. 1990; Sorrentino et al. 1991; Yoshinaga et al. 1991; Tajima et al. 1994; Dym et al. 1995; Schrans-Stassen et al. 1999). Initiation of cKIT expression marks the transition from undifferentiated A_aligned spermatogonia to differentiating A1 spermatogonia (Schrans-Stassen et al. 1999). Thus, based on whole mount analyses and molecular phenotyping in rodents, it is possible to distinguish stem/progenitor spermatogonia (A_single, A_paired and A_aligned; GFRα1⁺, PLZF⁺, NGN3^+/− and cKIT⁻) and differentiating spermatogonia (A1–4, Intermediate, B; GFRα1⁻, PLZF⁻, NGN3^+/− and cKIT⁺).

Mammalian spermatogenesis occurs in a synchronized, cyclic pattern where the cellular associations of differentiating germ cells and Sertoli cells are maintained in a progressive and repeated fashion (de Rooij & Russell 2000). Using this information, the seminiferous epithelium can be categorized into numerous discrete “stages” based upon the cellular complement observed in a given segment of seminiferous tubule. Thorough evaluation of these cellular associations have identified 12 discrete stages of the seminiferous epithelium in mice and 14 stages in rats [Table 1; (Oakberg 1956; Leblond & Clermont 1952)].

Table 1.

Stages of the cycle of the seminiferous epithelium in rodents and primates.

Species	Stages (#)	Duration (days)		Stages per Cross-section†	References
		one cycle	Spermatogenesis
Homo sapiens	6	16	64	2–4	Clermont, 1966; Clermont, 1963; Heller and Clermont, 1963; Amann, 2008
Pan troglodytes (Chimpanzee)	6	14	62.5	2–4 (1–5)	Smithwick & Young, 1996; Smithwick et al., 1996
Papio Anubis (Olive baboon)	12	11	42	1–3	Chowdhury and Steinberger, 1976; Chowdhury and Marshall, 1980
Macaca arctoides (Stump-tailed macaque)	12	11.6	46.4	1	Clermont and Antar, 1973; Clermont, 1972
Macaca fascicularis (Cynomolgus monkey)	12	10.5	42	1 to several	Fouquet and Dadoune, 1986; Dietrich et al., 1986.
Macaca mulata (Rhesus macaque)	12	10.5	42	1	Clermont and Leblond, 1959; de Rooij et al., 1986; Rosiepen et al., 1997.
Callithrix jacchus (Common marmoset)	9	10	37	1–5	Holt and Moore, 1984; Millar et al., 2001.
Mus musculus	12	8.62	34.5	1	Oakberg, 1956b; Oakberg, 1956a; Clermont and Trott, 1969; Oakberg, 1971; Kluin et al., 1982
Rattus norvenicus	14	13	51.6	1	Clermont and Perey, 1957; Leblond and Clermont, 1952a; Leblond and Clermont, 1952b; Huckins, 1971
Cricetulus griseus (Chinese hamster)	12	17	68	1	Oud and de Rooij, 1977; Clermont and Trott, 1969
Mesocricetus brandti (Turkish hamster)	8	8	32	1	Myoga et al., 1991
Mesocricetus auratus (Golden hamser)	13	8.74	35	1	Leblond and Clermont, 1952b; Miething, 1998

^†Evidence of discrete stages of the seminiferous epithelium in individual cross-sections of seminiferous tubules.

Morphometric whole-mount studies have demonstrated that the numbers of A_single in mice, rats and Chinese hamsters remain relatively constant throughout the spermatogenic cycle, apparently due to the balanced renewal of A_single and formation of A_paired [Fig. 1A, (Tegelenbosch & de Rooij 1993; Oakberg 1971; de Rooij 1973; Huckins 1971; Lok et al. 1982)]. Likewise, numbers of A_paired are relatively constant across the seminiferous cycle (Fig. 1A). In contrast, the density of A_aligned is cyclic and is lowest at stages IX–XI, after large A_aligned clones produce differentiating A1 spermatogonia, and highest at Stage VI of the subsequent cycle as A_aligned clones become larger prior to recruitment to A1 (Fig. 1A). Morphometric quantification of total undifferentiated spermatogonial numbers per testis indicates that there are roughly 35,000 A_single per testis, representing roughly 0.03% of all testicular germ cells (1.3% of spermatogonia or 10.6% of undifferentiated spermatogonia) (Tegelenbosch & de Rooij 1993).

Figure 1. Relative distribution of undifferentiated Type-A spermatogonia in mice and monkeys.

The relative number of undifferentiated spermatogonia in any given segment of seminiferous tubules at each stage of the seminiferous cycle are shown for (A) mice (Tegelenbosch & de Rooij 1993) and (B) monkeys (Fouquet & Dadoune 1986). For A, data for the relative number of A_single (Red squares, line), A_paired (Orange triangles, line), and A_aligned clones of 4 and 8+ cells (green circles, blue diamonds) are shown for C3H-101 F1 hybrid mice (Tegelenbosch & de Rooij 1993). The calculated relative density of undifferentiated Type-A spermatogonia at each stage was determined by dividing the value for each stage (number of spermatogonia per 1000 Sertoli cells) by the same value for Stage I (Tegelenbosch & de Rooij 1993). In B, data for the relative density of A_dark, A_transition and A_pale per stage is as reported for cynomolgus monkeys (Fouquet & Dadoune 1986). The calculated relative density of Type-A spermatogonia in each stage (number of spermatogonia per 100 Sertoli cells) was normalized to the value for Stage I (Fouquet & Dadoune 1986).

II. Identity and arrangement of undifferentiated spermatogonia in primates

Clermont and co-workers initially described two morphologically distinct types of undifferentiated spermatogonia in the testes of rhesus macaques in 1959 (Clermont & Leblond 1959) and designated these cells A₁ and A₂ [later renamed A_dark and A_pale, respectively; (Clermont & Antar 1973)]. Both cell types are present on the basement membrane of primate seminiferous tubules, but differ based on nuclear architecture and staining intensity with hematoxylin. Clermont proposed that A_dark were SSCs, which undergo self-renewing divisions to maintain the stem cell pool and give rise to A_pale that subsequently generate differentiating Type-B spermatogonia (Clermont & Leblond 1959).

Ten years after the initial description of A_dark and A_pale spermatogonia, Clermont revised his linear “A_dark stem cell - A_pale progenitor model” based on observations in the Vervet monkey (Cercopithecus aethiops) that A_dark failed to label with ³H-thymidine (Clermont 1969). Thus, since A_dark did not appear to self-renew under steady-state conditions, Clermont proposed that A_dark and A_pale represent reserve and active stem cells, respectively (Clermont 1969). In this “reserve stem cell” model, spermatogenesis is maintained by the “active” pool of A_pale SSCs under normal circumstances. A similar model has been proposed for human spermatogenesis, where A_dark and A_pale spermatogonia are both present and active A_pale proliferation maintains spermatogenesis by balancing production of differentiating B spermatogonia and renewing the A_pale pool [(Clermont 1966a; Clermont 1966b), reviewed in (Dym et al. 2009; Amann 2008)]. Alternatively, others have proposed that the low mitotic index of A_dark is specifically indicative of a “true SSC” phenotype, while the regular divisions of nearly all A_pale demonstrate these cells are “renewing progenitors” that amplify spermatogonial output to B1 [reviewed by (Ehmcke et al. 2006; Ehmcke & Schlatt 2006)].

Similar to rodents, twelve distinct stages of the seminiferous epithelium have been described for stump-tailed macaques, cynomolgus monkeys, rhesus macaques, and baboons, while marmosets and chimpanzees stages are reported to have nine and six stages, respectively [Table 1; (Clermont & Leblond 1959; Clermont & Antar 1973; Chowdhury & Steinberger 1976; Holt & Moore 1984; Fouquet & Dadoune 1986; de Rooij et al. 1986; Smithwick & Young 1996)]. Similar to chimpanzees, morphological examination of human spermatogenesis identified only six stages based on discrete cellular associations [Table 1, (Clermont 1966a; Clermont 1963; Heller & Clermont 1963); reviewed by (Amann 2008)]. The topological arrangement of stages in human seminiferous tubules is described as spiral or patchy where one cross-section may contain germ cells in 2 to 4 different stages (Amann 2008). This arrangement is different from the “linear” arrangement described for rodents where a given seminiferous tubule cross-section only contains one stage (Table 1). Different non-human primate species exhibit varying degrees of “linear” or “patchy” stage topography (Table 1).

As for rodents, numbers and distribution of undifferentiated spermatogonia along the seminiferous epithelium has been described for primates. Morphometric studies indicate that the adult rhesus testis contains roughly equal numbers of A_dark and A_pale (Marshall & Plant 1996). In the rhesus testis, A_dark are equally distributed along the length of the seminiferous tubule epithelium and do not fluctuate significantly between stages [Fig. 1B, red line; (Clermont & Leblond 1959; Fouquet & Dadoune 1986)]. Cells with an indeterminate “transition” phenotype also do not vary between stages [Fig. 1B, orange line; (Fouquet & Dadoune 1986)]. In contrast, A_pale numbers are not constant due to their more active stage-dependent proliferation resulting in peak numbers at mid-cycle (Stages VII–VIII) and a nadir late in the cycle (Stages X–XII) [Fig. 1B, blue line; (Fouquet & Dadoune 1986)]. A_pale divide between stages VII and IX of the of the seminiferous epithelium (Clermont 1969; Clermont & Antar 1973; Fouquet & Dadoune 1986; Ehmcke et al. 2005b; Simorangkir et al. 2009), although there is disagreement in the literature about whether A_pale divide once (Clermont & Leblond 1959; Clermont 1969; Simorangkir et al. 2009) or twice (Clermont & Antar 1973; Ehmcke et al. 2005a) during a cycle of the seminiferous epithelium. The profile of rhesus A_pale numbers across the cycle of the seminiferous epithelium (Fig. 1B, blue line) appears more similar to mouse A_aligned spermatogonia (Fig. 1A, green and blue lines), although there appears to be a difference between species in the timing of divisions.

There is limited information about the clonal arrangement of A_dark and A_pale spermatogonia in non-human primate seminiferous. Clermont and Leblond first reported pairs and “quartets” of A_dark and A_pale spermatogonia using maps of rhesus macaque spermatogonia from individual seminiferous tubules generated by plotting the relative positions of spermatogonia in numerous serial testis sections (Clermont & Leblond 1959). Using the camera lucida technique, Clermont subsequently reported the clonal arrangement of A_dark and A_pale spermatogonia in hematoxylin-stained segments of intact seminiferous tubules from adult vervet monkeys (Cercopithecus aethiops) (Clermont 1969). This approach enabled visualization of A_dark and A_pale spermatogonia and their topological arrangement. Clermont concluded from these studies that A_dark and A_pale existed only as cleary-demarkated even-numbered clusters, the highest frequency being pairs (20.7%) and groups of 4 (40.7%). It is difficult to draw any broad conclusions about the clonal organization of A_dark and A_pale in primates using these spermatogonial map and camera lucida data for two reasons. First, intercellular cytoplasmic bridges, which would definitively identify clones, are not reproduced in camera lucida drawings. Second, topographical guidelines to help establish clonality, such the 25μm criteria employed later in rodents [see (Huckins 1971)], were not applied to these camera lucida analyses.

de Rooij and colleagues reported clonal arrangement of Type-A spermatogonia in seminiferous tubules recovering after low-dose radiation in a large number of rhesus macaques (van Alphen et al. 1988). Clonal analysis was performed using morphological and topographical criteria similar to those employed for rodents (Huckins 1971; Oakberg 1971; de Rooij 1973). Results in the repopulating seminiferous epithelium demonstrated clones of 1, 2, 4, 8, and ≥16 of both A_dark and A_pale (van Alphen et al. 1988), but it is not clear whether these data equate to the normal clonal arrangement of spermatogonia during steady-state spermatogenesis. As proposed recently, the clonal arrangement of A_dark and A_pale in primates testes during steady-state spermatogenesis may relate to whether these cells exhibit functional identity as stem cells or progenitor spermatogonia [reviewed by (Ehmcke et al. 2006; Ehmcke & Schlatt 2006)]. The observation that isolated, single spermatogonia in S-phase were exceedingly rare in the rhesus testis prompted the theory that there are many nonproliferating single Type-A spermatogonia, which may be A_dark spermatogonia (Ehmcke et al. 2005b). Thus, additional studies are needed to clarify the kinetics of Type-A spermatogonial expansion in primates, including defining A_dark and A_pale clone size as it relates to stage of the seminiferous epithelium.

III. Kinetics of Type-A spermatogonial proliferation in primate testes

In support of the “reserve stem cell” model discussed above, a few studies have proposed mechanisms for how A_pale maintain spermatogenesis in the absence of significant A_dark proliferation. Based on morphometric evaluation in the Vervet monkey, Clermont reported that all A_pale divide between stages IX and X of the seminiferous epithelium (Clermont 1969). Half of the daughter population remains as A_pale (self-renews) and the other half differentiates to B1 spermatogonia (Clermont 1969). In the stump-tailed macaque (Macaca arctoides), Clermont observed two divisions of A_pale in each cycle of the seminiferous epithelium. A doubling A_pale division occurs at stage VII and a second, differentiating division occurs at stage IX to produce B1 spermatogonia (Clermont 1972; Clermont & Antar 1973). Similarly, using whole mount preparations of rhesus seminiferous tubules, Ehmcke and colleagues observed two mitotic A_pale events per cycle and proposed a “clone splitting” model of A_pale renewal and differentiation (Ehmcke et al. 2005a). In this model, larger clones of A_pale (e.g., 8 cells) produced after the first division at stage VII disintegrate into multiple smaller A_pale clones (e.g., 2 clones of 2 A_pale and 1 clone of 4 A_pale) that will again divide (at stage IX) to form clones of 4 or 8 B1 spermatogonia or more clones of 8 A_pale (self-renewal). These models attempt to explain how A_pale spermatogonia might balance self-renewing and differentiating divisions to maintain spermatogenesis with little contribution from A_dark.

Work from the de Rooij laboratory provided evidence that A_dark are mobilized following cytotoxic insult to the testis (van Alphen & de Rooij 1986). During the first 11 days after X-irradiation of rhesus testes, these investigators observed a near depletion of A_pale spermatogonia, with no significant change in the number of A_dark. A significant decrease in A_dark spermatogonia was observed 14 days after X-irradiation with a corresponding increase in A_pale. The authors concluded that the resting A_dark were activated into proliferating A_pale spermatogonia (van Alphen & de Rooij 1986). Thus, A_dark appear to fulfill the role of a “reserve stem cell”. However, whether A_dark spermatogonia also participate in steady state spermatogenesis remains an open question.

Since Clermont’s initial observation that A_dark spermatogonia do not divide (Clermont 1969) six additional studies have been conducted in various nonhuman primate species to describe the proliferating fraction of Type-A spermatogonia (Table 2). Four of the five studies observed ³H-Thymidine or BrdU label in A_dark spermatogonia within a few hours and/or several days after administration of a pulse label (Clermont & Antar 1973; Kluin et al. 1983; Fouquet & Dadoune 1986; Ehmcke et al. 2005b; Simorangkir et al. 2009). Similar to Clermont’s observation in the Vervet monkey, Simorangkir and colleagues did not observe labeling in A_dark (Simorangkir et al. 2009). However, these investigators observed labeling in a new “unclassified” category of Type-A spermatogonia (A_unc), which they acknowledged might be classified as A_dark by other investigators. It is not clear if A_unc are the same cells as those previously defined with an intermediate phenotype as A-transition (A_t), some of which also label with ³H-Thymidine (Fouquet & Dadoune 1986). Immunohistochemistry for the proliferating cell nuclear antigen (PCNA) has also determined rhesus and human A_dark spermatogonia failed to label, while only a fraction of A_pale in late stages of the cycle of the seminiferous epithelium were PCNA⁺ (Schlatt & Weinbauer 1994). Considering the lack of consensus about the cell cycling characteristics of A_dark spermatogonia, additional studies are needed to confirm whether 1) A_dark are quiescent and serve as reserve stem spermatogonia or 2) A_dark divide with sufficient (albeit low) frequency to maintain spermatogenesis under normal, steady state conditions.

Table 2.

Experimental evidence for A_dark and A_pale proliferation.

Study	Species€	Label	Animals, X-sections†	Time to Analysis	% Adark Labeled (stage)	% Apale Labeled (stage)	% At/Aunc Labeled (stage)
Clermont, 1969	Cercopithecus aethiops	3H-Thy	1, N/A	2.5hr	0	36.1% (VII–X)	N/A
Clermont & Antar, 1973	Macaca arctoides	3H-Thy	3, ≥400	3hr	0.06–0.09 (VII–X)	25.6–41.9 (VII–X)	N/A
			4, ≥400	12d 3hr	0	5.9–11.8 (VII–X)	N/A
Kluin et al., 1983	Macaca fascicularis	3H-Thy*	5, 60	1hr*	1.5 (9 of ~600 cells)	14.9 (88 of ~591 cells)	N/A
Fouquet & Dadoune, 1986	Macaca fascicularis	3H-Thy‡	2, 800	1hr	0	4–34 (VI–IX)	0.5–2.2 At (VII–IX)
			2, 800	9–10d	2.3–10.8 (IV–IV)	8.4–47.6 (IV–IX)	2.9–36.3 At (V–VIII)
Schlatt & Weinbauer, 1994	Macaca mulata	PCNA	2, 1.5×106 μm2	N/A	0	X–XI only	N/A
	Homo sapiens	PCNA	2, not indicated	N/A	0	V only	N/A
Ehmcke et al., 2005	Macaca mulata	BrdU	4,N/A#	3hr	0.77 or 18.39 (all stages)¥	27.52 (VII only)	N/A
Simorangkir et al., 2009	Macaca mulata	BrdU	4, N/A§	3hr	0 (IV–IX)	~13–24 (VI–XII)	~2% (VI–XII) Aunc
			4, N/A§	11d 3hr	0	Yes (VII–X)◇	Yes (VI–XI) Aunc◇

^€Cercopithecus aethiops: Vervet monkey; Macaca arctoides: Stump-tailed macaque; Macaca Fascicularis: Cynomolgus monkey (aka: crab-eating monkey, java monkey, Macaca irus); Macaca mulata: Rhesus monkey

^†Number of seminiferous tubule cross-sections is noted per animal evaluated, if available.

^*Testicular fragments (1–8mm³) were incubated in medium containing ³H-Thymidine for 1hr at 32°C after a 30min equilibration period.

^‡Label was administered into the testicular artery followed by two intratesticular injections of label at positions 2cm apart. Biopsy at 1hr recovered tissue at one intratesticular injection site, hemicastration at 9–10d was used to remove tissue at second injection site.

^#Although the precise number of seminiferous cross-sections was not reported, the number of microscopic fields was reported per animal (not discriminating between the two analysis points). Spermatogenesis of two animals included in this study was maintained by exogenous gonadotropin administration following a GnRH clamp for a separate study.

^¥A_dark labeling was classified as weak and strong, representing the two values noted, respectively.

^§Numbers of seminiferous tubule cross-sections were not reported, but 1053–1299 cells of each type were evaluated per testis.

^◇The labeling index of these cells was not reported at 11d 3hr after label administration.

The A_dark/A_pale “reserve stem cell” model of nonhuman primate SSCs is very similar to the A₀/A₁ “reserve stem cell” model that was originally advanced for rodents (Clermont & Bustos-Obregon 1968; Dym & Clermont 1970; Clermont & Hermo 1975; Bartmanska & Clermont 1983). However, this model was supplanted by an alternative model (Huckins 1971; Oakberg 1971), in which there is a single population of stem cells (A_single spermatogonia) that divides regularly, but infrequently, and gives rise to the entire spermatogenic lineage. This A_single model has gained wide (but not universal) acceptance in the field. Spermatogenesis is highly conserved (Fritz 1986), and thus, it is tempting to extrapolate results from studies of rodent SSCs to primates.

The identity, characteristics and behavior of primate SSCs, however, must be determined experimentally. As reviewed above, rodent SSCs can be identified using three approaches: 1) transplantation to observe functional capacity to establish and maintain spermatogenesis (Ogawa et al. 1997; Nagano & Brinster 1998), 2) molecular phenotype (expression of some or all of a battery of specific molecular markers), and 3) clonal arrangement [A_single; (de Rooij & Russell 2000)]. Until recently, almost nothing was known about SSCs in primate testes. Progress in the last few years has begun to address this deficit using rhesus-to-nude mouse xenotransplantation, molecular phenotyping of spermatogonia, and clonal analysis of spermatogonia in whole-mount aided by immunohistochemistry.

IV. An assay for studying primate SSCs

In rodents, SSC transplantation is the experimental “gold standard” for detecting spermatogonial stem cell activity because it demonstrates that a cell has the biological capacity to initiate and maintain spermatogenesis by balancing self-renewal and differentiation (Brinster & Avarbock 1994; Brinster & Zimmermann 1994). While this functional assay has been a powerful tool for characterizing SSCs in rodents, monkey-to-monkey SSC transplantation as a routine biological assay is not feasible. Nonhuman primates are a limited resource and studies on these large animals are confounded by their large size, long life span, high cost, and variability among out-bred individuals. Pioneering work from the Brinster, de Rooij, Dobrinski, and Griswold laboratories, however, provided the proof-in-principle that SSCs from a variety of species can be transplanted to the testes of immune deficient nude mice where they migrate to the seminiferous tubule basement membrane and proliferate to form chains of spermatogonia that persist long-term (Clouthier et al. 1996; Ogawa et al. 1999; Dobrinski et al. 1999a; Dobrinski et al. 2000; Nagano et al. 2001b; Nagano et al. 2002; Oatley et al. 2002; Izadyar et al. 2003; Honaramooz et al. 2002). Germ cells from closely related species (e.g., mouse, rat and hamster) produce chains or networks of spermatogonia by 2 weeks after transplantation (Fig. 2A–B), which give rise to extensive colonies of complete spermatogenesis by two months (Fig. 2C–D). In contrast, germ cells from primate species including rhesus macaques [Fig. 2E–F; (Hermann et al. 2007; Hermann et al. 2009)], baboons (Nagano et al. 2001b) and humans [Fig. 2G–H; (Nagano et al. 2002)] produce chains and patches of spermatogonia, similar to rodent colonies at two weeks, but do not produce complete spermatogenesis. The ability of mouse Sertoli cells to support the early stages of spermatogenesis from distantly related species represents remarkable evolutionary conservation. These patches of spermatogonia are maintained long-term and have been observed for several months to a year after transplantation (Nagano et al. 2001b; Nagano et al. 2002; Hermann et al. 2007; Hermann et al. 2009). As detailed below, these rudimentary spermatogonial patches in the xenotransplant paradigm may constitute an experimentally tractable bioassay for primate SSCs.

Figure 2. SSC transplantation from different donor species into busulfan-treated mouse testes.

(A–B) Donor GFP mouse testis cells (green) at 2 weeks after transplantation. Margins of recipient seminiferous tubules are marked by a dashed white line. (C–D) Donor GFP mouse testis cells (green) at 2 months after transplantation. Patches of transplanted donor (E–F) rhesus and (G–H) human testis cells in immune-deficient nude mouse seminiferous tubules were detected by whole-mount immunohistochemistry using the rhesus testis cell antibody. Scale bars = 50μm. Adapted from (Hermann et al. 2007; Hermann et al. 2009) and unpublished data.

To enable this xenotransplant assay, immune deficient nude mice are treated with busulfan to eliminate endogenous spermatogenesis, as previously described (Brinster & Avarbock 1994; Brinster & Zimmermann 1994). Donor testis cell suspensions can be generated by two-step enzymatic digestion (Bellve et al. 1977; Hermann et al. 2007) and transplanted into seminiferous tubules of nude mouse recipient testes by efferent duct injection (Nagano et al. 2001b; Nagano et al. 2002; Hermann et al. 2007; Maki et al. 2009). Donor-derived patches of spermatogonia can be identified 1–2 months after transplantation by staining with a donor species-specific antiserum, as previously described (Nagano et al. 2001b; Nagano et al. 2002; Hermann et al. 2009; Hermann et al. 2007). Alternatively, donor testis cells can be preloaded with a fluorescent marker (e.g., PKH26 or CFDA) prior to transplantation (Honaramooz et al. 2002; Maki et al. 2009). With the fluorescent loading approach, recipient testes are typically analyzed within 2–3 weeks after transplantation to minimize the dilution of the fluorescent dye through cell divisions.

While evolutionary distance between primates and mice presumably precludes establishment of complete spermatogenesis in the xenotransplant assay, colonization foci consisting of spermatogonial patches exhibit several features of SSCs. They arise from transplanted cells that migrate to the basement membrane of recipient seminiferous tubules and produce chains of spermatogonia that persist long-term (Nagano et al. 2001b; Nagano et al. 2002; Hermann et al. 2007; Hermann et al. 2009). For our studies in rhesus macaques, we have defined SSC-derived spermatogonial patches as 4 or more cells in discrete patches (within 100 μm length) on the basement membrane of recipient mouse seminiferous tubules that exhibit characteristic spermatogonial features (i.e., ovoid shape with high nuclear to cytoplasmic ratios). Nagano and co-workers used similar criteria for baboon and human SSCs, but defined patches as containing 10 or more cells within a 150 μm length of seminiferous tubule (Nagano et al. 2001b; Nagano et al. 2002). Co-staining for VASA or RBMY has been used to confirm that the xenotransplant spermatogonial patches were comprised of germ cells (Nagano et al. 2001b; Hermann et al. 2007). However, it is important to interpret xenotransplant results cautiously because some donor cell foci fail to exhibit spermatogonial features, and therefore, are not considered to arise from stem cells (Hermann et al. 2007). As more information becomes available, it may be reasonable to revise the criteria for defining spermatogonial patches in the xenotransplant assay.

Using the criteria detailed above, we employed rhesus-to-nude mouse xenotransplantation to characterize SSC activity in adult rhesus testes. Analysis of nude mouse recipient seminiferous tubules two months after transplantation revealed 4.64 patches/10⁶ viable donor adult rhesus testis cells transplanted (Hermann et al. 2007). In contrast, testis cells from males treated with busulfan (8 or 12 mg/kg) failed to produce any patches of spermatogonia. These data suggested that high-dose alkylating chemotherapy caused depletion of SSCs in rhesus testes, consistent with previous observations for mice (Kanatsu-Shinohara et al. 2003; Orwig et al. 2008). In a separate study, the xenotransplant assay demonstrated that spermatogonial patches were enriched in the THY-1⁺ fraction of rhesus testis cells (Hermann et al. 2009), with corresponding depletion in THY-1⁻ rhesus testis cells, similar to observations of mouse and rat SSCs (Kubota et al. 2003; Ryu et al. 2004). Evolutionary conservation of these biological readouts provides a partial validation of the xenotransplantation assay (Hermann et al. 2007; Hermann et al. 2009). Maki and colleagues have also utilized the xenotransplant technique and reported enhanced colonizing activity in an SSEA4⁺ subpopulation of rhesus testis cells (Maki et al. 2009). Continued FACS and xenotransplantation experiments will enable systematic characterization of primate SSCs and potentially lead to enrichment strategies with implications for future SSC-based treatments of male infertility.

V. Marker analysis for functional categorization of rhesus spermatogonia and identification of the putative stem cell pool

Until recently, little was known about the molecular characteristics of nonhuman primate spermatogonia, including SSCs. This contrasts with rodents, where decades of studies provide an extensive molecular phenotype of cell surface, cytoplasmic, and nuclear proteins that are expressed by rodent SSCs. Although no SSC-specific marker has been identified to date for any species, the combined expression profiles of multiple markers provides composite phenotypic information about stem, progenitor and differentiating spermatogonia in rodents that may be used to identify similar cells in other species, including primates. To this end, several recent studies have evaluated nonhuman primate testes for expression of various proteins known to mark SSCs and other stem cells (reviewed in Table 3). However, there is limited information about how these markers correlate with spermatogenic cell types in primates (e.g., A_dark, A_pale and B spermatogonia).

Table 3.

Molecular markers of germ cells and SSCs expressed in nonhuman primate testes.

Marker (aliases)	Reference	Species§	Approach	Expressed in Spermatogonia?	Overlap with
					Adark	Apale
cKIT	Hermann et al., 2009	Macaca mulata	IHC	Yes	0%	22.8%
	Maki et al., 2009	Macaca mulata	FCM	Nd	Nd	Nd
DAZL	Hermann et al., 2007	Macaca mulata	IHC	Staining weak/absent in spermatogonia.	Nd	Nd
GFRα1	Hermann et al., 2007	Macaca mulata	IHC	Rare B.M. cells‡	Nd	Nd
	Hermann et al., 2009	Macaca mulata	IHC	Yes	100%	100%
	Maki et al., 2009	Macaca mulata	ICC/IHC	Rare B.M. cells‡	Nd	Nd
α6-INTEGRIN (INTGA6)	Maki et al., 2009	Macaca mulata	FCM+ICC/XenoTP	Yes*	Nd	Nd
MAGE-A4	Mitchell et al., 2008	Callithrix jacchus	IHC	Yes in 0–6wk testis.	Nd	Nd
NANOS-1	Mitchell et al., 2008	Callithrix jacchus	IHC	Yes in 0–6wk testis.	Nd	Nd
NGN3 (NEUROG3)	Hermann et al., 2009	Macaca mulata	IHC	Yes	0%	48.5%
OCT-4 (POU5F1)	Mitchell et al., 2008	Callithrix jacchus	IHC	Rare cells in 0–6wk testis.	Nd	Nd
PLZF (ZBTB16)	Hermann et al., 2007	Macaca mulata	IHC	Rare B.M. cells‡	Nd	Nd
	Hermann et al., 2009	Macaca mulata	IHC	Yes	82%	100%
SSEA-4	Muller et al., 2008	Macaca mulata	IHC	Rare B.M. cells‡	Nd	Nd
	Muller et al., 2008	Macaca silenus	IHC	Rare B.M. cells‡	Nd	Nd
	Muller et al., 2008	Callithrix jacchus	IHC	Rare B.M. cells‡	Nd	Nd
	Maki et al., 2009	Macaca mulata	FCM+ICC/XenoTP	Yes*	Nd	Nd
SALL4	Hermann, Marshall and Orwig, unpublished.	Macaca mulata	IHC	Yes	Yes	Yes
THY-1 (CD90)	Maki et al., 2009	Macaca mulata	FCM+ICC/XenoTP	Yes*	Nd	Nd
	Hermann et al., 2009	Macaca mulata	FCM + ICC/qPCR/XenoTP	Yes†	Nd	Nd
TRA-1-81	Muller et al., 2008	Macaca mulata	IHC	Rare B.M. cells‡	Nd	Nd
	Muller et al., 2008	Macaca silenus	IHC	Rare B.M. cells‡	Nd	Nd
	Muller et al., 2008	Callithrix jacchus	IHC, FCM	Rare B.M. cells‡	Nd	Nd
VASA	Hermann et al., 2007	Macaca mulata	IHC	Weak in adult.	Nd	Nd
	Mitchell et al., 2008	Callithrix jacchus	IHC	Yes in 0–6wk testis.	Nd	Nd
	Hermann et al., 2009	Macaca mulata	IHC	Yes in juvenile.	100%	100%

^§Macaca mulata: Rhesus monkey; Macaca silenus: Lion-tailed macaque; Callithrix jacchus: Common marmoset.

^#Abbreviations for approaches; IHC – immunohistochemistry, FCM – flow cytometry/fluorescence activated cell sorting, XenoTP – xenotransplantation, ICC – immunocytochemistry, qPCR – quantitative PCR.

^‡Rare cells positive for the marker located on the basement membrane of testicular seminiferous tubule cross-sections.

^*Expression by spermatogonia determined by immunocytochemical staining for GFRα1 in sorted fractions of adult rhesus testis cells.

^†Expression by spermatogonia determined by immunocytochemical staining for VASA and qPCR for VASA, GFRα1, PLZF in juvenile rhesus testis cells sorted for THY-1.

In order to bridge the gap between molecular phenotype data and spermatogenic cell types, we recently investigated expression of rodent spermatogonial markers (GFRα1, PLZF, NGN3, and cKIT) in the rhesus testis and related our findings to classical descriptions of nuclear morphology [i.e., A_dark, A_pale and B spermatogonia; Fig. 3A–E; (Hermann et al. 2009)]. The expression profile of each marker in the rhesus testis was correlated with functional categories of rodent spermatogonia exhibiting similar phenotypes, including: stem (A_single and some A_paired; GFRα1⁺, PLZF⁺, NGN3^+/− and cKIT⁻), transit-amplifying progenitor (some A_paired and A_aligned; GFRα1⁺, PLZF⁺, NGN3⁺, cKIT^+/−) and differentiating (A1–4, Intermediate, B; GFRα1⁻, PLZF⁻, NGN3⁺ and cKIT⁺). We observed a remarkable degree of evolutionary conservation from rodents to primates in the succession of spermatogonial marker expression and their correlation with differentiation state (Fig. 3E). Assuming that molecular characteristics correlate with function and that these relationships are evolutionarily conserved, it may be possible to identify stem spermatogonia in primates. Note that most A_dark and ~50% of A_pale in the adult rhesus testis exhibit the phenotype GFRα1⁺, PLZF⁺, NGN3⁻, cKIT⁻ (Fig. 3E). As far as we can ascertain from the literature, this phenotype is restricted to A_single spermatogonia, which most will agree comprise at least part of the stem cell pool in mice. Moreover, since NGN3 marks 11.7% of functional stem cells in mice as well as A_aligned progenitor spermatogonia (Nakagawa et al. 2007), rhesus A_pale spermatogonia with the phenotype GFRα1⁺, PLZF⁺, NGN3⁺, cKIT⁻ could be either stem or progenitor (shaded as transition cells in Fig. 3E). Lastly, cKIT expression begins in longer-chain progenitors (i.e., A_aligned 8–16) in rodents and continues in differentiating types A1–4 spermatogonia (Schrans-Stassen et al. 1999). Some A_pale in the adult rhesus testis exhibit a transition phenotype (GFRα1⁺, PLZF⁺, NGN3⁺, cKIT⁺, like longer chain rodent A_aligned; Fig. 3E).

Figure 3. Correlation between molecular markers of rodent SSCs (GFRα1, PLZF, NGN3, cKIT) and morphological descriptions (A_dark, A_pale, B) of spermatogonia in the adult rhesus testis.

Sections of adult rhesus testes were evaluated by immunohistochemistry for GFRα1, PLZF, NGN3, and cKIT (Hermann et al. 2009). Subsequently, sections were counterstained by the PAS-Hematoxylin method to reveal nuclear morphology and identify A_dark and A_pale spermatogonia, as well as differentiating Type-B spermatogonia. (A–D) Representative staining for GFRα1 is shown. The image in (A) shows part of one seminiferous tubule (scale bar = 50μm). Enlargements are also shown of representative A_dark (B), A_pale (C) and B4 spermatogonia (D) (scale bar = 10μm). For all spermatogonia that could be definitively classified as A_dark or A_pale, the percentage that were labeled for GFRα1 is shown. (E) Colored bars (GFRα1, blue; PLZF, green; NGN3, black; cKIT, red) indicate the extent of marker expression in the adult spermatogenic lineage based on recently published data (Hermann et al. 2009) (rhesus, Top) or previously published mouse studies [reviewed in (Hermann et al. 2009)] (mice, Bottom). Colored boxes indicate functional descriptors “Stem” (orange), “Progenitor” (yellow) and “Differentiating” (violet), based on rodent data and may identify rhesus spermatogonia with corresponding phenotype and function. The transitions from stem to progenitor or progenitor to differentiating are noted by gradient shading in the middle grey bar between these functional categories. The following abbreviations are used for rodents: A_s = A_single, A_pr = A_paired, A_al = A_aligned. Adapted from (Hermann et al. 2009).

Based on the conservation of molecular markers from rodents to primates, we have proposed that the stem cell pool in the rhesus testis comprises all A_dark and at least 50% of A_pale spermatogonia and that the stem cell pool is considerably larger in rhesus than mouse testes [see (Hermann et al. 2009) for detailed discussion]. In contrast to the large SSC pool, the relative size of the progenitor pool (GFRα1⁺, PLZF⁺, NGN3⁺, cKIT⁻) appeared much smaller in adult macaques than in rodents. Thus, it appears that rodents and primates employ different strategies to meet a similar biological demand [adult rodent and adult rhesus testes have similar sperm output per gram of testis per day (Sharpe 1994; Gupta et al. 2000; Thayer et al. 2001)]. Rodents may have few SSCs and more transit-amplifying progenitors while rhesus testes may have more SSCs and fewer transit-amplifying progenitors.

Progress studying SSCs and the spermatogenic lineage in rodents and nonhuman primates has begun to stimulate investigations of the biology and regenerative potential of human SSCs [reviewed by (Dym et al. 2009)]. One recent study confirmed that α6-INTEGRIN, CD133, SSEA4, VASA, DAZL, and TSPYL2 are expressed in human testis cells or histological section (Conrad et al. 2008). For this review, we have also conducted a comparative analysis of the consensus SSC marker, PLZF, in mouse (Fig. 4A–B), rhesus monkey (Fig. 4C–D) and human (Fig. 4E–F) testes. PLZF staining in human testes was restricted to a subpopulation of cells on the basement membrane of the seminiferous epithelium (like mouse and monkey) and the frequency of PLZF⁺ cells was more similar to monkey than mouse. Other differences (e.g., expression of OCT-4, TSPY, cKIT) have been reported in marker expression between rodent and human spermatogonia, suggesting phenotypic differences in markers of stem cells and their progeny [see review by (Dym et al. 2009)]. Perhaps this suggests that the dynamics of the stem/progenitor spermatogonial pools in humans is similar to monkeys. Additional studies are necessary to elaborate on these findings. For future studies, it appears that human-to-nude mouse xenotransplantation can also be optimized as a bioassay for human SSCs [(Nagano et al. 2002); and see Fig. 2G and H].

Figure 4. Comparative immunohistochemical analysis reveals species-specific staining profiles for the stem/progenitor marker PLZF.

To begin translating knowledge of rodent and monkey SSCs to humans, we have initiated comparative marker analysis using immunohistochemistry for the transcription factor PLZF in sections from (A–B) mouse, (D–E) rhesus macaque, and (G–H) human testes. Images of sections incubated with non-immune isotype control IgGs are also shown for (C) mouse, (F) monkey, and (I) human to demonstrate non-specific background staining and tissue autofluorescence. White asterisks in A & C indicate the non-specific fluorescent signal observed in interstitial space between seminiferous tubules. Images are shown from (A, C, D, F, G, I) low magnification and (B, D, F) high magnification. PLZF immunoreactivity was observed as a nuclear fluorescent signal (green) in all three species. Sections were counterstained with DAPI (blue). Scale bar = 50μm. (Hermann, Hansel and Orwig, unpublished).

VI. Future Directions

The considerable degree of phenotypic similarity between A_dark and A_pale spermatogonia in the adult rhesus testis raises questions about the distinct functional classification of A_dark and A_pale as reserve and renewing stem cells, respectively (Hermann et al. 2009). One possibility is that dark and pale nuclear morphologies correspond with stage of the cell cycle (i.e., G₀ vs. G₁/S/G₂/M) and not distinct stem cell populations. Experiments are ongoing to determine if nuclear morphology correlates with cell cycle stage. In addition, expression of NGN3 encompassed a transition from cKIT⁻ to cKIT⁺ within A_pale spermatogonia, suggesting that initiation of NGN3 coincides with monkey spermatogenic differentiation. Future studies will begin to dissect this transition in A_pale to identify regulatory networks that instruct spermatogonial differentiation in primates.

In rodents, spermatogonial clone size is associated with spermatogenic differentiation state; a spermatogonial clone differentiates as it becomes larger. As discussed above (see Section II), there is limited information about the clonal arrangement of Type-A spermatogonia in primates (Clermont 1969). We and others have begun to characterize markers of undifferentiated (e.g., PLZF, GFRα1) versus differentiating (cKIT) spermatogonia in primates. Thus, coupling immunohistochemical staining with spermatogonial clone size analysis in whole mount preparations of seminiferous tubules would provide valuable information correlating differentiation state with clone size in primates. Clonal analyses of this nature could elucidate the point in spermatogonial amplification (i.e., clone size) at which a differentiated phenotype begins to emerge. To this end, we have initiated studies to evaluate spermatogonial clones that exhibit undifferentiated (PLZF⁺; Fig 5A–B) and differentiating (cKIT⁺; Fig. 5C–D) phenotypes using whole mount immunohistochemistry. We have taken the liberty of labeling some of these clones as single (S), paired (P) and aligned (Al) to stimulate thinking about how rhesus spermatogenic lineage development might compare/contrast with the rodent (Fig. 5).

Figure 5. Clonal organization of undifferentiated and differentiating spermatogonia in adult rhesus seminiferous tubules.

Determining the clonal arrangement of undifferentiated and differentiating spermatogonia may be possible using whole mount immunohistochemistry in intact seminiferous tubules. In separate experiments, (A–B) undifferentiated (PLZF⁺, Green) or (C–D) differentiating (cKIT⁺, Red) spermatogonia were detected in adult rhesus seminiferous tubules. Some clones are identified in each panel as single (S), pairs (P) or aligned (Al). Scale bars = 25μm. (Hermann and Orwig, unpublished). Note: this is not a co-staining experiment.

There is a growing body of literature suggesting that rodent SSCs exhibit some degree of phenotypic, and perhaps functional, heterogeneity. For instance, several studies have shown Neurogenin 3 (Ngn3) is expressed by a subset of spermatogonia with stem cell properties, suggesting subpopulations of SSCs with different functional roles or degrees of differentiation (Nakagawa et al. 2007; Yoshida et al. 2007; Zheng et al. 2009). A recent report suggests that GFRa1 expression is heterogeneous among A_single spermatogonia in mice and among human A_dark and A_pale (Grisanti et al. 2009). Moreover, a recent study suggested that some spermatogonia expressing the differentiation marker cKIT retained some degree of stem cell capacity and could repopulate the seminiferous epithelium under certain circumstances (Barroca et al. 2009). Additional studies are needed to validate these suggestions of stem cell heterogeneity and flexibility, expound on their implications for spermatogenesis, and define whether similar phenomena occur in primate testes.

Another emerging concept in the field is that there may be intraclonal heterogeneity among the earliest progeny of SSCs. Striking results have been observed in three separate studies where some undifferentiated spermatogonial clones (A_paired and A_aligned) exhibit this type of heterogeneity and some individual cells within a clone appear different from their clonal partners (Zheng et al. 2009; Grisanti et al. 2009; Luo et al. 2009). One interpretation of these data is that individual clones demonstrate asymmetry that could produce new SSCs when larger spermatogonial clones divide. These are infrequent observations (1.7–5%) but reminiscent of the “clone splitting” model of Type-A spermatogonial self-renewal and differentiation in primates [reviewed by (Ehmcke & Schlatt 2006)]. To date there is no other data supporting a mechanism of asymmetric division among clones of undifferentiated spermatogonia in rodents, but asymmetric division of germline stem cells is well established in flies [reviewed by (Fuller & Spradling 2007)].

VII. Conclusions

For several decades, rodent and primate spermatogenesis have been considered largely dissimilar with important biological differences in the identity and behavior of SSCs (Plant & Marshall 2001). Concepts of rodent and primate spermatogenesis and SSC biology, however, were based largely on different experimental methodologies (Fig. 6A–C). Knowledge of primate testis biology lagged behind rodents, due in part to the dramatic differences in the relative volume of research conducted in these species, which are experimentally and evolutionarily disparate. Research on primate spermatogenesis (nonhuman and human) is poised for accelerated growth with improved access to validated reagents (e.g., antibodies for SSC markers) and experimental tools (e.g., xenotransplantation and xenografting) (Jahnukainen et al. 2006; Muller et al. 2008; Hermann et al. 2007; Maki et al. 2009; Hermann et al. 2009). The molecular characteristics of A_dark and A_pale spermatogonia are beginning to emerge, allowing their alignment with subpopulations of undifferentiated rodent spermatogonia (i.e., A_single, A_paired, A_aligned; Fig. 6D–E), and identification of putative monkey SSCs (Fig. 3E). A_dark spermatogonia exhibit the most undifferentiated phenotype (GFRα1⁺, PLZF⁺, and cKIT⁻). Additional studies will be required to determine whether A_dark spermatogonia are truly reserve stem cells or whether these cells divide with sufficient frequency to participate in steady state spermatogenesis of the adult monkey testis. While many A_pale also exhibit this undifferentiated phenotype (GFRα1⁺, PLZF⁺, and cKIT⁻), some A_pale appear phenotypically more similar to committed progenitor spermatogonia in rodents (i.e., A_{aligned8–16}; GFRα1⁺, PLZF⁺, cKIT⁺). This linear developmental ordering bears some resemblance to the “A_single” model of rodent spermatogenesis (Fig. 6D–E). Questions remain about whether A_dark and A_pale are 1) different stem cell populations, 2) parts of the same cell population, perhaps at different stages of the cell cycle, or 3) stem cells and progenitors, respectively (Fig. 6E). In addition, more studies are needed to determine whether clone size correlates with differentiation state in primates, as it does in rodents (Fig. 6A) and whether clone size correlates with “dark” and “pale” descriptions of spermatogonial nuclear morphology (Fig. 6B–C). There is now increasing experimental momentum towards identifying and characterizing primate SSCs and the mechanisms by which they self-renew and differentiate to produce spermatogenesis. This momentum is fueled by both the biological insights that it provides and the possible implications for treating human male infertility.

Figure 6. Current conceptual models of rodent and primate spermatogenesis.

(A) Immunohistochemical staining for PLZF (green) was performed using whole-mount preparations of adult rat seminiferous tubules and clones of PLZF⁺ spermatogonia were identified as A_single, A_paired, or A_aligned using the 25μm criteria (Huckins 1971; de Rooij & Russell 2000). Scale bar = 50μm. Sections of rhesus macaque testes were stained using the periodic acid-Schiff method and counterstained with Gills hematoxylin (Simorangkir et al. 2003) to reveal nuclear morphology and identify (B) A_dark and (C) A_pale spermatogonia. Scale bars = 10μm.

(D) Rodent undifferentiated spermatogonia are noted (bracket) including stem spermatogonia (SSCs) comprised of A_singles and some A_paired spermatogonia that will complete cytokinesis to produce new A_singles and maintain the stem cell pool. Transit-amplifying progenitors include some A_paired and A_aligned spermatogonia (chains of 4–16 cells). Whole mount and transplantation analyses provided phenotypes for cells in these categories: stem (A_single and some A_paired; GFRα1⁺, PLZF⁺, and cKIT⁻), progenitor (some A_paired and A_aligned; GFRα1⁺, PLZF⁺, cKIT^+/−) and differentiating (A1–4, Intermediate, B; GFRα1⁻, PLZF⁻, and cKIT⁺). (E) In primate testes, undifferentiated (Type-A) spermatogonia are designated A_dark and A_pale based on nuclear architecture and staining intensity with hematoxylin. Recent progress has provided information about the molecular phenotype of A_dark and A_pale spermatogonia that allow alignment with rodent spermatogonia exhibiting a similar phenotype. The number of A_pale generations is still not clear and may be resolved by future whole-mount marker and clone size analysis (dotted arrows with question marks).