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PMCID: PMC2945412

The C. elegans adult male germline: stem cells and sexual dimorphism


The hermaphrodite C. elegans germline has become a classic model for stem cell regulation, but the male C. elegans germline has been largely neglected. This work provides a cellular analysis of the adult C. elegans male germline, focusing on its predicted stem cell region in the distal gonad. The goals of this study were two-fold: to establish the C. elegans male germline as a stem cell model and to identify sex-specific traits of potential relevance to the sperm/oocyte decision. Our results support two major conclusions. First, adult males do indeed possess a population of germline stem cells (GSCs) with properties similar to those of hermaphrodite GSCs (lack of cell cycle quiescence, lack of reproducibly oriented divisions). Second, germ cells in the mitotic region, including those most distal within the niche, exhibit sex-specific behaviors (e.g. cell cycle length) and therefore have acquired sexual identity. Previous studies demonstrated that some germ cells are not committed to a sperm or oocyte cell fate, even in adults. We propose that germ cells can acquire sexual identity without being committed to a sperm or oocyte cell fate.

Keywords: Stem cell, stem cell niche, cell cycle, sexual dimorphism, germline sex determination, sexual identity


The hermaphrodite germline of Caenorhabditis elegans provides a well-defined and extensively studied model for stem cell controls and the sperm/oocyte cell fate decision (Kimble and Crittenden, 2007), but the C. elegans male germline has largely been ignored. Yet C. elegans XX hermaphrodites and XO males are as distinct from each other as XX females and XY males in other phyla, with sex-specific differences in both morphology and gene expression in virtually all tissues (Wolff and Zarkower, 2008). Understanding controls of sexual dimorphism can provide key insights into how regulatory pathways can be modified and evolve (Williams and Carroll, 2009). We therefore embarked on an analysis of the adult C. elegans male germline, focusing on its predicted stem cell region at the distal end.

The gross organization of the adult germline is similar in the two C. elegans sexes (Figs. 1A and B) (Kimble and Crittenden, 2007). Most relevant to this work is the “mitotic region,” which resides at the distal end of the gonad and contains virtually all mitotically-dividing germ cells (Crittenden et al., 2006; Crittenden et al., 1994; Maciejowski et al., 2006). Maintenance of the adult mitotic region in both sexes requires continuous Notch signaling (Austin and Kimble, 1987; Kimble and Crittenden, 2007). Importantly, the hermaphrodite mitotic region, sometimes called the proliferative zone, contains germline stem cells (GSCs) that both self-renew and generate differentiating gametes. The GSCs were initially inferred from the ability of the mitotic region to maintain itself while contributing to the population of differentiating gametes (Crittenden et al., 2006). Stem cell function was then demonstrated more explicitly by regeneration of a functional germline from the adult stem cell pool after starvation (Angelo and Van Gilst, 2009).

Fig. 1
Adult hermaphrodite and male DTCs and germline organization. (A, B) A single somatic hermaphrodite distal tip cell (hDTC) (red) and two somatic male distal tip cells (mDTCs) (blue) reside at the distal ends of the adult hermaphrodite and male gonads, ...

Adult hermaphrodite germ cells move proximally from the mitotic region into the “transition zone,” where they enter early meiotic prophase (Dernburg et al., 1998; Francis et al., 1995). Entry into meiotic S-phase occurs within the mitotic region, but only at its proximal edge some distance from the stem cell niche (Crittenden et al., 2006; Hansen et al., 2004; Jaramillo-Lambert et al., 2007). Germ cells continue moving proximally as they progress through meiotic prophase and either differentiate as oocytes or undergo programmed cell death (Crittenden et al., 2006; Gumienny et al., 1999; Jaramillo-Lambert et al., 2007; Jaramillo-Lambert and Engebrecht, 2010). Adult hermaphrodite oocytes are typically fertilized by mature sperm that were made during larval development.

The adult male germline has been explored in less depth. Male germlines produce sperm continuously and do not undergo programmed cell death (Gumienny et al., 1999; Jaramillo-Lambert and Engebrecht, 2010). A mitotic region occupies the distal gonad, but germline self-renewal has not been investigated. Germ cells move proximally in the adult male germline from the mitotic region into differentiated progeny (Jaramillo-Lambert et al., 2007), but the rate of movement has not been estimated. Moreover, germ cell cycles have not been analyzed in adult male germlines. Therefore, a gap clearly exists in our knowledge of adult male germlines.

Germ cells reside within a sexually dimorphic somatic gonad, which includes regulatory cells as well as ducts for germ cell maturation and exit (Hubbard and Greenstein, 2000). Each hermaphrodite gonadal arm has a somatic “hermaphrodite distal tip cell (hDTC)” at its distal end (Figs. 1A and C, arrows) and additional hermaphrodite-specific somatic gonadal tissues more proximally (e.g. sheath cells proximal to the mitotic region); the single male gonadal arm possesses two somatic “male DTCs (mDTCs)” near its distal end (Figs. 1B and D, arrows) and additional male-specific somatic gonadal tissues (e.g. vas deferens) more proximally. The hDTCs drive GSC proliferation at the expense of differentiation and they also shape the developing gonad, whereas mDTCs are specialized for GSC proliferation (Kimble and White, 1981). In hermaphrodites, sheath cells support robust proliferation during larval development (Killian and Hubbard, 2005; Korta and Hubbard, 2010; McCarter et al., 1997; McGovern et al., 2009), but males do not have sheath cells. Therefore, the somatic gonadal controls of GSC proliferation are sexually dimorphic with respect to DTC number, DTC function and the presence or absence of sheath cells.

A comparison of adult male and hermaphrodite mitotic regions has the potential to impact our thinking about germline sexual identity and the sperm/oocyte decision. Previous results have begun to address those issues. First and perhaps most importantly, the adult C. elegans germline appears to be sexually labile. Using temperature-sensitive mutants, RNAi or chemical inhibitors to manipulate germline sex regulators, adult spermatogenic germlines can be induced to make oocytes and adult oogenic germlines can be triggered to make sperm (Barton and Kimble, 1990; Barton et al., 1987; Chen et al., 2000; Morgan et al., 2010; Otori et al., 2006; Schedin et al., 1994). Thus some adult germ cells are sexually labile. Second, commitment to sperm or oocyte differentiation occurs roughly when germ cells enter the meiotic cell cycle, at least in larvae (Barton and Kimble, 1990). If meiotic entry also coincides with sexual commitment in adult germlines, which is not yet known, one would predict that germ cells in the proximal mitotic region or transition zone, where germ cells transition into the meiotic cell cycle, would have become committed to the sperm or oocyte fate. Third, sex-specific gene expression is seen throughout the germline, even within the mitotic region (see below) (Jones et al., 1996; Starostina et al., 2007; Thompson et al., 2005). These three complementary lines of evidence lead to the idea that some germ cells, likely those in the mitotic region, are not committed to the sperm or oocyte cell fate even though they show sex-specific gene expression.

Three dual regulators of cell cycle and sperm/oocyte specification are expressed in sex-specific patterns in the mitotic region. The TRA-1/GLI transcription factor is more abundant in hermaphrodite than male distal gonads (Starostina et al., 2007). However, its role in the gonad is complex. TRA-1 promotes proliferation in the somatic gonad (Mathies et al., 2004), but a similar role in the germline has not been reported; in the germline, TRA-1 promotes oogenesis (Zarkower, 2006) as well as maintenance of sperm fate specification (Hodgkin et al., 1987; Schedl et al., 1989). Thus, the sex-specific abundance of TRA-1 in the distal germline is difficult to interpret functionally, but its sex-specific expression suggests that germ cells in this region have acquired a sexual identity. In the proximal mitotic region and transition zone, the GLD-1/STAR translational repressor is higher in hermaphrodites than males (Jones et al., 1996). Yet GLD-1 drives entry into the meiotic cell cycle in both sexes and is expressed in the proximal mitotic region in both sexes (Hansen et al., 2004; Kadyk and Kimble, 1998). Additionally, GLD-1 promotes oocyte specification and has a non-essential role in sperm specification (Francis et al., 1995; Kim et al., 2009). Finally, the FOG-1/CPEB RNA-binding protein is also present in the proximal mitotic region and transition zone of males but not in adult hermaphrodites (Thompson et al., 2005). FOG-1/CPEB promotes mitotic proliferation in spermatogenic germlines and sperm fate specification in both XX and XO animals (Barton and Kimble, 1990; Thompson et al., 2005). The role of FOG-1 in proliferation led us to explore the mitotic cell cycle for sexual identity at the level of cell behavior rather than gene expression.

This work focuses on the mitotic region in adult males. Our goals were to establish a stem cell model that was related to but distinct from the hermaphrodite GSC model and to identify sex-specific features of potential relevance to the sperm/oocyte decision. Our results provide evidence for the existence of adult male GSCs with properties similar to those of hermaphrodite GSCs (lack of cell cycle quiescence, lack of reproducibly oriented divisions). They also demonstrate that the length of the mitotic cell cycle is sex-specific throughout the mitotic region, including germ cells adjacent to the DTC within the niche. We conclude that all germ cells possess sexual identity, defined as the possession of sex-specific traits, and propose that sexual identity is not equivalent to commitment to a sperm or oocyte cell fate.


Nematode strains

Strains were maintained at 20°C as described (Brenner, 1974). We used the wild-type strain, N2, and the following mutants: LG I: fog-1(q785) (this work); LG II: tra-2(q276) (Okkema and Kimble, 1991); LG III: tra-1(e1488) (Hodgkin, 1987), tra-1(e1732) (Hodgkin, 1987); LG V: him-5(e1490) (Hodgkin et al., 1979). In addition, we used qIs56, an insertion on chromosome V of a transgene carrying Plag-2::GFP, a DTC-specific marker (Siegfried and Kimble, 2002), and fluorescently labeled hT2[qIs48] (Siegfried and Kimble, 2002) to balance fog-1(q785), eDp6 to balance tra-1(e1732), and mnC1 to balance tra-2(q276). tra-1(e1488) was maintained as a homozygote. fog-1(q785) was isolated in a deletion screen as described (Kraemer et al., 1999); fog-1(q785) removes 1041 base pairs, starting at the end of exon 1 and ending in the middle of intron 4 (positions 3,211,119 to 3,212,159 of the genomic sequence) and inserts AATTTTT at the site of the deletion. XX and XO fog-1(q785) homozygotes make oocytes only, and fog-1(q785) heterozygotes are feminized with all XO animals making both sperm and oocytes (n=48) and 11% XX animals making only oocytes (n=106). fog-1(q785) appears to be a null allele: it removes part of the first RRM domain, which is required for FOG-1 function (Jin et al., 2001), and fog-1L mRNA was not detectable by RT-PCR using oligo dT. By contrast, fog-1L mRNA was found in wild-type, and unc-54 mRNA was present in wild-type and fog-1(q785) animals.

Mitotic region analyses

We examined both unmated males (grown without hermaphrodites from L4 until 24 hours after L4) and mating males (grown with hermaphrodites from L4 until 24 hours after L4) for our mitotic region and cell cycle analyses. Data is from unmated males except where noted. Mitotic region morphology, MI, SI, G2+M+G1 interval, pulse-chase data, and movement rate were similar in unmated males and males from mating plates.

We analyzed mitotic regions in dissected, 4′,6-diamidino-2-phenylindole (DAPI)-stained, adult (24 hours after L4) gonads (Crittenden and Kimble, 2008). Germ cell position is designated relative to the distal end in germ cell diameters (gcd). The first row of germ cells in the transition zone is defined as the first row from the distal end with multiple crescent-shaped DAPI-stained nuclei (Crittenden and Kimble, 2008; Dernburg et al., 1998; Francis et al., 1995); the first pachytene region row is defined as the first row from the distal end with pachytene nuclei (Crittenden and Kimble, 2008). To count total germ cell numbers, we marked boundaries of the mitotic region and transition zone on a computer screen using Openlab software and counted nuclei in each focal plane within the desired region. Due to its size, each mitotic region was counted in two halves. Most germ “cells” reside at the periphery of the syncytial germline tissue and are connected by cytoplasmic bridges to a core of cytoplasm, the “rachis,” that extends along the distal-proximal axis of the gonad; in addition, some germ “cells” exist in “chains” that span, but do not seem to block, the rachis of the mitotic region of hermaphrodites. In this work, such germline “chains” were defined as two or more adjacent nuclei spanning the rachis, but most chains had more than two nuclei.

Bromodeoxyuridine (BrdU) and EdU labeling

To label germlines with BrdU or EdU, animals were placed on plates with labeled MG1693 E. coli for defined times and, in some cases, chased by feeding unlabeled OP50 E. coli, as described (Crittenden and Kimble, 2008). After labeling with BrdU, worms were washed twice in phosphate-buffered saline with 5% BSA and 0.1% Triton X 100 to remove any residual label. We extruded and fixed gonads, then incubated with DAPI at 0.5 μg/mL and anti-BrdU antibodies at 1:2.5 (B44, Becton-Dickinson, San Jose, CA) as described (Crittenden and Kimble, 2008) or used the Click-iT EdU Imaging Kit (Invitrogen) to conjugate EdU to an Alexa Fluor azide. We then scored the number of BrdU-positive nuclei as well as the total number of nuclei in each row along the distal to proximal axis. Data analysis and graphs were done using Microsoft Excel.

Mitotic index

We determined the mitotic index (MI) in germlines labeled with DAPI and antibodies to phosphohistone H3 (PH3), each diluted 1:200 (Upstate Biotechnology, Lake Placid, NY) as described (Crittenden et al., 2006). We counted the number of PH3-positive nuclei at each position along the distal to proximal axis. To determine the average MI, we divided the total number of PH3+ nuclei by the total number of nuclei within the mitotic region; to determine the MI at each position on the distal to proximal axis, we divided the number of PH3+ nuclei by the average number of nuclei at each position. Using Microsoft Excel, we determined confidence intervals at 95% and graphed the mitotic index by position from the distal end of the germline as described (Crittenden et al., 2006).

Laser microsurgery

Laser ablations were done as described (Bargmann and Avery, 1995), using XO qIs56 him-5 males or XX qIs56 hermaphrodites. The him-5 strain is used in this and other experiments to increase the percentage of males in the worm population (Hodgkin et al., 1979). Ablations were validated by nuclear and cellular morphology 3–8 hours after microsurgery, and confirmed by lack of DTC-specific Plag-2::GFP expression when germlines were scored two days later. As controls, we also examined animals that underwent a variety of treatment regimens, but were not subjected to laser microsurgery. Some animals were left on a plate to mature for two days, others were mounted only in M9 buffer, and another group was mounted in .25 mM levamisole in M9 buffer. Importantly, control animals were mounted on slides for approximately the same amount of time as animals undergoing laser microsurgery.

Orientation of germ cell divisions

DAPI-stained gonads from either N2 or qIs56 him-5 males twenty-four hours after L4 stage were scored for the position of the two mDTCs by either nuclear morphology using DAPI staining (N2 and qIs56 him-5) and/or Plag-2::GFP (qIs56 him-5). Next, we scored the orientation of metaphase plates with respect to the distal-to-proximal germline axis and to the position of the mDTC nuclei as described (Crittenden et al., 2006).

Image acquisition

Widefield images were acquired on a Zeiss axioimager with a Hamamatsu Orca digital camera using Openlab software (Improvision). Confocal images (Figs. 2 and and5)5) were acquired on a Zeiss LSM510 confocal microscope. Z-series (Fig. 2A–C) were projected in ImageJ (Rasband, 1997–2009). Images were processed in Adobe Photoshop.

Fig. 2
Evidence for male GSCs and their cellular properties. (A–C) Projections of confocal z-series of male gonads. Dashed line, MR/TZ boundary. Scale bar in (A) represents 15 microns in A and B; scale bar in (C) represents 15 microns in C. (A–B) ...
Fig. 5
Data used to estimate cell cycle lengths in male and hermaphrodite germlines. (A, B) S-phase indices. (A) After a 15-minute pulse of EdU, gonads were stained with DAPI (blue) and an Alexa Fluor azide to detect EdU (red). Many cells in the mitotic region ...


Germline stem cells in adult males

Adult males have germline stem cells

Stem cells are defined by their ability to self-renew and generate differentiated progeny; they can accomplish that dual task from a stem cell pool or by asymmetric cell divisions (Morrison and Kimble, 2006). To ask if the adult male germline is capable of self-renewal, we monitored the total number of germ cells in the mitotic region at progressively later stages of adulthood. All were scored in DAPI-stained gonads from mating males. The numbers were roughly the same throughout adulthood: 213 ± 12 (n=13) at 24 hours after L4; 209 ± 11 (n=15) at 48 hours after L4; 211 ± 12 (n=17) at 96 hours after L4. Therefore, the adult male germline maintains its mitotic region with essentially the same germ cell number despite continuous production of sperm.

Previous work showed that Cy3-dUTP-labeled germ cells in the mitotic region move into differentiating progeny in male germlines (Jaramillo-Lambert et al., 2007). We confirmed this result by labeling germ cells in the mitotic region with a short pulse of the thymidine analog, EdU (Fig. 2A). Immediately after a 30 minute EdU incubation of adult males (24 hr after L4), most labeling was restricted to the mitotic region (Fig. 2A); however, that labeling moved proximally into the meiotic region with time (Fig. 2B). We conclude that the adult male germline contains GSCs responsible for both self-renewal and generation of differentiated progeny.

Adult male GSCs actively cycle and lack reproducibly oriented divisions

Adult male GSCs must exist within the mitotic region but lineage tracing is not yet feasible in C. elegans germlines. Two hallmarks of stem cells in some but not all systems are cell cycle quiescence and reproducibly oriented divisions (Morrison and Kimble, 2006; Yamashita and Fuller, 2008). In an attempt to recognize individual stem cells in the adult male germline, we scored mitotic regions for these two stem cell hallmarks. To detect quiescent germ cells, we labeled adult males (24 hours after L4 stage) with EdU for 24 hours, with the idea that quiescent germ cells would not be labeled. However, all germ cell nuclei were labeled in the mitotic region (Fig. 2C). Labeled cells in our experiments are likely to be in S phase since repair-associated DNA synthesis incorporates insignificant levels of thymidine analog relative to replicating nuclei (Taupin, 2007; A. Dernburg, personal communication). We conclude that cell cycle quiescence is not a hallmark of adult male GSCs, which is confirmed with additional experiments below.

To ask if any germ cell divisions are reproducibly oriented in the C. elegans male, we scored the division planes in germ cells throughout the mitotic region in wild-type and him-5 XO males, using either DAPI or a GFP transgene to mark mDTCs. No division planes were oriented reproducibly with respect to either the gonadal axis (Fig. 2D) or position of mDTC nuclei (Figs. 2E and F). We conclude that reproducibly oriented divisions are not a hallmark of GSCs in the adult male mitotic region.

“Actual” GSCs are likely to reside at the distal end of the adult male gonad

“Actual” GSCs are those that self-renew and generate differentiated progeny, as opposed to those with potential for that dual task. Identification of the “actual” GSCs in the C. elegans germline awaits the development of lineage tracing techniques. However, two simple attributes of a stem cell compartment provide clues that were used in the hermaphrodite germline to predict their position (Crittenden et al., 2006). First, stem cells usually reside at a position that lacks neighboring cells that could contribute progeny to the tissue (Aherne et al., 1977). If germ cells within the male mitotic region move from the mitotic region to more proximal domains (Figs. 2A and B; Jaramillo-Lambert et al., 2007), the “actual” GSCs should reside at the distal-most end. Second, stem cells typically reside next to the regulatory cells forming their niche (Li and Xie, 2005). The two mDTCs provide the niche for larval GSCs (Kimble and White, 1981), and both larval and adult germlines rely on Notch signaling for self-renewal (Austin and Kimble, 1987). To confirm the idea that the two mDTCs provide the niche in adult males, both were laser ablated in XO L4 males (n=2) or young adult males 24 hr past L4 (n=2). After the mDTCs were killed, germline self-renewal was abolished: all germ cells left the mitotic cell cycle and entered the meiotic cell cycle (Fig. 3C, line 6). No effect on germline self-renewal was seen in unablated controls (n ≥ 3 for each control) (Fig. 3C, lines 1–3). We conclude that the mDTCs provide the GSC niche in adults. The typical positions of the two mDTCs in adult males (24 hours past L4) were at or near the distal end: the more distal mDTC was at row 1 [range of 1–2 for N2 (n=56), range of 1–2 for qIs56; him-5 (n=74)] and the more proximal mDTC was at row 3–4 [average at 4 (range of 2–5) for N2 (n=56), average at 3 (range of 1–6) for qIs56 him-5 (n=74)]. The GSC niche in adult males is therefore at or very near the distal end. We suggest that the “actual” GSCs are also at the distal end.

Fig. 3
mDTC number controls length of the adult male mitotic region. (A, B) Fluorescence micrographs of the distal gonad from an unablated male (A) or an animal with one mDTC ablated (B). All nuclei are labeled with DAPI (red); mDTCs are marked with GFP (green) ...

We confirmed that the hDTC provides the GSC niche in adult hermaphrodites at 24 hours past L4, the stage typically used to study GSC self-renewal in adults. Laser ablation of a single hDTC at this stage (n=7) resulted in a loss of mitotic germ cells that mimicked effects seen earlier for larval hDTCs and adults 12 hours past L4 (Kimble and White, 1981; Nadarajan et al., 2009), whereas controls had no effect (n ≥ 2 for each control).

mDTC number controls length of the adult male mitotic region

Previous experiments showed that either of the two mDTCs could support germline proliferation (Kimble and White, 1981), but those ablations were done in young larvae, and only scored for presence or absence of a functioning adult germline. Here we ablated a single mDTC in XO L4 males and measured mitotic region length two days later. When either the distal or proximal mDTC was killed, mitotic region length was significantly shorter than in unablated controls (Figs. 3A–C). We conclude that both mDTCs are required to maintain the normal length of the adult male mitotic region.

Sexual dimorphism in the distal gonad and its control

Sex-specific dimensions and “chains”

To investigate sexual dimorphism in the distal gonad, we first compared its overall morphology in DAPI-stained gonads dissected from staged adults (24 hrs past mid-L4). We measured lengths in germ cell diameters (gcd) and the number of germ cell nuclei per row of the mitotic region and transition zone (germ cells and their nuclei were about the same size in the two sexes) (Figs. 4A and B). The dimensions were sexually dimorphic: lengths along the gonadal axis were longer in males than hermaphrodites (Fig. 4C). The number of germ cells in a given row varied along the gonadal axis, but overall, males had fewer germ cells per row than hermaphrodites (approximately 7–8 germ cells in males versus 12–13 germ cells in hermaphrodites) (Fig. 4D). The number of germ cells per row correlated with the width of the gonad; male gonads were thin and hermaphrodite gonads were wide (Figs. 4D and 4E, lines 1 and 2). Despite their different dimensions, total germ cell numbers in the mitotic regions and transition zones of the two sexes were not significantly different (p=0.49 for the mitotic region, p=0.75 for the transition zone) (Fig. 4C). In addition to this sexual dimorphism in overall dimensions of the distal gonad, “chains” of germ cells extended across the cytoplasmic core, or rachis, in hermaphrodite but not male distal gonads (Figs. 4A and B). We conclude that the distal gonad is sexually dimorphic with respect to both overall morphology and the presence or absence of germ cell “chains”.

Fig. 4
Sexual dimorphism in the distal gonad. (A, B) DAPI-stained distal gonads dissected from adults 24 hours after L4. Hermaphrodite distal gonads (A) are wider than male distal gonads (B). In both sexes, germ cell nuclei reside at the periphery of the gonad; ...

Sexual dimorphism in the distal gonad correlates with sex of the somatic gonad

We next asked whether sexual dimorphism in the distal gonad relied on the ratio of X chromosomes to autosomes (X:A) (Meyer, 2005), the somatic gonad (Zarkower, 2006) or germline-intrinsic cues (e.g. FOG-1) (Kimble and Crittenden, 2007).

The X:A ratio normally determines whether the animal will be hermaphrodite or male (Madl and Herman, 1979), but sex determination mutants circumvent its control. To ask if the X:A ratio controls the distal gonad, we examined XX tra-2(q276) mutant males, which are morphologically indistinguishable from XO wild-type males (Okkema and Kimble, 1991). The XX tra-2(q276) distal gonad appeared male: mitotic region length was 28 ± 2 gcd (n=10) and germ cell chains were absent (n=10) (Fig. 4E, lines 1–3). Therefore, the X:A ratio does not directly regulate the sex-specific morphology of the distal gonad.

To ask if somatic gonadal sex might be critical for distal gonad morphology, we examined two unusual tra-1 mutants. XX tra-1(e1488) mutants typically possess a hermaphrodite somatic gonad within a male body, and XX tra-1(e1732) mutants have a male somatic gonad within a hermaphrodite body (Hodgkin, 1987). This tra-1 approach can only be used as a guide since these animals are not true genetic mosaics. Phenotypes can vary, but we carefully selected individuals that had opposite sexual morphologies in body and gonad and that were making oocytes. The morphology of the distal gonad matched the sex of the somatic gonad rather than the sex of the body wall for both tra-1 mutants (Fig. 4E, lines 1–2, 4–5).

To ask if germline sex might influence distal gonad morphology, we examined XX and XO fog-1 mutants, which make only oocytes in somatically normal males and hermaphrodites (Barton and Kimble, 1990). The distal gonads of XX fog-1 null mutants had germ cell chains but XO fog-1 mutants did not (XX, n=190; XO, n=88), suggesting that germline sex does not dictate the generation of these chains in the distal gonad. The number of cells per row of XX and XO fog-1 distal gonads were similarly typical of hermaphrodites and males, respectively (Fig. 4E, lines 1–2, 6–7). The fog-1 XX mitotic region length was similar to that of wild-type XX animals. The fog-1 XO mitotic region was longer than the fog-1 XX mitotic region (p=8.6×10−15), albeit shorter than wild-type males (p=8.4×10−15). The simplest explanation is that somatic gonadal sex normally controls distal gonad morphology and the presence of germ cell chains.

Mitotic cell cycle length is shorter in male than hermaphrodite germ cells

To further examine sex-specific properties in the wild-type distal gonad, we compared basic features of the mitotic cell cycle in oogenic hermaphrodite and spermatogenic male adult germlines. We first examined cell cycle length. To this end, we determined the fraction of cells in S-phase (S-phase index) and the time required to complete the G2-M-G1 interval, both in the mitotic region as a whole and by position along the gonadal axis. Our results were then used to estimate average cell cycle lengths in both male and hermaphrodite germ cells, as done previously for hermaphrodites using this same method (Crittenden and Kimble, 2008; Crittenden et al., 2006) or another method (Jaramillo-Lambert et al., 2007).

S-phase index (SI) provides a measure of the percentage of cells undergoing DNA replication. We treated synchronized young adults (24 hrs after L4) with BrdU for 15 minutes, and then stained their gonads with DAPI to see all nuclei and with antibodies to BrdU to visualize S-phase nuclei. The average S-phase indices were about the same: 50.2% for males and 49.3% for hermaphrodites (Fig. 5B, dashed lines). This average S-phase index for hermaphrodites was comparable to that seen before (Crittenden et al., 2006; Jaramillo-Lambert et al., 2007).

The SI varied with position along the gonadal axis, but was in the same range as the average (~40–60%). Most important to this work, germ cells in rows 1 and 2 at the distal-most end labeled well with both BrdU (Fig. 5B) and EdU (Fig. 5A), as seen previously with BrdU for hermaphrodites (Crittenden et al., 2006). At the other end of the mitotic region, most BrdU-labeled germ cells in the rows near the boundary between the mitotic region and transition zone (Fig. 5B, rows ~17–20 in hermaphrodites, rows ~22–27 in males) are likely in meiotic S-phase (see below).

The G2-M-G1 interval (time in hours from early G2 to early S-phase) can be estimated by measuring the time required to label all germ cell nuclei with BrdU. To this end, animals were treated continuously with BrdU for increasing lengths of time and then fixed and stained to score percent of germ cells labeled. Males required ~5 hours to label all germ cells in the mitotic region (Fig. 5C, blue data points), whereas hermaphrodites required ~10 hours (Fig. 5C, pink data points). This hermaphrodite G2-M-G1 interval corresponds well with previous results (Crittenden et al., 2006). The G2-M-G1 intervals were essentially the same by position along the distal-proximal axis for each sex (Figs. 5D and E). Again, this similarity by position corresponds well with previous results (Crittenden et al., 2006). Most importantly, our results demonstrate that the G2-M-G1 interval is shorter in male than hermaphrodite germ cells. They also confirm that all germ cells within the mitotic region are actively cycling in both sexes.

Cell cycle length can be estimated from measurements of S-phase index plus G2-M-G1 interval (Crittenden and Kimble, 2008; Crittenden et al., 2006). Male germ cells have a ~50% S-phase index on average and G2-M-G1 intervals of ~5 hours on average. Those two values indicate that the male germ cell cycle length is ~10 hours on average. For comparison, hermaphrodite germ cells have a ~50% S-phase index, G2-M-G1 intervals of ~10 hours and, therefore, an estimated cell cycle length of ~20 hours on average, a value that corresponds well with a previous estimate (Crittenden et al., 2006). We note that these estimates may suffer from technical biases. For example, measurements of S-phase index depend on reagent incorporation and sensitivity, and G2-M-G1 intervals include some minimal time in S-phase to visualize labeling. To avoid effects of technical biases, germlines of the two sexes were consistently prepared and scored side-by-side. We conclude that the mitotic cell cycle length is shorter on average in adult male germ cells than in adult hermaphrodite germ cells.

G2-M-G1 intervals were consistently shorter in males than hermaphrodites along the gonadal axis (Figs. 5D and E). We therefore estimated cell cycle lengths by position, as done for the average cell cycle lengths. For all rows, including rows 1 and 2, cell cycle length was shorter in males than hermaphrodites. We conclude that the sex-specific difference in cell cycle length extends throughout the mitotic region, including the distal germ cells within the stem cell niche.

Proximal germ cell movement is faster in males than hermaphrodites

Previous work showed that Cy3-dUTP-labeled germ cells in the mitotic region move proximally into differentiating progeny faster in male than hermaphrodite germlines (Jaramillo-Lambert et al., 2007). We confirmed that sex-specific difference in proximal movement with BrdU labeling and also measured the rates of movement (Fig. 6). Specifically, we pulsed mating adults with BrdU for 30 minutes, chased them for increasing times, dissected and stained gonads, and scored the position of the proximal border of BrdU-positive nuclei (Fig. 6). Male germ cells moved faster than hermaphrodite germ cells, approximately 2–3 rows per hour in males (Fig. 6, blue data points) compared to roughly one row per hour in hermaphrodites (Fig. 6, pink data points) (Crittenden et al., 2006). Taken together, the faster mitotic cell cycles, the more rapid proximal movement, and the increased rates of meiotic progression in male compared to hermaphrodite germlines (Jaramillo-Lambert et al., 2007; this work) raise the question of whether these rates are linked and how this link might be regulated.

Fig. 6
Proximal rates of movement are sexually dimorphic. Adult males and hermaphrodites (24 hours after L4) were pulsed with BrdU for 30 minutes and then chased for the indicated times. Dissected gonads were stained with DAPI and anti-BrdU at each time point. ...

M-phase indices in male and hermaphrodite mitotic regions

Finally, we examined the mitotic index (percentage of dividing cells) (MI) in adult male germlines. To this end, we stained dissected gonads from synchronized adults (24 hours past L4) with both DAPI to visualize all nuclei and an antibody to phosphohistone H3 to mark late G2- and M-phase cells (Hendzel et al., 1997). Hermaphrodite MIs were calculated in parallel for comparison with the male MIs and with previous data (Crittenden et al., 2006; Maciejowski et al., 2006). Average MIs were higher in male than hermaphrodite germlines (Figs. 7A and B, dashed lines); the MI varied with position along the gonadal axis in males, much as had been seen in hermaphrodites. The distal-most germ cells had a lower MI in males (3.3% for rows 1–2 vs 6.3% for rows 3–10 p=0.0002) (Fig. 7A, gray vs. green shading) as they did in hermaphrodites (3.5% for rows 1–2 vs 5.4% for rows 3–10 p=0.0002) (Fig. 7B, pink bars) (Crittenden et al., 2006; Maciejowski et al., 2006). A second MI dip appeared at row 10 in males (row 9 vs. row 10 p=0.04) (Fig. 7A). More proximally, the male MI declined after row 21 (5.9% average MI for rows 3–21 vs 1.1% for rows 22–27 p=6.8×10−24) (Fig. 7A). A comparison of MI and SI by position (Figs. 5B and and7A)7A) shows that PH3-positive M-phase cells were infrequent in the proximal mitotic region and extremely rare in the transition zone (Fig. 7A); by contrast, BrdU-positive S-phase cells were frequent in both domains (Fig. 5B). Given the proximal movement of male germ cells (Jaramillo-Lambert et al., 2007; this work) and the steady SI in the proximal mitotic region (Fig. 5B), we suggest that the declining MI in the proximal mitotic region coincides with entry into meiotic S-phase. A similar conclusion has also been put forward for the hermaphrodite germline based on similar reasoning (Crittenden et al., 2006; Hansen et al., 2004; Jaramillo-Lambert et al., 2007; Maciejowski et al., 2006).

Fig. 7
Mitotic index of adult C. elegans germlines. (A, B) Graphs of mitotic index (MI) with respect to position along the distal-proximal axis. The average MI is shown as a horizontal dashed line; the dashed line ends at the last row of the average mitotic ...

Germline mitotic index is influenced by downstream events

The average MI for the mitotic region in males was 4.8 ± 0.4% (Figs. 7A and B, blue dashed line), but 3.4 ± 0.2% in hermaphrodites (Fig. 7B, pink dashed line). This sex-specific MI is intriguing because one of the germline sex regulators, FOG-1, promotes mitotic proliferation (Thompson et al., 2005) in addition to its role in driving the sperm fate (Barton and Kimble, 1990). We measured MI in XO fog-1 null mutants, which are oogenic, expecting that it would be similar to the XX oogenic MI. However, the average MI of an XO fog-1 oogenic germline was 1.6 ± 0.5% (n=75) (Fig. 7C, line 4), lower than that in both XO wild-type spermatogenic and XX wild-type oogenic germlines. The MI of an unmated XX fog-1 germline was also low, 1.8 ± 0.3% (n=150), a number comparable to that of XO fog-1 males (Fig. 7C, lines 3–4). One explanation of these lower MIs might be that FOG-1 is required for normal mitotic proliferation in both sexes; alternatively, the lower MIs may reflect downstream events. Normally, fog-1 mutants accumulate oocytes within the gonad due to the absence of sperm; however, when sperm are introduced by mating with males, oocytes are fertilized and leave the gonad (Barton and Kimble, 1990). We therefore scored mated fog-1 mutants. The MI found in the mitotic regions of mated XX fog-1 mutants increased to 3.2 ± 0.3% (n=87), which is comparable to the 3.4% MI of self-fertile wild-type hermaphrodites (Fig. 7C, lines 1–2). Therefore, the lower MI typical of fog-1 mutants is likely to result from decreased oocyte use or lack of sperm, which feeds back on the mitotic region to lower its mitotic activity. This result is consistent with previous work demonstrating that the germlines of unmated Fog animals progress through meiosis more slowly than those of animals that have been mated (Jaramillo-Lambert et al., 2007). Consistent with the idea that there is communication between the distal and proximal ends of the germline, recent work showed that distal Notch signaling and proximal MSP signaling coordinately regulate cytoplasmic flow and oocyte growth (Nadarajan et al., 2009).


This work characterizes cellular properties of immature germ cells in adult C. elegans males and compares them to similar properties in adult C. elegans hermaphrodites. We make two major conclusions. First, adult males possess germline stem cells (GSCs), a finding that expands C. elegans as a model for GSC control. Second, immature germ cells have sex-specific cell cycles, a finding that bears on our understanding of germline sex determination. This discussion places these two findings in context, both within the C. elegans field and more broadly.

The C. elegans adult male as a model for GSC control

Cellular studies initially provided evidence for the existence of GSCs at the distal end of adult hermaphrodite germlines (Crittenden et al., 2006). Germ cell number was maintained despite constant movement of germ cells from the mitotic region and despite their continuous loss to oogenesis or cell death (Crittenden et al., 2006). Hermaphrodite GSCs exist within a stem cell pool of actively dividing germ cells that lack reproducibly oriented cell divisions (Cinquin et al., 2010; Crittenden et al., 2006). Indeed, starved hermaphrodites have a pool of ~35 germ cells that can regenerate a functioning germline upon subsequent refeeding (Angelo and Van Gilst, 2009).

Here we demonstrate that XO spermatogenic germlines also possess GSCs and that those GSCs have properties similar to those initially found in XX oogenic germlines (no quiescent cells, no reproducibly oriented divisions). We also show that the two male DTCs provide a niche for GSC maintenance in adults, and that DTC number regulates the length of the mitotic region. These results lay a necessary foundation for future analyses of adult male GSCs and comparisons of GSC molecular controls in both sexes. For example, Notch signaling regulates GSC proliferation in larvae of both sexes, GSC self-renewal in adult hermaphrodites and continued mitotic divisions at the expense of meiotic entry in adult males (Austin and Kimble, 1987). Our experiments showing that adult males must possess GSCs permit the conclusion that Notch signaling controls germline self-renewal in adults of both sexes.

Many questions remain regarding the male GSC pool and its niche. For example, how many GSCs are in the pool and which ones are the “actual” stem cells? And can they survive starvation to regenerate a functional germline as in hermaphrodites (Angelo and Van Gilst, 2009)? As for the niche, do the hDTCs and mDTCs use the same DSL ligands to trigger the GLP-1/Notch receptor? The hDTCs use LAG-2 and APX-1 to control the hermaphrodite GSCs (Henderson et al., 1994; Lambie and Kimble, 1991; Nadarajan et al., 2009), but DSL ligands controlling male GSC maintenance have not been explored, except that lag-2 appears to be expressed much more weakly in mDTCs than in hDTCs (Chesney et al., 2009). What effect might the use of different DSL ligands have on Notch signaling and GSC maintenance? C. elegans can now be used to explore how the distinct hDTC and mDTC niches regulate GSCs and the transition from the stem cell state to meiotic entry and early differentiation as sperm or oocyte.

Germ cell sexual identity is not equivalent to commitment as sperm or oocyte

The sperm/oocyte decision is not well understood in any organism (Casper and Van Doren, 2006; Kimble and Page, 2007). One experimental approach for elucidation of this broadly conserved cell fate decision has been to analyze mutants that cause a clean sexual transformation of germ cells from sperm to oocyte or vice versa. That genetic approach identified regulators of the sperm or oocyte fate in C. elegans (e.g. FOG-1, Barton and Kimble, 1990) that have been placed into a molecular pathway of sperm/oocyte control (Ellis and Schedl, 2007; Kimble and Crittenden, 2007). Yet when, where and how the pathway’s terminal regulators govern the sperm/oocyte fate remain unknown. An alternative approach has been to identify the earliest sex-specific characteristics of germ cells and then to analyze their control. In mammals, for example, germ cells enter the meiotic cell cycle in fetal XX ovaries but not in fetal XY testes. This early difference relies on the male-specific degradation of retinoic acid, a signaling molecule that drives germ cells into the meiotic cell cycle in both sexes (reviewed in Bowles and Koopman, 2007). Therefore, this earliest sex-specific difference in mammalian germ cells relies on the sex-specific control of a gender-neutral regulator of meiotic entry. Both approaches have been used in Drosophila (Casper and Van Doren, 2006; Hempel et al., 2008). However, Drosophila germline sex determination mutants often result in tumorous germlines and do not display clean sexual transformation; moreover the relationship between sex-specific germ cell markers in the embryo and controls of sperm or oocyte specification is not yet understood (Casper and Van Doren, 2009).

In C. elegans, the adult germline remains sexually labile (see Introduction and below) with some cells uncommitted to the sperm or oocyte fate. Germ cells at the distal end of the adult germline can be considered “earliest” because they require the most time to mature from the mitotic state through meiotic entry and progress through meiotic prophase to overt gamete differentiation. Previous studies discovered sex-specific molecular markers in the adult mitotic region (see Introduction) as well as sex-specific behaviors more proximally, including differences in progression through meiotic prophase and gametogenesis (Jaramillo-Lambert et al., 2007; Jaramillo-Lambert and Engebrecht, 2010; Shakes et al., 2009). This work identifies sex-specific morphology and cellular behavior in the adult mitotic region (Figs. 8A and B): hermaphrodite mitotic regions are shorter and wider than those in males; germ cell chains span the hermaphrodite but not the male mitotic region; and the cell cycle length is longer in germ cells of the hermaphrodite than the male mitotic region. This last sex-specific trait is arguably most informative, as we can infer from it that even the most immature GSCs have acquired a sexually dimorphic behavior. Therefore, all adult germ cells have acquired sexual identity.

Fig. 8
Germ cells in the adult mitotic region have acquired sexual identity. (A, B) Cartoons summarizing sex-specific characteristics of hermaphrodite (A) and male (B) distal gonads. Conventions: germ cells in mitotic cell cycle (yellow); predicted “actual” ...

The acquisition of sexual identity in all germ cells within the mitotic region raises the question of whether they are committed to a sexual fate. However, this seems unlikely. The adult germline can be sexually transformed from spermatogenic to oogenic and vice versa by manipulating sperm/oocyte regulators (Barton and Kimble, 1990; Barton et al., 1987; Chen et al., 2000; Morgan et al., 2010; Otori et al., 2006; Schedin et al., 1994). Those regulators must therefore act in the adult, either to initiate sperm or oocyte fate specification or to maintain commitment to the sperm or oocyte fate. In the larval germline, germ cells become committed to the sperm or oocyte fate around the time they enter the meiotic cell cycle (Barton and Kimble, 1990). The site of sperm/oocyte commitment has not yet been established in the adult germline, but by analogy to the larval germline, it is likely to be near the site of meiotic entry. By this reasoning, adult germ cells in the mitotic region are predicted to be sexually labile and their sexual identity is likely not equivalent to commitment to a sperm or oocyte fate. The idea that sexual identity is not equivalent to sexual commitment in C. elegans is consistent with recent findings in vertebrates. The sex-specific meiotic entry in vertebrate germ cells reveals sexual identity, but that meiotic entry is ultimately controlled by gender-neutral regulators (Anderson et al., 2008; Baltus et al., 2006; Bowles and Koopman, 2007). Moreover, vertebrate somatic gonadal cells with an overt sexual identity are not committed to a sexual fate (Uhlenhaut et al., 2009). Indeed, sexual plasticity is widespread (DeFalco and Capel, 2009).

We do not know what controls sexual identity in the mitotic region. One possibility is that the sex determination pathway could function at the distal end to control germline sex. The sex determination pathway involves signaling between cells (Zarkower, 2006), so one attractive idea is that the mDTC expresses the secreted ligand of the pathway to drive the male fate in distal germ cells. Alternatively, the control could be indirect and rely on coordination with later sex-specific events such as meiotic progression and proximal movement that feed back on the mitotic region (Jaramillo-Lambert et al., 2007; this work). Similar mechanisms could also drive sex-specific gene expression in the mitotic region.

The sex-specific mitotic cell cycle lengths in adult C. elegans germ cells are reminiscent of sex-specific cell cycle properties in mammals and Drosophila. Murine germ cells undergo mitotic arrest in fetal testes and resume mitotic proliferation later in juvenile testes, whereas germ cells in fetal ovaries enter the meiotic cell cycle and arrest at meiotic prophase I (reviewed in Kocer et al., 2009). In Drosophila, germ cells proliferate in male but not female embryonic gonads (Wawersik et al., 2005) and germline tumors form when female germ cells are likely transformed towards a more masculine fate (Hempel et al., 2008). Here we show that C. elegans germ cells divide more quickly in spermatogenic than oogenic germlines. A common thread is that mitotic proliferation is more robust in male than female germ cells. We speculate that the regulation of sex-specific mitotic proliferation may be molecularly linked to controls of sex-specific meiotic cell cycles, which generate sperm after two rapid meiotic divisions and oocytes after an arrested meiotic division coupled to cell growth. The idea that sex-specific mitotic cell cycles may provide a molecular launching pad for sex-specific meiotic cell cycles and gametogenesis is consistent with the concept that the meiotic cell cycle is a modification of the more ancient mitotic cell cycle (e.g. Wilkins and Holliday, 2009) and suggests that understanding the regulation of sex-specific cell cycles may be critical for unraveling controls of the sperm/oocyte decision.


We thank Peggy Kroll-Connor and Beth Thompson for isolating fog-1(q785), Kimble lab members for helpful discussions, and Anne Helsley-Marchbanks and Laura Vanderploeg for help preparing the manuscript and figures. We are particularly grateful to David Greenstein for extensive comments on the manuscript and to Abby Dernburg for communicating unpublished results. We received MG1693 from the E. coli stock center. D.E.M. was supported by NIH Training Grant GM007215 in Molecular Biosciences. NIH GM069454 supported this work. J.K. is an investigator of the Howard Hughes Medical Institute.


Distal Tip Cell
hermaphrodite DTC
male DTCs
Germline Stem Cell
Mitotic Region
Transition Zone
Mitotic Index
S-phase Index
germ cell diameters


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