Reproduction disrupts stem cell homeostasis in testes of aged male Drosophila via an induced microenvironment

Stem cells rely on instructive cues from their environment. Alterations in microenvironments might contribute to tissue dysfunction and disease pathogenesis. Germline stem cells (GSCs) and cyst stem cells (CySC) in Drosophila testes are normally maintained in the apical area by the testicular hub. In this study, we found that reproduction leads to accumulation of early differentiating daughters of CySCs and GSCs in the testes of aged male flies, due to hyperactivation of Jun-N-terminal kinase (JNK) signaling to maintain self-renewal gene expression in the differentiating cyst cells. JNK activity is normally required to maintain CySCs in the apical niche. A muscle sheath surrounds the Drosophila testis to maintain its long coiled structure. Importantly, reproduction triggers accumulation of the tumor necrosis factor (TNF) Eiger in the testis muscle to activate JNK signaling via the TNF receptor Grindelwald in the cyst cells. Reducing Eiger activity in the testis muscle sheath suppressed reproduction-induced differentiation defects, but had little effect on testis homeostasis of unmated males. Our results reveal that reproduction in males provokes a dramatic shift in the testicular microenvironment, which impairs tissue homeostasis and spermatogenesis in the testes.


Introduction
The adult mammalian testis generates millions of mature sperm every day [1,2]. Spermatogenesis begins when germline stem cell (GSC) progeny enter differentiation rather than remaining in a self-renewal state [3]. GSCs are maintained by secreted factors from the surrounding testicular niche, which is composed of different types of somatic cell populations in different species [4,5]. Failure by GSCs to balance self-renewal and differentiation impedes sperm production and consequently reproduction. In mammalian seminiferous tubules, Sertoli cells which are associated with the developing germ cells are the key contributors to the GSC niche [5]. In Drosophila testes, cyst stem cells (CySCs) are located in close proximity to the GSCs and are required to maintain GSC self-renewal and prevent differentiation [6][7][8][9]. Additionally, a cluster of 10-15 somatic cells located at the apical tip of the Drosophila testis, the hub, is crucial for self-renewal of both the GSCs and CySCs that surround it [10,11]. Division of a GSC or CySC generates a daughter stem cell that remains close to the hub, thereby maintaining the stem cell fate, while the other daughter cell is displaced from the hub to initiate differentiation [10][11][12][13]. Consequently, both GSCs and CySCs are restricted to the apical region, generating an apical-to-basal gradient of germ cell differentiation.
A sheath of smooth muscles surrounds both the mammalian seminiferous tubules and the Drosophila testes [10,14,15]. Contraction of this peritubular smooth muscle facilitates the transport of sperm and testicular fluid in the seminiferous tubules [16,17]. The peritubular cells also modulate the functions of Sertoli cells and GSCs via paracrine signaling, thereby acting as an additional niche in the mammalian testis [5,18,19]. In Drosophila, the testis sheath, which is composed of smooth muscles and the pigment cells, appears to be essential for the long coiled structure of the testes [20,21]. Mutations that affect sheath development result in smaller or abnormally-shaped testes [22,23]. However, in these cases, germ cells and somatic cells (including hub cells and cyst cells) are still observed in the apical region, suggesting that unlike mammalian peritubular cells, the testis sheath is not essential for early spermatogenesis in Drosophila [22].
It has been observed in diverse organisms that reproduction is associated with a reduction in future fertility [24][25][26]. In this study, we found that reproduction induces ectopic expression of the self-renewal protein Zfh-1 in the cyst lineage cells of aged testes. As a result, cyst cell differentiation is disrupted and CySC-like cells over-proliferate, leading to overproduction and accumulation of undifferentiated germ cells away from the hub. We found that the tumor necrosis factor-Jun N-terminal kinase (TNF-JNK) signaling pathway is hyperactivated and that genetic reduction of TNF-JNK signaling activity suppresses these defects. Interestingly, reproduction induces substantial accumulation of the TNF superfamily ligand Eiger in the testis muscle that activates TNF receptor Grindelwald (Grnd), which is specifically expressed in cyst cells following reproduction. Our study uncovers a cost of reproduction through ectopic stem cell self-renewal during spermatogenesis, and also reveals unexpected conservation from Reproduction reduces the lifespan of male flies [24]. Since we found that the survival rate of 5w-old mated males was much lower than that of 5w-old unmated males (S1 Fig), it is possible that the ectopic Zfh-1-positive cells in testes of mated males is the result of accelerated aging. To test whether aging is the major causative factor of the ectopic Zfh-1 phenotype, we Canton-S (J) testes with ectopic Zfh-1 expression. Extra Zfh-1-positive cells (levels 1-3) were observed in testes from single male mated with 6 females (1 vs. 6) and 1-2 males (1 vs. 1-2) but rarely seen in the testes from unmated males. The asterisks indicate the significance comparing total testes from week-5 and -6 males in 1 vs. 6 to 1 vs. 1-2 mating ratio. N = number of testes scored. P-values shown in C, I, and J were calculated with Chi-squared test. � p<0.05, �� p<0.01, and ��� p<0.001.
https://doi.org/10.1371/journal.pgen.1008062.g001 examined testes from unmated 9w-and 10w-old males (i.e. when survival rates declined rapidly to those comparable to 4w-and 5w-old mated flies, respectively) (N = 99) (S1 Fig). Ectopic Zfh-1-positive cells were observed in only 8% of the testes of 9w-old unmated males, and none of the testes of 10w-old unmated males had the ectopic cells (columns 7 and 8 in Fig 1I). Similarly, none of the testes of 8w-and 10w-old Canton-S unmated males exhibited ectopic Zfh-1 expression (columns 7 and 8 in Fig 1J). Our results suggest that accelerated aging is unlikely to be solely responsible for the reproduction-induced phenotypes we observed in the testes of mated male flies.
To further characterize the phenotypes in a larger sample size, we assessed the effects of reproduction on spermatogenesis using a mass-mating scheme. Male flies of different genetic backgrounds (w 1118 , Canton-S, yw and Oregon R) were examined. We found that when 10 males were supplied with 20 virgin females every week, they also exhibited age-dependent ectopic Zfh-1 expression in 44% (N = 104), 23% (N = 125), 20% (N = 101) and 11% (N = 104) of the testes, respectively, from mated 5w-old w 1118 , Canton-S, yw, and Oregon R flies (Fig 2B-2H). The differences in the percentages of the ectopic Zfh-1 expression among these strains might not be due to differential mating frequencies, since we found that the young males of w 1118 , CS and OR strains would mate to females of comparable numbers within 24 hours (S2 Fig). None or very few testes of 1w-and 3w-old mated males possessed ectopic Zfh-1-positive cells (Fig 2E-2H). Ectopic Zfh-1 expression was rarely observed in the testes of unmated males (N = 30), regardless of age and genetic backgrounds (Fig 2A and 2E-2H). Interestingly, 5w-old males that had mated from weeks 1 to 3 exhibited more level-2 and level-3 Zfh-1 phenotypes than those that had mated from weeks 3 to 5 (Fig 2I), suggesting that reproduction in early life has a stronger effect to induce ectopic Zfh-1 phenotypes.
We evaluated the possible correlation between reproductive rates and extra Zfh-1 cells by varying the number of females available for mating. When we reduced the mating system to 1-2 females per isolated male each week (Fig 1I and 1J), on average 20% of testes of w 1118 mated males (N = 85) and 30% of those of Canton-S mated males (N = 153) at 5w-and 6w-old also showed ectopic Zfh-1 expression, although this was a significantly lower proportion than exhibited by males mated with six females (Fig 1I and 1J). Consistently, we observed lower percentages of extra Zfh-1-positive cells in the testes of male w 1118 and Canton-S flies mated with females in a 5:1 ratio (25 males versus five females) than for males mated at a 1:2 ratio (10 males versus 20 females) ( Fig 2J).
These results indicate that reproduction can lead to ectopic Zfh-1 expression in the testes of aged male flies and that the levels are correlated with reproductive rate. Since the testes of males in mass mating exhibited reproduction-induced phenotypes similar to those in singlemale mating, we conducted mass-mating experiments in most of the following studies to achieve larger samples sizes.

Reproduction induces an accumulation of early germ cells by Dppdependent BMP signaling activation
Zfh-1 maintains CySCs by preventing them from entering differentiation [6]. To examine whether cyst cell differentiation was blocked in the ectopic Zfh-1-positive cells, we assessed expression of Dad-lacZ [a lacZ enhancer trap line for Daughter against dpp (Dad) [36]] and Eyes absent (Eya). Dad is a bone morphogenetic protein (BMP) signaling target [36], and Dad-lacZ expression was normally initiated in early differentiating cyst cells associated with fourto eight-cell spermatogonia clusters (arrows Fig 2K and 2K'). Eya was expressed later in the more mature cyst cells associated with late spermatogonia (arrowheads in Fig 2K and 2K'). In testes of mated males exhibiting ectopic Zfh-1 expression at level 1, most of the ectopic Zfh-1-positive cells were also positive for Dad-lacZ (100%) and Eya (78%) (column 3 in Fig 2L and  2M). In testes exhibiting level 2 and level 3 phenotypes, Eya expression was blocked in many of the ectopic Zfh-1-positive cells (83% and 100% for level 2 and level 3 phenotypes, respectively, Fig 2M), whereas Dad-lacZ expression was unaffected ( Fig 2L). We even observed Zfh-1 expression in Eya-positive cells distant from the hub (arrowheads in Fig 2N), suggesting that differentiation had not yet been severely disrupted. These data indicate that most of the ectopic Zfh-1-positive cells in testes exhibiting level 2 and level 3 phenotypes had initiated cell differentiation but were unable to proceed to stages when Eya is activated. Only CySCs actively divide in the cyst cell lineage [37]. In testes scored as level 3, some ectopic Zfh-1-positive cells proceeded through the S phase and incorporated EdU (arrows in Fig 2O), suggesting that reproduction may lead to the induction and/or maintenance of CySC characteristics away from the hub.
Signals from the cyst cell lineage are critical for GSC self-renewal and for differentiation of spermatogonia into spermatocytes [6-8, 30-32, 38-41]. We found that germ cell differentiation was severely disrupted in the majority of testes exhibiting ectopic Zfh-1-positive cells, as demonstrated by the accumulation of extra early germ cells at the expense of spermatocytes (Fig 1G and 1H, and Figs 2C, 1D and 3K). We categorized the phenotype of early germ cell accumulation into three grades (1, 2, and 3) (see the Methods section). In contrast to 1-3-day old and unmated 5w-old males whose early germ cells are restricted to the apical region of the testes (Fig 3A-3B') [35], testes categorized as grade 1 and 2 exhibited expansion of early germ cells, most of which were spermatogonia as evidenced by the presence of thin, branched fusomes (arrows in Fig 3C-3D') [42]. Testes categorized as grade 3 were full of early germ cells, some of which contained spectrosomes that are characteristic of GSCs and Gbs [43] ( Fig  3E and arrowheads in Fig 3E'). Bag of marbles (Bam) protein accumulation controls timing of the switch from spermatogonia proliferation to differentiation [43][44][45][46]. In 5w-old unmated males, we detected Bam in the 4-16 cell stages of spermatogonia in the apical region ( Fig 3F  and 3F') [44]. Onset of Bam accumulation appeared to be delayed or Bam levels were markedly reduced in testes of grades 1 and 2 ( Fig 3G-3H'). Anti-Bam immunostaining generated almost no signal in grade 3 testes (Fig 3I and 3I'). Thus, Bam expression is suppressed in the testes of mated males exhibiting early germ cell expansion. Single germ cells positive for the mitosis marker phospho-Histone H3 (pH3) were also observed away from the hub in grade 3 testes (arrows in Fig 3J), suggesting that GSC-or Gb-like cells are present in grade 3 testes.
Apart from its activity in cyst cells, BMP signaling is activated in GSCs to maintain selfrenewal and to suppress bam transcription [43,47]. We wondered whether BMP signaling is  . Interestingly, depletion of Dpp or Gbb from cyst lineage cells by RNA interference showed that dpp is required for undifferentiated germ cell expansion in the testes of mated males and that gbb is dispensable for this phenotype (Fig 4C and 4E). Unlike the marked suppression of early germ cell expansion, knockdown of dpp or gbb did not significantly suppress ectopic Zfh-1 expression in testes of mated males (Fig 4B and 4D). Therefore, our results show that Dpp-mediated BMP signaling hyperactivation via reproduction leads to delayed or blocked early germ cells differentiation.

JNK signaling is hyperactivated in the somatic cyst cells in testes of mated males
We found that reproduction induces hyperactivation of JNK signaling in the somatic cyst cells of the testes from mated males. Examination of the JNK signaling reporter puc-lacZ A251 found that it is expressed at high-levels in early cyst cells (Fig 5A and arrowheads in Fig 5B) [48]. Levels of puc-lacZ A251 expression in cyst cells were markedly reduced by RNAi knockdown of JNK, encoded by basket (bsk), in cyst lineage ( JNK signaling activation in cyst cells was also found via the EGFP reporter of TRE (TPA response element), that are the binding sites for the JNK effector AP-1 dimer (Fig 5I) [49].
While the testes of unmated males had approximately 20 puc-lacZ-positive cyst cells ( Fig  5C), the numbers of puc-lacZ-positive cells were increased (more than 50 cells, see Methods) in more than 60% of the testes of mated males aged 4 to 7 weeks (Fig 5D-5F and column 2 in 5G). We quantified anti-β-galactosidase staining intensity of single puc-lacZ-positive early cyst cells to analyze levels of JNK signaling activity. As shown in Fig 5H, staining intensities were markedly higher in cyst cells of the testes of 4w-old mated males compared to those of agematched unmated males. Reproduction-mediated JNK signaling hyperactivation is not solely induced by accelerated aging, as puc-lacZ levels in the cyst cells of unmated males cultured at 29˚C to hasten aging [50,51] were much lower than those of mated males cultured at 25˚C (Fig 5H), even though the adult survival rates were comparable (S3H Fig). Similarly, elevated TRE-EGFP signals were also observed in testicular cyst cells in mated males compared to the unmated males (Fig 5I-5K). Together, these results indicate that reproduction activates JNK signaling in the testicular cyst cells of aged males.
Next, we investigated whether JNK signaling hyperactivation is the major cause of ectopic Zfh-1 expression and the early germ cell expansion induced by reproduction. RNAi-mediated silencing of JNK signaling components-including D-JNKK/Hemipterous (Hep), D-JNK/ mark the hubs. (A-B') Dot spectrosomes and thin fusomes were observed only in the apical region of the testes from 1-3-day-old (A and A') and 5w-old unmated (B and B') males. (C-D') Thin fusomes were found in the middle part of the grade 1 (arrows in C and C') and grade 2 (arrows D and D') testes that exhibit excess early germ cells. (E and E') Dot-like spectrosomes (arrowheads) were observed away from the hub in the grade 3 testes. (F-I') Testes from mated 5w-old w 1118 males immunostained for Bam and FasIII (green in F-I and white in F'-I'), co-stained for Vasa (red). Bam-positive regions are indicated with brackets. Asterisks mark the hubs. (G-I') Testes with expansion of early germ cells. Bam expression was markedly reduced and the onset of Bam expression was delayed in grade 1 and 2 testes (G and H). Anti-Bam signals were not detected in grade 3 testes (I). (J) Testis from mated 5w-old w 1118 male immunostained for pH3 (green), co-stained for Vasa (red). Arrows indicate the single germ cells undergoing mitosis. (K) Percentages of mated 5wold w 1118 testes exhibiting excess small germ cells. The percentages and severity of accumulation of early germ cells (Y-axis) were positively correlated with the degrees of ectopic Zfh-1 expression (X-axis). Mass-mating of 10 males and 20 females was conducted for all experiments. https://doi.org/10.1371/journal.pgen.1008062.g003 Basket (Bsk) and D-Fos/Kayak (Kay) (Fig 5A), in the cyst cell lineage via the eyaA3 ts system at 29˚C markedly suppressed ectopic Zfh-1 expression in the testes of mated males, as also found for overexpression of dominant-negative Bsk (referred to as Bsk DN ) ( Fig 6A). Consistent with the role of cyst cells in controlling germ cell differentiation, expansion of undifferentiated germ cells was also suppressed by reducing bsk activity in cyst cells ( Fig 6B). Conversely, forced expression of the constitutively active Hep (referred to as Hep CA ) in the cyst cell lineage for seven days resulted in testes being filled with numerous ectopic Zfh-1-positive cells and small germ cells (Fig 6C-6C"'). Only a few Eya-positive cells were present in these testes, indicating cyst differentiation was inhibited (Fig 6C"). Together, these results demonstrate that hyperactivation of JNK signaling in cyst cells is the major cause of the cyst cell and germ cell differentiation defects induced by reproduction.   It is shown by a recent paper that mating and starvation promotes spermatogonia de-differentiation to GSCs, and this requires JNK signaling activation in the germline [52]. Thus, we asked if JNK signaling activation in germ cells influences Zfh-1 expression. When JNK signaling was hyper-activated in germ cells by forced expression of Hep CA for 7 days, neither ectopic Zfh-1 expression nor early germ cell expansion was observed ( Fig 6D and 6D'). This may be due to the lack of downstream transcriptional factor AP-1 protein complex, Kay and Jra in the germ cell nucleus (Fig 5A and Fig 6F-6G'). In the testes of aged, mated males, block of JNK signaling activation in germ cells by mis-expression of Puc, a negative feedback regulator of JNK [53], did not significantly suppress ectopic Zfh-1 expression compared to the controls ( Fig 6E). Together, these results show that JNK signaling activation in the germ cells does not influence Zfh-1 expression in both mated and unmated conditions.

The testis sheath produces TNF Eiger
To elucidate the mechanism by which reproduction activates JNK signaling in testes of aged males, we sought to identify the ligand that triggers this response. In Drosophila, JNK signaling can be activated by the TNF-type ligand Eiger (Egr) [54,55] (Fig 5A). We were unable to examine the reproduction-induced phenotypes in null mutant egr 1 testes due to the early lethality of egr 1 mated males before week 4. However, the percentage of ectopic Zfh-1 expression was significantly decreased in the testes of heterozygous egr 1 /+ 5w-old flies compared to control mated w 1118 flies ( Fig 7A). Depletion of egr in the whole body using the UAS-egr RNAi BL line driven by the ubiquitously-expressing tub-Gal4/Gal80 ts (tub ts ) eliminated egr expression in 1w-old adult flies (S4A Fig), and suppressed the ectopic Zfh-1 expression and the expansion of early germ cells in the testes from mated males (Fig 7B and 7C). Together, these results indicate that egr is essential for the reproduction-induced phenotypes we observed in the testes of male flies.
To investigate the source of Egr, we analyzed egr expression using the enhancer trap egr-Gal4 line [56]. Strikingly, we found that egr was expressed in the outer layer of the testis, the testis sheath (Fig 7D and 7E). The testis sheath is composed of an outer layer of pigment cells and an inner layer of circular smooth muscle [20,21]. We specifically detected nuclear β-galactosidase in the smooth muscles (arrows in Fig 7D and 7E) and pigment cells (arrowheads in Fig 7E) of testes from egr>lacZ males, that express LacZ via the egr-Gal4 driver. By contrast, βgalactosidase was absent from their cyst cells and germ cells (Fig 7F and 7G). We detected the same expression pattern through direct detection of GFP fluorescence in egr>nGFP testes (arrows in Fig 7H). Smooth muscles enclose the testis, and the images obtained by optically thin sectioning showed that cyst cell cytoplasm lays right beneath the muscle cells (arrows in Fig 7I), indicating the close proximity of these two types of cells. To further confirm egr expression in the testis sheath, we depleted it using tara-Gal4 and fz2-Gal4, both of which were specifically expressed in the smooth muscle and pigment cells in the testes (Fig 7J-7M) [57], and examined egr expression levels in whole testes by qRT-PCR. Knockdown of egr by either one of the two Gal4 lines via UAS-egr RNAi BL markedly and significantly reduced the egr mRNA levels in testes (S4B and S4C Fig), together, indicating that egr is primarily expressed in the testis sheath.
immunostained for GFP (green in F and G, and white in F' and G'), co-stained for Vasa (blue in F and G), and for Eya and FasIII (red in F and G). Both Kay-GFP and Jra-GFP proteins expressed from the BAC genomic clones were detected in the nucleus of cyst cells, but not in the nucleus of germ cells. Arrows mark the CySCs, and small and large arrowheads indicate early and late cyst cells, respectively.  Next, we tested the role of egr in reproduction-induced testis defects. Knockdown of egr in testis sheath by tara-Gal4 or fz2-Gal4 at 29˚C via UAS-egr RNAi BL and the previously characterized UAS-egr RNAi V [58,59] dramatically reduced the numbers of ectopic Zfh-1-positive cells and suppressed the accumulation of early germ cells in the testes of mated male flies ( Fig  7N and 7O). When egr was overexpressed by tara-Gal4 in unmated males, Zfh-1 expression was induced in Eya-positive cyst cells, a phenotype similar to what we observed in the testes of mated males exhibiting level-1 ectopic Zfh-1 expression (Fig 7P and 7P', 2N, and column 3 in Fig 2M). In contrast, egr overexpression in the cyst cell lineage failed to induce ectopic Zfh-1 expression (Fig 7Q and 7Q'). Together, these results demonstrate that egr acts non-autonomously in the testis sheath to promote ectopic Zfh-1 expression in cyst cells.
Although we found that decreasing and increasing egr levels in the testis sheath, respectively, suppressed and promoted ectopic Zfh-1 expression in cyst cells, egr mRNA levels, as measured by qRT-PCR, were comparable in testes of mated and unmated flies (Fig 8A), suggesting that the reproduction-induced mechanism operates downstream of egr mRNA production. We assessed a GFP-tagged Egr protein reporter expressed from a genomic fosmid clone [60] and notably found a reproduction-induced accumulation of Egr-GFP in the cytoplasm of testicular muscles of mated flies as young as 3w-old (Fig 8C and 8D). In some cases, high levels of Egr-GFP filled the muscle fibers throughout the whole testes ( Fig 8D). By comparison, no specific Egr-GFP signals were detected in the testis muscles of 3w-old unmated Egr-GFP flies or 3w-old mated w 1118 flies (Fig 8B and 8E). Furthermore, knockdown of egr by tara-Gal4 in mated males abolished Egr-GFP signals in testicular muscles (Fig 8F and 8G), demonstrating the specificity of Egr-GFP signals in testicular muscles. Up-regulation of Egr-GFP in testis muscles was primarily induced by reproductive activity since it was observed in 35% (N = 79) of the testes of 3w-old males who mated with six females in single-male mating scheme (Fig 8H), whereas only 4% (N = 78) of testes from 4w-old males kept in solitude exhibited Egr-GFP up-regulation, despite having similar survival rates to the 3w-old mated males ( Fig 8H and S5 Fig). Egr-GFP up-regulation in mated males is unlikely to be solely caused by accelerated aging because 90% of the flies were still surviving after mating for three weeks (S5 Fig). None of the testes of 3w-old mated males in which Egr-GFP had accumulated in the testicular muscle showed ectopic expression of Traffic jam (Tj), a CySC and early cyst cell marker (Fig 8I and 8J), suggesting that Egr accumulation in the testis muscle precedes induction of ectopic early cyst cells. These results indicate that reproduction can lead to Egr protein accumulation in testicular muscle before onset of the differentiation defect in cyst cells.
Although egr expression, shown by egr-Gal4, was specifically detected in the testicular smooth muscle (Fig 7D-7G), careful examination of Egr-GFP protein patterns found that GFP signals were also detected in some of the cyst cells (Fig 8K and 8K'). We speculate that the Egr-GFP in the cyst cells might be derived from muscles. When egr in testis muscle was knocked down by tara-Gal4, the Egr-GFP signals in the cyst cells in mated males were abolished (Fig 8L  and 8L'), suggesting that the Egr protein detected on cyst cells is from testis muscles.

Reproduction does not elevate Egr production in the fat body
In addition to testicular muscles, examination of egr expression found that it was also expressed in tissues that lie very close anatomically to testes; it was expressed in ISC/EB in the midgut [61] and in the fat body (S6A and S6A' Fig). Knockdown of egr in ISC/EB by the esg-Gal4 ts system [62] did not suppress ectopic Zfh-1 expression in the testes from mated males (S6E Fig), suggesting that egr in intestine is not required for reproduction-induced Zfh-1 upregulation. However, knockdown of egr in fat body by lsp2-Gal4 [63,64]  In summary, these data indicate that egr in fat body is not essential for the reproduction-induced, fz2-Gal4-sensitive Zfh-1 up-regulation in the testes from mated males.

TNFR Grnd in cyst cells is required for reproduction-associated phenotypes
The proximity between the testis sheath and the cyst cells prompted us to ask whether Egr acts directly on the somatic cyst cells. To answer this question, we first examined expression of the TNF receptor Grnd by immunostaining [65]. In 1-to 3-day old testes, there was almost no anti-Grnd staining (Fig 9A and 9A'). Interestingly, Grnd was clearly detected in the cytoplasm of cyst cells in the testes of 3w-old mated flies, but not in age-matched unmated males (Fig 9B  and 9C'). Specific anti-Grnd staining in cyst cells was not observed in the testes of 3w-old mated grnd minos protein-null mutant flies, indicating the specificity of the staining pattern ( Fig  9D and 9D'). Quantification of anti-Grnd staining intensity in the cyst cell cytoplasm revealed that Grnd levels were significantly higher in the testes of mated males compared to those from unmated flies (Fig 9E). Grnd could also be detected in the extra Zfh-1 cells in the testes with level 1 ectopic Zfh-1 expression (Fig 9G and 9G'"). Further examination of grnd transcript levels by qRT-PCR also confirmed up-regulation of grnd in the testes of mated male flies (Fig 9F).
We then assessed whether Grnd is involved in the manifestation of the testis phenotypes induced by reproduction. Silencing of grnd in cyst cells by two independent RNAi lines [65] substantially reduced the numbers of ectopic Zfh-1-positive cells and suppressed the expansion of early germ cells in the testes of mated flies (Fig 9H and 9I). In contrast, depletion of another Drosophila TNFR, Wengen (Wgn) [66] (Fig 5A), did not rescue the testicular phenotypes induced by reproduction (Fig 9J). Ectopic Zfh-1 expression was even enhanced in one of the wgn RNAi lines. Thus, reproduction induces ectopic Zfh-1 expression to block spermatogenesis in aged males via Grnd activation in cyst cells.

JNK signaling is important for CySC maintenance in the testicular niche
Finally, we investigated the functions of JNK signaling and the upstream Egr-Grnd ligand receptor complex in regulation of self-renewal gene expression in cyst lineage in unmated males. JNK signaling activity in early cyst lineage was reduced by depletion of bsk or kay via c587 ts , or by forced expression of Bsk K53R that blocks endogenous bsk activity [52,67]. Reduction of JNK signaling activity for both five and ten days significantly decreased the total numbers of Zfh-1-positive cells (Fig 10A). In contrast, knockdown of puc in the cyst cell lineage for mated 3w-old egr-GFP males (10 vs. 20) immunostained with GFP (Green), co-stained with Arm (red). Egr-GFP was detected in some of the cyst cells (K and K'). Anti-GFP signals in cyst cells were abolished by depletion of egr in muscles by tara-Gal4 (L and L'). P-value was calculated with paired t test in A and Chi-squared test for H. ns.: p>0.05 and ��� p<0.001. https://doi.org/10.1371/journal.pgen.1008062.g008 Reproduction disrupts testicular stem cell homeostasis via an induced microenvironment  (Fig 10B). Prolonged puc knockdown for 10 days led to testes being completely filled with Zfh-1-positive cells and small germ cells (Fig 10C), thereby recapitulating the phenotypes observed under Hep CA expression (Fig 6C). Depletion of puc also led to more somatic cells undergoing S phase ( Fig 10D) and an accumulation of spectrosomecontaining GSC or Gb-like cells (arrowheads in Fig 10E). Taken together, our results support that JNK signaling normally maintains self-renewal gene expression in cyst lineage, and is negatively regulated by Puc.
Although at a lower level compared to early cyst cells, we also noticed the mild but specific expression of puc-lacZ B48 and TRE-EGFP in CySCs (arrows in Fig 10F and 10G'), suggesting that JNK signaling was also activated in CySCs. Analysis of the loss-of-function bsk 1 and bsk 2 mutants by a GFP-labeled Mosaic Analysis with a Repressible Cell Marker (MARCM) approach revealed that autonomous JNK signaling is required for CySC maintenance in the testicular niche. CySC clones are identifiable as GFP-positive, Zfh-1-expressing cells located one cell diameter away from the hub [68]. We dissected testes of control FRT 40A and the bsk mutant FRT 40A bsk 1 and FRT 40A bsk 2 flies at 2, 4-5, 6-7 and 10 days after clone induction (ACI). In FRT 40A controls, percentages of testes exhibiting CySC clones were maintained over time, ranging from 67% (N = 21) at 2 days ACI to 63% (N = 19) at 10 days ACI (arrows in Fig  10H and 10J, and Table 1). However, the percentages of testes possessing CySC clones were dramatically reduced over time in FRT 40A bsk 1 flies, declining from 79% (N = 14) at 2 days ACI to only 6% (N = 16) at 6-7 days ACI (Fig 10I and 10K and Table 1). We also observed loss of CySC clones for the bsk 2 allele that encodes a truncated protein [69] (Table 1). In some FRT 40A bsk 1 /+ testes that lacked GFP-labeled CySCs, we noted differentiating GFP-positive cyst cells away from the hub (arrowhead in Fig 10K). Since CySCs are the only cells that undergo mitosis-mediated recombination in the cyst cell lineage [37], these findings indicate that bsk mutant CySCs produce progeny that differentiate.
We then examined the roles of Egr and Grnd in testes in unmated males. Examination of puc-lacZ showed comparable LacZ expression in cyst cells between egr 1 and heterozygous controls (Fig 11A and 11B). In testes from both grnd minos null males and grnd minos /+ males, puc-lacZ expression was also detected at similar levels (Fig 11C and 11D). These results together suggest that egr and grnd are dispensable for endogenous JNK signaling activity in cyst cells. In both heterozygous egr 1 /+ and homozygous egr 1 mutants, there was an approximately 20% reduction in the Zfh-1-positive cell numbers in the testes of 31-day-old flies compared to 5-day-old flies ( Fig 11E). Ubiquitous knockdown of egr by the tub ts system led to a mild decrease in Zfh-1 cell numbers (Fig 11E). Zfh-1-positive cell numbers were maintained in the testes of grnd minos mutants (Fig 11E). To examine the role of grnd in CySC maintenance, grnd anti-Grnd signals in the testes from young males (1-3-day old) (A) and from 3w-old unmated males (C), Grnd was clearly detected in the cyst cell cytoplasm in testes of mated 3w-old males (B). Anti-Grnd signal was absent from the cyst cells in mated 3w-old grnd minos testis (D). (E) Box-and-whisker plots showing normalized anti-Grnd/anti-Arm intensity in cyst cells. All the relative anti-Grnd intensities were further normalized to the median value for the testes of 3w-old mated males. (F) Fold changes of grnd transcript levels in testes from mated and unmated 3w-old males. Transcript levels were measured by qRT-PCR. The mean grnd/rp49 levels in different samples were normalized to that in the unmated testes. N (biological repeat) = 4. Error bars represent SEM. (G-G"') A testis with level-1 ectopic Zfh-1 phenotype from mated 5w-old w 1118 male immunostained for Grnd (green in G, and white in G"'), co-stained for Zfh-1 (blue in G; white in G') and Arm (red in G; white in G"). Arrows indicate the anti-Grnd signals in the ectopic Zfh Reproduction disrupts testicular stem cell homeostasis via an induced microenvironment was depleted by expression of grnd RNAi in somatic clones. Unlike the dramatic reduction of CySC clones by lack of bsk within one week (Table 1), more than 60% of the CySC clone expressing grnd RNAi was maintained at 14-day ACI (Table 2). Together, our results suggest that egr and grnd are not essential to maintain CySCs and early cyst cells in the adult testes of unmated males.

Discussion
Both males and females of diverse organisms incur costs such as reduced lifespan and future fecundity from increased reproduction [24,25,[70][71][72][73][74]. Although recent studies have revealed molecular mechanisms underlying the reproductive tradeoff that modulates lifespan [75,76], little is known about how reproduction influences spermatogenesis and stem cell homeostasis in the testes of aged animals. Our study demonstrates that reproduction can disrupt differentiation of stem cell progeny, leading to an accumulation of immature cyst cells and germ cells (right panel in Fig 12A). Strikingly, these reproduction-induced phenotypes result from Egr accumulation in testicular smooth muscle, which hyperactivates JNK signaling in the differentiating cyst cells, thereby up-regulating the self-renewal protein Zfh-1 and BMP signaling in CySC and GSC daughter cells, respectively (Fig 12B). Though maintenance of Drosophila CySCs and GSCs normally depends on the apically-localized testicular hub, our investigation reveals a novel role of testicular muscles as an induced signaling center to influence spermatogenesis and stem cell homeostasis in Drosophila.

Combined effect of mating and aging in reproduction-induced phenotypes
Aging and reproduction influence each other. Reproduction reduces life span and accelerates aging through mechanisms such as compromising immune functions and decreasing protection from stress and damage [24,[75][76][77]. Aging reduces the numbers and activity of gonadal stem cells via both autonomous and non-autonomous mechanisms [35, [78][79][80]. Our studies show that reproduction blocks differentiation of early germ cell and early cyst cells in aged males but not in young males, and these phenotypes were rarely observed in very old (9w and 10w-old) males that never mated. Together, it reveals a combined effect of reproduction and aging in the reproduction-associated phenotypes, and adds a new layer of complexity in the interaction between aging and reproduction. Our study showed that the combined effect is at (H-K) Testes with control or bsk 1 clones marked by MARCM system. Asterisks indicate the hubs. Arrows indicate the GFP-positive CySC clones. Arrowheads mark the cyst cells with elongated cytoplasmic GFP. Scale bars = 50μm. (H and I) When dissected 2 days after clone induction (ACI), CySC clones (arrows) were observed in both FRT 40A control (H) and FRT 40A bsk 1 /+ (I) testes. (J and K) When dissected 5 days ACI, cyst cell clones with elongated cytoplasm (arrowheads) were observed in both FRT 40A control (J) and FRT 40A bsk 1 /+ (K) testes. CySC clones were lost in some FRT 40A bsk 1  least on the level of Egr production in the testis muscle. Insulin signaling is critical to regulate reproduction and aging [81][82][83][84]. Interestingly, Egr is released from fat body to inhibit insulin production in the brain during nutrient shortage [59]. Potential communication between Egr and insulin signaling in the testis sheath and the role of insulin signaling in Egr production during reproduction needs further study. Many studies have revealed cross talk between germ cells and somatic cells in the modulation of reproduction and aging, such as the regulation of germ cell division and differentiation by somatic cells, and modulation of organismal longevity, stress resistance, and trans-generational inheritance of survival advantages through germ cells [6,38,[85][86][87][88]. We found that  reproduction blocks future gamete production by signals from smooth muscles, revealing a somatic cell-mediated mechanism in negative feedback control of reproduction. Our results reveal that mating has a detrimental effect in germline homeostasis in aged males. What is the evolutionary reasoning for this mechanism? Potentially, this mechanism could prevent production of large numbers of offspring form the same father, thus maintaining genetic diversity in the population. Furthermore, GSCs have to divide many times in order to generate substantial amounts of sperm for reproduction, and the likelihood of having a sperm with genetic mutations is much higher in aged fathers than the young fathers [89].
We also recorded a positive correlation between the levels of ectopic Zfh-1 expression and the numbers of available females in both single-male mating and mass-mating experiments. Since male flies fight for access to virgin females [90,91], it is possible that the accumulated Reproduction induces overproduction of CySC-like cells and early cyst cells due to extra cell divisions and blocked differentiation in aged males. Reproduction also leads to over-proliferation and accumulation of early germ cells at the expense of spermatocyte differentiation. (B) Models of reproduction-induced TNF-JNK signaling hyperactivation in regulation of spermatogenesis. In unmated testes (top), low-level JNK signaling is activated in cyst cells independent of Egr. In testes from mated males (bottom), Egr is accumulated abundantly in smooth muscles as early as 3w-old to act on induced TNFR Grnd. High-level JNK signaling activity induced by Egr up-regulates Zfh-1 expression and block differentiation in cyst lineage, that can be readily detected in testes from 5w-old males. Defect in germ cell differentiation is mediated by dpp-dependent BMP signaling activation. https://doi.org/10.1371/journal.pgen.1008062.g012 Zfh-1 cells seen in mass-mated males are largely due to male aggression. However, we found that the frequency of ectopic Zfh-1 expression in the Canton-S strain is quite comparable between mass-mating (10 males vs. 20 females) and single-male mating (1 male vs. 1-2 females) systems. In the w 1118 strain, the frequency of Zfh-1-positive cell accumulation was higher in the mass-mating compared to the single-male mating system. Importantly, the frequency of ectopic Zfh-1 expression was much lower in a w 1118 mass-mating system involving 25 males and only five females, which presumably induces more fighting and aggressive behavior among males. Moreover, Egr-GFP accumulation in testicular muscles was found in males subjected to either the single-male mating or mass-mating system. Thus, our results indicate that the up-regulation of TNF-JNK-Zfh-1 observed in mass-mating male flies is primarily induced by reproductive activity.

JNK signaling activation in somatic cells and germline in Drosophila testis
Our study showed that reproduction hyperactivates JNK signaling in cyst lineage cells based on the following observations: (1) JNK signaling activity, assayed by reporters puc-lacZ and TRE-EGFP was up-regulated in early cyst cells in response to reproduction. (2) Knockdown of JNK signaling activity in cyst lineage suppressed mating-induced ectopic Zfh-1 expression and accumulation of early germ cells. (3) Forced activation of JNK signaling led to overproduction of Zfh-1-positive cells and early germ cells at the expanse of mature cyst cells and spermatocytes. (4) Reduction of TNF receptor Grnd, an upstream activator of JNK signaling [65] in the cyst lineage rescued reproduction-induced phenotypes. A recent paper showed that reproduction and starvation triggers spermatogonia de-differentiation to GSCs, a process which requires autonomous JNK signaling activation in germ cells [52]. While the JNK signaling activity in the cyst lineage can be easily observed by the JNK pathway reporters, the germline JNK signaling activation by reproduction and starvation can only be observed by lineage tracing via puc-Gal4. Thus, it appears that JNK signaling activation is normally maintained in the cyst lineage, and its activation in the germline is transient. We found that blockage or hyperactivation of JNK signaling in germ cells did not influence native nor ectopic Zfh-1 expression. In contrast, activating JNK signaling in cyst cells blocked germ cell differentiation. Our study also revealed that reproduction elevated TNFR Grnd expression in cyst cells. No detectable Grnd expression was observed in germ cells in mated males, suggesting that reproduction might activate somatic and germline JNK signaling in the testes via distinct mechanisms.
The cellular functions of JNK signaling are highly diverse [92][93][94][95]. During protein starvation, JNK signaling is upregulated in cyst cells to non-autonomously induce spermatogonia cell death [96]. Our data show that under normal nutritional conditions, hyperactivation of JNK signaling in cyst lineage cells by either mating or genetic manipulation leads to ectopic self-renewal gene expression and non-autonomous accumulation of early germ cells including GSC-like cells and spermatogonia. These results demonstrate that JNK signaling hyperactivation induces different cellular responses in cyst cells under different environmental conditions. Since JNK directly phosphorylates more than fifty different target proteins [97], it may activate a different set of downstream effectors in cyst cells in response to poor nutritional conditions [98,99]. Differential cellular responses may also be dependent on the activities of additional components. For example, JNK hyperactivation induces apoptosis in scribble-deficient cells in imaginal discs. In scribble mutant cells that also express oncogenic Ras or Notch, JNK signaling is tumorigenic [100]. Therefore, protein starvation may activate additional signaling pathways or induce cellular changes in cyst cells, acting in concert with JNK signaling to give rise to non-autonomous spermatogonial cell death.

TNF-JNK signaling modulates adult physiology
Though it is not essential for normal development, Egr plays important roles in maintaining Drosophila physiology and adult survival during damage or stress [58,59,64,101,102]. For example, egr 1 mutant flies die much faster than control flies when challenged with several types of bacteria [102], and Egr is secreted from the fat body to inhibit insulin production during amino acid starvation. While Egr in the sheath blocked differentiation in the testes of mated males, our results suggest that Egr is required to decrease reproduction-induced death given that all mated egr 1 mutants died before the age of four weeks. Thus, it is also likely that Egr is essential to resist reproduction-induced mortality in younger males, but comes at the expense of defective spermatogenesis in older mated males.
The UAS-RNAi lines targeting bsk (VDRC34138) [106][107][108] [113,114] and NIG-FLY 15507R-2 for UAS-kay RNAi N [114]], and puc (BL34392 for UAS-puc RNAi BL  Adult flies were maintained in culture vials of 24 mm diameter and 50 mm height in an incubator with a 12h: 12h light: dark cycle at 25˚C, unless otherwise indicated. For the mating experiments, flies were transferred to fresh vials twice per week. Females were replaced with virgin females every week. For the experiments expressing RNAi or transgenes with or without tub-Gal80 ts , flies were maintained at 18˚C-20 o C to suppress Gal4 expression during development, unless otherwise noted. Adult flies were then moved to 25˚C or 29˚C for Gal4-mediated expression. For depletion of dpp, gbb, hep, bsk, or kay by eyaA3 ts and nos-Gal4, 3w-old adult flies were incubated at 29˚C for 10 days before dissection. To silence egr by tub ts , tara-Gal4, fz2-Gal4, and to silence grnd by eyaA3 ts , 2w-old adult flies were incubated at 29˚C for 17 days before dissection.
Testes were dissected in 1xPBS, fixed in 4% paraformaldehyde and washed in 0.3% PBT. Before antibody staining, testes were permeabilized in 0.3% PBT containing 0.3% NaDOC for 30 minutes. Testes were incubated overnight at 4˚C with primary antibodies diluted in 0.3% PBT containing 0.3% BSA, followed by three washes and incubation with secondary antibody for 2 hours at room temperature. For staining with anti-Grnd antibody, testes were blocked overnight in 0.3% PBT containing 0.3% BSA at 4˚C, followed by overnight incubation with pre-absorbed anti-Grnd antibody (1:200) at 4˚C. Pre-absorption was achieved by incubating overnight the anti-Grnd antibody with 50-70 pairs of adult grnd minos testes in 50 μl 0.3% PBT containing 0.3% BSA at 4˚C.

Classification of reproduction-induced testes defects
Ectopic Zfh-1-positive cells were defined as cells localized at least 200 μm (four times the average distance from the testis tip to the basal end of the spermatogonia in the testes of 5w-old unmated males) away from the testis tip. Level 1 ectopic Zfh-1 expression was defined as testes having total numbers of Zfh-1-positive cells below 50 but exhibiting ectopic localization of Zfh-1 positive cells. Level 2 and 3 ectopic Zfh-1 expression were defined as total numbers of Zfh-1-positive cells being between 50-200 and more than 200, respectively.
The phenotype of accumulation of early germ cells was defined as instances where the distribution of early germ cells (small DNA-bright Vasa-positive cells) extended more than 200 μm from the testis tip to the basal end of the early germ cells. Phenotypic severity was categorized into grades 1, 2, and 3 according to distributions ranging between 200-400 μm (grade 1), 400-600 μm (grade 2), and more than 600 μm (grade 3). Increase of puc-lacZ cell numbers was classified as class 1 or 2 based on total numbers of puc-lacZ cells being between 50-200 and more than 200, respectively.

Mosaic analysis
The MARCM system was used to create positively marked clones for bsk 1 , bsk 2 and wild type control flies. We used flies of the following genotypes to generate clones: hsFLP, UAS-mCD8-GFP/Y;FRT 40A bsk 1 /FRT 40A tub-gal80;tub-gal4/+, hsFLP, UAS-mCD8-GFP/Y;FRT 40A bsk 2 /FRT 40A tub-gal80;tub-gal4/+, hsFLP, UAS-mCD8-GFP/Y;FRT 40A /FRT 40A tub-gal80;tub-gal4/+. Adult males of 1-3 days old were subjected to three heat shocks at 37˚C for 30 minutes. After heat shocks, flies were kept at 25˚C until dissection. The percentage of CySC clones at each time-point represents the number of testes with GFP-positive CySCs divided by the total number of testes with GFP-positive cyst lineage cells. The random clone system was used to create positively marked clones for grnd RNAi and the control. We used flies of the following genotypes to generate clones: hsFLP, act<y+<Gal4, UAS-GFP/UAS-grnd RNAi KK hsFLP, act<y+<Gal4, UAS-GFP/+ Adult males of 1-3 days old were subjected to three heat shocks at 37˚C for 30 minutes. After heat shocks, flies were kept at 25˚C until dissection. The percentage of CySC clones at each time-point represents the number of testes with GFP-positive CySCs divided by the total number of testes scored.

EdU labeling
Testes were dissected in Schneider's Drosophila medium (Invitrogen), followed by incubation in Schneider's medium containing 100 μM EdU (Invitrogen) for 30 minutes at room temperature. Testes were then fixed in 4% formaldehyde for 15 minutes and stained with primary antibodies as described above. To visualize EdU, testes were incubated with Alexa 555-azide for 30 minutes at room temperature.

Quantification of fluorescence intensity
To quantify fluorescence intensities, the testes were processed in parallel for the entire set, and the confocal images were obtained with the same confocal settings. To determine the fluorescence intensity, we selected the areas within single confocal sections using the Freehand tool in Image J software.
To quantify the anti-β-galactosidase fluorescence intensity of puc-lacZ, we considered the mean intensity of each cyst cell as being the mean intensity of a 25-35 μm 2 nuclear area after subtracting the background intensity. We measured 8-30 puc-lacZ-positive cells in each testis. All anti-β-galactosidase intensities were normalized to the median value for the testes of 4wold mated males.
To quantify fluorescence intensities of anti-GFP (TRE-EGFP), anti-Grnd and anti-Arm staining, we considered the mean fluorescence intensity of each cyst cell as being the mean intensity of a 30-40 μm 2 cytoplasmic area. The same areas were used to measure anti-GFP/ anti-Arm or anti-Grnd/anti-Arm intensities. On average, we measured 6 to 8 cyst cells next to 8-to 16-cell spermatogonia clusters in each testis. The relative anti-Grnd intensity of each cyst cell represents the mean anti-Grnd intensity (after subtraction of the background anti-Grnd intensity) divided by the mean anti-Arm intensity (after subtraction of the background anti-Arm intensity). The same normalization method was also applied to the analysis of anti-GFP intensity in cyst cells.

Statistical analysis
P values were generated using Chi-squared test to compare ectopic Zfh-1 expression, accumulation of early germ cells, extra puc-lacZ-positive cells, survival rate, and Egr-GFP expression between treatments. Statistically significant differences in immunofluorescence intensities, including for anti-GFP, anti-Grnd staining, and anti-β-galactosidase staining, were calculated by Mann-Whitney nonparametric test. Results for Zfh-1-positive cell number were calculated by student t test in Figs 10A and 11E. Results from qRT-PCR were statistically assessed by student t test in S4A and S4B Fig, and paired t test in Fig 8A, Fig 9F and S4C Fig. For   DNA (red). egr was expressed in adult fat body. (B-C') Fat bodies from 3w-old egr-GFP males immunostained for GFP (green in B and C; white in B' and C'), co-stained for DNA (red in B and C) and Arm (blue in B and C). Comparable, low-level GFP expression was observed in fat bodies from unmated (B and B') and mated (C and C') males. (D) Fat body from 1w-old fz2-Gal4>UAS-nGFP male immunostained for GFP (green in D and white in D'), co-stained for DNA (red in D). No nuclear GFP signals were detected in fat body. (E) Percentages of testes with ectopic Zfh-1 expression from mated 4w-old males. No or mild suppression was observed by egr knockdown, respectively, in ISC/EB via esg ts and in fat body via lsp2-Gal4. P values were calculated with Chi-squared test. ns: p>0.05, � p<0.05, �� p<0.01, and ��� p<0.001. Mass-mating was conducted in this experiment. (TIF) 25. Partridge