Stem cells and their role in tissue homeostasis are at the center of current research due to their potential in regenerative medicine. While most studies have focused on stem cell specification and stem cell daughter differentiation, fewer studies addressed stem cell activity. Among these studies, it is reported that oestrogen levels during pregnancy can cause blood stem cells in the mouse to divide more frequently [1]. In Drosophila melanogaster, stem cells can modulate their MIs in response to temperature and nutrition [2,3]. Recently, our laboratory showed that repeated mating depleted the sperm pool, indicating that mating creates a demand for sperm. Repeated mating also increased the frequency at which male GSCs divide, suggesting that stem cells can modulate their activity in response to a demand for specialized cells [4].

It remained an open question whether a demand for specialized cells specifically modulates stem cell mitotic activity, or whether the response to mating is more drastic and involves other cells of the tissue as well. In many tissues, the adult stem cells reside in a specific cellular micro-environment, or niche, where they produce new stem cells and stem cell daughters destined for differentiation [5,6]. The latter then undergo transit amplifying (TA) mitotic divisions to produce a pool of precursor cells prior to differentiation [7,8]. These transit amplifying stem cell daughters might also divide more frequently to contribute to the increased production of specialized cells in response to a demand.

In Drosophila males, GSCs are anchored around their niche, the somatic hub cells at the tip of the testes. A GSC division produces a new GSC and a gonialblast. In contrast to the outcome of a GSC division, the gonialblast divides into two daughter cells that appear to be equal in fate and remain interconnected by cytoplasmic bridges. The pair of cells will go through three more rounds of TA divisions, resulting in 16 interconnected cells. The interconnected, mitotically active germline cells are commonly referred to as spermatogonia. After ceasing mitosis, the 16 cells grow in size, and divide two more times by meiosis, and develop into 64 spermatids (Figure 1A). The germline cells of the different amplification and differentiation steps are arranged along the testis axis with the most immature cells in the apical region and the fully differentiated sperm at the base. Thus, the location, shape, and size of the germline cells allows for their identification [9,10].

To address if the transit amplifying GSC daughters also increase their MI upon mating, we used the same experimental procedures and analyses as previously described [4]. We examined high magnification images of the apical region of testes stained with antibodies against the hub, anti-Fasciclin III (FasIII), against the germline cells, anti-Vasa, and against a mitosis marker, anti-phosphorylated Histone H3 (pHH3; Figure 1B, 1B’). The Vasa-positive gonialblasts and spermatogonia lie basal to the GSCs that are next to the hub, but apical to the larger, later stage germline cells (note white line in Figure 1B separating the mitotic germline cells from the pre-meiotic germline cells). To avoid errors in cell counts due to the three-dimensional structure of the testes, we picked one focal plane per testis in which the hub cells could be seen. In these images, only germline cells that clearly showed a circle of Vasa-staining as well as a non-stained nucleus in the middle were counted as GSC daughters. Among these, only the ones that clearly had pHH3-staining inside their nuclei were counted as dividing cells (arrows in Figure 1B, 1B’). Using this approach, we calculated the MIs of GSC daughters from wild-type males from five independent experiments. In this specific set of experiments, the GSC daughters in non-mated wild-type males had MIs ranging from 2.9 to 4.5, with a median at 3.5, while the GSC daughters in mated wild-type males had significantly higher MIs, ranging from 5.9 to 9, with a median at 7.5 (Figure 1C). We conclude that mating does not specifically increase the MI of the GSCs, but the MIs of all mitotically active germline cells within the Drosophila testes.

We previously demonstrated that the increase in the MI of the GSCs in response to mating was dependent on G-proteins and, potentially, on seven distinct G-Protein coupled receptors (GPCRs) [4]. Reducing the activity of one of the G-proteins, G-gamma, via a dominant negative construct specifically in the germline cells similarly eliminated the increase in MI of the GSC daughters when males were mated. Both, non-mated and mated animals expressing dominant negative G-gamma within their germline cells had MIs ranging between 1.9 and 4, with a medium around 3.2 (Figure 1D). This shows that the changes in MIs of the GSCs and their daughters upon mating both depend on G-protein signaling. Our findings will facilitate studying the molecular pathways regulating the MI in a larger group of cells, and likely expedite and ease future experiments.

It is not understood how G-protein signaling modulates germline divisions on a molecular level. However, based on the current literature, several scenarios appear possible. The cell cycle is subdivided into four phases: Gap1 (G1), Synthesis (S), Gap2 (G2), and Mitosis (M). Progression through these phases require a cell’s ability to pass through cell cycle checkpoints. One of these checkpoints, the G1-checkpoint or restriction point, acts at the end of G1 phase, and assures that nutritional and physiological conditions for cell division are met prior to the transition into S-phase [11-13]. Reducing or eliminating the expression of specific genes could place cells in a sub-optimal physiological condition, which may not interfere with the normal progression through the cell cycle but could block the cell’s top performance. In our case, it could block the increase in MI upon demand for sperm. We speculate that an example for a gene that could influence the physiology and nutritional status of the cell is the white gene. white encodes an ABC transporter that does not only transport molecules such as Tryptophan and cyclic GMP across the cell’s membrane but is also implicated in intracellular vesicular transport [14-19]. Not surprisingly, animals mutant for white failed to increase the MI of their GSCs in response to mating [20]. It is possible that one or several of the GPCRs with a role in increasing the MI act in a similar manner, and that their loss prevents an elevated progression through G1-phase of the cell cycle in mated males.

Among the GPCRs implicated in the increase in the MI were three of the highly conserved Serotonin (5-HT) receptors, 5-HT1A, 5-HT1B, and 5-HT7. A mitogen-like function of 5-HT had been suggested in several mammalian cell types, including fibroblasts, cells of the kidney, vascular endothelial cells, and cancer cells [21-24]. When rats were injected with 5-HT-antagonists, the numbers of cells in S-phase of the brain were significantly reduced, and this proliferative effect was dependent on the 5-HT1A receptor [25]. Together with our findings, this makes the highly conserved 5-HT signaling pathway a potential target in drug therapies aimed towards controlling cell divisions.

In several studies addressing the effect of 5-HT, the proliferative activity of cells was associated with increased phosphorylation of Map-Kinases, suggesting that 5-HT receptors modulate transcription via this downstream signal transducer cascade [26-28]. However, a direct link between 5-HT receptor stimulation and MAP-kinase activity is yet to be explored. GPCR stimulation can also modulate the activity of ion channels. Extensive research on G-protein signaling and ion channel conductivity has revealed that both, the G-alpha subunit and the G-beta/gamma dimer can modulate ion channel activity. G-alpha subunits can, for example, release Calcium-ions from the ER via Inositol phosphatase 3 which then act as co-factors for several regulatory proteins, and the G-beta/gamma complex can directly modulate inwardly rectifying Potassium-channels that control the resting membrane potential of the cell [29-34].

Interestingly, the membrane potential fluctuates throughout the cell cycle, with a hyper-polarized state during G2/M phase [35,36]. Several studies correlated membrane hyper-polarization with the failure to progress through G1/S. When the membrane of Chinese Hamster Ovary cells remained hyperpolarized, it inhibited DNA synthesis [37]. Likewise, the cell membrane needs to be depolarized for lymphocytes, Schwann cells, and fibroblasts to progress through G1/S [38-41]. The changes in membrane potential and progression through the cell cycle appears to depend on Calcium-, Potassium-, Sodium- and Chlorine-channels [36,42,43]. It is possible that one or more GPCRs required for the increase in MI in response to mating promote modulations of ion channel activities that allow for the cells to either progress though the cell cycle faster, or to enter the cell cycle more frequently. The exact mechanism how the different GPCRs modulate cell division remains to be investigated.

The authors declare no conflicts of interests.

The authors thank Leon McSwain, Alicia Hudson, and Manashree Malpe for technical assistance. This work was supported by NSF grants #0841419 and #1355009.