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G-Protein signaling accelerates stem cell divisions in Drosophila males

Manashree Malpe, Leon F. McSwain, Karl Kudyba, Chun L. Ng, Jennie Nicholson, Maximilian Brady, Yue Qian, Vinay Choksi, Alicia G. Hudson, Benjamin B. Parrott, Cordula Schulz

Preprint posted on 14 November 2019 https://www.biorxiv.org/content/10.1101/433623v2.full

Article now published in Scientific Reports at https://www.nature.com/articles/s41598-020-60807-8

When practice makes perfect: Repeated mating leads to an increase in germline stem cell division rate in the Drosophila testis, which involves the G-protein signaling pathway.

Selected by Nadia Edelsztein

Background:

Tissue homeostasis relies greatly on the ability of stem cells to keep a balance between their proliferative capacity to sustain a source of undifferentiated progenitor cells and the differentiation of their daughter cells. This ‘homeostasis-prone’ response, i.e.: the balance between proliferation and differentiation, is conserved between groups as different as humans and flies and is sensitive to changes in environment [1-5]. Upon injury or environmental challenges, such as temperature changes or nutrient availability, stem cells can alter their mitotic activity. For example, while mouse hematopoietic stem cells increase their division rate during pregnancy [1], Drosophila intestinal stem cells can divide more upon ablation of differentiated intestinal cells [2].

In the Drosophila testis when a germline stem cell (GSC) divides, it gives rise to two daughter cells, one of them remains as a GSC while the other one —the gonialblast— undergoes four rounds of mitotic divisions, generating 16 spermatogonia [6]. In turn, these spermatogonia enter a tissue-specific differentiation process characterized by size growth followed by two consecutive meiotic divisions and a final series of dramatic morphological changes that result in elongated spermatids. Each GSC division can only produce 64 spermatids [6]. Thus, a higher or lower sperm output may reflect GSC status and/or their division rate.

But what would happen if there was a ‘higher demand’ for sperm release? In this preprint the authors sought to elucidate whether mating and/or mating-frequency influences GSC division rate.

 

Key findings

  1. Repeated mating increases the percentage of dividing GSCs

The authors analyze the percentage of GSCs in mitosis (MIGSC) through immunofluorescent staining for the mitosis marker phosphorylated Histone-H3 in testes from mated and non-mated (isolated) wild type Drosophila males. They observe a significant increase in the MIGSC of mated males when compared to the isolated ones and describe the existence of variation within experimental groups regarding the number of GSCs present in each testis.

The Drosophila mating behaviour follows a series of events that are genetically controlled and includes visual, chemical, and physical contact. To pinpoint the cause for MIGSC increase, the authors dissected the components of the mating process by exposing males to virgin females —including visualization, smelling and touching—, allowing for courtship behaviour using males that cannot distinguish between males and females due to mutation of the fruitless (fru) gene and using decapitated virgin females. In none of these scenarios did the MIGSC increase relative to controls.

When males were housed with varying number (1, 2 or 3) of virgin females for 24 h, MIGSC did not increase. However, when males were exposed to 3 females for 48 h or 72 h, an increase in MIGSC was observed, suggesting a need for repetitive mating in order to increase GSC division rate. Interestingly, this change in division rate was reversible, as seen when males moved to solitude after the 3-day mating period abolished the increase in MIGSC.

 

  1. Sperm pool reduction is induced by mating

Sperm demand was assessed through comparison of seminal vesicles between non-mated and mated males. Making use of the genetic powerhouse that is Drosophila, sperm was labelled at the body —Don Juan-GFP— and at the head —ProtamineB-GFP (Mst35B-GFP) line—, thus allowing for total sperm and single sperm head count within the seminal vesicles, respectively.

Qualitatively, non-mated males had seminal vesicles filled with GFP-positive sperm heads both wide (class 1, as assessed by the authors) and thin (class 2), while mated males showed only few GFP-positive sperm and areas not filled with GFP (class 3). Furthermore, the authors introduce an automated procedure to calculate the volume occupied by Mst35B-GFP-positive sperm heads per seminal vesicle in all focal planes. With this approach, they observed that sperm heads of mated males took significantly less volume within the seminal vesicles than the sperm heads in non-mated males, and that the total volume occupied by sperm became smaller with every day of mating. The automated procedure also allowed them to estimate the number of sperm per seminal vesicle, being approximately 2000 in non-mated males versus 500 in males mated for 2-3 days.

 

  1. G-protein signaling and seven GPCRs participate in mating-induced MIGSC increase

Since mating in Drosophila has a strong behavioural component, suggesting a potential role for neuronal control in GSC divisions, the authors focused on a signaling pathway known to be stimulated during neural activity, the G-protein signaling pathway [7,8]. Classical G-protein coupled receptors (GPCRs) are typically associated with trimeric complexes of G-proteins (α,β,γ). Activation of the GPCRs leads to dissociation of Gα and the Gβ/γ complex. Gα and Gβ/γ then diffuse along the plasma membrane and activate downstream signal transducers [9,10]. In Drosophila there are six Gα, three Gβ, and two Gγ proteins, with few known examples associating a specific Drosophila G-protein with an upstream GPCR [9,10].

When abolishing G-protein signaling with a dominant negative version of Drosophila Gγ1, males showed no increase of MIGSC upon mating —contrary to control animals— showing that signaling via G-proteins is required for this MIGSC increase. To further validate participation of G-protein signaling, conserved signal transducers PKC98E (one of the Drosophila Protein Kinase C proteins) and Inositol-triphosphate 3-Kinase (IP3K) were abolished through RNAi. In these animals, mating did not increase MIGSC.

From tips of wild type testis, 35 classical GPCRs were identified through Next Generation Sequencing. Silencing expression of seven of them —Mth, Mth-l5, Octβ2R, Serotonin receptors 5-HT1A, 5-HT1B and 5-HT7, or CG12290 (a predicted GPCR)— in the germline with RNAi prevented MIGSC increase.

These results suggest that the regulation of GSC division rate is a very complex process that could involve the participation of multiple GPCRs that are needed either alone or in a concerted manner.

 

Personal thoughts:

I believe this work nicely shows how a classical and robust approach can still help elucidate novel functions of relevant pathways. By using controlled mating and the vastly used Gal4-UAS system, the authors were able to identify an increase in GSC division rate and a reduction in sperm pool when males participated in sequential mating. Furthermore, they show a novel role for GPCRs in regulating GSC division frequency.

Involvement of such a ubiquitous pathway as G-protein signaling could open up a whole new set of connections between systems and tissues. Is the brain telling the gonad what to do? Is the mechanical action of mating what triggers the G-protein signaling pathway that results in an increase of GSC division? What other signals could be participating?

 

Questions for the authors:

  1. When testing repeated mating for 48 or 72 h, have you considered using males with sperm markers to assess how many times each male mated, by looking at the female tract?
  2. Could sperm quality be different amongst single-mated, repeatedly-mated and repeatedly-mated-reversed-to-solitude males? Have you assessed sperm parameters (flagellar beat, wave, motility, etc)?

 

References:

[1] Nakada D, Oguro H, Levi BP, Ryan N, Kitano A, Saitoh Y, Takeichi M, Wendt GR, Morrison SJ. Oestrogen increases haematopoietic stem-cell self-renewal in females and during pregnancy. Nature. 505, 555–558 (2014).

[2] Amcheslavsky A, Jiang J & Ip YT Tissue damage-induced intestinal stem cell division in Drosophila. Cell Stem Cell 4, 49–61 (2009).

[3] Hsu HJ, LaFever L & Drummond-Barbosa D. Diet controls normal and tumorous germline stem cells via insulin-dependent and -independent mechanisms in Drosophila. Dev. Biol. 313, 700–712 (2008).

[4] McLeod CJ, Wang L, Wong C & Jones DL. Stem cell dynamics in response to nutrient availability. Curr. Biol. 20, 2100–2105 (2010).

[5] Parrott BB, Hudson A, Brady R & Schulz C. Control of germline stem cell division frequency – a novel, developmentally regulated role for epidermal growth factor signaling. PLoS One 7, e36460 (2012).

[6] Fuller MT. Spermatogenesis, in The development of Drosophila melanogaster, Vol. 1. (ed. Bate, M. & Martinez-Arias, A) 71–147 (Cold Spring Harbor Press, Cold Spring Harbor; 1993).

[7] Geppetti P, Veldhuis NA, Lieu T & Bunnett NW. G Protein-Coupled Receptors: Dynamic Machines for Signaling Pain and Itch. Neuron 88, 635–649 (2015).

[8] Lee D. Global and local missions of cAMP signaling in neural plasticity, learning, and memory. Front. Pharmacol. 6, 161 (2015).

[9] McCudden CR, Hains MD, Kimple RJ, Siderovski DP & Willard FS. G-protein signaling: back to the future. Cell Mol. Life Sci. 62, 551–577 (2005).

[10] Oldham WM & Hamm HE. Heterotrimeric G protein activation by G-protein-coupled receptors. Nat. Rev. Mol. Cell Biol. 9, 60–71 (2008).

Tags: fruit fly, germline, mating, stem cells, testis

Posted on: 23 April 2020 , updated on: 4 May 2020

doi: https://doi.org/10.1242/prelights.19159

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Author's response

Manashree Malpe and Cordula Schulz shared

Dear Nadia,

Thank you for your interest in our project and for the nice summary of our article you wrote. Most of our data are now published in Scientific Reports under the title: G-protein signaling is required for increasing germline stem cell division frequency in response to mating in Drosophila males. Please see our responses to your questions below.

When testing repeated mating for 48 or 72 h, have you considered using males with sperm markers to assess how many times each male mated, by looking at the female tract?

That is a good question. After mating, a Drosophila female stores the sperm in her two spermatothecae and the seminal receptacle, both parts of the uterus.  We have used the GFP-labeled sperm to count how many female uteri contained sperm at days one, two, and three of mating. We could clearly see sperm in 88% of the female reproductive tracts after 24 hours of mating. This number decrease to 73% after 48 hours, and to 25% after 72 hours. If we calculate from these numbers, then each male transferred sperm to four to five females, but likely the number of mating is higher. Considering the reduction in the amount of sperm in the male reproductive tract from day one to day three it is reasonable to assume that the ejaculates contained fewer to no sperm the more frequently they mated.

The most reliable tool for counting how many times a male mated is to video tape the flies. Due to the large amounts of flies necessary to detect significant differences in GSC divisions between non-mated and mated males, we were not able to do this (we also do not have cameras that fit the mating boxes)

Could sperm quality be different amongst single-mated, repeatedly-mated and repeatedly-mated-reversed-to-solitude males? Have you assessed sperm parameters (flagellar beat, wave, motility, etc)?

Those are also excellent questions. Unfortunately, we do not have answers to them. Please keep in mind that it takes 11 days for a cell to develop from a stem cell daughter to a mature sperm. Most of the sperm the males used in these experiments had been stored in their seminal vesicles and only a small portion of sperm has newly differentiated from immature spermatids during the three day-long experiment. It is definitively possible that the sperm quality in the ejaculate varies among the three days of mating, and/or changes after the mating challenge. If we had to speculate, we would assume that sperm that had been stored for longer time may be of lower quality compared to newly differentiated sperm. On the other hand, we challenge the males to ejaculate even though they have run out of mature sperm. It is possible that the ejaculate on day three contains immature spermatids. We are sure one could design experiments to test these and other interesting hypotheses.

We would like to point out another important factor in male fertility. It has been shown by the Wolfner, Noll, and other laboratories that Drosophila males do not only transfer sperm but also seminal fluid to the females during copulation. The peptides and proteins in the seminal fluid are essential for male fertility and female post-mating responses. Drosophila males only have enough seminal fluid for two to three mating events. Consistent with this, females that had been mated with males on days two and three of the experiment did not produce offspring even though they had sperm in their uteri. It would be interesting to know if the seminal fluid also impacts flagellar beat, wave, motility, etc.

In your view, what were the most important improvements in your study as a result of peer review

This question is very hard to answer as we received ample suggestions from colleagues and reviewers. For the latest version of the manuscript, we received several interesting comments on our stem cell counts and our statistics, as well as the presentation of the data. This made us re-analyse our data with mutliple tools and modify our images. Based on the comments we also re-phrased the manuscript, which helped narrow down our main message. In addition, one reviewer suggested early on in the review process to use the sperm-GFP lines which added an interesting twist to the story.

There are still many open questions to this story and our current manuscript is, hopefully, just the beginning towards understanding how stem cells can respond to a demand for specialized cells. We are honored that you share our enthusiasm and are grateful you picked our brains with your questions.

Best wishes

Manashree and Cordula

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