- Email: [email protected]
Mating induces developmental changes in the insect female reproductive tract
Available online at www.sciencedirect.com
ScienceDirect Mating induces developmental changes in the insect female reproductive tract I Carmel1, U Tram2 and Y Heifetz1 In response to mating, the Drosophila female undergoes a series of rapid molecular, morphological, behavioral and physiological changes. Studies in Drosophila and other organisms have shown that stimuli received during courtship and copulation, sperm, and seminal fluid are needed for the full mating response and thus reproductive success. Very little is known, however, about how females respond to these malederived stimuli/factors at the molecular level. More specifically, it is unclear what mechanisms regulate and mediate the mating response, how the signals received during mating are integrated and processed, and what network of molecules are essential for a successful mating response. Moreover, it is yet to be determined whether the rapid transition of the reproductive tract induced by mating is a general phenomenon in insects. This review highlights current knowledge and advances on the developmental switch that rapidly transitions the female from the ‘unmated’ to ‘mated’ state. Addresses 1 Department of Entomology, The Hebrew University, Rehovot, Israel 2 Department of Molecular Genetics, The Ohio State University, Columbus, OH, USA Corresponding author: Heifetz, Y ([email protected])
Current Opinion in Insect Science 2016, 13:106–113
model organisms are compared [7,8]. Numerous studies in Drosophila and other organisms have shown that sperm, seminal fluid and other signals received during mating alter female behavior and physiology postmating, and that these changes increase the reproductive success of both the male and female [9–24]. Seminal fluid is composed of sperm and components secreted by the accessory glands, ejaculatory duct, and ejaculatory bulb of the male reproductive tract. Each of these seminal fluid components play an essential role in male and female reproductive success [10,23,25]. Despite this wealth of knowledge, little is known about how females respond to these male-derived stimuli/factors at the molecular level and more specifically what mechanisms regulate and mediate the mating response, how the signals received during mating are integrated and processed, and what network of molecules are essential for a successful mating response. In this review, we will focus on the female response in the first 24 hours postmating, highlighting the developmental switch that transitions the female from an ‘unmated’ to ‘mated’ state. We will discuss possible mechanisms through which external male-delivered signals modulate this developmental switch.
The mating response of D. melanogaster
Introduction
In response to mating, the Drosophila female (Figure 1) exhibits an increase in ovulation and oviposition rate [26–29] a decrease in female receptivity to courting males [30]; storage of sperm [31,32]; and a decrease in female life span [26,33,34]. Many of the stimuli that initiate the female response are in the seminal fluid, a major component of which is the male accessory gland proteins (accessory glands proteins, Acps). While it is well-established that Acps are essential for triggering several postmating changes in the female [10,19,26,35], it is only recently, with the advent of cellular and molecular analyses of the female postmating, that we have begun to understand how these signals transform the female from an ‘unmated’ to ‘mated’ state.
The female reproductive system plays several crucial roles in organisms with internal fertilization: it provides an environment the egg and sperm require for maturation prior to gamete fusion, facilitates fertilization, and supports embryo development [1–4]. Drosophila melanogaster, as a model system for studying how essential biological processes are regulated, has played a major role in unraveling the molecular mechanisms of development and physiology [5,6]. Drosophila remains the most important model system to study insect development and is the main reference species to which other emerging insect
At the morphological level, mating induces immediately after the start of copulation a series of conformational and size changes in the uterus and the oviduct which in part aid sperm to storage and allow the release and movement of oocytes through the tract [21,36,37]. Additionally, mating induces tissue-wide differentiation in epithelia, muscle and nerve tissues. In many epithelia, one of the last steps of differentiation is the development of a layer of extracellular matrix (ECM) that covers the apical and/ or basal membranes and the associated development of
This review comes from a themed issue on Development and regulation Edited by Leslie Pick and Cassandra Extavour For a complete overview see the Issue and the Editorial Available online 11th March 2016 http://dx.doi.org/10.1016/j.cois.2016.03.002 2214-5745/# 2016 Elsevier Inc. All rights reserved.
Current Opinion in Insect Science 2016, 13:106–113
www.sciencedirect.com
Mating induces developmental changes Carmel, Tram and Heifetz
107
Phase I
MATING
Figure 1
Phase II
Phase III
Male and female derived components
The female takes control
Changes in: • Transcription (miRNA, mRNA) • Translation • Morphology • Receptivity
0h 0d
3h
• • • •
6h
Phase IV
Sperm storage Increased ovulation rate Increased eggs production Increased oviposition
24h
3d
4d
10d
Differentiation
Terminal differentiation
Maintenance
Priming
Preparations toward rapid production of eggs
Functional reproductive tract
Reproductive tract is poised for mating Current Opinion in Insect Science
Schematic model of the female Drosophila mating response. Phase I — the first few days post-eclosion, the reproductive system undergoes the first phase of differentiation, after which the reproductive tract is developmentally poised for a rapid response to an extrinsic cue (mating). Phase II — terminal differentiation is induced by stimuli (e.g. odorants, auditory, vision and tactile) perceived during courtship and largely by the transfer of seminal fluid proteins and sperm received during copulation. These stimuli and factors are necessary for the full female mating response [9–24]. In response to mating, the Drosophila female undergoes a series of rapid molecular, morphological, behavioral and physiological changes. During the initial phase, mating induces decrease or increase in the level of many miRNAs, mRNA transcripts and proteins. Other striking changes are also seen in reproductive tract morphology and female physiology. During this phase sperm reaches storage and ovulation begins. In addition, female receptivity to courting males decreases and persists for several days [9,17,19,21,37]. Phase III — a second switch point occurs at 6 hours postmating to sustain elevated fertilization and reproduction and is influenced by the female’s regulatory and physiological responses to seminal fluid components and newly stored sperm. At this time the female starts to produce and deposit eggs at high rate [20,23,37]; terminal differentiation continues along with the onset of increased egg production and deposition and terminates during this phase. Phase IV — Maintenance the developmental program of the reproductive tract has ended and the organ is ready to support high fertility. Different colors represent different phases of the mating response. Note that the schematic presents a clear difference between the different phases (donated by the dashed line), but the exact duration of each phase is unknown.
hemi-adherens junctions (HAJs). HAJs connect the cell cytoskeleton with the ECM and are formed in all cell surfaces that contact an ECM [38,39]. In the oviduct epithelia of mated females, the apical ECM (AECM) and the thin layer of cuticle above it have a ruffled appearance, unlike unmated females, suggesting that the AECM and cuticle have increased in surface area. It is possible that mating enhances or modulates apical secretion via the AECM. Increased apical secretion and surface of the AECM is likely to be essential for ovulation, activation and movement of eggs along the duct. Mating also increases secretion and/or deposition of the ECM in the basolateral membrane at the oviduct and brings the ECM to a threshold concentration that can support the development of HAJs [37]. Another striking postmating change observed in the oviduct epithelia is www.sciencedirect.com
the increase in the number of HAJs along the basolateral membrane in the upper oviduct. Because one function of the HAJ is to give shape and tension to cells and tissues [39], HAJs are particularly important in the upper oviduct, where the basolateral membrane is extensively infolded. Extensive infolding allows the expansion of epithelia during passage of an egg along the narrow duct. The infolded membrane also gives rise to a highly branched intercellular space that is filled with an ECM. The muscles of the upper oviduct appear more differentiated in mated than in unmated reproductive tracts; myofibrils and z-bodies are less dense, and there is little or no basal lamina. In addition, the number of nerve terminals or boutons innervating the lateral oviducts and the common oviduct increases 70% postmating. Interestingly, the increase is in type II boutons, which cause vesicle release Current Opinion in Insect Science 2016, 13:106–113
108 Development and regulation
Figure 2
OV
LO Upper RT CO
SR SP
Lower RT
AG UT VA VU Current Opinion in Insect Science
The Drosohila female reproductive tract. Typical of the female insect reproductive system, the Drosophila female reproductive tract consists of a pair of ovaries (OV) connected to a median common oviduct (CO) by two lateral oviducts (LO), a pouch-like uterus (UT) that leads to the vagina (VA), which opens to the exterior through the vulva (VU). The reproductive tract also includes specialized organs such as the sperm storage organs (a pair of spermathecae (SP) and one seminal receptacle (SR)) and a pair of female accessory glands (parovaria) (AG) [25]. The upper RT (turquoise) includes the lateral and common oviducts; the lower RT (light brown) includes the sperm storage organs, female accessory glands, the posterior part of the common oviduct and the anterior part of the uterus (the fertilization site).
of neurohormones, such as octopamine, from nerve termini, in the common and lateral oviducts (Figure 2, [37]) [40–44]. Overall, these changes facilitate sperm storage and egg ovulation, activation, and deposition. At the molecular level, mating induces a change in the expression level of many mRNAs and proteins postmating. Microarray analyses comparing unmated vs mated females or their reproductive tracts have revealed how and to which male-derived component the female responds; in these studies, differentially expressed genes (1.5-fold) were labeled mating-responsive genes. Comparing whole female bodies, 38 mating-responsive transcripts were identified at 2 hours postmating [24], and 46 mating responsive transcripts at 3 hours postmating, Current Opinion in Insect Science 2016, 13:106–113
35 of which were mediated by sperm, 5 by male accessory glands proteins (Acps) and 6 by other mating signals [23]. Furthermore, 4 Acps have been shown to contribute to changes in the transcriptome between 1 and 3 hours after mating [45]. Prominently up-regulated transcripts included those related to immune response and proteolysis (proteases) functional groups [23,24,45]. Comparison of the expression profiles of the lower region of the female reproductive tract [20] (lower RT (LRT), Figure 2), sperm storage organs [46], or oviducts [9] of mated and unmated females revealed how the reproductive tract itself responds to mating. Immediately after the end of copulation, a small number of transcripts (71) is differential expressed in the LRT, most of which were down-regulated [20]. At 6 hours postmating, a large peak of 451 differentially expressed transcripts was observed, 305 of which were up-regulated. Interestingly, at 3 hour postmating the behavior of the vast majority of mating-responsive genes were not in accordance with the behavior of their protein products. The prominent functional groups of the LRT matingresponsive genes were nucleic acid, catalytic and transport activity as well as genes related to immune response and proteolysis (proteases), as observed in the whole body analyses. A comparison between the sperm storage organs (spermathecae (SP) and seminal receptacle (SR); Figure 2) of unmated and mated females, revealed a change in gene expression at 3-hour and 6-hour postmating. In the SP, the long-term sperm storage organs which appear to be specialized in maintenance of sperm viability and motility, 101 and 48 mating-responsive transcripts were identified at 3-hour and 6-hour postmating. In the SR, which provides a microenvironment that maintains active, motile sperm and prepares sperm for fertilization, 1168 and 865 mating-responsive transcripts were identified at 3-hour and 6-hour postmating. The proportion of up-regulated and down-regulated transcripts was 1:1 in the SP and SR at both timepoints postmating. Furthermore, the composition of differently expressed transcripts differed between the spermathecae and seminal receptacle reflective of their different functions. SP were enriched for genes involved in proteolysis and metabolism and the SR exhibited an over-representation of genes involved in localization, signaling, and ion transport [46]. Examination of unmated and mated oviducts revealed 155 differentially expressed transcripts, of which 122 increased and 33 decreased by 1.5-fold or more after mating [9]. As seen in whole body and LRT, transcripts up-regulated postmating were enriched for immune response genes. Molecular profiling of the oviduct proteome demonstrated that proteins associated with cellular junctions, such as a-Spectrin and b-Spectrin, Coracle, and Neuroglian, increased postmating [37]. Thus, mating promotes changes in actin-based cytoskeletal organization in the reproductive system and induces www.sciencedirect.com
Mating induces developmental changes Carmel, Tram and Heifetz
immune-related transcripts such as antimicrobial peptides in the reproductive system and whole body that likely contribute to creating the optimal environment for successful fertilization. Furthermore, the expression levels of many proteolysis-related transcripts are significantly altered postmating in whole body and the reproductive system. The products of proteolysis-related genes could be interacting with male proteins to influence the rate and duration of postmating responses by affecting the processing of seminal fluid proteins [18,47–49]. Modulating expression of proteolysis regulators in mated females could control the terminal differentiation of the reproductive tract and/or other physiological processes such as protecting sperm from degradation and induction of egg-laying.
Female ‘poised’ for mating Together, these studies support a model in which the female reproductive tract undergoes a two-phase maturation [9,20,23,37,40,45]. Initially, newly eclosed Drosophila females undergo a 3-day period of reproductive maturation during which the reproductive tract becomes ‘poised’ (Figure 1). Then mating triggers a second phase of maturation (tissue remodeling and modulation) which enables the reproductive system to produce, fertilize, and deposit numerous eggs rapidly over several days [21,36,37]. Kapelnikov et al. [37] further proposed that the second phase of maturation involves both mating-independent (differentiation continues irrespective of mating) and mating-dependent processes (differentiation is poised and continues upon mating) and that both of these pathways are essential to produce a functional oviduct. For example, muscles in the lower oviduct are highly differentiated prior to mating, while muscles in the upper oviduct are less differentiated. Although the onset of muscle differentiation in the upper and lower RT is mating-independent, further differentiation of muscle in the upper oviduct is mating-dependent and terminal of differentiation in the lower RT unrelated to mating. Furthermore, analysis of cytoskeleton mating-responsive protein abundance following different mating regimes (unmated, once-mated, twice-mated), demonstrated that the expression level of some of the proteins (e.g. Muscle LIM protein at 84B) does not change with female age and are only responsive to the first mating while others (e.g. Coracle, Neuroglian and Hu li tai shao) respond to the first and second mating [37]. It is possible that mating-responsive proteins which respond only to the first mating are involved in the final maturation of the oviduct while proteins that remain responsive to later matings are also involved in maintenance and ongoing function of the oviduct. Hence, the response of the reproductive tract to an extrinsic cue (mating) is composed of rapid changes in the expression levels of mating responsive transcripts and proteins that are required for the final maturation of the reproductive system. www.sciencedirect.com
109
What is the advantage of delaying the terminal differentiation until after mating? Unmated females are capable of depositing eggs, though at a reduced rate compared to mated females of the same age. One possible interpretation for delaying the terminal differentiation until postmating is that reproduction is energetically costly and delaying maturation until sperm is available is advantageous to the female.
How does mating induce the rapid transition to the ‘mated state’? The female response to mating on the molecular level occurs very rapidly. Three recent genomic screens demonstrate that this rapid response may be mediated posttranscriptionally via microRNA [50,51,52]. MicroRNAs (miRNA) are non-coding RNA sequences of 21–24 nucleotides that regulate the expression of their ‘target genes’ by binding to complementary sites on the 30 UTR of their mRNA transcripts and reducing or eliminating the level of protein products, even on the order of hours or less [53–56]. Fricke et al. [50] examined the effect of Acp70 (sex peptide) on the miRNA profile of the mated Drosophila female. For that, they compared the miRNA profile of the abdomen and that of the head and thorax of females mated to males lacking sex peptide or to control males. Out of miRNAs identified by Fricke et al. [50], 4 were functionally analyzed. Females in which expression of any of the 4 miRNAs (miR-279, miR-317, miR-278, and miR-184) was reduced or eliminated exhibited altered receptivity compared to control females. Interesting, egg-laying and lifespan were not affected. They concluded that the rapid behavioral response to sex-peptide is subjected to posttranscriptional regulation by miRNAs. Zhuo et al. [51], using deep-sequencing of RNA extracted from Drosophila female whole bodies, identified 27 miRNAs and 474 mRNA that were expressed significantly differently between mated and unmated females. Overexpression of several mating-responsive miRNAs in females affected egg laying behavior, and altered the expression of their putative candidate target genes suggesting that miRNAs could mediate changes in gene expression postmating. Carmel et al. [52] found an enrichment of mRNAs that are targets of miRNAs among the mating-responsive genes identified in the LRT, suggesting miRNA involvement. Using microarray for miRNAs, Carmel et al. [52] found that mating-responsive miRNAs are expressed throughout the LRT prior to mating and that mating changed their expression levels in one or more specific regions. When they further reduced the function of all miRNAs by silencing Dicer-1 (a key component in miRNA biogenesis) in female whole body pre-mating, they found that these females exhibited a significant decrease in fertility. During the initial maturation period, females have already synthesized most of the transcripts and proteins needed to rapidly respond to mating and for the early Current Opinion in Insect Science 2016, 13:106–113
110 Development and regulation
stages of postmating reproductive physiology. Mating rapidly relieves the arrest, shifting the female from a posteclosion developmental phase toward the postmating end of the developmental program during which they reach reproductive maturity and prepare for maximum reproduction. Mating-responsive miRNAs may sharpen the transition from the unmated to the mated state by eliminating many target genes simultaneously and suppressing residual transcripts that are specific to the unmated stage which interfere with the final stage of differentiation of epithelial, muscle and nerve tissues. Mating-responsive miRNAs may possibly also relieve inhibition of factors that promote differentiation that were kept in check to prevent precocious expression. It is therefore suggested that mating-responsive miRNAs reprogram the female expression profile to rapidly initiate the final differentiation of the reproductive tract tissues, allowing sperm storage and leading to a functional reproductive system.
Conclusions Regulation by miRNAs has been found to be critical for the functionality of reproductive tissues in a wide range of animals, from mammals to Caenorhabditis elegans [57– 60] and insects [61,62,63]. Studies reviewed here raise the possibility that the mating response in D. melanogaster also involves post-translational regulation via miRNA [20,37,45]. The transition from unmated to mated state in the D. melanogaster female is rapid, involving changes in the expression level of many mRNA transcripts. This transition would require molecular mechanisms that can rapidly end one program (unmated) and initiate the next program (mated) while preventing transcriptional and translational errors in the subsequent state. In silico simulations predict that regulation by miRNAs is faster and consumes less energy than conventional regulation via transcription factors [64]. In vivo studies of miRNA kinetics in mouse retina support this prediction [65]. Indeed, numerous miRNAs that are differentially expressed between unmated and mated females have been identified [50,51,52] and have been shown to affect the female mating response. Because D. melanogaster is short-lived, the duration of the mating response (the time from the start of copulation to oviposition) affects fitness. A fast mating response increases the chance a female has to establish the next generation of offspring before a possible catastrophe. Similar to Drosophila, mating induces changes in the female of many other insect species [66]. In Anopheles gambiae, for example, mating stimulates the female to start to feed on blood, which is required for the production of mature oocytes [67], and male seminal fluid stimulates oviposition, sperm storage and changes in the morphology and expression profile of the female reproductive tract [68–71]. At 24 hours postmating, the reproductive system Current Opinion in Insect Science 2016, 13:106–113
of the Anopheles female undergoes prominent transcriptional changes, including a dramatic down-regulation of 13 protease genes [69]. At the morphological level, postmating changes in the vagina include elimination of extensive smooth endoplasmatic reticulum from the apical side of the cells and induction of the formation of large electron dense vacuoles of uniform granule in some apical cells along the vagina [69]. In addition, rough endoplasmatic reticulum at the basal side of the vaginal cells was greatly reduced. These morphological changes suggest decreased capacity for protein synthesis in vaginal cells and a possible transfer of components between the vaginal lumen and epithelial cells. The rapid changes occurring in the secretory machinery and/or the epithelial cells of the Anopheles vagina may reflect a change in the communication mode between epithelial cells and/or epithelial cells and the lumen, reminiscent of the changes observed in the Drosophila oviduct postmating. Together, these studies suggest that the mosquito female reproductive tract undergoes a two-phase maturation, in which a rapid mating response leads to a functional reproductive system. In addition to Drosophila and Anopheles, seminal fluid induces physiological and behavioral changes (switch from unmated to mated state) in the females of many other insects, including moths, beetles, and honeybees [66]. However, not much is known about the mating response kinetics and/or developmental status of the system. We hypothesize that the model we describe for Drosophila can be also found in other insects (either dipteran or non-dipteran) which are short-lived and have similar reproductive strategy. We propose that delaying the final maturation allows the female to conserve the energy required to invest in the development of the reproductive tissues until conditions are optimal. It will be interesting to examine if mating-induced developmental changes take place in other insects. For this, further studies comparing the morphology and expression profile of reproductive tissues in virgin and mated female at different times postmating are required. These studies will determine if the rapid transition of the reproductive tract induced by mating is a general phenomenon in insects. Because the mating signal of organisms with internal reproduction is composed of stimuli perceived during courtship and the receipt of sperm and seminal fluid, it is likely that this rapid unmated to mated switch may reflect the evolution of a mechanism to optimize reproductive capacity in early adulthood in short-lived animals. As female reproductive molecules are identified in more insect species and their functions are better understood, we will get a deeper understanding of the dynamics of male-female reproductive molecule interactions and of how these interactions maximize reproductive success. www.sciencedirect.com
Mating induces developmental changes Carmel, Tram and Heifetz
Acknowledgements We thank Zohar Nir-Amitin for the graphics; support by US-Israel Binational Agricultural Research and Development Fund, research Grant 3492 (to YH), The Chief Scientist Ministry of Agriculture grant 872-0055-10 (to YH) and US-Israel Binational Science Foundation grant 2009270 (to YH and Mark Siegal).
References and recommended reading Papers of particular interest, published within the period of review, have been highlighted as: of special interest of outstanding interest 1.
Fazeli A, Moein Vaziri N, Holt WV: Proteomics of the periconception milieu. Proteomics 2015, 15:649-655.
2.
Scott MA: A glimpse at sperm function in vivo: sperm transport and epithelial interaction in the female reproductive tract. Anim Reprod Sci 2000, 60–61:337-348.
3.
Bloch Qazi MC, Heifetz Y, Wolfner MF: The developments between gametogenesis and fertilization: ovulation and female sperm storage in Drosophila melanogaster. Dev Biol 2003, 256:195-211.
4.
Watkins AJ, Lucas ES, Fleming TP: Impact of the periconceptional environment on the programming of adult disease. J Dev Orig Health Dis 2010, 1:87-95.
5.
Arias AM: Drosophila melanogaster and the development of biology in the 20th century. Methods Mol Biol 2008, 420:1-25.
6.
Jennings B: Drosophila — a versatile model in biology & medicine. Mater Today 2011, 14:190-195.
7.
Schneider D: Using Drosophila as a model insect. Nat Rev Genet 2000, 1:218-226.
8.
Nassel DR: Neuropeptides in the nervous system of Drosophila and other insects: multiple roles as neuromodulators and neurohormones. Prog Neurobiol 2002, 68:1-84.
9.
Kapelnikov A, Zelinger E, Gottlieb Y, Rhrissorrakrai K, Gunsalus KC, Heifetz Y: Mating induces an immune response and developmental switch in the Drosophila oviduct. Proc Natl Acad Sci U S A 2008, 105:13912-13917.
10. Wolfner MF: ‘‘S.P.E.R.M.’’ (seminal proteins (are) essential reproductive modulators): the view from Drosophila. Soc Reprod Fertil Suppl 2007, 65:183-199. 11. Yapici N, Kim YJ, Ribeiro C, Dickson BJ: A receptor that mediates the post-mating switch in Drosophila reproductive behaviour. Nature 2008, 451:33-37. 12. Griffith LC, Ejima A: Multimodal sensory integration of courtship stimulating cues in Drosophila melanogaster. Ann N Y Acad Sci 2009, 1170:394-398. 13. Billeter JC, Levine JD: Who is he and what is he to you? Recognition in Drosophila melanogaster. Curr Opin Neurobiol 2013, 23:17-23. 14. Pavlou HJ, Goodwin SF: Courtship behavior in Drosophila melanogaster: towards a ‘courtship connectome’. Curr Opin Neurobiol 2013, 23:76-83. 15. Villella A, Hall JC: Neurogenetics of courtship and mating in Drosophila. Adv Genet 2008, 62:67-184. 16. Vosshall LB: Scent of a fly. Neuron 2008, 59:685-689. 17. Heifetz Y, Rivlin PK: Beyond the mouse model: using Drosophila as a model for sperm interaction with the female reproductive tract. Theriogenology 2010, 73:723-739. 18. LaFlamme BA, Ram KR, Wolfner MF: The Drosophila melanogaster seminal fluid protease ‘‘seminase’’ regulates proteolytic and post-mating reproductive processes. PLoS Genet 2012, 8:e1002435. This paper identified ‘seminase’ a trypsin-like serine protease that is transferred to the female in the seminal fluid during copulation. Seminase is located upstream in proteolytic cascades of several male-transferred www.sciencedirect.com
111
components and induce both the long and the short-term mating response of the female. The identification of seminase highlights the complexity of the seminal protein network and shows that the short and long-term mating responses are under the same control. 19. Kubli E, Bopp D: Sexual behavior: how Sex Peptide flips the postmating switch of female flies. Curr Biol 2012, 22:R520R522. 20. Mack PD, Kapelnikov A, Heifetz Y, Bender M: Mating-responsive genes in reproductive tissues of female Drosophila melanogaster. Proc Natl Acad Sci U S A 2006, 103:10358-10363. 21. Adams EM, Wolfner MF: Seminal proteins but not sperm induce morphological changes in the Drosophila melanogaster female reproductive tract during sperm storage. J Insect Physiol 2007, 53:319-331. 22. Prokupek AM, Eyun SI, Ko L, Moriyama EN, Harshman LG: Molecular evolutionary analysis of seminal receptacle sperm storage organ genes of Drosophila melanogaster. J Evol Biol 2010, 23:1386-1398. 23. McGraw LA, Gibson G, Clark AG, Wolfner MF: Genes regulated by mating, sperm, or seminal proteins in mated female Drosophila melanogaster. Curr Biol 2004, 14:1509-1514. 24. Lawniczak MK, Begun DJ: A genome-wide analysis of courting and mating responses in Drosophila melanogaster females. Genome 2004, 47:900-910. 25. Demerec M (Ed): Biology of Drosophila. New York: John Wiley & Sons; 1950. 26. Wolfner MF: Tokens of love: functions and regulation of Drosophila male accessory gland products. Insect Biochem Mol Biol 1997, 27:179-192. 27. Heifetz Y, Lung O, Frongillo EA Jr, Wolfner MF: The Drosophila seminal fluid protein Acp26Aa stimulates release of oocytes by the ovary. Curr Biol 2000, 10:99-102. 28. Heifetz Y, Yu J, Wolfner MF: Ovulation triggers activation of Drosophila oocytes. Dev Biol 2001, 234:416-424. 29. Ram KR, Wolfner MF: Sustained post-mating response in Drosophila melanogaster requires multiple seminal fluid proteins. PLoS Genet 2007, 3:e238. 30. Wolfner MF: The gifts that keep on giving: physiological functions and evolutionary dynamics of male seminal proteins in Drosophila. Heredity (Edinb) 2002, 88:85-93. 31. Neubaum DM, Wolfner MF: Wise, winsome, or weird? Mechanisms of sperm storage in female animals. Curr Top Dev Biol 1999, 41:67-97. 32. Tram U, Wolfner MF: Male seminal fluid proteins are essential for sperm storage in Drosophila melanogaster. Genetics 1999, 153:837-844. 33. Chapman T, Liddle LF, Kalb JM, Wolfner MF, Partridge L: Cost of mating in Drosophila melanogaster females is mediated by male accessory gland products. Nature 1995, 373:241-244. 34. Ueyama M, Fuyama Y: Enhanced cost of mating in female sterile mutants of Drosophila melanogaster. Genes Genet Syst 2003, 78:29-36. 35. Chapman T: Seminal fluid-mediated fitness traits in Drosophila. Heredity 2001, 87:511-521. 36. Mattei AL, Riccio ML, Avila FW, Wolfner MF: Integrated 3D view of postmating responses by the Drosophila melanogaster female reproductive tract, obtained by micro-computed tomography scanning. Proc Natl Acad Sci U S A 2015, 112:84758480. This is the first high-resolution micro-CT study that reveals how male and female molecules and anatomy interface to carry out and coordinate mating-dependent changes in the female’s reproductive physiology. 37. Kapelnikov A, Rivlin PK, Hoy RR, Heifetz Y: Tissue remodeling: a mating-induced differentiation program for the Drosophila oviduct. BMC Dev Biol 2008, 8:114. 38. Tepass U, Hartenstein V: The development of cellular junctions in the Drosophila embryo. Dev Biol 1994, 161:563-596. Current Opinion in Insect Science 2016, 13:106–113
112 Development and regulation
39. Harris TJ, Tepass U: Adherens junctions: from molecules to morphogenesis. Nat Rev Mol Cell Biol 2010, 11:502-514. 40. Heifetz Y, Wolfner MF: Mating, seminal fluid components, and sperm cause changes in vesicle release in the Drosophila female reproductive tract. Proc Natl Acad Sci U S A 2004, 101:6261-6266. 41. Avila FW, Bloch Qazi MC, Rubinstein CD, Wolfner MF: A requirement for the neuromodulators octopamine and tyramine in Drosophila melanogaster female sperm storage. Proc Natl Acad Sci U S A 2012, 109:4562-4567. 42. Rubinstein CD, Wolfner MF: Drosophila seminal protein ovulin mediates ovulation through female octopamine neuronal signaling. Proc Natl Acad Sci U S A 2013, 110:17420-17425. 43. Heifetz Y, Lindner M, Garini Y, Wolfner MF: Mating regulates neuromodulator ensembles at nerve termini innervating the Drosophila reproductive tract. Curr Biol 2014, 24:731-737. 44. Rezaval C, Nojima T, Neville MC, Lin AC, Goodwin SF: Sexually dimorphic octopaminergic neurons modulate female postmating behaviors in Drosophila. Curr Biol 2014, 24:725-730. 45. McGraw LA, Clark AG, Wolfner MF: Post-mating gene expression profiles of female Drosophila melanogaster in response to time and to four male accessory gland proteins. Genetics 2008, 179:1395-1408. 46. Prokupek AM, Kachman SD, Ladunga I, Harshman LG: Transcriptional profiling of the sperm storage organs of Drosophila melanogaster. Insect Mol Biol 2009, 18:465-475. 47. Heifetz Y, Vandenberg LN, Cohn HI, Wolfner MF: Two cleavage products of the Drosophila accessory gland protein ovulin can independently induce ovulation. Proc Natl Acad Sci U S A 2005, 102:743-748. 48. Ram RK, Sirot LK, Wolfner MF: Predicted seminal astacin-like protease is required for processing of reproductive proteins in Drosophila melanogaster. Proc Natl Acad Sci U S A 2006, 103:18674-18679. 49. Laflamme BA, Avila FW, Michalski K, Wolfner MF: A Drosophila protease cascade member, seminal metalloprotease-1, is activated stepwise by male factors and requires female factors for full activity. Genetics 2014, 196:1117-1129. 50. Fricke C, Green D, Smith D, Dalmay T, Chapman T: microRNAs influence reproductive responses by females to male sex peptide in Drosophila melanogaster. Genetics 2014, 198:16031619. This paper demonstrates that sex peptide induces post-transcriptional activity of miRNAs in the female. A comparison between miRNA profiles of females mated to males with and without sex peptide identified candidate miRNAs. Functional analysis of four selected miRNAs showed that mated females which lack these miRNAs had altered receptivity to courting males compared to control females. 51. Zhou S, Mackay T, Anholt RR: Transcriptional and epigenetic responses to mating and aging in Drosophila melanogaster. BMC Genomics 2014, 15:927. The study of Zhou et al. supports two important themes in our review: (1) the phenotypic plasticity of mating and (2) the presence of functional miRNAs in the mating response of the Drosophila female. Zhou et al. preformed a comprehensive deep-Seq screen to examine the effect of mating and/or aging on the transcriptome, miRNome and histone modification (using Chip-seq) of the Drosophila female whole body. 52. Carmel I, Nave M, Zelinger E Apel I, Keidar T, Schnakenberg S, Widmayer P, Breer H, Siegal M, Heifetz Y: miRNAs regulate the mating response in the lower RT of the Drosophila melanogaster female. Unpublished results. 53. Giraldez AJ, Mishima Y, Rihel J, Grocock RJ, Van Dongen S, Inoue K, Enright AJ, Schier AF: Zebrafish miR-430 promotes deadenylation and clearance of maternal mRNAs. Science 2006, 312:75-79. 54. Hornstein E, Shomron N: Canalization of development by microRNAs. Nat Genet 2006, 38(Suppl.):S20-S24. Current Opinion in Insect Science 2016, 13:106–113
55. Bushati N, Stark A, Brennecke J, Cohen SM: Temporal reciprocity of miRNAs and their targets during the maternal-to-zygotic transition in Drosophila. Curr Biol 2008, 18:501-506. 56. Bartel DP: microRNAs: target recognition and regulatory functions. Cell 2009, 136:215-233. 57. Esquela-Kerscher A, Johnson SM, Bai L, Saito K, Partridge J, Reinert KL, Slack FJ: Post-embryonic expression of C. elegans microRNAs belonging to the lin-4 and let-7 families in the hypodermis and the reproductive system. Dev Dyn 2005, 234:868-877. 58. Hu S-J, Ren G, Liu J-L, Zhao Z-A, Yu Y-S, Su R-W, Ma X-H, Ni H, Lei W, Yang Z-M: microRNA expression and regulation in mouse uterus during embryo implantation. J Biol Chem 2008, 283:23473-23484. 59. Revel A, Achache H, Stevens J, Smith Y, Reich R: microRNAs are associated with human embryo implantation defects. Hum Reprod 2011, 26:2830-2840. 60. Imbar T, Galliano D, Pellicer A, Laufer N: Introduction: microRNAs in human reproduction: small molecules with crucial regulatory roles. Fertil Steril 2014, 101:1514-1515. 61. Reich J, Snee MJ, Macdonald PM: miRNA-dependent translational repression in the Drosophila ovary. PLoS ONE 2009, 4:e4669. 62. Lucas KJ, Roy S, Ha J, Gervaise AL, Kokoza VA, Raikhel AS: microRNA-8 targets the Wingless signaling pathway in the female mosquito fat body to regulate reproductive processes. Proc Natl Acad Sci U S A 2014, 112:1440-1445. This study identifies the fat body specific action of miR-8 and its target in regulating mosquito reproduction. miR-8 plays an essential role in the female mosquito fat body controlling the secretory activity of yolk protein precursors required for oocyte development. 63. Yang CH, Rumpf S, Xiang Y, Gordon MD, Song W, Jan LY, Jan YN: Control of the postmating behavioral switch in Drosophila females by internal sensory neurons. Neuron 2009, 61:519-526. 64. Shimoni Y, Friedlander G, Hetzroni G, Niv G, Altuvia S, Biham O, Margalit H: Regulation of gene expression by small non-coding RNAs: a quantitative view. Mol Syst Biol 2007, 3. This study further supports the presence of miRNAs in rapid evolving processes such as mating. Using in silico simulation, Shimoni et al. compared the rate of repression by small RNA and ‘conventional’ repression using transcription factor repressor in E. coli. Using kinetic parameters based on experimental data the authors show that regulation by small RNA is advantageous. 65. Krol J, Busskamp V, Markiewicz I, Stadler MB, Ribi S, Richter J, Duebel J, Bicker S, Fehling HJ, Schu¨beler D et al.: Characterizing light-regulated retinal microRNAs reveals rapid turnover as a common property of neuronal microRNAs. Cell 2010, 141:618-631. The study provides experimental evidence for the fast kinetics of miRNAs. Krol et al. exposed mice retina cells to light and found an increase in the expression level of several miRNAs within 30 min. These light responsive miRNAs also showed a rapid decline, as they were down-regulated within 90 min upon exposure to darkness. One of the miRNAs that was downregulated in response to light induce an increase in the level of its target Sla1a1 within 2–3 hours of release from the dark signal (0.5–1.5 hours from the miRNA decrease). 66. Avila FW, Sirot LK, LaFlamme BA, Rubinstein CD, Wolfner MF: Insect seminal fluid proteins: identification and function. Annu Rev Entomol 2014, 56:21-40. 67. Clements AN: The Biology of Mosquitoes. New York: CABI Publishing; 2000. 68. Rogers DW, Baldini F, Battaglia F, Panico M, Dell A, Morris HR, Catteruccia F: Transglutaminase-mediated semen coagulation controls sperm storage in the malaria mosquito. PLoS Biol 2009, 7:e1000272. 69. Rogers DW, Whitten MM, Thailayil J, Soichot J, Levashina EA, Catteruccia F: Molecular and cellular components of the mating machinery in Anopheles gambiae females. Proc Natl Acad Sci U S A 2008, 105:19390-19395. www.sciencedirect.com
Mating induces developmental changes Carmel, Tram and Heifetz
70. Gabrieli P, Kakani EG, Mitchell SN, Mameli E, Want EJ, Mariezcurrena Anton A, Serrao A, Baldini F, Catteruccia F: Sexual transfer of the steroid hormone 20E induces the postmating switch in Anopheles gambiae. Proc Natl Acad Sci U S A 2014, 111:16353-16358.
www.sciencedirect.com
113
71. Shaw WR, Teodori E, Mitchell SN, Baldini F, Gabrieli P, Rogers DW, Catteruccia F: Mating activates the heme peroxidase HPX15 in the sperm storage organ to ensure fertility in Anopheles gambiae. Proc Natl Acad Sci U S A 2014, 111:5854-5859.
Current Opinion in Insect Science 2016, 13:106–113
ScienceDirect Mating induces developmental changes in the insect female reproductive tract I Carmel1, U Tram2 and Y Heifetz1 In response to mating, the Drosophila female undergoes a series of rapid molecular, morphological, behavioral and physiological changes. Studies in Drosophila and other organisms have shown that stimuli received during courtship and copulation, sperm, and seminal fluid are needed for the full mating response and thus reproductive success. Very little is known, however, about how females respond to these malederived stimuli/factors at the molecular level. More specifically, it is unclear what mechanisms regulate and mediate the mating response, how the signals received during mating are integrated and processed, and what network of molecules are essential for a successful mating response. Moreover, it is yet to be determined whether the rapid transition of the reproductive tract induced by mating is a general phenomenon in insects. This review highlights current knowledge and advances on the developmental switch that rapidly transitions the female from the ‘unmated’ to ‘mated’ state. Addresses 1 Department of Entomology, The Hebrew University, Rehovot, Israel 2 Department of Molecular Genetics, The Ohio State University, Columbus, OH, USA Corresponding author: Heifetz, Y ([email protected])
Current Opinion in Insect Science 2016, 13:106–113
model organisms are compared [7,8]. Numerous studies in Drosophila and other organisms have shown that sperm, seminal fluid and other signals received during mating alter female behavior and physiology postmating, and that these changes increase the reproductive success of both the male and female [9–24]. Seminal fluid is composed of sperm and components secreted by the accessory glands, ejaculatory duct, and ejaculatory bulb of the male reproductive tract. Each of these seminal fluid components play an essential role in male and female reproductive success [10,23,25]. Despite this wealth of knowledge, little is known about how females respond to these male-derived stimuli/factors at the molecular level and more specifically what mechanisms regulate and mediate the mating response, how the signals received during mating are integrated and processed, and what network of molecules are essential for a successful mating response. In this review, we will focus on the female response in the first 24 hours postmating, highlighting the developmental switch that transitions the female from an ‘unmated’ to ‘mated’ state. We will discuss possible mechanisms through which external male-delivered signals modulate this developmental switch.
The mating response of D. melanogaster
Introduction
In response to mating, the Drosophila female (Figure 1) exhibits an increase in ovulation and oviposition rate [26–29] a decrease in female receptivity to courting males [30]; storage of sperm [31,32]; and a decrease in female life span [26,33,34]. Many of the stimuli that initiate the female response are in the seminal fluid, a major component of which is the male accessory gland proteins (accessory glands proteins, Acps). While it is well-established that Acps are essential for triggering several postmating changes in the female [10,19,26,35], it is only recently, with the advent of cellular and molecular analyses of the female postmating, that we have begun to understand how these signals transform the female from an ‘unmated’ to ‘mated’ state.
The female reproductive system plays several crucial roles in organisms with internal fertilization: it provides an environment the egg and sperm require for maturation prior to gamete fusion, facilitates fertilization, and supports embryo development [1–4]. Drosophila melanogaster, as a model system for studying how essential biological processes are regulated, has played a major role in unraveling the molecular mechanisms of development and physiology [5,6]. Drosophila remains the most important model system to study insect development and is the main reference species to which other emerging insect
At the morphological level, mating induces immediately after the start of copulation a series of conformational and size changes in the uterus and the oviduct which in part aid sperm to storage and allow the release and movement of oocytes through the tract [21,36,37]. Additionally, mating induces tissue-wide differentiation in epithelia, muscle and nerve tissues. In many epithelia, one of the last steps of differentiation is the development of a layer of extracellular matrix (ECM) that covers the apical and/ or basal membranes and the associated development of
This review comes from a themed issue on Development and regulation Edited by Leslie Pick and Cassandra Extavour For a complete overview see the Issue and the Editorial Available online 11th March 2016 http://dx.doi.org/10.1016/j.cois.2016.03.002 2214-5745/# 2016 Elsevier Inc. All rights reserved.
Current Opinion in Insect Science 2016, 13:106–113
www.sciencedirect.com
Mating induces developmental changes Carmel, Tram and Heifetz
107
Phase I
MATING
Figure 1
Phase II
Phase III
Male and female derived components
The female takes control
Changes in: • Transcription (miRNA, mRNA) • Translation • Morphology • Receptivity
0h 0d
3h
• • • •
6h
Phase IV
Sperm storage Increased ovulation rate Increased eggs production Increased oviposition
24h
3d
4d
10d
Differentiation
Terminal differentiation
Maintenance
Priming
Preparations toward rapid production of eggs
Functional reproductive tract
Reproductive tract is poised for mating Current Opinion in Insect Science
Schematic model of the female Drosophila mating response. Phase I — the first few days post-eclosion, the reproductive system undergoes the first phase of differentiation, after which the reproductive tract is developmentally poised for a rapid response to an extrinsic cue (mating). Phase II — terminal differentiation is induced by stimuli (e.g. odorants, auditory, vision and tactile) perceived during courtship and largely by the transfer of seminal fluid proteins and sperm received during copulation. These stimuli and factors are necessary for the full female mating response [9–24]. In response to mating, the Drosophila female undergoes a series of rapid molecular, morphological, behavioral and physiological changes. During the initial phase, mating induces decrease or increase in the level of many miRNAs, mRNA transcripts and proteins. Other striking changes are also seen in reproductive tract morphology and female physiology. During this phase sperm reaches storage and ovulation begins. In addition, female receptivity to courting males decreases and persists for several days [9,17,19,21,37]. Phase III — a second switch point occurs at 6 hours postmating to sustain elevated fertilization and reproduction and is influenced by the female’s regulatory and physiological responses to seminal fluid components and newly stored sperm. At this time the female starts to produce and deposit eggs at high rate [20,23,37]; terminal differentiation continues along with the onset of increased egg production and deposition and terminates during this phase. Phase IV — Maintenance the developmental program of the reproductive tract has ended and the organ is ready to support high fertility. Different colors represent different phases of the mating response. Note that the schematic presents a clear difference between the different phases (donated by the dashed line), but the exact duration of each phase is unknown.
hemi-adherens junctions (HAJs). HAJs connect the cell cytoskeleton with the ECM and are formed in all cell surfaces that contact an ECM [38,39]. In the oviduct epithelia of mated females, the apical ECM (AECM) and the thin layer of cuticle above it have a ruffled appearance, unlike unmated females, suggesting that the AECM and cuticle have increased in surface area. It is possible that mating enhances or modulates apical secretion via the AECM. Increased apical secretion and surface of the AECM is likely to be essential for ovulation, activation and movement of eggs along the duct. Mating also increases secretion and/or deposition of the ECM in the basolateral membrane at the oviduct and brings the ECM to a threshold concentration that can support the development of HAJs [37]. Another striking postmating change observed in the oviduct epithelia is www.sciencedirect.com
the increase in the number of HAJs along the basolateral membrane in the upper oviduct. Because one function of the HAJ is to give shape and tension to cells and tissues [39], HAJs are particularly important in the upper oviduct, where the basolateral membrane is extensively infolded. Extensive infolding allows the expansion of epithelia during passage of an egg along the narrow duct. The infolded membrane also gives rise to a highly branched intercellular space that is filled with an ECM. The muscles of the upper oviduct appear more differentiated in mated than in unmated reproductive tracts; myofibrils and z-bodies are less dense, and there is little or no basal lamina. In addition, the number of nerve terminals or boutons innervating the lateral oviducts and the common oviduct increases 70% postmating. Interestingly, the increase is in type II boutons, which cause vesicle release Current Opinion in Insect Science 2016, 13:106–113
108 Development and regulation
Figure 2
OV
LO Upper RT CO
SR SP
Lower RT
AG UT VA VU Current Opinion in Insect Science
The Drosohila female reproductive tract. Typical of the female insect reproductive system, the Drosophila female reproductive tract consists of a pair of ovaries (OV) connected to a median common oviduct (CO) by two lateral oviducts (LO), a pouch-like uterus (UT) that leads to the vagina (VA), which opens to the exterior through the vulva (VU). The reproductive tract also includes specialized organs such as the sperm storage organs (a pair of spermathecae (SP) and one seminal receptacle (SR)) and a pair of female accessory glands (parovaria) (AG) [25]. The upper RT (turquoise) includes the lateral and common oviducts; the lower RT (light brown) includes the sperm storage organs, female accessory glands, the posterior part of the common oviduct and the anterior part of the uterus (the fertilization site).
of neurohormones, such as octopamine, from nerve termini, in the common and lateral oviducts (Figure 2, [37]) [40–44]. Overall, these changes facilitate sperm storage and egg ovulation, activation, and deposition. At the molecular level, mating induces a change in the expression level of many mRNAs and proteins postmating. Microarray analyses comparing unmated vs mated females or their reproductive tracts have revealed how and to which male-derived component the female responds; in these studies, differentially expressed genes (1.5-fold) were labeled mating-responsive genes. Comparing whole female bodies, 38 mating-responsive transcripts were identified at 2 hours postmating [24], and 46 mating responsive transcripts at 3 hours postmating, Current Opinion in Insect Science 2016, 13:106–113
35 of which were mediated by sperm, 5 by male accessory glands proteins (Acps) and 6 by other mating signals [23]. Furthermore, 4 Acps have been shown to contribute to changes in the transcriptome between 1 and 3 hours after mating [45]. Prominently up-regulated transcripts included those related to immune response and proteolysis (proteases) functional groups [23,24,45]. Comparison of the expression profiles of the lower region of the female reproductive tract [20] (lower RT (LRT), Figure 2), sperm storage organs [46], or oviducts [9] of mated and unmated females revealed how the reproductive tract itself responds to mating. Immediately after the end of copulation, a small number of transcripts (71) is differential expressed in the LRT, most of which were down-regulated [20]. At 6 hours postmating, a large peak of 451 differentially expressed transcripts was observed, 305 of which were up-regulated. Interestingly, at 3 hour postmating the behavior of the vast majority of mating-responsive genes were not in accordance with the behavior of their protein products. The prominent functional groups of the LRT matingresponsive genes were nucleic acid, catalytic and transport activity as well as genes related to immune response and proteolysis (proteases), as observed in the whole body analyses. A comparison between the sperm storage organs (spermathecae (SP) and seminal receptacle (SR); Figure 2) of unmated and mated females, revealed a change in gene expression at 3-hour and 6-hour postmating. In the SP, the long-term sperm storage organs which appear to be specialized in maintenance of sperm viability and motility, 101 and 48 mating-responsive transcripts were identified at 3-hour and 6-hour postmating. In the SR, which provides a microenvironment that maintains active, motile sperm and prepares sperm for fertilization, 1168 and 865 mating-responsive transcripts were identified at 3-hour and 6-hour postmating. The proportion of up-regulated and down-regulated transcripts was 1:1 in the SP and SR at both timepoints postmating. Furthermore, the composition of differently expressed transcripts differed between the spermathecae and seminal receptacle reflective of their different functions. SP were enriched for genes involved in proteolysis and metabolism and the SR exhibited an over-representation of genes involved in localization, signaling, and ion transport [46]. Examination of unmated and mated oviducts revealed 155 differentially expressed transcripts, of which 122 increased and 33 decreased by 1.5-fold or more after mating [9]. As seen in whole body and LRT, transcripts up-regulated postmating were enriched for immune response genes. Molecular profiling of the oviduct proteome demonstrated that proteins associated with cellular junctions, such as a-Spectrin and b-Spectrin, Coracle, and Neuroglian, increased postmating [37]. Thus, mating promotes changes in actin-based cytoskeletal organization in the reproductive system and induces www.sciencedirect.com
Mating induces developmental changes Carmel, Tram and Heifetz
immune-related transcripts such as antimicrobial peptides in the reproductive system and whole body that likely contribute to creating the optimal environment for successful fertilization. Furthermore, the expression levels of many proteolysis-related transcripts are significantly altered postmating in whole body and the reproductive system. The products of proteolysis-related genes could be interacting with male proteins to influence the rate and duration of postmating responses by affecting the processing of seminal fluid proteins [18,47–49]. Modulating expression of proteolysis regulators in mated females could control the terminal differentiation of the reproductive tract and/or other physiological processes such as protecting sperm from degradation and induction of egg-laying.
Female ‘poised’ for mating Together, these studies support a model in which the female reproductive tract undergoes a two-phase maturation [9,20,23,37,40,45]. Initially, newly eclosed Drosophila females undergo a 3-day period of reproductive maturation during which the reproductive tract becomes ‘poised’ (Figure 1). Then mating triggers a second phase of maturation (tissue remodeling and modulation) which enables the reproductive system to produce, fertilize, and deposit numerous eggs rapidly over several days [21,36,37]. Kapelnikov et al. [37] further proposed that the second phase of maturation involves both mating-independent (differentiation continues irrespective of mating) and mating-dependent processes (differentiation is poised and continues upon mating) and that both of these pathways are essential to produce a functional oviduct. For example, muscles in the lower oviduct are highly differentiated prior to mating, while muscles in the upper oviduct are less differentiated. Although the onset of muscle differentiation in the upper and lower RT is mating-independent, further differentiation of muscle in the upper oviduct is mating-dependent and terminal of differentiation in the lower RT unrelated to mating. Furthermore, analysis of cytoskeleton mating-responsive protein abundance following different mating regimes (unmated, once-mated, twice-mated), demonstrated that the expression level of some of the proteins (e.g. Muscle LIM protein at 84B) does not change with female age and are only responsive to the first mating while others (e.g. Coracle, Neuroglian and Hu li tai shao) respond to the first and second mating [37]. It is possible that mating-responsive proteins which respond only to the first mating are involved in the final maturation of the oviduct while proteins that remain responsive to later matings are also involved in maintenance and ongoing function of the oviduct. Hence, the response of the reproductive tract to an extrinsic cue (mating) is composed of rapid changes in the expression levels of mating responsive transcripts and proteins that are required for the final maturation of the reproductive system. www.sciencedirect.com
109
What is the advantage of delaying the terminal differentiation until after mating? Unmated females are capable of depositing eggs, though at a reduced rate compared to mated females of the same age. One possible interpretation for delaying the terminal differentiation until postmating is that reproduction is energetically costly and delaying maturation until sperm is available is advantageous to the female.
How does mating induce the rapid transition to the ‘mated state’? The female response to mating on the molecular level occurs very rapidly. Three recent genomic screens demonstrate that this rapid response may be mediated posttranscriptionally via microRNA [50,51,52]. MicroRNAs (miRNA) are non-coding RNA sequences of 21–24 nucleotides that regulate the expression of their ‘target genes’ by binding to complementary sites on the 30 UTR of their mRNA transcripts and reducing or eliminating the level of protein products, even on the order of hours or less [53–56]. Fricke et al. [50] examined the effect of Acp70 (sex peptide) on the miRNA profile of the mated Drosophila female. For that, they compared the miRNA profile of the abdomen and that of the head and thorax of females mated to males lacking sex peptide or to control males. Out of miRNAs identified by Fricke et al. [50], 4 were functionally analyzed. Females in which expression of any of the 4 miRNAs (miR-279, miR-317, miR-278, and miR-184) was reduced or eliminated exhibited altered receptivity compared to control females. Interesting, egg-laying and lifespan were not affected. They concluded that the rapid behavioral response to sex-peptide is subjected to posttranscriptional regulation by miRNAs. Zhuo et al. [51], using deep-sequencing of RNA extracted from Drosophila female whole bodies, identified 27 miRNAs and 474 mRNA that were expressed significantly differently between mated and unmated females. Overexpression of several mating-responsive miRNAs in females affected egg laying behavior, and altered the expression of their putative candidate target genes suggesting that miRNAs could mediate changes in gene expression postmating. Carmel et al. [52] found an enrichment of mRNAs that are targets of miRNAs among the mating-responsive genes identified in the LRT, suggesting miRNA involvement. Using microarray for miRNAs, Carmel et al. [52] found that mating-responsive miRNAs are expressed throughout the LRT prior to mating and that mating changed their expression levels in one or more specific regions. When they further reduced the function of all miRNAs by silencing Dicer-1 (a key component in miRNA biogenesis) in female whole body pre-mating, they found that these females exhibited a significant decrease in fertility. During the initial maturation period, females have already synthesized most of the transcripts and proteins needed to rapidly respond to mating and for the early Current Opinion in Insect Science 2016, 13:106–113
110 Development and regulation
stages of postmating reproductive physiology. Mating rapidly relieves the arrest, shifting the female from a posteclosion developmental phase toward the postmating end of the developmental program during which they reach reproductive maturity and prepare for maximum reproduction. Mating-responsive miRNAs may sharpen the transition from the unmated to the mated state by eliminating many target genes simultaneously and suppressing residual transcripts that are specific to the unmated stage which interfere with the final stage of differentiation of epithelial, muscle and nerve tissues. Mating-responsive miRNAs may possibly also relieve inhibition of factors that promote differentiation that were kept in check to prevent precocious expression. It is therefore suggested that mating-responsive miRNAs reprogram the female expression profile to rapidly initiate the final differentiation of the reproductive tract tissues, allowing sperm storage and leading to a functional reproductive system.
Conclusions Regulation by miRNAs has been found to be critical for the functionality of reproductive tissues in a wide range of animals, from mammals to Caenorhabditis elegans [57– 60] and insects [61,62,63]. Studies reviewed here raise the possibility that the mating response in D. melanogaster also involves post-translational regulation via miRNA [20,37,45]. The transition from unmated to mated state in the D. melanogaster female is rapid, involving changes in the expression level of many mRNA transcripts. This transition would require molecular mechanisms that can rapidly end one program (unmated) and initiate the next program (mated) while preventing transcriptional and translational errors in the subsequent state. In silico simulations predict that regulation by miRNAs is faster and consumes less energy than conventional regulation via transcription factors [64]. In vivo studies of miRNA kinetics in mouse retina support this prediction [65]. Indeed, numerous miRNAs that are differentially expressed between unmated and mated females have been identified [50,51,52] and have been shown to affect the female mating response. Because D. melanogaster is short-lived, the duration of the mating response (the time from the start of copulation to oviposition) affects fitness. A fast mating response increases the chance a female has to establish the next generation of offspring before a possible catastrophe. Similar to Drosophila, mating induces changes in the female of many other insect species [66]. In Anopheles gambiae, for example, mating stimulates the female to start to feed on blood, which is required for the production of mature oocytes [67], and male seminal fluid stimulates oviposition, sperm storage and changes in the morphology and expression profile of the female reproductive tract [68–71]. At 24 hours postmating, the reproductive system Current Opinion in Insect Science 2016, 13:106–113
of the Anopheles female undergoes prominent transcriptional changes, including a dramatic down-regulation of 13 protease genes [69]. At the morphological level, postmating changes in the vagina include elimination of extensive smooth endoplasmatic reticulum from the apical side of the cells and induction of the formation of large electron dense vacuoles of uniform granule in some apical cells along the vagina [69]. In addition, rough endoplasmatic reticulum at the basal side of the vaginal cells was greatly reduced. These morphological changes suggest decreased capacity for protein synthesis in vaginal cells and a possible transfer of components between the vaginal lumen and epithelial cells. The rapid changes occurring in the secretory machinery and/or the epithelial cells of the Anopheles vagina may reflect a change in the communication mode between epithelial cells and/or epithelial cells and the lumen, reminiscent of the changes observed in the Drosophila oviduct postmating. Together, these studies suggest that the mosquito female reproductive tract undergoes a two-phase maturation, in which a rapid mating response leads to a functional reproductive system. In addition to Drosophila and Anopheles, seminal fluid induces physiological and behavioral changes (switch from unmated to mated state) in the females of many other insects, including moths, beetles, and honeybees [66]. However, not much is known about the mating response kinetics and/or developmental status of the system. We hypothesize that the model we describe for Drosophila can be also found in other insects (either dipteran or non-dipteran) which are short-lived and have similar reproductive strategy. We propose that delaying the final maturation allows the female to conserve the energy required to invest in the development of the reproductive tissues until conditions are optimal. It will be interesting to examine if mating-induced developmental changes take place in other insects. For this, further studies comparing the morphology and expression profile of reproductive tissues in virgin and mated female at different times postmating are required. These studies will determine if the rapid transition of the reproductive tract induced by mating is a general phenomenon in insects. Because the mating signal of organisms with internal reproduction is composed of stimuli perceived during courtship and the receipt of sperm and seminal fluid, it is likely that this rapid unmated to mated switch may reflect the evolution of a mechanism to optimize reproductive capacity in early adulthood in short-lived animals. As female reproductive molecules are identified in more insect species and their functions are better understood, we will get a deeper understanding of the dynamics of male-female reproductive molecule interactions and of how these interactions maximize reproductive success. www.sciencedirect.com
Mating induces developmental changes Carmel, Tram and Heifetz
Acknowledgements We thank Zohar Nir-Amitin for the graphics; support by US-Israel Binational Agricultural Research and Development Fund, research Grant 3492 (to YH), The Chief Scientist Ministry of Agriculture grant 872-0055-10 (to YH) and US-Israel Binational Science Foundation grant 2009270 (to YH and Mark Siegal).
References and recommended reading Papers of particular interest, published within the period of review, have been highlighted as: of special interest of outstanding interest 1.
Fazeli A, Moein Vaziri N, Holt WV: Proteomics of the periconception milieu. Proteomics 2015, 15:649-655.
2.
Scott MA: A glimpse at sperm function in vivo: sperm transport and epithelial interaction in the female reproductive tract. Anim Reprod Sci 2000, 60–61:337-348.
3.
Bloch Qazi MC, Heifetz Y, Wolfner MF: The developments between gametogenesis and fertilization: ovulation and female sperm storage in Drosophila melanogaster. Dev Biol 2003, 256:195-211.
4.
Watkins AJ, Lucas ES, Fleming TP: Impact of the periconceptional environment on the programming of adult disease. J Dev Orig Health Dis 2010, 1:87-95.
5.
Arias AM: Drosophila melanogaster and the development of biology in the 20th century. Methods Mol Biol 2008, 420:1-25.
6.
Jennings B: Drosophila — a versatile model in biology & medicine. Mater Today 2011, 14:190-195.
7.
Schneider D: Using Drosophila as a model insect. Nat Rev Genet 2000, 1:218-226.
8.
Nassel DR: Neuropeptides in the nervous system of Drosophila and other insects: multiple roles as neuromodulators and neurohormones. Prog Neurobiol 2002, 68:1-84.
9.
Kapelnikov A, Zelinger E, Gottlieb Y, Rhrissorrakrai K, Gunsalus KC, Heifetz Y: Mating induces an immune response and developmental switch in the Drosophila oviduct. Proc Natl Acad Sci U S A 2008, 105:13912-13917.
10. Wolfner MF: ‘‘S.P.E.R.M.’’ (seminal proteins (are) essential reproductive modulators): the view from Drosophila. Soc Reprod Fertil Suppl 2007, 65:183-199. 11. Yapici N, Kim YJ, Ribeiro C, Dickson BJ: A receptor that mediates the post-mating switch in Drosophila reproductive behaviour. Nature 2008, 451:33-37. 12. Griffith LC, Ejima A: Multimodal sensory integration of courtship stimulating cues in Drosophila melanogaster. Ann N Y Acad Sci 2009, 1170:394-398. 13. Billeter JC, Levine JD: Who is he and what is he to you? Recognition in Drosophila melanogaster. Curr Opin Neurobiol 2013, 23:17-23. 14. Pavlou HJ, Goodwin SF: Courtship behavior in Drosophila melanogaster: towards a ‘courtship connectome’. Curr Opin Neurobiol 2013, 23:76-83. 15. Villella A, Hall JC: Neurogenetics of courtship and mating in Drosophila. Adv Genet 2008, 62:67-184. 16. Vosshall LB: Scent of a fly. Neuron 2008, 59:685-689. 17. Heifetz Y, Rivlin PK: Beyond the mouse model: using Drosophila as a model for sperm interaction with the female reproductive tract. Theriogenology 2010, 73:723-739. 18. LaFlamme BA, Ram KR, Wolfner MF: The Drosophila melanogaster seminal fluid protease ‘‘seminase’’ regulates proteolytic and post-mating reproductive processes. PLoS Genet 2012, 8:e1002435. This paper identified ‘seminase’ a trypsin-like serine protease that is transferred to the female in the seminal fluid during copulation. Seminase is located upstream in proteolytic cascades of several male-transferred www.sciencedirect.com
111
components and induce both the long and the short-term mating response of the female. The identification of seminase highlights the complexity of the seminal protein network and shows that the short and long-term mating responses are under the same control. 19. Kubli E, Bopp D: Sexual behavior: how Sex Peptide flips the postmating switch of female flies. Curr Biol 2012, 22:R520R522. 20. Mack PD, Kapelnikov A, Heifetz Y, Bender M: Mating-responsive genes in reproductive tissues of female Drosophila melanogaster. Proc Natl Acad Sci U S A 2006, 103:10358-10363. 21. Adams EM, Wolfner MF: Seminal proteins but not sperm induce morphological changes in the Drosophila melanogaster female reproductive tract during sperm storage. J Insect Physiol 2007, 53:319-331. 22. Prokupek AM, Eyun SI, Ko L, Moriyama EN, Harshman LG: Molecular evolutionary analysis of seminal receptacle sperm storage organ genes of Drosophila melanogaster. J Evol Biol 2010, 23:1386-1398. 23. McGraw LA, Gibson G, Clark AG, Wolfner MF: Genes regulated by mating, sperm, or seminal proteins in mated female Drosophila melanogaster. Curr Biol 2004, 14:1509-1514. 24. Lawniczak MK, Begun DJ: A genome-wide analysis of courting and mating responses in Drosophila melanogaster females. Genome 2004, 47:900-910. 25. Demerec M (Ed): Biology of Drosophila. New York: John Wiley & Sons; 1950. 26. Wolfner MF: Tokens of love: functions and regulation of Drosophila male accessory gland products. Insect Biochem Mol Biol 1997, 27:179-192. 27. Heifetz Y, Lung O, Frongillo EA Jr, Wolfner MF: The Drosophila seminal fluid protein Acp26Aa stimulates release of oocytes by the ovary. Curr Biol 2000, 10:99-102. 28. Heifetz Y, Yu J, Wolfner MF: Ovulation triggers activation of Drosophila oocytes. Dev Biol 2001, 234:416-424. 29. Ram KR, Wolfner MF: Sustained post-mating response in Drosophila melanogaster requires multiple seminal fluid proteins. PLoS Genet 2007, 3:e238. 30. Wolfner MF: The gifts that keep on giving: physiological functions and evolutionary dynamics of male seminal proteins in Drosophila. Heredity (Edinb) 2002, 88:85-93. 31. Neubaum DM, Wolfner MF: Wise, winsome, or weird? Mechanisms of sperm storage in female animals. Curr Top Dev Biol 1999, 41:67-97. 32. Tram U, Wolfner MF: Male seminal fluid proteins are essential for sperm storage in Drosophila melanogaster. Genetics 1999, 153:837-844. 33. Chapman T, Liddle LF, Kalb JM, Wolfner MF, Partridge L: Cost of mating in Drosophila melanogaster females is mediated by male accessory gland products. Nature 1995, 373:241-244. 34. Ueyama M, Fuyama Y: Enhanced cost of mating in female sterile mutants of Drosophila melanogaster. Genes Genet Syst 2003, 78:29-36. 35. Chapman T: Seminal fluid-mediated fitness traits in Drosophila. Heredity 2001, 87:511-521. 36. Mattei AL, Riccio ML, Avila FW, Wolfner MF: Integrated 3D view of postmating responses by the Drosophila melanogaster female reproductive tract, obtained by micro-computed tomography scanning. Proc Natl Acad Sci U S A 2015, 112:84758480. This is the first high-resolution micro-CT study that reveals how male and female molecules and anatomy interface to carry out and coordinate mating-dependent changes in the female’s reproductive physiology. 37. Kapelnikov A, Rivlin PK, Hoy RR, Heifetz Y: Tissue remodeling: a mating-induced differentiation program for the Drosophila oviduct. BMC Dev Biol 2008, 8:114. 38. Tepass U, Hartenstein V: The development of cellular junctions in the Drosophila embryo. Dev Biol 1994, 161:563-596. Current Opinion in Insect Science 2016, 13:106–113
112 Development and regulation
39. Harris TJ, Tepass U: Adherens junctions: from molecules to morphogenesis. Nat Rev Mol Cell Biol 2010, 11:502-514. 40. Heifetz Y, Wolfner MF: Mating, seminal fluid components, and sperm cause changes in vesicle release in the Drosophila female reproductive tract. Proc Natl Acad Sci U S A 2004, 101:6261-6266. 41. Avila FW, Bloch Qazi MC, Rubinstein CD, Wolfner MF: A requirement for the neuromodulators octopamine and tyramine in Drosophila melanogaster female sperm storage. Proc Natl Acad Sci U S A 2012, 109:4562-4567. 42. Rubinstein CD, Wolfner MF: Drosophila seminal protein ovulin mediates ovulation through female octopamine neuronal signaling. Proc Natl Acad Sci U S A 2013, 110:17420-17425. 43. Heifetz Y, Lindner M, Garini Y, Wolfner MF: Mating regulates neuromodulator ensembles at nerve termini innervating the Drosophila reproductive tract. Curr Biol 2014, 24:731-737. 44. Rezaval C, Nojima T, Neville MC, Lin AC, Goodwin SF: Sexually dimorphic octopaminergic neurons modulate female postmating behaviors in Drosophila. Curr Biol 2014, 24:725-730. 45. McGraw LA, Clark AG, Wolfner MF: Post-mating gene expression profiles of female Drosophila melanogaster in response to time and to four male accessory gland proteins. Genetics 2008, 179:1395-1408. 46. Prokupek AM, Kachman SD, Ladunga I, Harshman LG: Transcriptional profiling of the sperm storage organs of Drosophila melanogaster. Insect Mol Biol 2009, 18:465-475. 47. Heifetz Y, Vandenberg LN, Cohn HI, Wolfner MF: Two cleavage products of the Drosophila accessory gland protein ovulin can independently induce ovulation. Proc Natl Acad Sci U S A 2005, 102:743-748. 48. Ram RK, Sirot LK, Wolfner MF: Predicted seminal astacin-like protease is required for processing of reproductive proteins in Drosophila melanogaster. Proc Natl Acad Sci U S A 2006, 103:18674-18679. 49. Laflamme BA, Avila FW, Michalski K, Wolfner MF: A Drosophila protease cascade member, seminal metalloprotease-1, is activated stepwise by male factors and requires female factors for full activity. Genetics 2014, 196:1117-1129. 50. Fricke C, Green D, Smith D, Dalmay T, Chapman T: microRNAs influence reproductive responses by females to male sex peptide in Drosophila melanogaster. Genetics 2014, 198:16031619. This paper demonstrates that sex peptide induces post-transcriptional activity of miRNAs in the female. A comparison between miRNA profiles of females mated to males with and without sex peptide identified candidate miRNAs. Functional analysis of four selected miRNAs showed that mated females which lack these miRNAs had altered receptivity to courting males compared to control females. 51. Zhou S, Mackay T, Anholt RR: Transcriptional and epigenetic responses to mating and aging in Drosophila melanogaster. BMC Genomics 2014, 15:927. The study of Zhou et al. supports two important themes in our review: (1) the phenotypic plasticity of mating and (2) the presence of functional miRNAs in the mating response of the Drosophila female. Zhou et al. preformed a comprehensive deep-Seq screen to examine the effect of mating and/or aging on the transcriptome, miRNome and histone modification (using Chip-seq) of the Drosophila female whole body. 52. Carmel I, Nave M, Zelinger E Apel I, Keidar T, Schnakenberg S, Widmayer P, Breer H, Siegal M, Heifetz Y: miRNAs regulate the mating response in the lower RT of the Drosophila melanogaster female. Unpublished results. 53. Giraldez AJ, Mishima Y, Rihel J, Grocock RJ, Van Dongen S, Inoue K, Enright AJ, Schier AF: Zebrafish miR-430 promotes deadenylation and clearance of maternal mRNAs. Science 2006, 312:75-79. 54. Hornstein E, Shomron N: Canalization of development by microRNAs. Nat Genet 2006, 38(Suppl.):S20-S24. Current Opinion in Insect Science 2016, 13:106–113
55. Bushati N, Stark A, Brennecke J, Cohen SM: Temporal reciprocity of miRNAs and their targets during the maternal-to-zygotic transition in Drosophila. Curr Biol 2008, 18:501-506. 56. Bartel DP: microRNAs: target recognition and regulatory functions. Cell 2009, 136:215-233. 57. Esquela-Kerscher A, Johnson SM, Bai L, Saito K, Partridge J, Reinert KL, Slack FJ: Post-embryonic expression of C. elegans microRNAs belonging to the lin-4 and let-7 families in the hypodermis and the reproductive system. Dev Dyn 2005, 234:868-877. 58. Hu S-J, Ren G, Liu J-L, Zhao Z-A, Yu Y-S, Su R-W, Ma X-H, Ni H, Lei W, Yang Z-M: microRNA expression and regulation in mouse uterus during embryo implantation. J Biol Chem 2008, 283:23473-23484. 59. Revel A, Achache H, Stevens J, Smith Y, Reich R: microRNAs are associated with human embryo implantation defects. Hum Reprod 2011, 26:2830-2840. 60. Imbar T, Galliano D, Pellicer A, Laufer N: Introduction: microRNAs in human reproduction: small molecules with crucial regulatory roles. Fertil Steril 2014, 101:1514-1515. 61. Reich J, Snee MJ, Macdonald PM: miRNA-dependent translational repression in the Drosophila ovary. PLoS ONE 2009, 4:e4669. 62. Lucas KJ, Roy S, Ha J, Gervaise AL, Kokoza VA, Raikhel AS: microRNA-8 targets the Wingless signaling pathway in the female mosquito fat body to regulate reproductive processes. Proc Natl Acad Sci U S A 2014, 112:1440-1445. This study identifies the fat body specific action of miR-8 and its target in regulating mosquito reproduction. miR-8 plays an essential role in the female mosquito fat body controlling the secretory activity of yolk protein precursors required for oocyte development. 63. Yang CH, Rumpf S, Xiang Y, Gordon MD, Song W, Jan LY, Jan YN: Control of the postmating behavioral switch in Drosophila females by internal sensory neurons. Neuron 2009, 61:519-526. 64. Shimoni Y, Friedlander G, Hetzroni G, Niv G, Altuvia S, Biham O, Margalit H: Regulation of gene expression by small non-coding RNAs: a quantitative view. Mol Syst Biol 2007, 3. This study further supports the presence of miRNAs in rapid evolving processes such as mating. Using in silico simulation, Shimoni et al. compared the rate of repression by small RNA and ‘conventional’ repression using transcription factor repressor in E. coli. Using kinetic parameters based on experimental data the authors show that regulation by small RNA is advantageous. 65. Krol J, Busskamp V, Markiewicz I, Stadler MB, Ribi S, Richter J, Duebel J, Bicker S, Fehling HJ, Schu¨beler D et al.: Characterizing light-regulated retinal microRNAs reveals rapid turnover as a common property of neuronal microRNAs. Cell 2010, 141:618-631. The study provides experimental evidence for the fast kinetics of miRNAs. Krol et al. exposed mice retina cells to light and found an increase in the expression level of several miRNAs within 30 min. These light responsive miRNAs also showed a rapid decline, as they were down-regulated within 90 min upon exposure to darkness. One of the miRNAs that was downregulated in response to light induce an increase in the level of its target Sla1a1 within 2–3 hours of release from the dark signal (0.5–1.5 hours from the miRNA decrease). 66. Avila FW, Sirot LK, LaFlamme BA, Rubinstein CD, Wolfner MF: Insect seminal fluid proteins: identification and function. Annu Rev Entomol 2014, 56:21-40. 67. Clements AN: The Biology of Mosquitoes. New York: CABI Publishing; 2000. 68. Rogers DW, Baldini F, Battaglia F, Panico M, Dell A, Morris HR, Catteruccia F: Transglutaminase-mediated semen coagulation controls sperm storage in the malaria mosquito. PLoS Biol 2009, 7:e1000272. 69. Rogers DW, Whitten MM, Thailayil J, Soichot J, Levashina EA, Catteruccia F: Molecular and cellular components of the mating machinery in Anopheles gambiae females. Proc Natl Acad Sci U S A 2008, 105:19390-19395. www.sciencedirect.com
Mating induces developmental changes Carmel, Tram and Heifetz
70. Gabrieli P, Kakani EG, Mitchell SN, Mameli E, Want EJ, Mariezcurrena Anton A, Serrao A, Baldini F, Catteruccia F: Sexual transfer of the steroid hormone 20E induces the postmating switch in Anopheles gambiae. Proc Natl Acad Sci U S A 2014, 111:16353-16358.
www.sciencedirect.com
113
71. Shaw WR, Teodori E, Mitchell SN, Baldini F, Gabrieli P, Rogers DW, Catteruccia F: Mating activates the heme peroxidase HPX15 in the sperm storage organ to ensure fertility in Anopheles gambiae. Proc Natl Acad Sci U S A 2014, 111:5854-5859.
Current Opinion in Insect Science 2016, 13:106–113