Insect Biochemistry and Molecular Biology 63 (2015) 152e158
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Panning for sperm gold: Isolation and purification of apyrene and eupyrene sperm from lepidopterans Timothy L. Karr*, James R. Walters Department of Ecology and Evolution, 2041 Haworth Hall, 1200 Sunnyside Avenue, Lawrence, KS 66045, USA
a r t i c l e i n f o
a b s t r a c t
Article history: Received 9 March 2015 Received in revised form 1 June 2015 Accepted 10 June 2015 Available online 30 June 2015
We describe a simple and straightforward procedure for the purification and separation of apyrene and eupyrene forms of lepidopteran sperm. The procedure is generally applicable to both butterfly and moth species with results varying according to the relative amounts of sperm produced and size of sperm storage organs. The technique relies upon inherent differences between eupyene sperm bundles and free apyrene sperm morphology. These differences allow for separation of the sperm morphs by repeated “panning” of sperm bundles into the center of a plastic dish. The purified eupyrene sperm bundles can then be removed and apyrene sperm collected from the supernatant by centrifugation. Efficacy of the purification process was confirmed by light microscopy and gel electrophoresis of the resulting fractions. Both one- and two-dimensional gel electrophoresis identified significant protein differences between the fractions further suggesting that the panning procedure effectively separated eurpyrene from apyrene sperm. The panning procedure should provide a convenient and accessible technique for further studies of sperm biology in lepidopterans. © 2015 Elsevier Ltd. All rights reserved.
Keywords: Lepidopteran Sperm Purification Proteome
1. Introduction Sperm have long been the focus of physiologists, morphologists and cell biologists interested in cellular function and evolution. A typical sperm cell consists of an axoneme based sperm tail, a mitochondria (or mitochondrial derivative) energy source, and a head structure containing a highly condensed haploid content of DNA. This ‘typical’ sperm has been considered a premier example and model system for the study of cellular development, specialization and differentiation. Historically, sperm have also served a central role in the development of evolutionary theory, particularly as it relates to sexual selection, adaptation and sperm competition (Birkhead, 2009). The spermatozoa has also been held up as a paradigm for “form denotes function” as sperm morphology clearly informs on sperm function and vice versa. However, the sperm biology of the Lepidopterans presents a unique challenge to this prevailing paradigm because nearly every species in this order of insects produces two distinct types of sperm within a common testis. This phenomenon termed, “dichtomous spermatogenesis” (Meves, 1903) results in a
* Corresponding author. E-mail address: [email protected] (T.L. Karr). http://dx.doi.org/10.1016/j.ibmb.2015.06.007 0965-1748/© 2015 Elsevier Ltd. All rights reserved.
nucleated sperm containing a haploid content of DNA (eupyrene) and another type (apyrene) that completely lacks a nucleus and nuclear DNA. Characteristic differences in developmental pathways are typically observed during the early stages of spermatogenesis and produce the two distinct groups of encased sperm bundles containing the apyrene and eupyrene sperm. The sheath cells surrounding the apyrene sperm bundles break down in the testis and enter the seminal vesicles as individualized sperm whereas €nder et al., 2005). eupyrene sperm bundles remain intact (Friedla The production of sperm with varying morphological characteristics has also been termed “sperm heteromorphism”; this term is used to describe the phenomenon for a wide variety of other taxa including insects, rotifers, sea urchins and nematodes (Swallow and €nder et al., 2005). However, sperm hetWilkinson, 2002; Friedla eromorphism in taxa other than Lepidoptera usually refers to differences in sperm length or size and not in the presence/absence of nuclear DNA. As previously reviewed (Snook, 1997), sperm heteromorphism (polymegaly) in members of the Drosophila obscura group is characterized by nucleated sperm of distinct length categories (Beatty and Burgoyne, 1971; Bressac and Hauschteck-Jungen, 1996). However, only the longer sperm morph is used in fertilization and therefore the two sperm morphs are not functionally equivalent (Snook et al., 1994; Snook and Karr, 1998). Other morphometric parameters such as sperm size in the nematode
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worm Caenorhabditis elegans are involved in the determinants of reproductive success (LaMunyon and Ward, 1998) and some of these determinants are under selective pressure (LaMunyon and Ward, 1999). Indeed, sperm morphological traits have been observed to effect reproduction at all levels of analysis including, e.g., factors such as motility, sperm quality and quantity, fertilizability and storage (Pitnick, 2009). Thus it is clear that sperm form and function can vary across diverse taxa and Lepidopterans represent a particularly extreme example of this variation. In all of its various forms, sperm heteromorphism presents an evolutionary conundrum-how and why has this type of gametogenesis evolved? What are the function(s) for the observed variation in sperm morphs in fertilization and/or reproduction in general? The perplexing nature of this phenomenon is most clearly apparent in Lepidopterans that produce an entire class of a nucleated sperm incapable of participating, directly, in the all-important act of fertilization, karyogamy and re-establishment of the diploid state. The majority of empirical data on lepidopteran sperm heteromorphism has historically come from morphological studies of spermatogenesis based on light and electron microscopy €nder et al., 2005). A number of functional and evolutionary (Friedla hypotheses have been proposed to explain the existence of sperm heteromorphy. Due to the obvious fact that apyrene are devoid of a nucleus and lack genetic material to contribute to the zygote, these hypotheses typically propose functions for apyrene sperm that are peripheral to the primary reproductive role played by eupyrene sperm. Major ideas put forward include a role in sperm competition (Cook and Wedell, 1999), facilitation of eusperm (Holman and Snook, 2008; Sahara and Takemura, 2003), provisioning or nutritive functions (Boggs and Gilbert, 1979) and mediation of postcopulatory sexual selection (LaMunyon and Eisner, 1994). These, and other scenarios for the evolution of sperm heteromorphy have been comprehensively reviewed (Friedl€ ander et al., 2005; Swallow and Wilkinson, 2002). However, beyond the obvious impact that fertilizing sperm have on organismal fitness, the genetic and cellular processes involved in origin and maintenance of sperm heteromorphism remain poorly understood. Systems level analyses of sperm (e.g., RNA profiling and proteomics) have provided a wealth of new knowledge on the fundamental building blocks of this remarkable cell (Dorus et al., 2006; Fischer et al., 2012; Wasbrough et al., 2010; Zareie et al., 2013). Application of these modern “omics” technologies is providing new and exciting avenues to study the developmental biology of spermatogenesis and the molecular evolutionary basis of sperm competition and sperm evolution [detailed in several recent reviews (Amaral et al., 2014; Baker et al., 2012; Dacheux and Dacheux, 2014; Dorus et al., 2008, 2012)]. These technical advances offer great promise for insight into the mysteries posed by sperm heteromorphism, but have not yet been so employed. One major limitation in this effort has been the lack of high quality whole genome sequences necessary for comprehensive proteomic analyses. However, genome sequences for several Lepidopteran taxa are now publicly available, (Heliconius Genome Consortium, 2012; You et al., 2013; International Silkworm Genome Consortium, 2008), with several additional species expected in the near future. Thus the door is now open for detailed molecular genetic and functional genomic studies of this diverse group of insects. To date, the absence of an efficient and accessible method for the separation of apyrene and eupyrene sperm suitable for biochemical, proteomic or functional analyses stands as one of the largest barriers to a deeper understanding of the cellular and molecular basis of sperm heteromorphy in Lepidoptera and other taxa. A prior published study described the successful separation of apyrene and eupyrene sperm from Bombyx mori using a discontinuous density gradient sedimentation technique (Osanai et al., 1989). While
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successful, the approach involves rather sophisticated centrifugation methods and analyses not commonly available to standard entomology laboratories and has therefore not found wide application and usage. Here we describe a simple and straightforward procedure, applicable to both butterflies and moths, for the separation and purification of apyrene and eupyrene forms of lepidopteran sperm using only simple tools and reagents, a dissecting microscope and table top centrifuge. Recently, high throughput discovery proteomics have been successfully applied to the Manduca sexta sperm proteomes (Whittington et al., 2015). This detailed analysis of the total “A þ E” sperm proteomes clearly paves the way for future in depth functional and genomic analyses of this important lepidopteran. Future studies of M. sexta sperm proteomes and other lepidopterans should benefit by use of the panning techniques described in this report. 2. Materials and methods 2.1. Lepidopterans used in this study Two butterfly species, the Monarch (Danaus plexippus, kind gift of Monarchwatch.org, Lawrence, KS) and the American Painted Lady (Vanessa virginiensis, obtained from Monarchs Forever, Bixby, OK) and a moth, the tobacco horned-worm (Manduca sexta from Carolina Biological, Burlington, NC) were used in this study. 2.2. Bulk sperm isolation 5e10 day old adult males were euthanized and the reproductive tract, associated organs and connective tissue dissected from abdomens using small surgical scissors and fine tipped forceps. To facilitate sperm removal, the testis, ductus deferens and the vesicula seminalis (seminal vesicles) were removed from the surrounding connective tissue and placed into 100 mm plastic petri dishes containing phosphate buffered saline (PBS). Sperm and seminal fluids were removed by making a small incision near the mid-todistal section which released the contents due to residual pressure within the seminal vesicle. Both apyrene and eupyrene sperm were commingled within an envelope of highly viscous fluid which remained relatively intact as it exited the seminal vesicle thus facilitating its easy removal to the petri dish using a pipettor fitted with P200 tip (the tip was cut back approximately 5 mm to enlarge the opening). In addition, for bulk purification of mixed samples of apyrene and eupyrene sperm, seminal vesicle contents were deposited into 1.5 ml microcentrifuge tubes and the contents spun down in a Sorvall benchtop centrifuge 2 min at 15 K rpm. Following supernatant removal, resulting pellets were resuspended in 1 ml PBS. This process was repeated 2X resulting in a purified mixture of apyrene and eupyrene sperm (termed, “AE” sperm). 2.3. Panning and purification of eupyrene sperm All stages of the panning process are performed using a stereo dissecting microscope. Seminal vesicle contents containing AE sperm are used to prepare purified apyrene (“A”-) and eupyrene (“E”-) sperm using a novel panning technique (this technique is described in detail below and in the Supplemental Movie). To begin the process, seminal vesicle contents were transferred to a 100 mm plastic petri dish containing approximately 10 ml of PBS and dispersed by gentle mixing using a P1000 pipettor. (N.B. Noteexperience suggests the use of plastic petri dishes yield optimal results over glass petri dishes). The dispersed contents are allowed to settle for approximately 2e3 min and the manual panning process begins with gentle circular rotation of the dish for
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approximately 10e15 s followed by a 30 s recovery period. This process is repeated 2e3X until eupyrene sperm bundles are observed in the center of the dish. E-sperm are then transferred into a second 60 mm petri dish using a P200 pipettor. E-sperm are continually collected from the original petri dish by repeated cycles of panning and transfer into the second dish and the process repeated again. Purified E-sperm bundles are then transferred to 1.5 ml microcentrifuge tubes, spun down for 2 min at 5 K rpm and resuspended in PBS and the process repeated 2X. A-sperm were then collected from the original 100 mm dish (depleted of E-sperm bundles) into either a 15 ml centrifuge using a P1000 pipettor for storage at 4 C, or transferred into a 60 mm petri dish for further immediate processing. Panning of the transferred A-sperm was then used to capture any residual of E-sperm bundles into the center, and A-sperm supernatant collected from the periphery (away from any contaminating E-sperm bundles) and transferred into 1.5 ml tubes. The tubes were then spun 5 min at 15 K rpm and pellets resuspended in PBS and the process repeated 3X. Final pellets of each of the three fractions, AE, A- and E-sperm, were frozen at 20 C. Supplementary video related to this article can be found at http://dx.doi.org/10.1016/j.ibmb.2015.06.007. 2.4. Gel electrophoresis 1-dimensional SDS-PAGE-Sperm samples were solubilized in 2X LDS sample buffer as per manufacturers instructions (Invitrogen, Inc) and quantified using the EZQ Protein Quantitation Kit (Invitrogen, Inc). Protein fluorescence was measured using a Typhoon Trioþ (Amersham Biosciences/GE Healthcare) equipped with a 488 nm laser and 610 nm bandpass filter. ImageQuant TL software was used to analyze fluorescence data. A standard curve was generated using fluorescence data from control samples of known concentration and used to determine sperm sample concentration. A 1 mm 10% NuPAGE Novex Bis-Tris Mini Gel was set up using the XCell SureLock Mini-Cell system (Invitrogen) as per manufacturer instructions for reduced samples. 25 mg from each of three fractions containing a co-mixture of A- and E-sperm, purified A- and Esperm were loaded and samples electrophoresed for 35 min at 200 V. Following electrophoresis, the gel was stained using SimplyBlue SafeStain (Invitrogen, Inc) and destained as per manufacturer instructions. Gels were then imaged using a BioRad gel imager and images imported into Adobe Photoshop and Illustrator for labeling and annotation. 2-Dimensional IEF-PAGE- Two-dimensional electrophoresis was performed according to the carrier ampholine method of isoelectric focusing (Burgess-Cassler et al., 1989; O'Farrell, 1975) by Kendrick Labs, Inc. (Madison, WI). Isoelectric focusing was carried out in a glass tube of inner diameter 2.3 mm using 2.0% pH 3e10 isodalt Servalytes (Serva, Heidelberg, Germany) for 9600 V-hrs. One ug of an IEF internal standard, tropomyosin, was added to each sample. This protein migrates as a doublet with lower polypeptide spot of MW 33,000 and pI 5.2; The enclosed tube gel pH gradient plot for this set of Servalytes was determined with a surface pH electrode. After equilibration for 10 min in buffer “O” (10% glycerol, 50 mM dithiothreitol, 2.3% SDS and 0.0625 M tris, pH 6.8), each tube gel was sealed to the top of a stacking gel that overlaid a 10% acrylamide slab gel (0.75 mm thick). SDS slab gel electrophoresis was carried out for about 4 h at 15 mA/gel. The following proteins (Sigma Chemical Co., St. Louis, MO and EMD Millipore, Billerica, MA) were used as molecular weight standards: myosin (220,000), phosphorylase A (94,000), catalase (60,000), actin (43,000) carbonic anhydrase (29,000) and lysozyme (14,000). These standards appear at the basic edge of the Coomassie Brilliant Blue R-250stained gel. The Coomassie Blue R-250 stained gel was dried
Fig. 1. Separation of apyrene and eupyrene sperm using a panning purification method. (A) Dissected reproductive tract showing the paired testis (T) connected to the paired seminal vesicles at its base (arrowhead) seen coiled downward away from the testis and then back up to the distal end (arrow) where seminal vesicle contents are observed (bracket) following puncture with a fine needle. (B) Higher magnification view of the released contents near the puncture site in panel A shows the two sperm morphs. The eupyrene bundles are highly birefringent coiled structures (arrowheads) embedded in a viscous proteinaceous material containing the free apyrene sperm which are seen as the wispy material surrounding the eupyrene sperm bundles. This viscous material is easily removed from the seminal vesicle and further purified using a panning method as further described in detail in the Supplementary movie. (C) The result of the panning procedure are highly purified eupyrene sperm bundles with characteristic coiled bundles.
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Fig. 2. Light microscopy images of purified sperm fractions. Each of three fractions obtained from the panning procedure, combined apyrene/eupyrene, eupyrene and apyrene, were washed a final time in 1ug/ml of DAPI and mounted on microscope slides. Sperm were imaged and using a Leica DMRE microscope equipped with Nomarski contrast and epifluorescence optics. Images were recorded using a Leica DFC500 camera and imported into Leica software. (AeC) respectively, showing Monarch butterfly (D. plexippus) sperm mixed A- and E-sperm, purified E-sperm, and purified A-sperm. (DeF) showing respectively the same categories as above for the Painted Lady butterfly (V. virginiensis). (GeI) showing respectively the same categories for the tobacco horned-worm (M. sexta). Note the bright white signal (G, arrows) showing the DAPI (nuclear DNA) signal at the tips of the eupyrene sperm bundles. No DAPI signal was observed in apyrene sperm (not shown). Bar ¼ 20 ym.
Fig. 3. SDS gel electrophoresis of purified sperm fractions from three lepidopterans, two butterflies, D. plexippus and V. virginiensis and the moth M. sexta. Each lane represents protein bands present in purified fractions of whole sperm (E/A), eupyrene sperm (E) and apyrene (A) sperm. Visual inspection of the gels reveals obvious differences in either the presence/absence or band intensities between the samples. Similar comparisons for the other two show clear differences in V. virginiensis and M. sexta. Ticks on left represent molecular weight markers (from top to bottom) of 250-, 150-, 100-, 75-, 50-, 37-, 25,- 20-, 15- and 10 kDa, respectively.
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between sheets of cellophane with the acid edge to the left. 2.5. Image analysis using a negative-image overlay technique We developed a simple, semi-quantitative method for 2D gel comparisons as follows. TIFF files of the E- and A-sperm 2D gels were imported into Photoshop (Adobe, Inc) in separate layers. The top layer image file is chosen (image order can be reversed and results compared) and the pixels values are reversed using the “invert” function. This creates a negative image of the upper layer which obscures the layer below. The gel image in the lower layer can be made visible by reducing the opacity of the inverted image. The level of opacity reduction is performed manually to optimize the visibility of the two layers. The top layer can then be manually moved around the center of the images to optimize the alignment of the spots that appear coincident. This utilizes the human brain's ability as an “image processor”. This process works best in alignment of regional areas of the gel as overall match is poor due to well known factors (gel stretching, cathodic drift, etc …). An example comparing A- and E-sperm is shown in Suppl. Fig. 1. 3. Results & discussion 3.1. The conundrum of sperm heteromorphism in the Lepidoptera Spermatogenesis in Lepidopterans produces two sperm morphs, a nucleated form that contains a haploid complement of DNA (eupyrene or E-sperm as used here), and an anucleated form that does not contain DNA (apyrene or A-sperm as used here). The purpose and persistence of a reproductive strategy that produces an entire class of sperm (A-sperm) incapable of participating directly in the transfer of genetic material, is unknown. Numerous hypotheses have been proposed to explain the presence and persistence of sperm heteromorphism, but no consensus has been reached as to either the evolutionary origins nor the functional €nder significance of apyrene sperm (discussed in detail in (Friedla et al., 2005)). One impediment to a deeper understanding of the lepidopteran system has been the absence of the molecular and cellular processes that generate the two sperm morphs. To better understand the biochemical makeup of E- and A-sperm, we developed a novel purification procedure that yields highly enriched fractions of each. The method exploits differences in the physical properties of the two morphs: apyrene sperm are smaller and released into the ductus deferens as individual sperm whereas the larger eupyrene sperm remain in bundles (Fig. 1). Two different criteria were used to establish the efficacy of the purification process; light microscopy to visualize sperm morphology and gel electrophoresis to monitor protein differences between A- and E-sperm. Taken together these results demonstrate the overall effectiveness of the panning procedure as discussed below. 3.2. Effective purification of eupyrene sperm via a novel panning method The Monarch butterfly (D. plexippus) was chosen for development of the panning process due to the high levels of sperm stored in the seminal vesicles (Fig. 1A). Seminal vesicle fluid is highly viscous facilitating transfer out of the seminal vesicle (Fig. 1B) and into either centrifuge tubes for purification of bulk AE sperm, or into another plastic dish for subsequent panning and purification. Once transferred and dispersed in an appropriate volume of buffer in a plastic dish, separation by panning (detailed in Suppl. Movie 1) is facilitated by differences in A- and E-sperm structure: A-sperm are released into the ductus deferens individually whereas E-sperm
Fig. 4. Two-dimensional gel electrophoresis of D. plexippus purified sperm fractions. Each panel shows protein spot patterns present in purified fractions of (Eupyrene/ Apyrene), (Eupyrene) and (Apyrene) sperm. Spot pattern differences between the three samples were analyzed using a negative-image overlay method (Supplemental Fig. 1). Regions showing distinct differences are shown (boxes, circles and ovals) with solid lines indicating presence, and dashed lines, absence of protein spots. The arrowhead on the stained gels marks the position of the internal standard protein, tropomyosin.
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remain bundled and embedded in a dense proteinaceous matrix that retains its integrity during dissection, transfer and panning (Fig. 1). Successive panning and transfer effectively separates Esperm (Fig. 1C; Suppl. Movie 1). We further assessed the utility of the panning technique using another butterfly, V. virginiensis, and a moth, Manduca sexta (Fig. 2). In each case, the panning method was effective in separating A-from E-sperm.
3.3. Gel electrophoresis reveals significant protein differences between A- and E-sperm To further characterize sperm, the three fractions of sperm (purified as described above) were subjected to 1-dimensional SDSPAGE and the protein patterns compared (Fig. 3). Cursory examination of the gels clearly shows a number of differences between each fraction. Although the precise number and identity of the differences apparent in these gels cannot be ascertained at this level of analysis, they do reflect differences in sperm morphs purified by the panning method, consistent with the 2D gels results described below. Two-dimensional IEF-PAGE separates proteins by isoelectric point and molecular weight thus providing a more detailed analysis of differences observed between the fractions. Visual inspection of the gels shows clear differences in protein spot patterns. Overall, compared to the A-sperm gel, E-sperm are more complex containing 239 clearly visible spots in E-sperm and 179 spots in Asperm (measured by manual spot counts). A more direct comparison using an negative-image overlay technique (see Materials and Methods and Supplementary file 1) among the three 2D gel images revealed clear regional differences between A- and E-sperm (Fig. 4). As anticipated, the AE-sperm gel contained all proteins recognized in either the A- and E-sperm and, as expected from the spot counts, the A-sperm gel contained most proteins absent from either the AE- or E-sperm gels (dashed boxes, Fig. 4C). Thus we can conclude that in general E-sperm are more biologically complex at the protein level.
4. Conclusion The panning technique provides a simple and efficient method for obtaining highly purified samples of apyrene and eupyrene sperm. The method was effective in separating the two sperm morphs as measured by both microscopy and gel electrophoresis. As such this work provides a solid foundation for future studies on the cellular and molecular basis of sperm dimorphism in Lepidopterans. For example, work in progress is using this methodology as input for high throughput shotgun proteomic experiments to determine the A- and E-sperm proteomes. Knowledge of the differences, and similarities, between these two sperm proteomes will facilitate future studies designed to understand the functional genomic and molecular evolutionary biology of this fascinating system.
Acknowledgments We are grateful for the expert knowledge, guidance and resources provided by Dr. Chip Taylor, Monarchwatch.org. We also thank Desiree Harpel, Channing Shives and Ann Ryan for invaluable technical assistance and animal rearing and maintenance. Meg Jamieson filmed and edited the supplementary video, for which we are extremely grateful. This work was supported by start-up funds to JRW from the University of Kansas.
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Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.ibmb.2015.06.007. References Amaral, A., Castillo, J., Ramalho-Santos, J., Oliva, R., 2014. The combined human sperm proteome: cellular pathways and implications for basic and clinical science. Hum. Reprod. Update 20, 40e62. Baker, M.A., Nixon, B., Naumovski, N., Aitken, R.J., 2012. Proteomic insights into the maturation and capacitation of mammalian spermatozoa. Syst. Biol. Reprod. Med. 58, 211e217. Beatty, R.A., Burgoyne, P.S., 1971. Size classes of the head and flagellum of drosophila species. Cytogenetics 10, 177e185. Birkhead, T.R., 2009. In: Hosken, D.J., Pitnick, S.S. (Eds.), Sperm Biology: an Evolutionary Perspective. Elsevier/Academic Press. Boggs, C.L., Gilbert, L.E., 1979. Male contribution to egg production in butterflies: evidence for transfer of nutrients at mating. Science 206, 83e84. Bressac, C., Hauschteck-Jungen, E., 1996. Drosophila subobscura females preferentially select long sperm for storage and use. J. Insect. Physiol. 42, 323e328. Burgess-Cassler, A., Johansen, J.J., Santek, D.A., Ide, J.R., Kendrick, N.C., 1989. Computerized quantitative analysis of coomassie-blue-stained serum proteins separated by two-dimensional electrophoresis. Clin. Chem. 35, 2297e2304. Cook, P.A., Wedell, N., 1999. Non-fertile sperm delay female remating. Nature 397, 486. Dacheux, J.L., Dacheux, F., 2014. New insights into epididymal function in relation to sperm maturation. Reproduction 147, R27eR42. Dorus, S., Busby, S.A., Gerike, U., Shabanowitz, J., Hunt, D.F., Karr, T.L., 2006. Genomic and functional evolution of the Drosophila melanogaster sperm proteome. Nat. Genet. 38, 1440e1445. Dorus, S., Freeman, Z.N., Parker, E.R., Heath, B.D., Karr, T.L., 2008. Recent origins of sperm genes in drosophila. Mol. Biol. Evol. 25, 2157e2166. Dorus, S., Skerget, S., Karr, T.L., 2012. Proteomic discovery of diverse immunity molecules in mammalian spermatozoa. Syst. Biol. Reprod. Med. 58, 218e228. Fischer, B.E., Wasbrough, E., Meadows, L.A., Randlet, O., Dorus, S., Karr, T.L., Russell, S., 2012. Conserved properties of drosophila and human spermatozoal mRNA repertoires. Proc. Biol. Sci. 279, 2636e2644. Friedl€ ander, M., Seth, R.K., Reynolds, S.E., 2005. Eupyrene and apyrene sperm: dichotomous spermatogenesis in lepidoptera advances. Insect. Physiol. 32, 206e308. Heliconius Genome Consortium, 2012. Butterfly genome reveals promiscuous exchange of mimicry adaptations among species. Nature 487, 94e98. Holman, L., Snook, R.R., 2008. A sterile sperm caste protects brother fertile sperm from female-mediated death in drosophila pseudoobscura. Curr. Biol. 18, 292e296. International Silkworm Genome Consortium, 2008. The genome of a lepidopteran model insect, the silkworm Bombyx mori. Insect. Biochem. Mol. Biol. 38, 1036e1045. LaMunyon, C.W., Eisner, T., 1994. Spermatophore size as determinant of paternity in an arctiid moth (Utetheisa ornatrix). Proc. Natl. Acad. Sci. U. S. A. 91, 7081e7084. LaMunyon, C.W., Ward, S., 1998. Larger sperm outcompete smaller sperm in the nematode caenorhabditis elegans. Proc. Biol. Sci. 265, 1997e2002. LaMunyon, C.W., Ward, S., 1999. Evolution of sperm size in nematodes: sperm competition favours larger sperm. Proc. Biol. Sci. 266, 263e267. Meves, F., 1903. Über oligopyrene und apyrene spermien und ihre Entstehung nach Beobachtungen an Paludina un Pygaera. Arch. Mikrosk. Anat. Entwicklungsmech 61, 1e84. O'Farrell, P.H., 1975. High resolution two-dimensional electrophoresis of proteins. J. Biol. Chem. 250, 4007e4021. Osanai, M., Kasuga, H., Aigaki, T., 1989. Isolations of eupyrene sperm bundles and apyrene spermatozoa from seminal fluid of the silkmoth, Bombyx mori. J. Insect Physiology 35, 401e408. Pitnick, S., 2009. Sperm morphological diversity. In: Birkhead, T.R., Hosken, D.J., Pitnick, S. (Eds.), Sperm Biology: an Evolutionary Perspective. Elsevier Science & Technology, Oxford, UK, pp. 69e149. Sahara, K., Takemura, Y., 2003. Application of artificial insemination technique to eupyrene and/or apyrene sperm in bombyx mori. J. Exp. Zool. A. Comp. Exp. Biol. 297, 196e200. Snook, R.R., Markow, T.A., Karr, T.L., 1994. Functional nonequivalence of sperm in drosophila pseudoobscura. Proc. Natl. Acad. Sci. U. S. A. 91, 11222e11226. Snook, R.R., 1997. Is the production of multiple sperm types adaptive? Evolution 51, 797e808. Snook, R.R., Karr, T.L., 1998. Only long sperm are fertilization-competent in six sperm-heteromorphic drosophila species. Curr. Biol. 8, 291e294. Swallow, J.G., Wilkinson, G.S., 2002. The long and short of sperm polymorphisms in insects. Biol. Rev. Camb. Philos. Soc. 77, 153e182. Wasbrough, E.R., Dorus, S., Hester, S., Howard-Murkin, J., Lilley, K., Wilkin, E., Polpitiya, A., Petritis, K., Karr, T.L., 2010. The Drosophila melanogaster sperm proteome-II (DmSP-II). J. Proteomics 73, 2171e2185. Whittington, E., Zhao, Q., Borziak, K., Walters, J.R., Dorus, S., 2015. Characterisation of the Manduca sexta sperm proteome: genetic novelty underlying sperm composition in lepidoptera. Insect Biochem. Mol. Biol. 62, 183e193.
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http://dx.doi.org/10.1016/j.ibmb.2015.02.01. You, M., Yue, Z., He, W., Yang, X., Yang, G., Xie, M., Zhan, D., Baxter, S.W., Vasseur, L., Gurr, G.M., Douglas, C.J., Bai, J., Wang, P., Cui, K., Huang, S., Li, X., Zhou, Q., Wu, Z., Chen, Q., Liu, C., Wang, B., Li, X., Xu, X., Lu, C., Hu, M., Davey, J.W., Smith, S.M., Chen, M., Xia, X., Tang, W., Ke, F., Zheng, D., Hu, Y., Song, F., You, Y., Ma, X., Peng, L., Zheng, Y., Liang, Y., Chen, Y., Yu, L., Zhang, Y., Liu, Y., Li, G., Fang, L., Li, J.,
Zhou, X., Luo, Y., Gou, C., Wang, J., Wang, J., Yang, H., Wang, J., 2013. A heterozygous moth genome provides insights into herbivory and detoxification. Nat. Genet. 45, 220e225. Zareie, R., Eubel, H., Millar, A.H., Baer, B., 2013. Long-term survival of high quality sperm: insights into the sperm proteome of the honeybee Apis mellifera. J. Proteome. Res. 12, 5180e5188.
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Panning for sperm gold: Isolation and purification of apyrene and eupyrene sperm from lepidopterans Timothy L. Karr*, James R. Walters Department of Ecology and Evolution, 2041 Haworth Hall, 1200 Sunnyside Avenue, Lawrence, KS 66045, USA
a r t i c l e i n f o
a b s t r a c t
Article history: Received 9 March 2015 Received in revised form 1 June 2015 Accepted 10 June 2015 Available online 30 June 2015
We describe a simple and straightforward procedure for the purification and separation of apyrene and eupyrene forms of lepidopteran sperm. The procedure is generally applicable to both butterfly and moth species with results varying according to the relative amounts of sperm produced and size of sperm storage organs. The technique relies upon inherent differences between eupyene sperm bundles and free apyrene sperm morphology. These differences allow for separation of the sperm morphs by repeated “panning” of sperm bundles into the center of a plastic dish. The purified eupyrene sperm bundles can then be removed and apyrene sperm collected from the supernatant by centrifugation. Efficacy of the purification process was confirmed by light microscopy and gel electrophoresis of the resulting fractions. Both one- and two-dimensional gel electrophoresis identified significant protein differences between the fractions further suggesting that the panning procedure effectively separated eurpyrene from apyrene sperm. The panning procedure should provide a convenient and accessible technique for further studies of sperm biology in lepidopterans. © 2015 Elsevier Ltd. All rights reserved.
Keywords: Lepidopteran Sperm Purification Proteome
1. Introduction Sperm have long been the focus of physiologists, morphologists and cell biologists interested in cellular function and evolution. A typical sperm cell consists of an axoneme based sperm tail, a mitochondria (or mitochondrial derivative) energy source, and a head structure containing a highly condensed haploid content of DNA. This ‘typical’ sperm has been considered a premier example and model system for the study of cellular development, specialization and differentiation. Historically, sperm have also served a central role in the development of evolutionary theory, particularly as it relates to sexual selection, adaptation and sperm competition (Birkhead, 2009). The spermatozoa has also been held up as a paradigm for “form denotes function” as sperm morphology clearly informs on sperm function and vice versa. However, the sperm biology of the Lepidopterans presents a unique challenge to this prevailing paradigm because nearly every species in this order of insects produces two distinct types of sperm within a common testis. This phenomenon termed, “dichtomous spermatogenesis” (Meves, 1903) results in a
* Corresponding author. E-mail address: [email protected] (T.L. Karr). http://dx.doi.org/10.1016/j.ibmb.2015.06.007 0965-1748/© 2015 Elsevier Ltd. All rights reserved.
nucleated sperm containing a haploid content of DNA (eupyrene) and another type (apyrene) that completely lacks a nucleus and nuclear DNA. Characteristic differences in developmental pathways are typically observed during the early stages of spermatogenesis and produce the two distinct groups of encased sperm bundles containing the apyrene and eupyrene sperm. The sheath cells surrounding the apyrene sperm bundles break down in the testis and enter the seminal vesicles as individualized sperm whereas €nder et al., 2005). eupyrene sperm bundles remain intact (Friedla The production of sperm with varying morphological characteristics has also been termed “sperm heteromorphism”; this term is used to describe the phenomenon for a wide variety of other taxa including insects, rotifers, sea urchins and nematodes (Swallow and €nder et al., 2005). However, sperm hetWilkinson, 2002; Friedla eromorphism in taxa other than Lepidoptera usually refers to differences in sperm length or size and not in the presence/absence of nuclear DNA. As previously reviewed (Snook, 1997), sperm heteromorphism (polymegaly) in members of the Drosophila obscura group is characterized by nucleated sperm of distinct length categories (Beatty and Burgoyne, 1971; Bressac and Hauschteck-Jungen, 1996). However, only the longer sperm morph is used in fertilization and therefore the two sperm morphs are not functionally equivalent (Snook et al., 1994; Snook and Karr, 1998). Other morphometric parameters such as sperm size in the nematode
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worm Caenorhabditis elegans are involved in the determinants of reproductive success (LaMunyon and Ward, 1998) and some of these determinants are under selective pressure (LaMunyon and Ward, 1999). Indeed, sperm morphological traits have been observed to effect reproduction at all levels of analysis including, e.g., factors such as motility, sperm quality and quantity, fertilizability and storage (Pitnick, 2009). Thus it is clear that sperm form and function can vary across diverse taxa and Lepidopterans represent a particularly extreme example of this variation. In all of its various forms, sperm heteromorphism presents an evolutionary conundrum-how and why has this type of gametogenesis evolved? What are the function(s) for the observed variation in sperm morphs in fertilization and/or reproduction in general? The perplexing nature of this phenomenon is most clearly apparent in Lepidopterans that produce an entire class of a nucleated sperm incapable of participating, directly, in the all-important act of fertilization, karyogamy and re-establishment of the diploid state. The majority of empirical data on lepidopteran sperm heteromorphism has historically come from morphological studies of spermatogenesis based on light and electron microscopy €nder et al., 2005). A number of functional and evolutionary (Friedla hypotheses have been proposed to explain the existence of sperm heteromorphy. Due to the obvious fact that apyrene are devoid of a nucleus and lack genetic material to contribute to the zygote, these hypotheses typically propose functions for apyrene sperm that are peripheral to the primary reproductive role played by eupyrene sperm. Major ideas put forward include a role in sperm competition (Cook and Wedell, 1999), facilitation of eusperm (Holman and Snook, 2008; Sahara and Takemura, 2003), provisioning or nutritive functions (Boggs and Gilbert, 1979) and mediation of postcopulatory sexual selection (LaMunyon and Eisner, 1994). These, and other scenarios for the evolution of sperm heteromorphy have been comprehensively reviewed (Friedl€ ander et al., 2005; Swallow and Wilkinson, 2002). However, beyond the obvious impact that fertilizing sperm have on organismal fitness, the genetic and cellular processes involved in origin and maintenance of sperm heteromorphism remain poorly understood. Systems level analyses of sperm (e.g., RNA profiling and proteomics) have provided a wealth of new knowledge on the fundamental building blocks of this remarkable cell (Dorus et al., 2006; Fischer et al., 2012; Wasbrough et al., 2010; Zareie et al., 2013). Application of these modern “omics” technologies is providing new and exciting avenues to study the developmental biology of spermatogenesis and the molecular evolutionary basis of sperm competition and sperm evolution [detailed in several recent reviews (Amaral et al., 2014; Baker et al., 2012; Dacheux and Dacheux, 2014; Dorus et al., 2008, 2012)]. These technical advances offer great promise for insight into the mysteries posed by sperm heteromorphism, but have not yet been so employed. One major limitation in this effort has been the lack of high quality whole genome sequences necessary for comprehensive proteomic analyses. However, genome sequences for several Lepidopteran taxa are now publicly available, (Heliconius Genome Consortium, 2012; You et al., 2013; International Silkworm Genome Consortium, 2008), with several additional species expected in the near future. Thus the door is now open for detailed molecular genetic and functional genomic studies of this diverse group of insects. To date, the absence of an efficient and accessible method for the separation of apyrene and eupyrene sperm suitable for biochemical, proteomic or functional analyses stands as one of the largest barriers to a deeper understanding of the cellular and molecular basis of sperm heteromorphy in Lepidoptera and other taxa. A prior published study described the successful separation of apyrene and eupyrene sperm from Bombyx mori using a discontinuous density gradient sedimentation technique (Osanai et al., 1989). While
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successful, the approach involves rather sophisticated centrifugation methods and analyses not commonly available to standard entomology laboratories and has therefore not found wide application and usage. Here we describe a simple and straightforward procedure, applicable to both butterflies and moths, for the separation and purification of apyrene and eupyrene forms of lepidopteran sperm using only simple tools and reagents, a dissecting microscope and table top centrifuge. Recently, high throughput discovery proteomics have been successfully applied to the Manduca sexta sperm proteomes (Whittington et al., 2015). This detailed analysis of the total “A þ E” sperm proteomes clearly paves the way for future in depth functional and genomic analyses of this important lepidopteran. Future studies of M. sexta sperm proteomes and other lepidopterans should benefit by use of the panning techniques described in this report. 2. Materials and methods 2.1. Lepidopterans used in this study Two butterfly species, the Monarch (Danaus plexippus, kind gift of Monarchwatch.org, Lawrence, KS) and the American Painted Lady (Vanessa virginiensis, obtained from Monarchs Forever, Bixby, OK) and a moth, the tobacco horned-worm (Manduca sexta from Carolina Biological, Burlington, NC) were used in this study. 2.2. Bulk sperm isolation 5e10 day old adult males were euthanized and the reproductive tract, associated organs and connective tissue dissected from abdomens using small surgical scissors and fine tipped forceps. To facilitate sperm removal, the testis, ductus deferens and the vesicula seminalis (seminal vesicles) were removed from the surrounding connective tissue and placed into 100 mm plastic petri dishes containing phosphate buffered saline (PBS). Sperm and seminal fluids were removed by making a small incision near the mid-todistal section which released the contents due to residual pressure within the seminal vesicle. Both apyrene and eupyrene sperm were commingled within an envelope of highly viscous fluid which remained relatively intact as it exited the seminal vesicle thus facilitating its easy removal to the petri dish using a pipettor fitted with P200 tip (the tip was cut back approximately 5 mm to enlarge the opening). In addition, for bulk purification of mixed samples of apyrene and eupyrene sperm, seminal vesicle contents were deposited into 1.5 ml microcentrifuge tubes and the contents spun down in a Sorvall benchtop centrifuge 2 min at 15 K rpm. Following supernatant removal, resulting pellets were resuspended in 1 ml PBS. This process was repeated 2X resulting in a purified mixture of apyrene and eupyrene sperm (termed, “AE” sperm). 2.3. Panning and purification of eupyrene sperm All stages of the panning process are performed using a stereo dissecting microscope. Seminal vesicle contents containing AE sperm are used to prepare purified apyrene (“A”-) and eupyrene (“E”-) sperm using a novel panning technique (this technique is described in detail below and in the Supplemental Movie). To begin the process, seminal vesicle contents were transferred to a 100 mm plastic petri dish containing approximately 10 ml of PBS and dispersed by gentle mixing using a P1000 pipettor. (N.B. Noteexperience suggests the use of plastic petri dishes yield optimal results over glass petri dishes). The dispersed contents are allowed to settle for approximately 2e3 min and the manual panning process begins with gentle circular rotation of the dish for
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approximately 10e15 s followed by a 30 s recovery period. This process is repeated 2e3X until eupyrene sperm bundles are observed in the center of the dish. E-sperm are then transferred into a second 60 mm petri dish using a P200 pipettor. E-sperm are continually collected from the original petri dish by repeated cycles of panning and transfer into the second dish and the process repeated again. Purified E-sperm bundles are then transferred to 1.5 ml microcentrifuge tubes, spun down for 2 min at 5 K rpm and resuspended in PBS and the process repeated 2X. A-sperm were then collected from the original 100 mm dish (depleted of E-sperm bundles) into either a 15 ml centrifuge using a P1000 pipettor for storage at 4 C, or transferred into a 60 mm petri dish for further immediate processing. Panning of the transferred A-sperm was then used to capture any residual of E-sperm bundles into the center, and A-sperm supernatant collected from the periphery (away from any contaminating E-sperm bundles) and transferred into 1.5 ml tubes. The tubes were then spun 5 min at 15 K rpm and pellets resuspended in PBS and the process repeated 3X. Final pellets of each of the three fractions, AE, A- and E-sperm, were frozen at 20 C. Supplementary video related to this article can be found at http://dx.doi.org/10.1016/j.ibmb.2015.06.007. 2.4. Gel electrophoresis 1-dimensional SDS-PAGE-Sperm samples were solubilized in 2X LDS sample buffer as per manufacturers instructions (Invitrogen, Inc) and quantified using the EZQ Protein Quantitation Kit (Invitrogen, Inc). Protein fluorescence was measured using a Typhoon Trioþ (Amersham Biosciences/GE Healthcare) equipped with a 488 nm laser and 610 nm bandpass filter. ImageQuant TL software was used to analyze fluorescence data. A standard curve was generated using fluorescence data from control samples of known concentration and used to determine sperm sample concentration. A 1 mm 10% NuPAGE Novex Bis-Tris Mini Gel was set up using the XCell SureLock Mini-Cell system (Invitrogen) as per manufacturer instructions for reduced samples. 25 mg from each of three fractions containing a co-mixture of A- and E-sperm, purified A- and Esperm were loaded and samples electrophoresed for 35 min at 200 V. Following electrophoresis, the gel was stained using SimplyBlue SafeStain (Invitrogen, Inc) and destained as per manufacturer instructions. Gels were then imaged using a BioRad gel imager and images imported into Adobe Photoshop and Illustrator for labeling and annotation. 2-Dimensional IEF-PAGE- Two-dimensional electrophoresis was performed according to the carrier ampholine method of isoelectric focusing (Burgess-Cassler et al., 1989; O'Farrell, 1975) by Kendrick Labs, Inc. (Madison, WI). Isoelectric focusing was carried out in a glass tube of inner diameter 2.3 mm using 2.0% pH 3e10 isodalt Servalytes (Serva, Heidelberg, Germany) for 9600 V-hrs. One ug of an IEF internal standard, tropomyosin, was added to each sample. This protein migrates as a doublet with lower polypeptide spot of MW 33,000 and pI 5.2; The enclosed tube gel pH gradient plot for this set of Servalytes was determined with a surface pH electrode. After equilibration for 10 min in buffer “O” (10% glycerol, 50 mM dithiothreitol, 2.3% SDS and 0.0625 M tris, pH 6.8), each tube gel was sealed to the top of a stacking gel that overlaid a 10% acrylamide slab gel (0.75 mm thick). SDS slab gel electrophoresis was carried out for about 4 h at 15 mA/gel. The following proteins (Sigma Chemical Co., St. Louis, MO and EMD Millipore, Billerica, MA) were used as molecular weight standards: myosin (220,000), phosphorylase A (94,000), catalase (60,000), actin (43,000) carbonic anhydrase (29,000) and lysozyme (14,000). These standards appear at the basic edge of the Coomassie Brilliant Blue R-250stained gel. The Coomassie Blue R-250 stained gel was dried
Fig. 1. Separation of apyrene and eupyrene sperm using a panning purification method. (A) Dissected reproductive tract showing the paired testis (T) connected to the paired seminal vesicles at its base (arrowhead) seen coiled downward away from the testis and then back up to the distal end (arrow) where seminal vesicle contents are observed (bracket) following puncture with a fine needle. (B) Higher magnification view of the released contents near the puncture site in panel A shows the two sperm morphs. The eupyrene bundles are highly birefringent coiled structures (arrowheads) embedded in a viscous proteinaceous material containing the free apyrene sperm which are seen as the wispy material surrounding the eupyrene sperm bundles. This viscous material is easily removed from the seminal vesicle and further purified using a panning method as further described in detail in the Supplementary movie. (C) The result of the panning procedure are highly purified eupyrene sperm bundles with characteristic coiled bundles.
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Fig. 2. Light microscopy images of purified sperm fractions. Each of three fractions obtained from the panning procedure, combined apyrene/eupyrene, eupyrene and apyrene, were washed a final time in 1ug/ml of DAPI and mounted on microscope slides. Sperm were imaged and using a Leica DMRE microscope equipped with Nomarski contrast and epifluorescence optics. Images were recorded using a Leica DFC500 camera and imported into Leica software. (AeC) respectively, showing Monarch butterfly (D. plexippus) sperm mixed A- and E-sperm, purified E-sperm, and purified A-sperm. (DeF) showing respectively the same categories as above for the Painted Lady butterfly (V. virginiensis). (GeI) showing respectively the same categories for the tobacco horned-worm (M. sexta). Note the bright white signal (G, arrows) showing the DAPI (nuclear DNA) signal at the tips of the eupyrene sperm bundles. No DAPI signal was observed in apyrene sperm (not shown). Bar ¼ 20 ym.
Fig. 3. SDS gel electrophoresis of purified sperm fractions from three lepidopterans, two butterflies, D. plexippus and V. virginiensis and the moth M. sexta. Each lane represents protein bands present in purified fractions of whole sperm (E/A), eupyrene sperm (E) and apyrene (A) sperm. Visual inspection of the gels reveals obvious differences in either the presence/absence or band intensities between the samples. Similar comparisons for the other two show clear differences in V. virginiensis and M. sexta. Ticks on left represent molecular weight markers (from top to bottom) of 250-, 150-, 100-, 75-, 50-, 37-, 25,- 20-, 15- and 10 kDa, respectively.
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between sheets of cellophane with the acid edge to the left. 2.5. Image analysis using a negative-image overlay technique We developed a simple, semi-quantitative method for 2D gel comparisons as follows. TIFF files of the E- and A-sperm 2D gels were imported into Photoshop (Adobe, Inc) in separate layers. The top layer image file is chosen (image order can be reversed and results compared) and the pixels values are reversed using the “invert” function. This creates a negative image of the upper layer which obscures the layer below. The gel image in the lower layer can be made visible by reducing the opacity of the inverted image. The level of opacity reduction is performed manually to optimize the visibility of the two layers. The top layer can then be manually moved around the center of the images to optimize the alignment of the spots that appear coincident. This utilizes the human brain's ability as an “image processor”. This process works best in alignment of regional areas of the gel as overall match is poor due to well known factors (gel stretching, cathodic drift, etc …). An example comparing A- and E-sperm is shown in Suppl. Fig. 1. 3. Results & discussion 3.1. The conundrum of sperm heteromorphism in the Lepidoptera Spermatogenesis in Lepidopterans produces two sperm morphs, a nucleated form that contains a haploid complement of DNA (eupyrene or E-sperm as used here), and an anucleated form that does not contain DNA (apyrene or A-sperm as used here). The purpose and persistence of a reproductive strategy that produces an entire class of sperm (A-sperm) incapable of participating directly in the transfer of genetic material, is unknown. Numerous hypotheses have been proposed to explain the presence and persistence of sperm heteromorphism, but no consensus has been reached as to either the evolutionary origins nor the functional €nder significance of apyrene sperm (discussed in detail in (Friedla et al., 2005)). One impediment to a deeper understanding of the lepidopteran system has been the absence of the molecular and cellular processes that generate the two sperm morphs. To better understand the biochemical makeup of E- and A-sperm, we developed a novel purification procedure that yields highly enriched fractions of each. The method exploits differences in the physical properties of the two morphs: apyrene sperm are smaller and released into the ductus deferens as individual sperm whereas the larger eupyrene sperm remain in bundles (Fig. 1). Two different criteria were used to establish the efficacy of the purification process; light microscopy to visualize sperm morphology and gel electrophoresis to monitor protein differences between A- and E-sperm. Taken together these results demonstrate the overall effectiveness of the panning procedure as discussed below. 3.2. Effective purification of eupyrene sperm via a novel panning method The Monarch butterfly (D. plexippus) was chosen for development of the panning process due to the high levels of sperm stored in the seminal vesicles (Fig. 1A). Seminal vesicle fluid is highly viscous facilitating transfer out of the seminal vesicle (Fig. 1B) and into either centrifuge tubes for purification of bulk AE sperm, or into another plastic dish for subsequent panning and purification. Once transferred and dispersed in an appropriate volume of buffer in a plastic dish, separation by panning (detailed in Suppl. Movie 1) is facilitated by differences in A- and E-sperm structure: A-sperm are released into the ductus deferens individually whereas E-sperm
Fig. 4. Two-dimensional gel electrophoresis of D. plexippus purified sperm fractions. Each panel shows protein spot patterns present in purified fractions of (Eupyrene/ Apyrene), (Eupyrene) and (Apyrene) sperm. Spot pattern differences between the three samples were analyzed using a negative-image overlay method (Supplemental Fig. 1). Regions showing distinct differences are shown (boxes, circles and ovals) with solid lines indicating presence, and dashed lines, absence of protein spots. The arrowhead on the stained gels marks the position of the internal standard protein, tropomyosin.
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remain bundled and embedded in a dense proteinaceous matrix that retains its integrity during dissection, transfer and panning (Fig. 1). Successive panning and transfer effectively separates Esperm (Fig. 1C; Suppl. Movie 1). We further assessed the utility of the panning technique using another butterfly, V. virginiensis, and a moth, Manduca sexta (Fig. 2). In each case, the panning method was effective in separating A-from E-sperm.
3.3. Gel electrophoresis reveals significant protein differences between A- and E-sperm To further characterize sperm, the three fractions of sperm (purified as described above) were subjected to 1-dimensional SDSPAGE and the protein patterns compared (Fig. 3). Cursory examination of the gels clearly shows a number of differences between each fraction. Although the precise number and identity of the differences apparent in these gels cannot be ascertained at this level of analysis, they do reflect differences in sperm morphs purified by the panning method, consistent with the 2D gels results described below. Two-dimensional IEF-PAGE separates proteins by isoelectric point and molecular weight thus providing a more detailed analysis of differences observed between the fractions. Visual inspection of the gels shows clear differences in protein spot patterns. Overall, compared to the A-sperm gel, E-sperm are more complex containing 239 clearly visible spots in E-sperm and 179 spots in Asperm (measured by manual spot counts). A more direct comparison using an negative-image overlay technique (see Materials and Methods and Supplementary file 1) among the three 2D gel images revealed clear regional differences between A- and E-sperm (Fig. 4). As anticipated, the AE-sperm gel contained all proteins recognized in either the A- and E-sperm and, as expected from the spot counts, the A-sperm gel contained most proteins absent from either the AE- or E-sperm gels (dashed boxes, Fig. 4C). Thus we can conclude that in general E-sperm are more biologically complex at the protein level.
4. Conclusion The panning technique provides a simple and efficient method for obtaining highly purified samples of apyrene and eupyrene sperm. The method was effective in separating the two sperm morphs as measured by both microscopy and gel electrophoresis. As such this work provides a solid foundation for future studies on the cellular and molecular basis of sperm dimorphism in Lepidopterans. For example, work in progress is using this methodology as input for high throughput shotgun proteomic experiments to determine the A- and E-sperm proteomes. Knowledge of the differences, and similarities, between these two sperm proteomes will facilitate future studies designed to understand the functional genomic and molecular evolutionary biology of this fascinating system.
Acknowledgments We are grateful for the expert knowledge, guidance and resources provided by Dr. Chip Taylor, Monarchwatch.org. We also thank Desiree Harpel, Channing Shives and Ann Ryan for invaluable technical assistance and animal rearing and maintenance. Meg Jamieson filmed and edited the supplementary video, for which we are extremely grateful. This work was supported by start-up funds to JRW from the University of Kansas.
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Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.ibmb.2015.06.007. References Amaral, A., Castillo, J., Ramalho-Santos, J., Oliva, R., 2014. The combined human sperm proteome: cellular pathways and implications for basic and clinical science. Hum. Reprod. Update 20, 40e62. Baker, M.A., Nixon, B., Naumovski, N., Aitken, R.J., 2012. Proteomic insights into the maturation and capacitation of mammalian spermatozoa. Syst. Biol. Reprod. Med. 58, 211e217. Beatty, R.A., Burgoyne, P.S., 1971. Size classes of the head and flagellum of drosophila species. Cytogenetics 10, 177e185. Birkhead, T.R., 2009. In: Hosken, D.J., Pitnick, S.S. (Eds.), Sperm Biology: an Evolutionary Perspective. Elsevier/Academic Press. Boggs, C.L., Gilbert, L.E., 1979. Male contribution to egg production in butterflies: evidence for transfer of nutrients at mating. Science 206, 83e84. Bressac, C., Hauschteck-Jungen, E., 1996. Drosophila subobscura females preferentially select long sperm for storage and use. J. Insect. Physiol. 42, 323e328. Burgess-Cassler, A., Johansen, J.J., Santek, D.A., Ide, J.R., Kendrick, N.C., 1989. Computerized quantitative analysis of coomassie-blue-stained serum proteins separated by two-dimensional electrophoresis. Clin. Chem. 35, 2297e2304. Cook, P.A., Wedell, N., 1999. Non-fertile sperm delay female remating. Nature 397, 486. Dacheux, J.L., Dacheux, F., 2014. New insights into epididymal function in relation to sperm maturation. Reproduction 147, R27eR42. Dorus, S., Busby, S.A., Gerike, U., Shabanowitz, J., Hunt, D.F., Karr, T.L., 2006. Genomic and functional evolution of the Drosophila melanogaster sperm proteome. Nat. Genet. 38, 1440e1445. Dorus, S., Freeman, Z.N., Parker, E.R., Heath, B.D., Karr, T.L., 2008. Recent origins of sperm genes in drosophila. Mol. Biol. Evol. 25, 2157e2166. Dorus, S., Skerget, S., Karr, T.L., 2012. Proteomic discovery of diverse immunity molecules in mammalian spermatozoa. Syst. Biol. Reprod. Med. 58, 218e228. Fischer, B.E., Wasbrough, E., Meadows, L.A., Randlet, O., Dorus, S., Karr, T.L., Russell, S., 2012. Conserved properties of drosophila and human spermatozoal mRNA repertoires. Proc. Biol. Sci. 279, 2636e2644. Friedl€ ander, M., Seth, R.K., Reynolds, S.E., 2005. Eupyrene and apyrene sperm: dichotomous spermatogenesis in lepidoptera advances. Insect. Physiol. 32, 206e308. Heliconius Genome Consortium, 2012. Butterfly genome reveals promiscuous exchange of mimicry adaptations among species. Nature 487, 94e98. Holman, L., Snook, R.R., 2008. A sterile sperm caste protects brother fertile sperm from female-mediated death in drosophila pseudoobscura. Curr. Biol. 18, 292e296. International Silkworm Genome Consortium, 2008. The genome of a lepidopteran model insect, the silkworm Bombyx mori. Insect. Biochem. Mol. Biol. 38, 1036e1045. LaMunyon, C.W., Eisner, T., 1994. Spermatophore size as determinant of paternity in an arctiid moth (Utetheisa ornatrix). Proc. Natl. Acad. Sci. U. S. A. 91, 7081e7084. LaMunyon, C.W., Ward, S., 1998. Larger sperm outcompete smaller sperm in the nematode caenorhabditis elegans. Proc. Biol. Sci. 265, 1997e2002. LaMunyon, C.W., Ward, S., 1999. Evolution of sperm size in nematodes: sperm competition favours larger sperm. Proc. Biol. Sci. 266, 263e267. Meves, F., 1903. Über oligopyrene und apyrene spermien und ihre Entstehung nach Beobachtungen an Paludina un Pygaera. Arch. Mikrosk. Anat. Entwicklungsmech 61, 1e84. O'Farrell, P.H., 1975. High resolution two-dimensional electrophoresis of proteins. J. Biol. Chem. 250, 4007e4021. Osanai, M., Kasuga, H., Aigaki, T., 1989. Isolations of eupyrene sperm bundles and apyrene spermatozoa from seminal fluid of the silkmoth, Bombyx mori. J. Insect Physiology 35, 401e408. Pitnick, S., 2009. Sperm morphological diversity. In: Birkhead, T.R., Hosken, D.J., Pitnick, S. (Eds.), Sperm Biology: an Evolutionary Perspective. Elsevier Science & Technology, Oxford, UK, pp. 69e149. Sahara, K., Takemura, Y., 2003. Application of artificial insemination technique to eupyrene and/or apyrene sperm in bombyx mori. J. Exp. Zool. A. Comp. Exp. Biol. 297, 196e200. Snook, R.R., Markow, T.A., Karr, T.L., 1994. Functional nonequivalence of sperm in drosophila pseudoobscura. Proc. Natl. Acad. Sci. U. S. A. 91, 11222e11226. Snook, R.R., 1997. Is the production of multiple sperm types adaptive? Evolution 51, 797e808. Snook, R.R., Karr, T.L., 1998. Only long sperm are fertilization-competent in six sperm-heteromorphic drosophila species. Curr. Biol. 8, 291e294. Swallow, J.G., Wilkinson, G.S., 2002. The long and short of sperm polymorphisms in insects. Biol. Rev. Camb. Philos. Soc. 77, 153e182. Wasbrough, E.R., Dorus, S., Hester, S., Howard-Murkin, J., Lilley, K., Wilkin, E., Polpitiya, A., Petritis, K., Karr, T.L., 2010. The Drosophila melanogaster sperm proteome-II (DmSP-II). J. Proteomics 73, 2171e2185. Whittington, E., Zhao, Q., Borziak, K., Walters, J.R., Dorus, S., 2015. Characterisation of the Manduca sexta sperm proteome: genetic novelty underlying sperm composition in lepidoptera. Insect Biochem. Mol. Biol. 62, 183e193.
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http://dx.doi.org/10.1016/j.ibmb.2015.02.01. You, M., Yue, Z., He, W., Yang, X., Yang, G., Xie, M., Zhan, D., Baxter, S.W., Vasseur, L., Gurr, G.M., Douglas, C.J., Bai, J., Wang, P., Cui, K., Huang, S., Li, X., Zhou, Q., Wu, Z., Chen, Q., Liu, C., Wang, B., Li, X., Xu, X., Lu, C., Hu, M., Davey, J.W., Smith, S.M., Chen, M., Xia, X., Tang, W., Ke, F., Zheng, D., Hu, Y., Song, F., You, Y., Ma, X., Peng, L., Zheng, Y., Liang, Y., Chen, Y., Yu, L., Zhang, Y., Liu, Y., Li, G., Fang, L., Li, J.,
Zhou, X., Luo, Y., Gou, C., Wang, J., Wang, J., Yang, H., Wang, J., 2013. A heterozygous moth genome provides insights into herbivory and detoxification. Nat. Genet. 45, 220e225. Zareie, R., Eubel, H., Millar, A.H., Baer, B., 2013. Long-term survival of high quality sperm: insights into the sperm proteome of the honeybee Apis mellifera. J. Proteome. Res. 12, 5180e5188.
