Post‐Ejaculatory Modifications to Sperm (PEMS)

Biol. Rev. (2020), 95, pp. 365–392. 365 doi: 10.1111/brv.12569 Post-ejaculatory modiﬁcations to sperm (PEMS)

Scott Pitnick1∗ , Mariana F. Wolfner2 and Steve Dorus1 1Department of Biology, Center for Reproductive Evolution, Syacuse University, Syracuse, NY 13244, USA 2Department of Molecular Biology and Genetics, Cornell University, Ithaca, NY 14853, USA

ABSTRACT

Mammalian sperm must spend a minimum period of time within a female reproductive tract to achieve the capacity to fertilize oocytes. This phenomenon, termed sperm ‘capacitation’, was discovered nearly seven decades ago and opened a window into the complexities of sperm–female interaction. Capacitation is most commonly used to refer to a specific combination of processes that are believed to be widespread in mammals and includes modifications to the sperm plasma membrane, elevation of intracellular cyclic AMP levels, induction of protein tyrosine phosphorylation, increased intracellular Ca2+ levels, hyperactivation of motility, and, eventually, the acrosome reaction. Capacitation is only one example of post-ejaculatory modifications to sperm (PEMS) that are widespread throughout the animal kingdom. Although PEMS are less well studied in non-mammalian taxa, they likely represent the rule rather than the exception in species with internal fertilization. These PEMS are diverse in form and collectively represent the outcome of selection fashioning complex maturational trajectories of sperm that include multiple, sequential phenotypes that are specialized for stage-specific functionality within the female. In many cases, PEMS are critical for sperm to migrate successfully through the female reproductive tract, survive a protracted period of storage, reach the site of fertilization and/or achieve the capacity to fertilize eggs. We predict that PEMS will exhibit widespread phenotypic plasticity mediated by sperm–female interactions. The successful execution of PEMS thus has important implications for variation in fitness and the operation of post-copulatory sexual selection. Furthermore, it may provide a widespread mechanism of reproductive isolation and the maintenance of species boundaries. Despite their possible ubiquity and importance, the investigation of PEMS has been largely descriptive, lacking any phylogenetic consideration with regard to divergence, and there have been no theoretical or empirical investigations of their evolutionary significance. Here, we (i) clarify PEMS-related nomenclature; (ii) address the evolutionary origin, maintenance and divergence in PEMS in the context of the protracted life history of sperm and the complex, selective environment of the female reproductive tract; (iii)describe taxonomically widespread types of PEMS: sperm activation, chemotaxis and the dissociation of sperm conjugates; (iv) review the occurence of PEMS throughout the animal kingdom; (v) consider alternative hypotheses for the adaptive value of PEMS; (vi) speculate on the evolutionary implications of PEMS for genomic architecture, sexual selection, and reproductive isolation; and (vii) suggest fruitful directions for future functional and evolutionary analyses of PEMS.

Key words: spermatozoa, morphogenesis, capacitation, hyperactivation, motility, seminal proteins, female reproductive tract, post-copulatory sexual selection, sperm competition, fertility.

CONTENTS I. Introduction ...... 366 II. Deﬁning PEMS and suggested nomenclature ...... 367 III. Elements of the life history of sperm ...... 368 (1) Sperm maturation rarely ends in the testes ...... 368 (2) The female reproductive tract is a complex, interactive and selective environment ...... 369 IV. Taxonomically widespread forms of PEMS ...... 370 (1) Sperm activation and chemotaxis ...... 370 (2) Dissociation of sperm conjugates ...... 370

* Author for correspondence (Tel.: +315 443 5128; E-mail: [email protected]).

V. A survey of PEMS throughout the kingdom Animalia ...... 371 VI. Hypotheses for the adaptive signiﬁcance of PEMS ...... 372 (1) H1: economy of sperm transfer ...... 375 (2) H2: protecting sperm from stress during transfer and storage ...... 375 (3) H3: aiding sperm reaching a critical location in the FRT ...... 375 (4) H4: aiding sperm to remain in a critical location in the FRT ...... 376 (5) H5: enhancing sperm longevity ...... 376 (6) H6: delivering male-derived materials to the female in a temporally and/or spatially critical manner 376 (7) H7: priming sperm for extragenic contributions to early embryogenesis ...... 378 (8) H8: female assessment of sperm quality ...... 379 VII. Evolutionary implications of PEMS ...... 380 (1) Genomic consequences of PEMS ...... 380 (2) PEMS and sexual selection ...... 380 (3) PEMS and reproductive isolation ...... 382 VIII. Future directions ...... 382 IX. Conclusions ...... 384 X. Acknowledgments ...... 384 XI. References ...... 384 XII. Supporting Information ...... 392

I. INTRODUCTION (Roldan & Gomendio, 2009) may not have been possible (Visconti, 2009). Between the 1870s and the 1950s, numerous research Capacitation in mammals has subsequently been inves- programs employed an in vitro approach to the study of tigated intensively [see online Supporting Information, fertilization in echinoderms, amphibians and mammals. Appendix S1 (Section VII.5); Gervasi & Visconti, 2016]. The These studies were fruitful in elucidating, for example, term is now used widely to refer to a combination of cellular processes that include specific molecular modifications to the interactions between sperm and eggs, and between sperm and + the cumulus cells surrounding the egg in mammals. However, sperm plasma membrane, increased Ca2 permeability, the and despite numerous erroneous claims to the contrary elevation of intracellular cyclic AMP levels, hyperactivation (reviewed by Austin, 1951; but see Moricard, 1950 as of motility, the induction of sperm protein tyrosine phospho- discussed in Austin, 1951), the goal of achieving fertilization rylation and, eventually, the acrosome reaction (Vadnais, in vitro in a mammal was stymied. The breakthrough came Galantino-Homer, & Althouse, 2007; Gadella & Boerke, in a pair of studies published by C. R. ‘Bunny’ Austin 2016; Gervasi & Visconti, 2016). These modifications occur and Min Chueh Chang in 1951. Working with rabbits naturally within the FRT, but can also be induced in vitro. and rats, and seemingly inspired by previous investigations Although capacitation is frequently suggested to be exclusive of the timing of fertilization relative to insemination (i.e. to mammals, evidence suggests that this phenomenon is more delays were not attributable to the time required by sperm taxonomically widespread (e.g. Nixon et al., 2016a, 2019b). to reach the site of fertilization, nor for large numbers of This point depends in part, of course, on the definition of sperm to accumulate there; e.g. Hammond, 1934) both ‘capacitation’. Numerous reports in the literature describe Austin (1951) and Chang (1951) showed that sperm must an array of post-ejaculatory modifications to sperm (PEMS) spend some minimum threshold amount of time within occurring in the FRT in a diversity of internally fertilizing the female reproductive tract (FRT) before fertilization can taxa, of which capacitation as described for mammals repre- occur. Specifically, sperm introduced into the periovarian sac sents but one example. Because there is no cohesive field of of the rat or the Fallopian tubes of the rabbit required several study encompassing such modifications, the descriptions are hours to lapse before fertilization was observed (Austin, mostly anecdotal and lack consistent nomenclature. Never- 1951; Chang, 1951). However, fertilization occurred swiftly theless, there is cause to postulate that critical modifications and efficiently if sperm had first spent 5 h within the uterus of to sperm within the FRT are the rule rather than the excep- another rabbit (Chang, 1951). Both authors concluded that tion for nearly all taxa with internal fertilization. Moreover, sperm must be physiologically modified within the FRT in recent evidence suggests female-induced modifications to some manner necessary to acquire the capacity to fertilize sperm, mediated by contact with ovarian/oviductal fluid oocytes. In the following year, Austin (1952) confirmed the released with eggs and forming a boundary layer around results under the condition of natural insemination in rats, them, may be widespread among externally fertilizing and he coined the term ‘capacitation’. Without the discovery species as well (Evans & Sherman, 2013). Alterations to of capacitation, the application of in vitro fertilization to assist sperm form and function resulting from sperm–female inter- reproduction by humans experiencing fertility problems actions may therefore have derived from deeply ancestral (Pacey, 2009) and by threatened and endangered species processes.

The depth of knowledge about the biology of PEMS varies being triggered by non-sperm seminal fluid constituents, or greatly among taxa, with the highest resolution studies having by being intrinsic to sperm (i.e. ‘programmed’ modifications been directed at capacitation (including hyperactivation) in that do not occur until after ejaculation). Alternatively, mammals, the attachment of proteins manufactured by male PEMS may be female-mediated, resulting, for example, accessory reproductive glands to the head and/or flagellum from (i) female-derived proteins, carbohydrates or lipids of sperm and their cleavage from sperm within the FRT binding to sperm, or female-derived exosomes fusing with of Drosophila, the shedding of extracellular envelopes by sperm (Aalberts, Stout, & Stoorvogel, 2014; Corrigan et al., lepidopteran sperm, and the activation of frog and toad 2014); (ii) female-mediated cleavage or dissolution of sperm sperm by egg jelly. These systems stand out for having components; and/or (iii) post-translational modifications to been the subject of molecular genetic analyses and, in sperm proteins. Sperm also undergo changes within the FRT rare cases, the subject of experimental approaches (e.g. as a consequence of aging, degradation or destruction that RNA interference (RNAi) knockdown or CRISPR–Cas9 has been shown to occur within the FRT across diverse knockouts; e.g. Fricke, Bretman, & Chapman, 2009; Findlay taxa (e.g. Brinton, Burgdorfer & Oliver, 1974; Picard, et al., 2014). By contrast, studies of PEMS in other taxa 1980; Longo et al., 1993; Viscuso et al., 1996; Burighel & are predominantly descriptive, including ultrastructural, Martinucci, 1994a; Sutovsky, 2003; Pizzari et al., 2008). We gross morphological or behavioural data (e.g. flagellar do not consider such phenomena to be PEMS. Rather, beat frequency and swimming trajectory) acquired using PEMS include those highly regulated and consistently transmission electron, scanning electron or light microscopy observed phenomena that are necessary for sperm to to compare sperm that have or have not had the opportunity progress (e.g. within the FRT), survive and/or eventually to interact with the FRT. Importantly, studies to discern fertilize ova. whether PEMS occur have not been carried out for the The majority of PEMS will be associated with alterations overwhelming majority of taxa. to sperm behaviour, such as hyperactivation in eutherian Given that PEMS are potentially widespread and an mammals and the widespread phenomena of activation important determinant of reproductive outcomes, yet in most and chemotaxis (see Section IV.1). However, one should cases understudied and poorly understood, we had seven not conversely presume that all changes to sperm behaviour goals in crafting this review. First, we clarify PEMS-related involve PEMS, since many determinants of sperm behaviour, nomenclature. Second, we promote a perspective of sperm including flagellar conformation, beat frequency and having a more protracted life history and maturational velocity, can all be influenced by interactions with the trajectory than traditionally considered, while highlighting environment (e.g. temperature, viscosity, architecture) and the FRT as the principle selective environment for sperm hence may arise without biochemical, physiological or over the majority of their life history in species with structural modifications to sperm (Werner & Simmons, 2008; internal fertilization. Third, we describe two taxonomically Yang & Lu, 2011; Lüpold & Pitnick, 2018). widespread types of PEMS: sperm activation and the The abbreviation ‘PEMS’ applies equally well to dissociation of sperm conjugates. Fourth, we review the all animals irrespective of their mode of reproduction occurrence and diversity of PEMS throughout the animal (e.g. external, internal, spermcasting, direct or indirect kingdom. Fifth, we consider alternative hypotheses for the spermatophore transfer) and is not restricted to the single, adaptive value of PEMS. Sixth, we address the evolutionary operational criterion of any specific modification being implications of PEMS for genomic architecture, sexual critical to sperm achieving the capacity for fertilization (i.e. selection and speciation. Finally, we suggest fruitful directions capacitation, sensu stricto). This latter attribute is important, for future functional and evolutionary analyses of PEMS. because it is hypothetically possible for sperm to have the Whereas our goal was to be exhaustive in reviewing examples capacity to fertilize despite failure to undergo some PEMS. of PEMS throughout the animal kingdom, the expansive Moreover, most investigations, across diverse animal taxa, biology related to PEMS restricted us to providing citations do not include explicit demonstrations that the modifications that could serve as entry points to further exploration for to sperm are required for fertilization competency, despite numerous, more general aspects of reproductive biology. it being evident that most of the examples would meet this criterion. For example, fertilization would not be possible in any species with conjugated sperm or sperm surrounded by II. DEFINING PEMS AND SUGGESTED a glycocalyx or outer vestment prior to successful execution NOMENCLATURE of the PEMS. In other cases, sperm would not migrate properly to the site of storage/fertilization or experience In order to facilitate a more cohesive and taxon-independent prolonged survival within the FRT without activation or field of enquiry, we encourage adoption of ‘PEMS’ when other PEMS. Hence, demonstrating capacitation, sensu referring to biochemical, physiological and/or structural stricto, is unlikely to be a priority for most investigators [but modifications to sperm occuring after ejaculation but note that a specific PEMS being a necessary prerequisite excluding modifications to sperm that are attributable to for fertilization has been shown for some non-mammalian sperm–egg interactions (Karr, Swanson, & Snook, 2009). species: e.g. hydrozoa (O’Rand, 1972, 1974); frogs (Shivers These modifications may be male-mediated, for example & James, 1970a); toads (Krapf et al., 2007, 2009)].

We suggest the canonical adoption of the term spermiogenesis, is defined as the morphogenesis of haploid, ‘capacitation’ to refer to the specific case of PEMS that round spermatids into spermatozoa within the testes (Gilbert involves molecular modifications to the sperm plasma & Barresi, 2016). It is widely recognized that sperm may membrane, increased Ca2+ permeability, the elevation of become modified or complete maturation after leaving the intracellular cyclic AMP levels, hyperactivation of motility, testes. In mammals, the epididymides are specialized for the induction of sperm protein tyrosine phosphorylation sperm modification, with proteins, glycoproteins and RNA and, eventually, the acrosome reaction that is widespread in potentially being added, lost and modified and the lipid com- eutherian mammals (e.g. Gervasi & Visconti, 2016; see ponent of membranes being altered as sperm pass through Section VII.5c in Appendix S1) and has recently been epididymides of monotremes (e.g. Djakiew & Jones, 1983; shown also to occur in a crocodile (Nixon et al., 2016a, Nixon et al., 2011, 2016b), marsupials (e.g. Temple-Smith & 2019b; see Section VII.4 in Appendix S1). This suggestion is Bedford, 1980) and eutherian mammals (e.g. Bedford, 1979; consistent with the term’s application by many contemporary Baker et al., 2005; Sullivan & Saez, 2013; Aalberts, Stout, & reproductive biologists, and underlies the commonly Stoorvogel, 2014; Skerget et al., 2015; Machtinger, Laurent, proferred opinion that capacitation is a phenomenon & Baccarelli, 2016; Sharma et al., 2018; Nixon et al., 2019a). predominantly restricted to mammals (e.g. Nixon et al., Although less thoroughly studied, similar changes to sperm 2016a, 2019b). This explicitly restrictive use of ‘capacitation’ may occur within the Wolffian duct of reptiles (e.g. Esponda will avoid confusion that has arisen by some studies & Bedford, 1987) and birds (e.g. Esponda & Bedford, 1985; applying the term more generally to PEMS. For example, Morris et al., 1987; Nixon et al., 2014) and the seminal ‘capacitation’ was used to refer to sperm modification in vesicles of insects (e.g. Riemann & Giebultowicz, 1992; T. L. the FRT in taxa including ticks (Oliver & Brinton, 1971), Karr, personal communication). For insects, sperm may be spiders (Brown, 1985), cockroaches (Hughes & Davey, additionally modified further downstream in the ejaculatory 1969), grasshoppers (Longo et al., 1993), flies (Makielski, duct as they are combined with secretions from the male 1966), butterflies (Friedlander,¨ Jeshtadi, & Reynolds, accessory reproductive glands and other secretory organs 2001), prosobranch snails (Bojat, Sauder, & Haase, 2001), (see Section V.3h in Appendix S1). Finally, as evidenced by pulmonate snails (Selmi, Bigliardi, & Giusti, 1989), octopuses descriptions of PEMS in diverse taxa (see Section V), the (Tosti et al., 2001) and frogs (Shivers & James, 1970a). The final steps in sperm maturation for many species take place inconsistent application of ‘capacitation’ may explain why within the female. The precise nature of sperm maturation no general term for the phenomenon of PEMS is applied in processes occurring in males and females is expected to be most relevant studies on non-mammalian species, whereas evolutionarily dynamic as they are largely determined by other studies use expressions such as ‘‘capacitation-like’’, sperm–female interactions, which are themselves expected ‘‘reminiscent of capacitation’’, ‘‘sperm reaction’’ and ‘‘sperm to evolve rapidly (Pitnick, Wolfner, & Suarez, 2009b). conditioning’’. Finally, some PEMS have been described for Among all cell types present in metazoan taxa, sperm mammal species that occur independently of, or in addition have a truly unique biology. They are the only cells that to, the traditionally described modifications associated with are cast forth from the soma to spend their lives in a capacitation (e.g. rotation of the sperm head into the ‘foreign’ environment. In the case of species with internal ‘thumbtack’ or ‘T’ orientation in Australian marsupials and fertilization, the ‘free-living’ portion of a sperm’s life takes the dissociation of sperm pairs in New World marsupials or place inside the FRT and can last for hours, days, months the conjugatated sperm of some flying squirrels, rodents and or years. A robust understanding of the biology of PEMS primates; Monclus & Fornes, 2016). Additional mammalian thus requires explicit consideration of the protracted life PEMS undoubtedly remain to be discovered, and delineating history of sperm from a behavioural ecology perspective. which qualify as capacitation is unlikely to advance a general How are sperm designed to maximize their survival as they understanding of the phenomena of sperm modification. navigate a spatially and temporally heterogeneous selective One goal of this review is to demonstrate that capacitation environment? Resolving structure–function relationships in mammals is simply one example of a much larger from an evolutionary perspective requires examination of phenomenon common to most, if not all, animals with fitness variation in the context of the underlying mechanisms internal fertilization (and some with external fertilization) at play within the selective environment (or an appropriate and to encourage a more cohesive field of investigation into proxy), and these criteria have rarely been met in studies of the molecular, cellular and evolutionary biology of PEMS. spermatozoa (Lüpold & Pitnick, 2018). Given the protracted life history of sperm, and the fitness consequences of properly executing PEMS and otherwise III. ELEMENTS OF THE LIFE HISTORY OF being properly designed for compatibility with the FRT, SPERM there are several reasons why it might be adaptive for sperm to complete their maturation within the FRT. First, the function of some PEMS may be to deliver male-derived (1) Sperm maturation rarely ends in the testes materials to the female and, hence, sperm changes associated Spermatogenesis is the origin and development of spermato- with delivery cannot be completed until sperm have reached zoa from germ cells. The post-meiotic portion of this process, the proper place and time within the FRT (see Section VI.6).

Second, sperm must perform numerous functions within the mediated by FRT traits (morphological, cellular, biochemical FRT, and there may be no single optimal design serving and immune) that interact directly with the ejaculate all functions (see Section VI for a full discussion). Selection to influence the migration of sperm, their maintenance may have shaped the maturational trajectory of sperm to and modification, and their relative competitiveness for match their functional life history, as it has with evolution of fertilization. Because the secretory biology of the FRT the soma. Further, it would be advantageous to coordinate has historically been understudied and generally is poorly the timing of PEMS with critical events occurring within characterized and understood, the specific mechanisms the FRT, and hence for sperm to use FRT characteristics underlying the majority of these interactions have yet to as proximate triggers for the modifications. Third, because be determined. Fortunately, interest in the molecular and of FRT variation within species (e.g. Lüpold et al., 2013), it functional biology of FRT secretions is rapidly growing and, may be adaptive for sperm to delay their maturation until thanks to recent advances in proteomics and transcriptomics, they are in the FRT and then exhibit some plasticity to our understanding of sperm–FRT interactions is expanding conform to the specific biochemical, physiological and/or (McDonough et al., 2016). morphogical FRT conditions in which they find themselves. Taxonomically diverse examples of sperm–female Adaptive plasticity in sperm form and function has been interactions critical to sperm performance and survival demonstrated for diverse taxa, including insects, ascidians, within the FRT are being revealed. Genetic manipulation fish and birds (e.g. Pizzari et al., 2003; Rudolfsen et al., 2006; of the secretory cells of the spermathecae and parovaria Thomas & Simmons, 2007; Crean & Marshall, 2008; Ota in Drosophila melanogaster supports the importance of FRT et al., 2010), albeit not explicitly with regard to PEMS. secretions for fertility, sperm storage and normal ovulation (Anderson, 1945; Allen & Spradling, 2008; Schnakenberg, (2) The female reproductive tract is a complex, Matias, & Siegal, 2011). In both the honeybee, Apis mellifera, interactive and selective environment andinthebollweevil,Anthonomus grandis,secretionsofthe There is tremendous variation among species in female spermathecal gland have similarly been shown to contribute reproductive ecology, remating behaviour and FRT to sperm activation and their continued motility (Koeniger, morphology, physiology and biochemistry (Eberhard, 1996; 1970; Ruttner & Koeniger, 1971; Villavaso, 1975). The Pitnick, Wolfner, & Suarez, 2009b; Orr & Brennan, 2015; sperm of A. mellifera can survive for decades within the McDonough et al., 2016). There is also growing evidence FRT, and recent proteomic analyses have revealed that of extensive within-population genetic variation in FRT spermathecal fluid contains a large, integrated network of traits that influence sperm performance and fate (Lüpold proteins that includes enzymes of energy metabolism and et al., 2012, 2016) and that such traits diversify rapidly (e.g. antioxidant defence (Baer et al., 2009a, 2009b;Polandet al., Simmons & Fitzpatrick, 2019). As a consequence, the FRT 2011). Ovarian fluid in a fish with internal fertilization, the may generate diversifying selection on sperm, including guppy Poecilia reticulata, has been shown experimentally to PEMS. For example, one of the most robust patterns in reduce the temporal decline in sperm viability (Gasparini comparative reproductive biology is the co-diversification & Evans, 2013). In the Chinese soft-shelled turtle, Pelodiscus of sperm and FRT morphology, as observed in diverse sinensis, spermatogenesis is seasonal and, following spermia- taxa including several families of flies and beetles, moths, tion, sperm spend many months within the male epididymis snails, frogs, birds and mammals (reviewed in Pitnick, and the female oviduct, respectively (Zhang et al., 2008). Wolfner, & Suarez, 2009b). In fact, among species of diving The epithelial cells of both tissues have distinctive secretory beetles (Dytiscidae), the evolutionary remodelling of different functions that are believed to contribute to the protection components of the FRT explains a significant amount of the and nourishment of sperm (Han et al., 2008; Bian et al., variation in sperm length, sperm-head shape, the presence 2013). In mammals, the complex epithelial folds, channels, or absence of conjugation, and conjugate size and length microgrooves and mucous of the FRT create a highly (Higginson et al., 2012b). selective environment through which sperm must navigate, The selective forces underlying widespread sperm–FRT significantly reducing the population of sperm that enter the co-diversification are not well understood. Each sex-specific oviduct from the uterotubal junction (Coy et al., 2012; Holt trait can theoretically generate selection on the other (i.e. & Fazeli, 2015, 2016a; Tung et al., 2015). coevolution) as a consequence of both natural/ecological The proteomics and transcriptomics of the mammalian selection (Reinhardt, Dobler, & Abbott, 2015b)and oviduct microenvironment has revealed anatomic regions post-copulatory sexual selection (Birkhead, Møller, & with distinct, hormonally regulated molecular profiles (Buhi, Sutherland, 1993; Keller & Reeve, 1995; Eberhard, 1996; Alvarez, & Kouba, 2000). Recent transcriptome studies of Yasui, 1997; Snook, 2005; Pitnick, Wolfner, & Suarez, 2009b; the oviduct in the pig and human have established the Orr & Brennan, 2015; Firman et al., 2017; Lüpold & Pitnick, sensitivity of oviduct epithelial cells and secretions to respond 2018). There is also some empirical evidence, albeit limited, differentially to the presence of sperm (Alminana et al., suggesting that FRT design may evolve first, with sperm form 2014; Artemenko et al., 2015). The oviductal epithelium then evolutionarily tracking such changes (i.e. compensatory of eutherian mammals also plays an important role in evolution; Miller & Pitnick, 2002; Higginson et al., 2012b). sperm storage and capacitation, including hyperactive Regardless, virtually all such selection is expected to be motility (Coy et al., 2012). Interestingly, proteins involved in

Biological Reviews 95 (2020) 365–392 © 2019 Cambridge Philosophical Society 370 Scott Pitnick and others oviduct–sperm binding, carbohydrates in the apical cells of (metabolism and motility) and chemotaxis, which describes the epithelium and glycosylated proteins in the sperm head, changes in sperm flagella waveform (and hence swimming all exhibit pronounced variation among species, suggesting path) in order to move up a chemoattractant gradient, may be species specificity in the biochemistry of this ejaculate–female further stimulated by molecular signals (e.g. small peptides interaction (Suarez, 2008; Talevi & Gualtieri, 2010). Finally, and other molecules) that are released from unfertilized across taxa as diverse as polychaete worms, scale insects, eggs, the FRT epithelium or found in ovarian fluid or jelly mites and ticks, crustacea, clams, snails, ascidians, frogs, surrounding eggs and then bind to receptors on the sperm’s snakes, birds and eutherian mammals, the epithelium of surface (Miller, 1985; Ward & Kopf, 1993; Eisenbach, 1999; the FRT has been observed to interact directly with sperm Morisawa & Yoshida, 2005; Eisenbach & Giojalas, 2006; through sperm binding or embedding (reviewed in Pitnick, Watanabe et al., 2010; Evans & Sherman, 2013). Wolfner, & Suarez, 2009b). Perhaps less well known are the myriad examples of sperm Recognizing this protracted life history of sperm with motility being initiated by more dramatic structural PEMS maturation spanning both sexes, we predict molecular and following insemination. For example, in spiders, many insects biochemical continuity between the male reproductive tract and some crustacea, sperm do not become motile within the (MRT) and the FRT, which should be manifest in patterns of FRT until a rigid outer sheath, coat or glycocalyx has been sex- and organ-specific gene expression. We further predict removed (e.g. Alberti, 1990, 2000; Lupetti, Mercati, & Dallai, that such continuity will be evolutionarily dynamic with 2001; Friedlander,¨ Seth, & Reynolds, 2005; Matzke-Karasz, variation across taxa correlated with diversification in the Smith, & Heb, 2017), or until an accessory membrane extent of PEMS. We develop this concept and its genomic has been degraded to permit the flagellum to unkink or consequences in greater detail below (see Section VII.1). uncoil (Dallai, 1972; Dallai et al., 2003, 2004). In the fungus gnat, Sciara coprophila, sperm activation is associated with the evacuation of a large portion of the mitochondrial derivative (Makielski, 1966; Phillips, 1966a, 1966b). The sperm of IV. TAXONOMICALLY WIDESPREAD FORMS many tick species must undergo a dramatic metamorphosis OF PEMS and elongation within the FRT before motility is possible (Oliver & Brinton, 1971; Brinton, Burgdorfer, & Oliver Jr., In addition to capacitation by eutherian mammal sperm, 1974). These examples are described in Appendix S1. other specific classes of PEMS that are generally well known include sperm activation, chemotaxis, and the dissociation (2) Dissociation of sperm conjugates of sperm conjugates. Because these phenomena have been widely investigated and the subject of previous reviews, Sperm conjugation refers to the phenomenon of inseminated we only briefly describe them below before proceeding sperm being physically bound to one another (reviewed to detailed descriptions of the myriad, lesser-known and by Immler, 2008; Pitnick, Hosken, & Birkhead, 2009a; often taxonomically restricted forms of PEMS (Section V). Higginson & Pitnick, 2011; Monclus & Fornes, 2016). Note that in the taxon-specific descriptions of PEMS (see Conjugation can be primary, with all ‘sibling’ descendants of Appendix S1), we mention sperm activation and dissociation each spermatogonium remaining attached to one another of conjugates when they co-occur with other forms of PEMS. rather than dissociating at the end of spermatogenesis. However, we have excluded taxonomic groups from Section Alternatvely, conjugation can be secondary, with sperm V for which the only known PEMS are those associated with individualizing within the testes and later, within the sperm activation, chemotaxis and conjugate dissociation. epididymides, seminal vesicles or the FRT, binding together in a species-specific manner (Higginson & Pitnick, 2011). Conjugation has arisen independently numerous times (1) Sperm activation and chemotaxis across diverse taxa, including annelid and polychaete For many internally and externally fertilizing species, sperm worms, gastropod molluscs, myriapods, spiders, insects, and activation (i.e. the acquisition of full motility) is only achieved monotreme, marsupial and placental mammals (Immler, after spawning/ejaculation, whereas sperm within the male 2008; Pitnick, Hosken, & Birkhead, 2009a; Higginson & reproductive tract are observed to be immotile or only weakly Pitnick, 2011; Higginson et al., 2012a; Monclus & Fornes, motile. The cell signalling mechanisms underlying sperm 2016). It is manifested in diverse ways (e.g. Fig. 1), from paired activation have been the subject of intense investigation and sperm (e.g. nearly all species of New World opossum; Biggers numerous reviews (e.g. Ward & Kopf, 1993; Darszon et al., & Creed, 1962; Moore & Taggart, 1995) to loosely formed 1999; Morisawa & Yoshida, 2005; Miller et al., 2016; Tosti sperm trains involving up to hundreds of sperm (e.g. some &Men´ ezo,´ 2016) and so will only be briefly addressed here. species of muroid rodent; Immler et al., 2007) to conjugates Motility is triggered by the binding of ligands to sperm comprising thousands of sperm that possess a nearly receptors and/or the opening or closing of ion channels. crystalline exactness in their structural organization (e.g. Exposure of sperm to a variety of cations or to changes in the diving beetle Hygrotus sayi; Higginson & Pitnick, 2011). osmotic pressure following dilution in fresh water, salt water Ultrastructural analyses have further revealed a diversity of or within the environment of the FRT have been shown cellular, extracellular and mechanical mechanisms by which to initiate motility across diverse taxa. Sperm chemokinesis conjugation is achieved (reviewed by Afzelius & Dallai,

dissociation of conjugates prior to fertilization clearly repre- sents a dramatic example of structural PEMS. Such dissociation rarely occurs prior to conjugates arriving in the female’s sperm-storage organ(s), and in many cases only after prolonged storage or immediately prior to fertilization (Rodger & Bedford, 1982; Higginson & Pitnick, 2011). The mechanisms by which sperm within conjugates dissociate from one another are generally unknown and postulated to involve an active female secretion (Higginson & Pitnick, 2011). Never- theless, the only identiﬁed candidate (in the moth Bombyx mori) is a product of the male’s ejaculatory duct (Osanai, Kasuga, & Aigaki, 1989a; Aigaki et al., 1994; Osanai & Isono, 1997).

V. A SURVEY OF PEMS THROUGHOUT THE KINGDOM ANIMALIA

The occurrence of PEMS has been convincingly demonstrated for a multitude of diverse taxa (described in detail in Appendix S1), including hydras, bryozoans, clams (see Fig. 2C, D), snails, octopuses (see Fig. 3), polychaete worms (see Fig. 2A, B), ticks (see Fig. 4), spiders (see Fig. 5), crustaceans, insects [e.g. springtails (see Fig. 6), jumping bristletails (see Fig. 7), grasshoppers, cockroaches, beetles (see Fig. 1), honeybees, butterfles and flies (see Figs 8 and 9)], tunicates (see Fig. 10), fish, salamanders, frogs and toads, turtles (see Fig. 11), crocodiles, birds, monotremes, marsupials (see Fig. 12) and placental mammals. It is important to note that, among the taxon-specific PEMS described in Appendix S1, there is tremendous variation in the extent to which systems have been investigated and in the experimental tools employed. Consequently, we have a relatively sophisticated understanding of the cellular and molecular mechanisms underlying PEMS in model systems such as eutherian mammals (i.e. mouse, rat, rabbit and human) and the fruit fly Drosophila melanogaster. By contrast, our understanding of PEMS for the majority of taxa is restricted to what can be inferred from ultrastructural com- parisons between sperm obtained from the MRT and FRT. In the descriptions provided (see Appendix S1), we have striven to be explicit about methods and to share authors’ Fig. 1. Sperm conjugates of the whirligig beetle, Dineutus sp.: conclusions and interpretations of their findings. We describe (A) conjugates from the male ejaculatory duct under differential more generalized, taxon-specific aspects of the reproductive interference contrast microscopy; (B) conjugates stained with biology whenever it was deemed necessary to understand the 4 ,6-diamidino-2-phenylindole (DAPI) to show the organization described PEMS. of sperm heads along the spermatostyle; (C) conjugate from the Several general conclusions can be drawn from this female spermatheca in the process of sperm dissociation; (D) survey (Appendix S1). First, mammals can exhibit multiple bundle of spermless spermatostyles from the spermatheca of a types of PEMS, meaning modifications in addition to wild-caught female. Photomicrographs by S. Pitnick. the well-studied capacitation. Second, modifications to the plasma membrane of sperm and those related to the 1987; Hayashi, 1997; Higginson & Pitnick, 2011; Monclus acrosome reaction are by no means restricted to mammals. & Fornes, 2016). Third, some forms of PEMS, such as conjugate dissociation Whereas the adaptive value of conjugation is unknown in are taxonomically widespread (albeit they may be relatively most cases, it often operationally facilitates social cooperation rare, being found in relatively few species within a taxon; among sperm for the purpose of movement through the FRT Higginson & Pitnick, 2011). Fourth, insects as a group are (reviewed by Immler, 2008; Pizzari & Foster, 2008; Higgin- especially diverse with regard to the different forms of PEMS son & Pitnick, 2011). For all taxa with sperm conjugation, the exhibited. Fifth, some forms of PEMS are particularly rare,

Fig. 2. Tranmission electron micrographs showing post-ejaculatory modifications to sperm (PEMS) in the form of digitate processes (examples indicated by arrows) formed from the sperm periacrosomal plasmalemma in order to strengthen contact with (A, B) the female spermathecal cell membrane in the polychaete worm, Spirorbis spirorbis, and (C, D) the female gill filament in the brooding clam, Mysella tumida. a, acrosome; f, flagellum; m, mitochondrion; n, nucleus; *, specialized contacts between sperm and spermathecal cell membranes with scalariform junctions. Adopted with permission from (A, B) Daly & Golding (1977); (C, D) O´ Foighil (1985b). Scale bars: A, 0.5 μm; C, 2 μm; D, 0.4 μm.

Fig. 3. Scanning electron micrographs of Octopus vulgaris spermatozoon collected from (A) the spermatophore, with an intact outer membrane covering the acrosome, and (B) from the female oviducal gland, following post-ejaculatory modifications to sperm (PEMS) to reveal the corkscrew-shaped acrosome. Inset in A shows magnification of the acrosomal region. Arrows indicate indentations separating the acrosome, nuclear and midpiece regions. Adopted with permission from Tosti et al. (2001). Scale bars: A, 3.0 μm; B, 2.0 μm. including the attachment of seminal fluid proteins to sperm VI. HYPOTHESES FOR THE ADAPTIVE or the release of sperm-bound material into the FRT. It SIGNIFICANCE OF PEMS is important to recognize that such rarity may strictly be a function of limited investigation of these phenomena Our understanding of variation in PEMS across taxa is because they are difficult to observe and to study. too incomplete to draw conclusions about its evolutionary

Fig. 4. Schematic diagram of different morphological stages of post-ejaculatory modifications to sperm (PEMS) of the tick, Amblyomma dissimili. mp, motile processes. Adopted with permission from Reger (1963). diversification. Despite an extensive literature on capacita- kingdom. Modifications to sperm structure and physiology tion in mammals, our knowledge of its comparative biology vary tremendously, as well as in the degree and nature of is scant (but see Fan, Lefebvre, & Manjunath, 2006; Lefeb- interaction(s) with the FRT that induce the modifications vre et al., 2007). Because there is variation among mammal (albeit female contributions are largely unknown). It is thus species in temporal and mechanistic aspects of the formation likely that PEMS have arisen in response to a diversity of of the sperm reservoir, sperm longevity, and in sperm–egg evolutionary selection pressures, and the nature of selection interactions during fertilization (Holt & Fazeli, 2016b), there underlying the evolutionary maintenance of PEMS may vary may be correlated variation among species in the timing among related species. In considering alternative hypotheses and molecular mechanisms of capacitation. In general, the- to explain the evolutionary origin and maintenance ory would predict that PEMS diverge rapidly. Reproductive of species-specific PEMS, it is helpful to recognize traits, especially those involved in male–female interactions, that, for internally fertilizing species, sperm perform tend to evolve quickly (e.g. Swanson & Vacquier, 2002; multiple pre-fertilization actions within the female. Between Haerty et al., 2007), post-copulatory sexual selection is known insemination and fertilization, sperm must successfully to be a powerful agent of diversification (Simmons, 2001; migrate and/or be transported to specialized sperm-storage Pitnick & Hosken, 2010), and sperm and FRT traits are organs [e.g. the spermatheca(e) and/or seminal receptacle] notorious for evolving rapidly (Pitnick et al., 2009a, 2009b; or a site of quasi-specialized, short-term storage (e.g. the Simmons & Fitzpatrick, 2019). Congeneric species have been sperm reservoir in mammals), successfully compete with demonstrated to have diverged in sperm–FRT interactions competitor sperm for a position within the sperm-storage site in the case of internal fertilization (Howard et al., 2009; and/or to engage in fertilization, survive and remain viable Manier et al., 2013a, 2013b, 2013c), and in sperm–ovarian in storage for a period lasting from hours to decades, and fluid interactions in the case of external fertilization (Yeates exit the storage site and migrate to the site of fertilization et al., 2013) that critically determine reproductive outcomes. at the proper time, all before interacting with an oocyte Further, there can be high heritability of both sperm to form a zygote. Additionally, sperm (or seminal fluid) traits and FRT traits that determine sperm handling (Sim- components may provide material support in the form of mons & Moore, 2009; Lüpold et al., 2012, 2013, 2016), nutrients that may increase the number of eggs produced, and there is even adaptive, within-population variation in egg size, or otherwise enhance egg defence or embryonic sperm–ovarian fluid interactions pertaining to chemoattrac- viability (Simmons & Parker, 1989; Gwynne, 2008). Sperm tion in echinoderms and molluscs (Evans & Sherman, 2013). (and/or seminal fluid) may also provide signals influencing a The broad survey of PEMS in diverse taxa presented in multitude of female physiological functions that impact, for Section V does not reveal rates and patterns of diversification. example, oogenesis, ovulation, immune function, feeding and It does, however, suggest that PEMS may have arisen remating (Ravi Ram et al., 2005; Poiani, 2006; Avila et al., independently numerous times throughout the animal 2011). These functions have been shown (or are expected)

Fig. 5. Sperm of the spider Caponina alegre (A–D) before and (E) after post-ejaculatory modifications to sperm (PEMS). (A–D) Reconstruction of a synspermium. The image stack used for the three-dimensional reconstruction is stored in MorphDBase (https:// www.morphdbase.de?P_Michalik_20120927-M-3.1). (A) Numerous membrane-bound vesicles are attached to the vesicular area, enclosing the spermatozoa. (B) The main cell components of the four fused spermatozoa are coiled within the vesicular area of the syncytium, but not twisted around each other. (C) The prominent extremely elongated nucleus of one spermatozoon is coiled 2.5 times around the centre of the syncytium into which the axoneme finally opens. (D) Cross-section through a synspermium showing the arrangement of the coiled sperm components in the periphery of the syncytium, leaving the centre only filled with the vesicular area. (E) Schematic drawing of the main components of a post-PEMS, mature spermatozoon. AC, acrosomal complex (acrosomal vacuole and acrosomal filament); AF, acrosomal filament; Ax, axoneme; IF, implantation fossa; peN, post-centriolar elongation of nucleus; prcN, precentriolar region of nucleus. Adopted with permission from Lipke & Michalik (2012). to involve some degree of ejaculate-female interaction (Ravi spermatogenesis within the male, with a division of labour Ram & Wolfner, 2007; Pitnick, Wolfner, & Suarez, 2009b) among sperm types, each specialized to perform different and may be associated with or reliant upon PEMS. functions within the female (Swallow & Wilkinson, 2002; PEMS should be expected to arise if ‘one size does not fit Till-Bottraud et al., 2005). Third, selection can fashion a tra- all’ regarding the optimal design of sperm for the execution of jectory that includes multiple, sequential phenotypes that are all of the above activities. Selection shapes the development specialized for stage-specific functionality (i.e. PEMS). To the of organisms such that phenotypes change throughout an extent that these are alternative evolutionary outcomes, we organism’s life history (e.g. insect holometabolism). When predict comparative studies to reveal fewer PEMS in species optimal sperm design differs for different functions, selection with sperm heteromorphism. But note that, as demonstrated can respond in three different ways. First, it can favour a by some species of Lepidoptera, the second and third single, best compromise phenotype (a ‘jack of all trades and strategies can co-occur (see Section V.3g in Appendix S1). master of none’), which could be achievable in the male Note that none of the following nine hypotheses are reproductive tract. Second, it can favour heteromorphic mutually exclusive, and that multiple selection pressures

Fig. 6. (A) Scanning electron micrograph (SEM) of two rolled spermatozoa showing the long extra-acrosomal structure (‘peduncle’) of the collembolan Allacma fusca. Inset, SEM of a single rolled sperm showing the acrosome and peduncle. (B) Schematic reconstruction of a spermatozoon of Orchesella villosa. Sperm components form several spires within the same plasma membrane surrounding material within an ‘extracellular’ cavity. A, acrosome; EAS, extra-acrosomal structure; Ex, extracellular cavity; sp, spermatozoon. Adopted with permission from (A) Fanciulli et al. (2017); (B) Dallai et al. (2004). may shape PEMS in any given species. In fact, the known (2) H2: protecting sperm from stress during biology of many PEMS is consistent with the predictions of transfer and storage multiple of the hypotheses described below. However, we are Another hypothesis for PEMS relates to the fact that sperm unaware of any experimental tests of any of these hypotheses, may be subject to considerable stress, both physical (i.e. or of comparative tests of hypotheses that contrast the shearing forces) during ejaculation and chemical (e.g. reactive reproductive biology of related species that differ in the oxygen species) during storage in the FRT. Transport presence or form of PEMS. through certain regions of the FRT may also be highly selective of sperm due to physical barriers, chemical (1) H1: economy of sperm transfer barriers and leukocytic/phagocytotic responses to copulation One hypothesis for the origin of PEMS is that males can (Birkhead, Møller, & Sutherland, 1993; Arnqvist & Rowe, transfer many more sperm per copulation if a substantive 2005; Suarez, 2006). Due to the often lengthy interval portion of the growth component of sperm morphogenesis between insemination and fertilization (Birkhead & Møller, occurs post-insemination. This hypothesis, proposed by 1993; Neubaum & Wolfner, 1999; Orr & Brennan, 2015; Brinton, Burgdorfer, & Oliver Jr. (1974), is consistent with Holt & Fazeli, 2016b), sperm may further be subject to the PEMS of some tick species, where sperm increase in size oxidative damage in the FRT (Reinhardt et al., 2015a, up to tenfold within the FRT (Fig. 4; e.g. Mothes & Seitz, 2015b). Sperm are largely transcriptionally quiescent and 1981). Enhancing efﬁciency of sperm transfer has also been thus unable to deploy a full repertoire of repair mechanisms postulated as an explanation for sperm conjugation (Dallai to respond to these stresses (Dorus & Karr, 2009). If the & Afzelius, 1985; Afzelius & Dallai, 1987). This hypothesis optimal design of sperm for transfer, transport and/or storage is unlikely to serve as a general explanation, however, as differs from that for fertilization, then selection may have we are not aware of any other taxa in which PEMS involve favoured PEMS. Throughout their life history within the increases in sperm size, and it is an unlikely explanation for FRT, sperm may have to modify attributes that enhance sperm conjugation (Higginson & Pitnick, 2011). We are also their survival during the early stages in the reproductive skeptical of this selective explanation for tick PEMS, as it is process in order to achieve and retain the capacity to fertilize. typically the storage capacity of females that is limiting rather Hypothesis 2 is generally supported by a diversity of PEMS, than the number of sperm transferred. Hypothesis 1 predicts including delayed activation of motility, the rigid glycocalyx that, for taxa with PEMS involving an increase in sperm size of many insect sperm, the encapsulation of spider sperm, and or the tight packaging of sperm for transfer, there will be a thickened periacrosomal membranes that limit premature negative association between the expression of PEMS and acrosomal reactions and capacitation in mammals. the degree of female-biased sexual size dimorphism (as small male size may limit investment per ejaculate). (3) H3: aiding sperm reaching a critical location in Another variation of Hypothesis 1, proposed by the FRT Matzke-Karasz, Smith, & Heb (2017) as a possible adaptive explanation for the PEMS of ostracod crustacea (i.e. shedding Independent of protection from stressors (H2), PEMS may of an outer, ﬁbrous coat) is enhanced organization of be hypothesized as adaptations to enhance sperm transport. transferred and stored sperm in the case of giant sperm Two well-studied PEMS support this hypothesis. First, and a small FRT. hyperactivation in eutherian mammals has been interpreted

competition to occupy limited sperm-storage space within the FRT (e.g. Miller & Pitnick, 2002; Pattarini et al., 2006), and such selection can drive the evolution of extreme sperm traits (L¨upold et al., 2016). That PEMS are important for this is supported by the numerous PEMS that appear to enhance sperm storage, in some cases through fusing with, binding to, or embedding in the epithelial cells of the FRT. Examples include loss of most of the acrosomal membrane from the sperm of the octopus, O. vulgaris,toexposea screw-shaped acrosome, thus permitting sperm to drill into the epithelial cells of the spermatheca (Froesch & Marthy, 1975; Tosti et al., 2001), growth of long slender digitations from the periacrosomal plasma membrane from sperm after embedding in epithelial cells in the gastropod snail, C. montanum (Giusti & Selmi, 1985) and the polychaete worms, S. spirorbis (Daly & Golding, 1977) and P. remota (Alikunhi, 1951; Westheide, 1988), growth of ﬁne, thread-like extensions of the periacrosomal plasmalemma of sperm in the clam, M. tumida, that aid in attachment to the female’s gills (O´ Foighil, 1985b), and adaptations for binding to the oviduct epithelium to form the sperm reservoir in eutherian mammals (Suarez, 2002).

(5) H5: enhancing sperm longevity As discussed in Section III.2, females of most taxa have specialized organs for sperm storage with associated secretory cells or glands that provide an environment conducive to the long-term viability of sperm. As a consequence, sperm can survive within the FRT from days to decades, depending on the species. The strength of selection for sperm longevity, Fig. 7. Schematic drawing of a spermatozoon of the jumping and hence any associated PEMS that may extend longevity, bristletail, Machilis distincta, from the female spermatheca but will depend upon the sperm-storage capacity of females, rate prior to post-ejaculatory modifications to sperm (PEMS). of sperm use (which depends on fecundity and sperm use Adopted with permission from Dallai (1972). efficiency), female remating interval, and the mechanisms of sperm competition (see Section VII.2). Putative examples as an adaptation to assist sperm in their release from the of PEMS that may have been selected to enhance sperm oviductal sperm reservoir and movement through mucous longevity include those associated with sperm remaining in secretions in reaching the oocyte (Suarez & Pacey, 2006). The either an inactive or a reduced metabolic state. For example, second example is sperm conjugation and their dissociation the sperm of spiders remain coiled and inactive within after reaching the site of sperm storage or fertilization capsules for prolonged periods within the spermathecae (e.g. (Higginson & Pitnick, 2011). Based on general hydrodynamic Brown, 1985). Note that any species for which PEMS include and biomechanic principles, conjugation is predicted to sperm activating shortly after ejaculation or upon reaching enhance sperm motility because it increases force generation the sperm-storage organs do not support this hypothesis. with proportionately less drag (e.g. Woolley et al., 2009). Any cases of sperm transitioning to an increased flagellar It should be noted, however, that only a few studies beat frequency may support this hypothesis. If a higher have quantified the motility of sperm conjugates and these active state is required to fertilize an egg successfully, or have resulted in inconclusive or mixed results (reviewed by to compete for fertilization, then remaining in a state of Higginson & Pitnick, 2011). reduced activity until the right time might be an adaptation to enhance sperm longevity. (4) H4: aiding sperm to remain in a critical location in the FRT (6) H6: delivering male-derived materials to the In virtually all species with internal fertilization, sperm must female in a temporally and/or spatially critical manner be properly stored within a specialized organ or region of the FRT in order eventually to have an opportunity Seminal fluid is biochemically complex and rapidly evolving, to encounter an oocyte. Moreover, in many cases sperm as natural and sexual selection can drive ejaculate evolution competition between males predominantly distills down to to serve a multitude of functions. It provides direct material

Fig. 8. (A) Schematic of a ‘mature’ sperm from the testis (top) and the female spermatheca 2 days after insemination (bottom) in the fungus gnat, Sciara coprophila. Discontinuities in the diagrams indicate that the cell is much longer relative to the width than depicted. (B) Changes undergone by the axial filament complex during storage in the female reproductive tract. (C, D) Transmission electron micrograph of transverse section through the subnuclear portion of a ‘mature’ sperm from (C) the male testis and (D) the female spermatheca. A, acrosome; AF, axial filament complex; B, dense body; MC, mitochondrial crystalloid; MH, mitochondrial homogeneous material; N, nucleus. Adopted with permission from (A, B, D) Phillips (1966a); (C) Dallai, Bernini, & Giusti (1973). support for sperm and contributes to the FRT environment after transfer either because they may not be transported (and the female as a whole) to facilitate sperm motility and from the site of insemination to the location within the survival. Seminal fluid proteins (SFPs) and other constituents female where they can best function or because they are including small molecules, and exosomes or related vesicles, processed, degraded, or ejected relatively rapidly (notable may also provision females with nutrients or hormones used exceptions include several members of the sex peptide to make eggs or for the female’s own somatic maintenance, pathway in Drosophila;Penget al., 2005; Singh et al., 2018). and they can modify female gene expression, physiology One solution to this problem is to incorporate the molecules and (in insects, at least) behaviour in myriad ways that may into sperm or bind them to the sperm for regulated help or harm the female (Simmons & Parker, 1989; Pitnick, transport/release/cleavage (i.e. PEMS) within the FRT. Spicer, & Markow, 1997; Wolfner, 1997; Markow, Coppola, Another potential problem, arising in the case of costly & Watts, 2001; McGraw et al., 2004; Arnqvist & Rowe, 2005; nutritive donations, is that males run the risk of having Poiani, 2006; Gwynne, 2008; Avila et al., 2011; Baldini et al., their mate incorporate the material into eggs that are then 2013; Aalberts, Stout, & Stoorvogel, 2014; Bromfield et al., fertilized by another male’s sperm (Markow, 1988). One 2014; Corrigan et al., 2014; Sirot et al., 2015; Droge-Young solution to this problem would be to provision sperm with et al., 2016). the material for delivery to the oocyte upon fertilization, There may be temporal or functional constraints, however, although such modifications could hamper sperm motility, on the efficacy of molecules within seminal plasma. It competitiveness or ability to fertilize. However, we are aware seems likely that many may have local effects in the FRT of only a single investigation to test this hypothesis, which

Fig. 9. A model of molecular post-ejaculatory modifications to sperm (PEMS) in Drosophila melanogaster.Anetworkofseminal proteins is required for sex peptide (SP) to bind stably to sperm within the female seminal receptacle. Coloured shapes indicate proteins produced in the male accessory glands. CG1652 and CG1656 require fellow network proteins CG9997 and Antr to be transferred to females. Once deposited in females, Sems and CG17575 are required for SP and CG1656 to localize to the seminal receptacle (SR), the major site of female sperm storage. In the SR, SP and CG1656 bind sperm within 2 h of the start of mating. Also, within the female reproductive tract (FRT), the presence of CG1652 and CG1656 slows the rate at which CG9997 is processed from a 45 kDa form to a 36 kDa form. One additional network protein, Intrepid, is not shown, since its position in the pathway is presently unknown. Loss of any one of these network proteins prevents SP accumulation on sperm in the SR. Following the events shown, the SP C-terminus is cleaved from stored sperm over time. Colours indicate predicted protein functional classes: red/ orange/yellow are serine proteases and protease homologs; pink/purple are cysteine-rich secretory proteins; green are C-type lectins. Adopted with permission from Singh et al. (2018). did not support it. Karr & Pitnick (1996) examined the was favoured so that sperm could cooperate in the delivery of amount of spermic material entering the oocyte in 12 species the rods to the female’s spermatheca (Fig. 1). Finally, the best of Drosophila, exhibiting sperm lengths ranging from 36 to support for the hypothesis that PEMS result from selection 58,290 μm. Whereas the entire sperm enters the oocyte in on sperm as delivery vehicles comes from investigations of D. most of the species, they found that only a small fraction melanogaster. For example, of the estimated 200 SFPs that are of the sperm enters the oocyte in multiple, independent transferred to females during insemination in D. melanogaster lineages that have evolved giant sperm. Thus, at least in (Findlay et al., 2008; Findlay, MacCoss, & Swanson, 2009; Drosophila, larger sperm do not appear to have evolved Avila et al., 2011), sex peptide and several other SFPs have due to selection for post-fertilization provisioning (Karr & been seen to bind to sperm (Fig. 9; Peng et al., 2005; Ravi Pitnick, 1996). Alternatively, the nutritive material could Ram & Wolfner, 2009; Singh et al., 2018; E. Whittington, A. be incorporated into sperm and then released within the Singh, S. Pitnick, M. F. Wolfner & S. Dorus, unpublished female’s spermatheca(e), which would at least require that a data) although their retention times on sperm differ. female stores a male’s sperm (i.e. does not eject the sperm from the FRT; e.g. Manier et al., 2010) in order to secure the donation. (7) H7: priming sperm for extragenic contributions Several of the PEMS described in Section V provide to early embryogenesis general support for Hypothesis 6. First, this explanation Inherent in several of the previous hypotheses is the notion was suggested by Dallai et al. (2004) to explain the bizarre that adaptations specific to sperm transfer, storage or arrangement of collembolan sperm, with the flagellum coiled survival may not be conducive to fertilization, leading to the around a central, extracellular cavity containing testicular evolution of additional sperm phenotype modifcations prior secretions, and the PEMS occurring inside the spermatheca to encountering an oocyte. A related consideration is that releasing the secretions (Fig. 6). A second possible example some aspects of the pre-fertilization sperm phenotype may be is provided by the PEMS of the fungus gnat, S. coprophila, detrimental to post-fertilization zygote viability. The entire during which nearly all of the non-paracrystalline component sperm enters the oocyte during fertilization in most animal of the mitochondrial derivative (comprising about 50% of species (Karr, 1996; Karr & Pitnick, 1996; Krawetz, 2005; the sperm’s volume) is released into the spermatheca (Fig. 8; Karr, Swanson, & Snook, 2009), with the structure derived Makielski, 1966; Phillips, 1966a, 1966b). A third example from the flagellum being of considerable dimensions and may be the spermatostyles of some whirligig and carabid highly persistent throughout early embryogenensis in some beetles. Whereas these substantive rods have only been taxa (Karr, 1991; Pitnick & Karr, 1998). Some PEMS may interpreted as a proximate mechanism facilitating sperm have arisen to eliminate sperm proteins or organelles that conjugation (e.g. Higginson & Pitnick, 2011), the correct would be harmful to the zygote or otherwise impede early evolutionary interpretation may be that sperm conjugation development (Sutovsky & Song, 2017). Conversely, some

and sperm quality (in this case, their ability to transform properly). Alternatively, the ‘genetic compatibility’ model suggests that females evolve mechanisms to discriminate among sperm based on the compatibility of their haplotypes with the female genome (Jennions, 1997; Neff & Pitcher, 2005). The best evidence in support of Hypothesis 8 comes from studies of differential chemoattraction of sperm by eggs in the externally fertilizing marine mussel, Mytilus galloprovincialis. Using an elegant method to quantify variation in female-induced acrosome reaction and sperm surface glycan modifications (Kekal¨ ainen¨ et al., 2015), Kekal¨ ainen¨ & Evans (2016) show that the extent to which both processes occur depends upon specific male–female interactions. Further, variation in the response of sperm to chemoattractant cues from different female egg clutches has been shown in M. galloprovincialis to correlate with both fertilization rates (Evans et al., 2012) and offspring fitness (Oliver & Evans, 2014). This example may represent a more widespread phenomenon of sexual selection co-opting self-incompatibility systems to facilitate fertilization by genetically compatible gametes (Kao & McCubbin, 1996; Swanson & Vacquier, 2002; Gillingham et al., 2009; Palumbi, 2009; Evans & Sherman, 2013). With regard to internal fertilization, the FRT is immunologically highly active, particularly after mating and ejaculate transfer (Wira et al., 2005). Moreover, sperm are replete with proteins that function in immunity, and it has been suggested that immunity systems may provide a means for discerning among respective gametes (Dorus, Skerget, & Karr, 2012). It is certainly plausible that sexual selection could co-opt self-incompatibility or immunological systems for use in discriminating among sperm according to other axes of Fig. 10. Schematic drawing illustrating post-ejaculatory quality (Birkhead, Møller, & Sutherland, 1993; Eberhard, modifications to sperm (PEMS) of the tunicate, Diplosoma 1996; Holt & Fazeli, 2016a). listerianum. Head of a spermatozoon from the male’s sperm In vivo experiments support the ‘good sperm’ over duct (left) and from the female’s ovarian fertilization canal the ‘genetic compatibility’ hypothesis to explain the (right). et, endoplasmic tubules; fl, flagellum; m, mitochondrion; motility-related PEMS of leaf-cutter ants. It is ancestral dg, dense groove. Adopted with permission from Burighel & to all ant species that queens only mate during the single day Martinucci (1994a). of their nuptial flight, after which they have the potential to live for several decades and to produce thousands to PEMS may represent females providing substances to sperm millions of offspring (den Boer, Baer, & Dreier, 2009a;den that are beneficial to fertilization or early embryogenesis. Boer, Boomsma, & Baer, 2009b). Despite the potentially long interval between insemination and fertilization, A. colombica queens have been shown to fertilize close to 100% (8) H8: female assessment of sperm quality of their eggs (den Boer, Baer, & Dreier, 2009a). Given Another hypothesis for the existence of PEMS is that this unique biology, and because many more sperm are they could have arisen through selection on females to transferred to females than they are capable of storing enhance zygote viability and fitness through ‘sperm choice’ in their spermatheca, it was postulated that females have (Birkhead, 1998). According to this hypothesis, females a mechanism of selectively storing only sperm with high modify sperm as a mechanism to distinguish high- from viability (Liberti, Baer, & Boomsma, 2016). The greater low-quality sperm. Those sperm that were able successfully than 50% enhancement in sperm swimming performance to undergo PEMS, or in the most timely manner, would observed in these ants following exposure to FRT extract successfully traverse the FRT and participate in fertilization, is a PEMS that was interpreted as a mechanism of sperm whereas unmodified sperm would not (perhaps via a targeted choice (Liberti, Baer, & Boomsma, 2016). Notably, this degradation mechanism). According to the ‘good sperm’ mechanism did not discriminate between sperm of brothers model (Yasui, 1997), females accrue indirect genetic benefits and unrelated males (relative to the queen; Liberti, Baer, & through positive covariation between male genetic condition Boomsma, 2016).

Fig. 11. Schematic illustrating post-ejaculatory modiﬁcations to the head of the spermatozoon of the Chinese soft-shelled turtle, Pelodiscus sinensis. Adopted with permission from Zhang et al. (2015).

VII. EVOLUTIONARY IMPLICATIONS OF PEMS between tissues and/or sexes because they have relatively limited functional constraints within their source tissue and are already evolutionarily optimized to function in the (1) Genomic consequences of PEMS extracellular milieu of the FRT (regardless of whether they are expressed in males or females). Another common feature The unique biology of sperm, whose prolonged life history of the life history of sperm in males and females is the includes PEMS in the FRT, requires physiological and extensive remodelling of the plasma membrane, including biochemical continuity across the sexes. This predicts an changes in protein composition, post-translational protein evolutionarily dynamic relationship between gene expression modification and biochemistry (such as the removal of sterols within and between the male and female reproductive prior to capacitation). It is easy to envision that the occurence systems. For example, the extension of mammalian sperm of these modifications (and the underlying mechanisms upon maturation to include the dynamic processes that occur in which they rely) could switch, in a bidirectional manner, the epididymis would be expected to require the co-option between the sexes over evolutionary time. Similar scenarios and deployment of genes ancestrally restricted to the testis could also pertain to sperm metabolism, including the (as well as the evolution of entirely new genes). As the intrinsic metabolic capacity conferred by males relative to the duration of sperm maturation evolutionarily expands (to contribution of female-derived factors to sustain or promote include more extensive PEMS) and contracts (e.g. sperm metabolic processes in the FRT. maturation completed within the male), we predict correlated patterns of sex-biased gene expression evolution amongst (2) PEMS and sexual selection spermiogenesis/PEMS-related loci. We note that sex-biased gene expression evolves rapidly, particularly for male-biased Any traits that impact fertilization success will be subject to genes (Haerty et al., 2007; Zhang et al., 2007; Harrison strong selection. It is clear from the descriptions above that et al., 2015). An informative example of this involves the PEMS are likely to contribute to successful fertility in many creation of a novel SFP (a serine endopeptidase) through species. Because females of most species mate with multiple the duplication of an existing secreted FRT gene (Sirot males within a reproductive cycle (Taylor, Price, & Wedell, et al., 2014). Molecular analyses across seven Drosophila 2014), post-copulatory sexual selection is pervasive (Parker, species indicated a switch in sex-biased expression of 1970; Simmons, 2001), including sexual selection generated one of the duplicates from the ancestral pattern of FRT by conflict between the sexes over paternity (Parker, 1979; expression to the male accessory gland (Sirot et al., 2014). Arnqvist & Rowe, 2005). Post-copulatory sexual selection We further propose that secreted reproductive proteins is often credited as the principal agent responsible for may be particularly amenable to expressional switches the rapid evolutionary diversification of seminal fluid and

Fig. 12. Differential interference contrast micrographs of (A, B) many sperm stored within the deep portion of an isthmic crypt, and of (C, D) a single sperm released from storage of a female of the dasyurid marsupial, Sminthopsis crassicaudata, mated about 24–26 h previously. (A, C) The female is preovulatory and all sperm are spear shaped, with the anterior midpiece of the tail lying within lateral folds of the head; (B, D) the female is post-ovulatory and all sperm are T-shaped with the head angulated or perpendicular to the tail (the flagellum in panel D was oscillating and so appears blurred). Adopted with permission from Bedford & Breed (1994). ovarian fluid composition, sperm morphology and FRT the contribution of male × female interactions to critical design (Lahnsteiner, Weismann, & Patzner, 1995; Snook, reproductive outcomes (e.g. Clark, Begun, & Prout, 1999; 2005; Poiani, 2006; Pitnick et al., 2009a, 2009b). Relatively Clark, Dermitzakis, & Civetta, 2000; Tregenza & Wedell, few studies have quantified genetic variation in ejaculate and 2000; Nilsson, Fricke, & Arnqvist, 2003; Oh & Badyaev, FRT traits (Simmons & Moore, 2009), but those that have 2006; Bjork et al., 2007; Ravi Ram & Wolfner, 2007; Chow, revealed substantial within-population genetic variation in Wolfner, & Clark, 2010; Evans & Sherman, 2013; Lüpold sex-specific components known to contribute to variation et al., 2016; Suarez, 2016). in competitive fertilization success (e.g. Chow, Wolfner, & Failure to modify sperm properly could be a general Clark, 2010; Lüpold et al., 2012, 2013; Zhang, Clark, & and widespread mechanism by which females discriminate Fiumera, 2013). against the sperm of less-preferred males, as has been shown PEMS may similarly prove to be a widespread contributor to be the case with sperm chemoattraction (Kekal¨ ainen¨ to post-copulatory sexual selection on males and females. & Evans, 2016) and with female sperm ejection and sperm However, to our knowledge there have been no experimental digestion (Manier et al., 2013b; Firman et al., 2017). Similarly, tests of this postulate. There is limited knowledge of the two kinds of studies implicate a role for ovarian fluid in mechanisms by which sperm and constituents of seminal fluid mediating sexual selection within populations. First, ovarian interact with the female and, hence, of the actual targets of fluid has been shown to affect differentially the behaviour post-copulatory sexual selection. Post-mating interactions are of sperm from males exhibiting alternative reproductive notoriously cryptic and likely to be mediated predominantly tactics (Alonzo, Stiver, & Marsh-Rollo, 2016; Butts et al., by molecular interactions between the sexes (Eberhard, 1996; 2017; Lehnert et al., 2017a, 2017b). Second, the chemical Pitnick, Wolfner, & Suarez, 2009b; McDonough et al., 2016; composition of ovarian fluid differs among females within Firman et al., 2017; Lüpold & Pitnick, 2018). Indeed, there is a populations (Rosengrave et al., 2008; Johnson et al., 2014), growing paradigm in sexual selection theory that emphasizes with the influence of ovarian fluid on sperm behaviour

Biological Reviews 95 (2020) 365–392 © 2019 Cambridge Philosophical Society 382 Scott Pitnick and others being significantly influenced by female identity, male (2013) demonstrated that such co-diversification results in identity and female × male interaction (Rosengrave et al., species-specific PEMS (i.e. modifications to sperm flagellar 2008). Establishing whether these modifications to sperm beat and swimming trajectory) that limit the likelihood of following their exposure to ovarian fluid constitute PEMS hybrid fertilization. We predict that sperm and FRT traits awaits identification of molecular mechanisms underlying the involved in PEMS will tend to coevolve rapidly. If true, then interactions. However, Gasparini & Pilastro (2011) present PEMS may represent a powerful and possibly widespread experimental data for the guppy, Poecilia reticulata, suggesting mechanism of PMPZ reproductive isolation. The failure to that a female preference for genetically unrelated males is execute PEMS successfully could restrict the ability of sperm mediated by PEMS that influence sperm velocity. Finally, to be stored, to experience sustained longevity and/or to selection on males would favour traits that reduce the proper achieve the capacity to fertilize. An illustrative example of execution of PEMS by competitor sperm (i.e. resulting this is the rapid diversification and species specificity of the from subsequent mating by the female). Such selection sperm glycosylation system found in amphibian egg jelly has also been postulated to explain mate guarding in the (Coppin, Maes, & Strecker, 2002). golden-orb-weaving spider, Nephila clavipes (Brown, 1985).

(3) PEMS and reproductive isolation VIII. FUTURE DIRECTIONS The rapid diversification of interacting sex-specific traits by sexual selection is believed to be a widespread agent of reproductive isolation, and hence to play an important role Based on our extensive survey of the literature, PEMS in the formation of new species and the maintenance of appear to be widespread throughout the animal kingdom species boundaries (Coyne & Orr, 2004; Ritchie, 2007; and there is strong support in well-studied systems that Kraaijeveld, Kraaijeveld-Smit & Mann, 2011). Whereas they serve as important determinants of reproductive both empirical and theoretical studies have overwhelmingly outcomes. As such, PEMS warrant much wider experimental addressed either pre-copulatory or post-zygotic isolating attention. We propose three investigative goals to advance mechanisms, there is growing recognition of the potential our understanding of the importance of this general importance of post-mating/pre-zygotic (PMPZ) reproductive phenomenon. First, comparative analyses of PEMS across isolation resulting from reproductive incompatibilities or strategically selected taxonomic groups and populations fertilization biases that occur between the start of copulation are needed to assess the evolution of PEMS. Second, and successful karyogamy (Coyne & Orr, 2004; Howard the application of complementary techniques to link the et al., 2009; McDonough et al., 2016). In fact, this may be the structural and molecular bases of PEMS is required. Last, only kind of isolation restricting gene flow in some species and most challenging, will be the functional characterization (e.g. Howard et al., 1998; Ahmed-Braimah & McAllister, of PEMS, including elucidating male–female interactions 2012). Moreover, its occurrence is taxonomically widespread, that are required for the induction or mediation of PEMS. having been documented in internal fertilizers, broadcast There is much to be gained from investigating PEMS in spawners, and plants (as ‘conspecific pollen precedence’). related species to facilitate comparative analyses within a Identification of the mechanisms that restrict gene flow phylogenetic framework (e.g. Adams, 2014; Fuentes-G et al., is a central and long-standing goal of speciation research 2016). With the exception of sperm capacitation studies in (Dobzhansky, 1937; Mayr, 1942). Although the causes mammalian models, few comparative data relating to PEMS of PMPZ reproductive isolation are unknown for most are available despite their importance to establishing patterns systems, recent progress with two model systems, fruit of PEMS macroevolution. For example, hypotheses about flies and crickets, suggest that underlying mechanisms the adaptive diversification in PEMS could be tested by will tend to be multivariate and multifarious (e.g. Manier examining the direction and rate variation of phenotypic et al., 2013a, 2013b, 2013c;Tyleret al., 2013, Avila et al., divergence in PEMS-related characters throughout clades 2011). To our knowledge, the only study to date that relative to discrete transitions in traits putatively generating has explicitly investigated a possible role of PEMS in selection on PEMS, such as mode of fertilization, features reproductive isolation has been with the congeneric fish, of the mating system and aspects of female reproductive Salmo salar and S. trutta. Ovarian fluid composition is highly biology. Importantly, if interacting FRT components are divergent in fish, exhibiting substantial differences among identified (see below), then the comparative approach would species (Lahnsteiner, Weismann, & Patzner, 1995) and also facilitate examination of co-diversification of interacting even between members of different geographic populations sperm and FRT traits underlying PEMS. Where genomic within species (Beirao˜ et al., 2015). Studies comparing ovarian resources are available for the species examined, important fluid-induced modification to sperm exposed to ovarian fluid questions related to the molecular evolution of PEMS derived from the either the same or different populations or eventually could be addressed. Do genes underlying PEMS congeneric species provide strong support for the conclusion (both male and female expressed) evolve faster or slower that ovarian fluid composition and interacting sperm traits than other reproductive genes? Do genes underlying PEMs are evolutionarily co-diversifying (Yeates et al., 2013; Beirao˜ coevolve and can rate-covariation methods be used to identify et al., 2015; Devigili et al., 2018). Moreover, Yeates et al. PEMS interacting loci (Clark & Aquadro, 2010; Findlay

Biological Reviews 95 (2020) 365–392 © 2019 Cambridge Philosophical Society Sperm modification 383 et al., 2014)? Answers to these questions require a far greater could be used to inventory sperm composition across the understanding of the molecular basis of PEMS. entire life history of sperm in the FRT. The consequent Intraspecific analyses of PEMS should also be pursued, determination of gains, losses and modifications of sperm particularly where this could lend itself to quantitative (or sperm-associated seminal or female) proteins will provide genetic analyses. Advancing our understanding of the molecular precision to our understanding of the sequence of evolutionary diversification of PEMS requires an evaluation changes in sperm as they reside in the FRT. Combining of the degree to which PEMS variation is attributable to this approach with cryo-electron-tomography (Nicastro, males, females or male × female interactions and, ideally, McIntosh, & Baumeister, 2005) to reveal structural changes how this relates to variation in competitive fertilization in the sperm and in the positions or shapes of complexes success (Birkhead, 1998). Some PEMS are likely to on or in them, and methods for single-cell measurement contribute to the variation in reproductive success attributed of energy consumption (Chen et al., 2015) in sperm will to differential ‘genetic compatibility’ between males and further extend the molecular precision of what is involved in females (e.g. inbreeding avoidance; Jennions, 1997; Neff & PEMS. This in turn will provide a foundation for systematic Pitcher, 2005). The failure of PEMS to be executed properly efforts to examine the in vivo functional importance of in certain male–female combinations may provide a novel PEMS, in organisms where genetic manipulation is possible, explanation for idiopathic infertility, as first suggested by and establish molecular networks governing post-copulatory Chang (1951; see also Matzuk & Lamb, 2008). Along sperm–female interactions critical to sperm viability and these lines, PEMS-related molecules and mechanisms may fertility (McDonough et al., 2016). prove to be useful candidate targets for the development of The ultimate goal of assigning specific functions to PEMS long-sought non-hormonal and reversible contraceptives. will require molecular advances in our understanding of The failure of sperm successfully to undergo PEMS within PEMS in taxa where genetic manipulation (RNAi and the FRT of hetero-population or heterospecific females (e.g. CRISPR) and functional assays are tractable (e.g. Bassett following a hybrid mating) could be a widespread mechanism & Liu, 2014; Mohr et al., 2014). These methods can be used contributing to PMPZ reproductive isolation (Howard et al., to remove (or deplete) proteins identified in proteomic studies 2009). We are unaware of any investigations of this possibility. such as those described above (or other proteins suggested The ideal research program would include a fully factorial to be involved in PEMS based on other data), from their cross design using pairs of sibling species that will engage in tissue of synthesis (testis, male reproductive glands, or FRT reciprocal hybrid matings, or else are amenable to artificial tissues). They can also be used to prevent production of insemination, to examine whether PEMS occur in the same other potential effectors of PEMS, such as extracellular way in interspecific as in intraspecific crosses, and to explore vesicles that deliver molecules to sperm after the latter have whether the failure of PEMS to be executed in hybrid left the testis and/or entered the female (e.g. Aalberts, Stout, inseminations contributes to gametic isolation (e.g. Howard & Stoorvogel, 2014; Corrigan et al., 2014; Fereshteh et al., et al., 2009; Manier et al., 2013b). Combining this approach 2019). Effects of removal of the proteins or other effectors with molecular approaches (see below) in a hybridizing model can then be examined in terms of the ability of sperm from system with genomic resources (e.g. sibling Drosophila species), (or in) knockdown/knockout animals to undergo PEMS, especially when followed up with functional experiments, by assaying fertility, sperm fate, and sperm morphology would be exceptionally powerful. after sperm have left the testis and entered the FRT. The vast majority of empirical data supporting PEMS Knockdown/out of molecules needed for PEMS would come from microscopy and ultrastructure studies. There is be expected to decrease or abolish fertility (or perhaps to a pressing need to link such observations with underlying result in abnormalities like polyspermy), to modulate sperm molecular changes. Applying a combination of fine-structure competitive success, and possibly to result in visibly abnormal and proteomic analyses to compare the sperm from male sperm structures or morphologies, or inappropriate fates or seminal vesicles and sperm that have experienced prolonged targeting of sperm. For example, such genetic studies have storage in the FRT will prove extremely valuable, especially already been useful for examining the function of proteins if applied to taxonomically diverse species. Such studies that bind to sperm and are subsequently cleaved or otherwise would further our understanding of the variation in the released from sperm into the FRT in the well-studied case structural and molecular nature of PEMS. Proteomic studies of Drosophila sex peptide (Peng et al., 2005; Ravi Ram & have played a transformative role in achieving a refined Wolfner, 2009; Singh et al., 2018). Once genetic studies have molecular understanding of sperm composition, with sperm pinpointed molecules that are necessary for PEMS, further having been characterized using proteomics for humans studies can define those molecules’ active regions, partners, and several model organisms, including the mouse, rat, and evolutionary dynamics. Although such studies are likely macaque, fruit fly, mosquito, honeybee and worm, as to be easiest in traditional genetic model organisms, it is well as for some non-model species (e.g. Dorus et al., expected that analogous studies could be carried out in any 2006; Baker et al., 2008; Poland et al., 2011; Skerget et al., organism for which sperm proteomics (or other molecular) 2013; Amaral et al., 2014; Ma et al., 2014; Whittington analyses can be carried out and in which RNAi and CRISPR et al., 2015; Degner et al., 2019). As methods for single-cell techniques are useable, such as insects beyond D. melanogaster. analysis improve (Budnik et al., 2018), single-cell proteomics It will likely prove interesting, albeit challenging, to use such

Biological Reviews 95 (2020) 365–392 © 2019 Cambridge Philosophical Society 384 Scott Pitnick and others approaches to examine the material voided from sperm complex and protracted life histories that are shaped by within the FRT of species such as collembolans (Dallai et al., selection generated by the female reproductive tract. 2004) and the fungus gnat S. coprophila (Phillips, 1966a), (2) Post-ejaculatory modifications to sperm (PEMS) are and perhaps on the spermatostyles that accumulate in the a widespread, if not a ubiquitous, component of the life female spermatheca of some whirligig and carabid beetles history of sperm in internally fertilizing species. The proper after primary sperm conjugates have disassociated (Breland implementation of most PEMS requires interactions between & Simmons, 1970; Sasakawa, 2007). sperm and female reproductive tract molecules and cells. Whereas the functional analysis of specific proteins that (3) The widely investigated phenomenon of capacitation bind to or are removed from sperm is certainly tractable in eutherian mammals is only one example of a much in many model and non-model organisms, manipulating more widespread phenomenon. PEMS have likely evolved more complex PEMS, such as major structural transitions or independently numerous times throughout the kingdom sperm–sperm associations, is likely to prove much more Animalia. A diversity of selection pressures likely underlies challenging. However, single-cell proteomics may again the evolutionary origin, maintenance and diversification of come to our aid. It will be necessary to isolate and PEMS. We advance numerous hypotheses for the adaptive characterize the female-derived molecules responsible for the value of PEMS, but note that few data currently exist to test induction of complex PEMS. Such studies would represent or otherwise discriminate among them. major advances towards achieving an understanding (4) Few studies have considered PEMS from an of male–female interactions that mediate reproductive evolutionary perspective. Such research initiatives are outcomes. This is important from many fundamental warranted as variation in PEMS has important implications perspectives: our understanding of basic reproductive biology for our understanding of sex-specific gene expresssion and fertility, of the cell biology of gametes and their and of post-copulatory sexual selection. Moreover, the interaction with the reproductive tract environment and rapid evolution of ejaculate–female interactomes underlying with each other, and of how evolutionary forces shaped these PEMS has the potential to generate reproductive isolation interactions and, in turn, may have been constrained by between divergent populations or incipient species and hence them. In addition, understanding sperm traits and how they to be a wideapread ‘engine of speciation’. are modified post-ejaculation will likely have direct practical implications. It could provide information that will improve conditions for in vitro sperm maintenance/storage or for X. ACKNOWLEDGMENTS assisted reproductive technologies, thus benefitting human reproductive biomedicine or assisting in the recovery or support of threatened species. Alternatively, this infomation We would like to thank Sharleen Buel for technical assistance could reveal potential new targets for human contraception and Susan Suarez, Kirill Borziak, Ethan Degner, Tim or for biological control programs against pest or vector Karr, Erin McCullough, Caitlin McDonough, Jane Pascar, insects. Zeeshan Syed and Emma Whittington for fruitful advice, Finally, we hope this review serves to remind readers of the discussion, bringing relevant literature to our attention, benefits of carrying out descriptive studies of the fascinating and/or helpful comments on earlier drafts. We are also reproductive biology of diverse taxa. Such pursuits used to be grateful for the detailed and perceptive comments from two far more popular than they are now. As one referee pointed anonymous reviewers. We are especially indebted to the out, ‘the rise in molecular and developmental genetics proved myriad biologists whose curiosity and creative exploration of so seductive as to suck all the oxygen out of the room’, variation in reproductive systems made this synthesis possible in the sense of focusing much research emphasis onto a and a joy to write. This work was supported by a generous small number of model organisms. Especially in light of gift by Mike and Jane Weeden to Syracuse University and by the mechanistic insights arising from such research, it is grants from the Eunice Shriver National Institute for Child important and instructive to return to examining the varied Health and Human Development (R21-HD088910 to S.D., reproductive strategies used across the animal kingdom. As S.P. and M.F.W. and R01-HD038921 to M.F.W.) and the sagely noted by Grimaldi & Engel (2007, p. 647), ‘‘a theory National Science Foundation (DEB-1655840 to S.D., S.P. is only as good as what it explains and the evidence (i.e. and M.F.W.), descriptions) that supports it.’’

XI. REFERENCES

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(Received 10 March 2019; revised 12 October 2019; accepted 16 October 2019; published online 18 November 2019)