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Modification of Crocodile Spermatozoa Refutes the Tenet That Post-testicular Sperm Maturation Is Restricted To Mammals*

  • Brett Nixon
    Correspondence
    To whom correspondence should be addressed:Priority Research Centre for Reproductive Science, School of Environmental and Life Sciences, The University of Newcastle, Callaghan, NSW 2308, Australia. Tel.:+61-2-4921-6977; Fax:+61-2-4921-6308;
    Affiliations
    From the ‡Priority Research Centre for Reproductive Science, School of Environmental and Life Sciences, The University of Newcastle, Callaghan, NSW 2308, Australia;

    Hunter Medical Research Institute, New Lambton Heights, NSW 2305, Australia;
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  • Stephen D. Johnston
    Affiliations
    School of Agriculture and Food Science, The University of Queensland, Gatton, QLD 4343, Australia;
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  • David A. Skerrett-Byrne
    Affiliations
    Hunter Medical Research Institute, New Lambton Heights, NSW 2305, Australia;
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  • Amanda L. Anderson
    Affiliations
    From the ‡Priority Research Centre for Reproductive Science, School of Environmental and Life Sciences, The University of Newcastle, Callaghan, NSW 2308, Australia;
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  • Simone J. Stanger
    Affiliations
    From the ‡Priority Research Centre for Reproductive Science, School of Environmental and Life Sciences, The University of Newcastle, Callaghan, NSW 2308, Australia;
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  • Elizabeth G. Bromfield
    Affiliations
    From the ‡Priority Research Centre for Reproductive Science, School of Environmental and Life Sciences, The University of Newcastle, Callaghan, NSW 2308, Australia;

    Hunter Medical Research Institute, New Lambton Heights, NSW 2305, Australia;
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  • Jacinta H. Martin
    Affiliations
    From the ‡Priority Research Centre for Reproductive Science, School of Environmental and Life Sciences, The University of Newcastle, Callaghan, NSW 2308, Australia;

    Hunter Medical Research Institute, New Lambton Heights, NSW 2305, Australia;
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  • Philip M. Hansbro
    Affiliations
    Hunter Medical Research Institute, New Lambton Heights, NSW 2305, Australia;

    Priority Research Centre for Healthy Lungs, Faculty of Health and Medicine, The University of Newcastle, Newcastle, NSW 2308, Australia;
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  • Matthew D. Dun
    Affiliations
    Hunter Medical Research Institute, New Lambton Heights, NSW 2305, Australia;

    Priority Research Centre for Cancer Research, Innovation and Translation, School of Biomedical Sciences and Pharmacy, Faculty of Health and Medicine, The University of Newcastle, Callaghan, NSW 2308, Australia
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  • Author Footnotes
    This article contains supplemental Tables.
Open AccessPublished:August 02, 2018DOI:https://doi.org/10.1074/mcp.RA118.000904
      Competition to achieve paternity has contributed to the development of a multitude of elaborate male reproductive strategies. In one of the most well-studied examples, the spermatozoa of all mammalian species must undergo a series of physiological changes, termed capacitation, in the female reproductive tract before realizing their potential to fertilize an ovum. However, the evolutionary origin and adaptive advantage afforded by capacitation remains obscure. Here, we report the use of comparative and quantitative proteomics to explore the biological significance of capacitation in an ancient reptilian species, the Australian saltwater crocodile (Crocodylus porosus,). Our data reveal that exposure of crocodile spermatozoa to capacitation stimuli elicits a cascade of physiological responses that are analogous to those implicated in the functional activation of their mammalian counterparts. Indeed, among a total of 1119 proteins identified in this study, we detected 126 that were differentially phosphorylated (± 1.2 fold-change) in capacitated versus, noncapacitated crocodile spermatozoa. Notably, this subset of phosphorylated proteins shared substantial evolutionary overlap with those documented in mammalian spermatozoa, and included key elements of signal transduction, metabolic and cellular remodeling pathways. Unlike mammalian sperm, however, we noted a distinct bias for differential phosphorylation of serine (as opposed to tyrosine) residues, with this amino acid featuring as the target for ∼80% of all changes detected in capacitated spermatozoa. Overall, these results indicate that the phenomenon of sperm capacitation is unlikely to be restricted to mammals and provide a framework for understanding the molecular changes in sperm physiology necessary for fertilization.

      Graphical Abstract

      The molecular processes leading to fertilization remain among the key unresolved questions in the field of reproductive biology. Based on studies of the mammalian lineage, it is widely accepted that terminally differentiated spermatozoa develop the capacity to fertilize an ovum during sequential phases of post-testicular maturation as they pass through the male (epididymis) (
      • Zhou W.
      • De Iuliis G.N.
      • Dun M.D.
      • Nixon B.
      Characteristics of the epididymal luminal environment responsible for sperm maturation and storage.
      ) and female reproductive tracts (
      • Aitken R.J.
      • Nixon B.
      Sperm capacitation: a distant landscape glimpsed but unexplored.
      ). The latter of these is termed capacitation and is defined as a time-dependent process during which spermatozoa experience a suite of biochemical and biophysical changes that collectively endow them with the ability to recognize and fertilize an ovum (
      • Aitken R.J.
      • Nixon B.
      Sperm capacitation: a distant landscape glimpsed but unexplored.
      ,
      • Baker M.A.
      • Nixon B.
      • Naumovski N.
      • Aitken R.J.
      Proteomic insights into the maturation and capacitation of mammalian spermatozoa.
      ). A distinctive characteristic of this phase of functional maturation is that it occurs in the complete absence of nuclear gene transcription and de novo, protein synthesis. Instead, the regulation of capacitation rests almost exclusively with convergent signaling cascades that transduce extracellular signals to effect extensive post-translational modification of the intrinsic sperm proteome (
      • Gervasi M.G.
      • Visconti P.E.
      Chang's meaning of capacitation: A molecular perspective.
      ). In this context, differential protein phosphorylation has emerged as a dominant molecular switch, which regulates sperm-oocyte recognition and adhesion, the ability to undergo acrosomal exocytosis, and the propagation of an altered pattern of movement referred to as hyperactivation (
      • Stival C.
      • Puga Molina Ldel C.
      • Paudel B.
      • Buffone M.G.
      • Visconti P.E.
      • Krapf D.
      Sperm capacitation and acrosome reaction in mammalian sperm.
      ).
      A curiosity of the capacitation cascade is its evolutionary origin and the adaptive advantage that is afforded by such an elaborate form of post-testicular sperm maturation. Indeed, studies of the spermatozoa of sub-therian vertebrate species such as those of the aves, have failed to document a process equivalent to capacitation (
      • Howarth Jr., B.
      An examination for sperm capacitation in the fowl.
      ,
      • Howarth Jr., B.
      Fertilizing ability of cock spermatozoa from the testis epididymis and vas deferens following intramagnal insemination.
      ,
      • Howarth Jr., B.
      • Palmer M.B.
      An examination of the need for sperm capacitation in the turkey, Meleagris gallopavo.
      ); with spermatozoa of fowls and turkeys appearing refractory to the need for any such physiological changes during their residence in the female reproductive system (
      • Howarth Jr., B.
      An examination for sperm capacitation in the fowl.
      ,
      • Howarth Jr., B.
      • Palmer M.B.
      An examination of the need for sperm capacitation in the turkey, Meleagris gallopavo.
      ). Similarly, in the quail it has been shown that most testicular sperm can bind to a perivitelline membrane and acrosome react with no additional advantage being afforded by exposure to capacitation stimuli (
      • Nixon B.
      • Ewen K.A.
      • Krivanek K.M.
      • Clulow J.
      • Kidd G.
      • Ecroyd H.
      • Jones R.C.
      Post-testicular sperm maturation and identification of an epididymal protein in the Japanese quail (Coturnix coturnix japonica,).
      ). Although it has been suggested that reptilian spermatozoa also experience minimal post-testicular maturation, this paradigm has recently been challenged by functional analysis of ejaculated spermatozoa from the Australian saltwater crocodile (Crocodylus porosus,) (
      • Nixon B.
      • Anderson A.L.
      • Smith N.D.
      • McLeod R.
      • Johnston S.D.
      The Australian saltwater crocodile (Crocodylus porosus) provides evidence that the capacitation of spermatozoa may extend beyond the mammalian lineage.
      ). In this context, exposure to capacitation stimuli, which elevate intracellular levels of cyclic AMP (cAMP), promoted a significant enhancement of the motility profile recorded in crocodile spermatozoa. Notably, dilution in capacitation medium also enhances the post-thaw survival of cryopreserved crocodile spermatozoa (
      • Johnston S.D.
      • Qualischefski E.
      • Cooper J.
      • McLeod R.
      • Lever J.
      • Nixon B.
      • Anderson A.L.
      • Hobbs R.
      • Gosalvez J.
      • Lopez-Fernandez C.
      • Keeley T.
      Cryopreservation of saltwater crocodile (Crocodylus porosus) spermatozoa.
      ). Conversely, crocodile spermatozoa are rendered quiescent upon incubation in bicarbonate-free media formulated to suppress the capacitation of eutherian spermatozoa (
      • Nixon B.
      • Anderson A.L.
      • Smith N.D.
      • McLeod R.
      • Johnston S.D.
      The Australian saltwater crocodile (Crocodylus porosus) provides evidence that the capacitation of spermatozoa may extend beyond the mammalian lineage.
      ). We contend that such changes may reflect physiological demands imposed by the transferal of sperm storage responsibilities from the male to the female reproductive tract, and the attendant need to alternatively silence and reactivate spermatozoa to enhance their longevity and fertilization competence, respectively. Nevertheless, the mechanistic basis of these opposing responses remain obscure, as does the identity of the proteins implicated in their regulation.
      Here, we have used mass spectrometry-based proteomics to generate a comprehensive protein inventory of mature crocodile spermatozoa and subsequently explore signatures of capacitation via quantitative phosphoproteomic profiling strategies. Our data confirm that the phosphorylation status of the crocodile sperm proteome is substantially modified in response to capacitation stimuli, thus refuting the tenet that this phenomenon is restricted to the mammalian lineage and providing a framework for understanding the molecular changes in sperm physiology necessary for fertilization.

      DISCUSSION

      It is well recognized the spermatozoa of all mammalian species only acquire functional maturity as they are conveyed through the male and female reproductive tracts. Despite decades of research however, the evolutionary origin, and adaptive advantage, of these elaborate forms of post-testicular maturation remain obscure. Here, we have exploited quantitative proteomics coupled with phosphopeptide-enrichment strategies to explore the crocodile sperm proteome and identify signatures of post-translational modification associated with the functional activation of these cells. Our data confirm that the phosphorylation status of the crocodile sperm proteome is substantially modified in response to stimuli formulated to elevate intracellular levels of the second messenger cAMP; thus supporting the necessity for capacitation-like changes in promoting the fertility of these cells. Moreover, we have established that the enhanced motility profile of capacitated crocodile spermatozoa is likely fueled by aerobic metabolism of selective membrane fatty acid substrates.
      Although spermatozoa have now been successfully recovered from several reptilian species, systematic attempts to modulate the physiology of these cells are rare and global proteomic analyses are currently lacking. Thus, in completing the first comprehensive proteomic assessment of reptilian spermatozoa, we have been able to initiate comparative analyses with the curated proteomes from representative mammalian (human, (
      • Amaral A.
      • Castillo J.
      • Ramalho-Santos J.
      • Oliva R.
      The combined human sperm proteome: cellular pathways and implications for basic and clinical science.
      )) and avian (rooster, (
      • Labas V.
      • Grasseau I.
      • Cahier K.
      • Gargaros A.
      • Harichaux G.
      • Teixeira-Gomes A.P.
      • Alves S.
      • Bourin M.
      • Gerard N.
      • Blesbois E.
      Qualitative and quantitative peptidomic and proteomic approaches to phenotyping chicken semen.
      )) spermatozoa with a view to furthering our understanding of this cell's complex biological machinery. These analyses confirmed the presence of some 84% of the identified crocodile spermatozoa proteins within the human sperm proteome; a level of conservation that suggests the core proteomic architecture of spermatozoa from these distantly related vertebrate species are broadly comparable. Working within the limitations imposed by incomplete coverage, functional classification of crocodile sperm proteins that are not currently annotated in the human sperm proteome revealed enrichment in the molecular function category of catalytic activity, and the biological process of metabolism. In addition, a substantial number of these proteins mapped to the membrane domain. Based on these data, we infer specialization of the surface, and possibly the metabolic characteristics, of crocodile spermatozoa.
      The former explanation is consistent with evidence that the plasma membrane of crocodile spermatozoa displays exceptionally high tolerance to anisotonic osmotic stress (
      • Johnston S.D.
      • Lever J.
      • McLeod R.
      • Qualischefski E.
      • Brabazon S.
      • Walton S.
      • Collins S.N.
      Extension, osmotic tolerance and cryopreservation of saltwater crocodile (Crocodylus porosus,) spermatozoa.
      ). Indeed, crocodile spermatozoa retain high levels of plasma membrane integrity (>50%) during exposure to osmotic excursions of between 25–1523 mOsm kg−1 (
      • Johnston S.D.
      • Lever J.
      • McLeod R.
      • Qualischefski E.
      • Brabazon S.
      • Walton S.
      • Collins S.N.
      Extension, osmotic tolerance and cryopreservation of saltwater crocodile (Crocodylus porosus,) spermatozoa.
      ). Such characteristics are perhaps a physiological necessity owing to the potential for these cells to encounter dilution into fresh, or brackish, water following ejaculation into the cloacal chamber of the female. Alternatively, a high tolerance to anisotonic media could be linked to sperm storage in this species, as it appears to be in microbats (
      • Crichton E.G.
      Sperm storage and fertilization.
      ). Although, the preservation of plasma membrane integrity in the face of extreme osmotic challenge undoubtedly reflects its lipid architecture, such properties may be augmented by the synergistic action of ion transport and drug efflux proteins in the lipid bilayer. An interesting example of one such protein is that of the testis anion transporter 1 (SLC26A8), an anion exchanger that mediates chloride, sulfate and oxalate transport and has been postulated to fulfil critical functions in the male germ line (
      • Toure A.
      • Lhuillier P.
      • Gossen J.A.
      • Kuil C.W.
      • Lhote D.
      • Jegou B.
      • Escalier D.
      • Gacon G.
      The testis anion transporter 1 (Slc26a8) is required for sperm terminal differentiation and male fertility in the mouse.
      ). Noteworthy in the context of our study, SLC26A8 has been implicated in the formation of a molecular complex involved in the regulation of chloride and bicarbonate ions fluxes during induction of sperm capacitation (
      • Rode B.
      • Dirami T.
      • Bakouh N.
      • Rizk-Rabin M.
      • Norez C.
      • Lhuillier P.
      • Lores P.
      • Jollivet M.
      • Melin P.
      • Zvetkova I.
      • Bienvenu T.
      • Becq F.
      • Planelles G.
      • Edelman A.
      • Gacon G.
      • Toure A.
      The testis anion transporter TAT1 (SLC26A8) physically and functionally interacts with the cystic fibrosis transmembrane conductance regulator channel: a potential role during sperm capacitation.
      ). Thus, an increased understanding of the functional relationships between the proteomic composition of the crocodile sperm membrane and their ability to survive osmotic excursions may ultimately help inform protocols to address the emerging need for the successful cryopreservation of crocodile spermatozoa (
      • Johnston S.D.
      • Qualischefski E.
      • Cooper J.
      • McLeod R.
      • Lever J.
      • Nixon B.
      • Anderson A.L.
      • Hobbs R.
      • Gosalvez J.
      • Lopez-Fernandez C.
      • Keeley T.
      Cryopreservation of saltwater crocodile (Crocodylus porosus) spermatozoa.
      ).
      Consistent with energy-production being a key attribute in the support of motility needed for spermatozoa to ascend the female reproductive tract and achieve fertilization, metabolic enzymes were identified as one of the dominant functional categories represented among the crocodile sperm proteome. Notably, enzymes mapping to glycolysis, oxidative phosphorylation and lipid metabolism were each highly enriched in the crocodile sperm proteome, with those of the latter category including proteins implicated in lipid catabolism, modification, and transport. Among these proteins we identified carnitine palmitoyl transferase 1 (CPT1A), an enzyme that catalyzes the rate-limiting reaction of beta-oxidation of fatty acids (
      • Lee K.
      • Kerner J.
      • Hoppel C.L.
      Mitochondrial carnitine palmitoyltransferase 1a (CPT1a) is part of an outer membrane fatty acid transfer complex.
      ), and one that experienced among the highest fold changes (2.27 increase) of accumulation into the detergent labile (soluble) fraction of capacitated sperm lysates. From its position in the outer mitochondrial membrane, CPT1 catalyzes the formation of long-chain acylcarnitines from their respective CoA esters and thus commits them to β-oxidation within the mitochondrial matrix (
      • Lee K.
      • Kerner J.
      • Hoppel C.L.
      Mitochondrial carnitine palmitoyltransferase 1a (CPT1a) is part of an outer membrane fatty acid transfer complex.
      ). It follows that pharmacological inhibition of CPT1A reduces flux through β-oxidation and, in species such as the horse, this manifests in the form of compromised sperm motility (
      • Swegen A.
      • Curry B.J.
      • Gibb Z.
      • Lambourne S.R.
      • Smith N.D.
      • Aitken R.J.
      Investigation of the stallion sperm proteome by mass spectrometry.
      ). The fact that this response occurs independently of any attendant loss of vitality, has been taken as evidence that stallion spermatozoa are able to effectively use endogenous fatty acids as an energy substrate to support motility (
      • Swegen A.
      • Curry B.J.
      • Gibb Z.
      • Lambourne S.R.
      • Smith N.D.
      • Aitken R.J.
      Investigation of the stallion sperm proteome by mass spectrometry.
      ). Although we have not yet had the opportunity to test this hypothesis directly in crocodile spermatozoa, we did secure several lines of correlative evidence that these cells use a similar metabolic strategy. Thus, capacitated crocodile spermatozoa experienced a selective depletion of palmitoleic acid [a monounsaturated fatty acid substrate known to enhance the motility profile of spermatozoa from species as diverse as sheep and fowls (
      • Eslami M.
      • Ghasemiyan H.
      • Zadeh Hashem E.
      Semen supplementation with palmitoleic acid promotes kinematics, microscopic and antioxidative parameters of ram spermatozoa during liquid storage.
      ,
      • Rad H.M.
      • Eslami M.
      • Ghanie A.
      Palmitoleate enhances quality of rooster semen during chilled storage.
      )], as well as a significant reduction in motility following the uncoupling of oxidative phosphorylation. Moreover, we identified CPT1A as a substrate for differential phosphorylation in noncapacitated versus, capacitated crocodile spermatozoa. This finding takes on added significance in view of the demonstration that CPT1 catalytic activity can be selectively modulated by a mechanism of cAMP-dependent phosphorylation/dephosphorylation in somatic cells (
      • Harano Y.
      • Kashiwagi A.
      • Kojima H.
      • Suzuki M.
      • Hashimoto T.
      • Shigeta Y.
      Phosphorylation of carnitine palmitoyltransferase and activation by glucagon in isolated rat hepatocytes.
      ,
      • Pegorier J.P.
      • Garcia-Garcia M.V.
      • Prip-Buus C.
      • Duee P.H.
      • Kohl C.
      • Girard J.
      Induction of ketogenesis and fatty acid oxidation by glucagon and cyclic AMP in cultured hepatocytes from rabbit fetuses. Evidence for a decreased sensitivity of carnitine palmitoyltransferase I to malonyl-CoA inhibition after glucagon or cyclic AMP treatment.
      ,
      • Guzman M.
      • Geelen M.J.
      Activity of carnitine palmitoyltransferase in mitochondrial outer membranes and peroxisomes in digitonin-permeabilized hepatocytes. Selective modulation of mitochondrial enzyme activity by okadaic acid.
      ). It is therefore conceivable that the differential phosphorylation of CPT1A witnessed in crocodile spermatozoa may serve as a physiological switch to divert their metabolism either toward, or away from, fatty acid oxidation. Because fatty acid metabolism is conducive to long-term sustained release of energy, this strategy could assist with prolonging in vivo, sperm storage before ovulation (
      • Davenport M.
      Evidence of possible sperm storage in the caiman, Paleosuchus palpebrosus.
      ,
      • Gist D.H.
      • Bagwill A.
      • Lance V.
      • Sever D.M.
      • Elsey R.M.
      Sperm storage in the oviduct of the American alligator.
      ), while also proving advantageous in the context of enabling crocodile spermatozoa to negotiate the many meters of female reproductive tract before arriving at the site of fertilization (
      • Gist D.H.
      • Bagwill A.
      • Lance V.
      • Sever D.M.
      • Elsey R.M.
      Sperm storage in the oviduct of the American alligator.
      ).
      Beyond its putative impact on CPT1A activity, elevation of intracellular cAMP also elicited the phosphorylation of numerous alternative substrates implicated in sperm motility initiation and maintenance. Notably, these proteins included peptides mapping to the alpha and beta regulatory subunits of protein kinase A (PKA), a promiscuous cAMP-dependent serine/threonine kinase. In eutherian spermatozoa, PKA is widely acknowledged as the central hub of the canonical capacitation cascade owing to its ability to integrate cAMP signaling with the downstream tyrosine kinase signaling pathways that underpin the functional activation of the cell (
      • Aitken R.J.
      • Nixon B.
      Sperm capacitation: a distant landscape glimpsed but unexplored.
      ). Consistent with data from our own immunolocalization studies (
      • Nixon B.
      • Anderson A.L.
      • Smith N.D.
      • McLeod R.
      • Johnston S.D.
      The Australian saltwater crocodile (Crocodylus porosus) provides evidence that the capacitation of spermatozoa may extend beyond the mammalian lineage.
      ), PKA primarily resides in the axoneme, a structure that forms an integral part of the motility apparatus of the sperm flagellum. Indeed, PKA is effectively anchored within this specific subcellular location by interaction between a docking domain present in the enzyme's regulatory subunit and that of scaffolding proteins of the protein kinase A anchoring protein (AKAP) family (
      • Wong W.
      • Scott J.D.
      AKAP signalling complexes: focal points in space and time.
      ,
      • Luconi M.
      • Carloni V.
      • Marra F.
      • Ferruzzi P.
      • Forti G.
      • Baldi E.
      Increased phosphorylation of AKAP by inhibition of phosphatidylinositol 3-kinase enhances human sperm motility through tail recruitment of protein kinase A.
      ); multiple members of which also displayed differential phosphorylation in capacitating crocodile spermatozoa (i.e., AKAP4, AKAP5, AKAP8, AKAP10/11). This sequestration of PKA ensures that the enzyme is juxtaposed with its relevant axonemal protein targets, while simultaneously segregating its activity to prevent indiscriminate phosphorylation of alternative substrates.
      These data are entirely consistent with the demonstration that the bulk of the crocodile sperm proteins identified as undergoing differential capacitation-associated phosphorylation were those harbored within the sperm flagellum; with prominent examples including fibrous sheath CABYR-binding protein, outer dense fiber proteins (ODF2, ODF3, ODF4, ODF5), cilia-and flagella-associated proteins (CFAP57, CFAP58), fibrous sheath-interacting proteins (FSIP3, FSIP4/5), microtubule-associated protein, tubulins (TUBA, TUBB), and dynein (DYNLL1). They also accord with our previous observations of a rapid and sustained increase in the rate of motility as being among the principle changes witnessed in capacitating crocodile spermatozoa (10). Although the conservation of phospho-substrates documented above suggests conservation of the core activation pathways employed by reptilian and eutherian spermatozoa, it is also apparent that downstream signaling events show some degree of divergence. Thus, unlike eutherian sperm capacitation in which tyrosine phosphorylation appears to exert overriding control (
      • Gervasi M.G.
      • Visconti P.E.
      Chang's meaning of capacitation: A molecular perspective.
      ), we identified comparatively few tyrosine phosphorylated peptides in capacitated crocodile spermatozoa. Such findings agree with our previous immunoblotting studies in which we also documented only relatively subtle changes in tyrosine phosphorylation status, save for a small subset of very high molecular weight proteins (
      • Nixon B.
      • Anderson A.L.
      • Smith N.D.
      • McLeod R.
      • Johnston S.D.
      The Australian saltwater crocodile (Crocodylus porosus) provides evidence that the capacitation of spermatozoa may extend beyond the mammalian lineage.
      ). With the increased resolution afforded by the MS strategy employed herein, we have now affirmed the identity of at least one of these proteins as dynein; a microtubule-dependent force-generating ATPase that plays a pivotal role in axonemal microtubule sliding and hence the propagation of sustained flagellum beating (
      • Loreng T.D.
      • Smith E.F.
      The Central Apparatus of Cilia and Eukaryotic Flagella.
      ).
      Although the identification of relatively few phosphotyrosine substrates represents a departure from the widely accepted models of eutherian sperm capacitation, our findings do more closely approximate those experienced in somatic cells wherein, phosphorylation of serine, threonine and tyrosine amino acids occurs at an estimated ratio of 1000:100:1 (
      • Raggiaschi R.
      • Gotta S.
      • Terstappen G.C.
      Phosphoproteome analysis.
      ). In seeking to reconcile these apparently incongruous results, it is perhaps noteworthy that a subset of the serine/threonine substrates identified herein are instead regulated by tyrosine phosphorylation in the spermatozoa of mammalian species, thus raising the possibility of lineage specific expansion of the role of tyrosine kinases in the spermatozoa of higher vertebrates. Illustrative of this phenomenon, we identified the fibrous sheath calcium-binding tyrosine phosphorylation-regulated protein (CABYR) as comprising as many as 17 differentially phosphorylated peptides, not one of which features a phospho-tyrosine residue. As its name suggests, this represents a marked departure from the homologue characterized in mouse (
      • Naaby-Hansen S.
      • Mandal A.
      • Wolkowicz M.J.
      • Sen B.
      • Westbrook V.A.
      • Shetty J.
      • Coonrod S.A.
      • Klotz K.L.
      • Kim Y.H.
      • Bush L.A.
      • Flickinger C.J.
      • Herr J.C.
      CABYR, a novel calcium-binding tyrosine phosphorylation-regulated fibrous sheath protein involved in capacitation.
      ,
      • Li Y.F.
      • He W.
      • Kim Y.H.
      • Mandal A.
      • Digilio L.
      • Klotz K.
      • Flickinger C.J.
      • Herr J.C.
      CABYR isoforms expressed in late steps of spermiogenesis bind with AKAPs and ropporin in mouse sperm fibrous sheath.
      ) and human spermatozoa (
      • Li Y.F.
      • He W.
      • Mandal A.
      • Kim Y.H.
      • Digilio L.
      • Klotz K.
      • Flickinger C.J.
      • Herr J.C.
      • Herr J.C.
      CABYR binds to AKAP3 and Ropporin in the human sperm fibrous sheath.
      ), the former of which harbors as many as seven potential tyrosine phosphorylation motifs that are subject to extensive phosphorylation during in vitro, capacitation (
      • Naaby-Hansen S.
      • Mandal A.
      • Wolkowicz M.J.
      • Sen B.
      • Westbrook V.A.
      • Shetty J.
      • Coonrod S.A.
      • Klotz K.L.
      • Kim Y.H.
      • Bush L.A.
      • Flickinger C.J.
      • Herr J.C.
      CABYR, a novel calcium-binding tyrosine phosphorylation-regulated fibrous sheath protein involved in capacitation.
      ). Characterization of the adaptive significance of such changes remains as an intriguing focus for future research.
      In summary, we have exploited an advanced proteomic platform to improve our understanding of sperm biology in a model reptilian species, the Australian saltwater crocodile. Through the identification of recognized hallmarks of the capacitation cascade, our collective data affirm the hypothesis that crocodile sperm do engage a network of signaling pathways, centering on PKA activity, to promote their functional activation (
      • Nixon B.
      • Anderson A.L.
      • Smith N.D.
      • McLeod R.
      • Johnston S.D.
      The Australian saltwater crocodile (Crocodylus porosus) provides evidence that the capacitation of spermatozoa may extend beyond the mammalian lineage.
      ). In doing so, these data challenge the widely promulgated view that post-testicular sperm maturation is limited to the mammalian lineage.

      DATA AVAILABILITY

      The data set (supplemental Dataset S1) analyzed here have been deposited in the Mass Spectrometry Interactive Virtual Environment (MassIVE) database (Project ID: MassIVE MSV000082258), and are publicly accessible at: https://massive.ucsd.edu/ProteoSAFe/dataset.jsp?task=8acd6725da734f6f89bbd64460d03686.

      Acknowledgments

      We thank the staff at Koorana Crocodile Farm, and in particular John Lever and Robbie McLeod, for assistance with collection of crocodile semen. We are also grateful for the technical assistance of Tamara Keeley and Ed Qualischefski.

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