Table S1: Summary of seminal characteristics of ejaculates collected by manual massage from Australian saltwater crocodiles.

Transcription

1 Electronic supplementary material Table S1: Summary of seminal characteristics of ejaculates collected by manual massage from Australian saltwater crocodiles. Seminal characteristic Mean ± SEM Range Volume (ml) 1.15 ± Sperm concentration ( 10 9 ml -1 ) 2.6 ± Mass activity (0 5) a 1.4 ± % Motile 87 ± Rate of motility (0 5) b 4.2 ± a Mass activity is defined as the amount of swirling (or wave motion) present in the undiluted semen sample and is a function of both sperm concentration and individual sperm motility. The scale for this estimate ranges from: 0 (no swirl no oscillation of individual sperm), 1 (no swirl - generalized oscillation of individual sperm only), 2 (very slow distinct swirl), 3 (slow distinct swirl), 4 (moderately fast distinct swirl), 5 (fast distinct swirl - appearance of good quality semen). b The rate of sperm movement being determined using the following criteria: 0 (no sperm movement), 1 (slight tail undulation without forward motion), 2 (slow tail undulation with slow or stop and start forward motion), 3 (forward progression at a moderate speed), 4 (rapid forward progression), 5 (very rapid progression in which cells are difficult to follow visually).

2 Table S2: Identification of substrates for protein tyrosine phosphorylation following exposure of crocodile spermatozoa to capacitation stimuli. Protein a Symbol Species MW pi MASCOT Peptides (kda) Score b Cytoskeletal function Dynein, axonemal, heavy chain 17 DNAH17 Cathartes aura (Turkey vulture) Filamin A, alpha FLNA Corvus brachyrhynchos (American crow) Outer dense fibre of sperm tails 2 ODF2 Anas platyrhynchos (Mallard) Molecular chaperoning activity Heat shock protein 90kDa alpha (cytosolic), HSP90AA1 Corvus brachyrhynchos class A member 1 (American crow) Heat shock 70kDa protein 5 (glucose- HSPA5 Phoenicopterus ruber regulated protein, 78kDa) ruber

3 (American flamingo) Mitochondrial function Aconitase 2, soluble ACO2 Phalacrocorax carbo (Great cormorant) Cellular signalling RAB2A, member RAS oncogene family RAB2A Corvus brachyrhynchos (American crow) Phosphatidylethanolamine binding protein 1 PEBP1 Taeniopygia guttata (Zebra finch) a Protein identifications were based on alignment of sequenced peptides with those of the Archosauria crown group comprising birds and crocodilians within the UniprotKB database. Proteins have been grouped according to putative function. b The MASCOT Score is a probabilistic scoring algorithm for protein identification ( The recorded MASCOT Score for each protein represents the summed score for all matching peptides, with positive protein identification assigned on the basis of a MASCOT score >35 (corresponding to a 95% confidence level).

4 Figure S1 (a) DAPI PSA Tubulin (b) Acrosome Nucleus Mid-piece Principal-piece Figure S1. Structural characterization of Australian saltwater crocodile spermatozoa. (a) Following isolation, populations of ejaculated crocodile spermatozoa were prepared for labelling of key functional domains with PSA (acrosomal cap, green), DAPI (nucleus, blue), and tubulin (principal-piece of flagellum, red). (b) Alternatively, spermatozoa were lysed in SDS extraction buffer and the extracted proteins resolved by SDS-PAGE alongside those of mouse and human spermatozoa. The separated proteins were subsequently stained with silver reagent.

5

6 Figure S2. Localisation of proteins of interest in crocodile spermatozoa. Following preparation, populations of non-capacitated (NC) and capacitated (CAP) crocodile spermatozoa they were fixed and labelled with either (a) anti-phosphotyrosine or (b) anti-phospho-pka substrate antibodies (green). Alternatively, crocodile spermatozoa were labelled with antibodies against the target proteins of (c) PKA (red) or (d) ODF2 (red) or (e) adenylate cyclase 10 (soluble) (ADCY10) (green). (f) The specificity of the anti-adcy10 antibody was confirmed by immunoblotting of crocodile and mouse sperm lysates as well as a homogenate of mouse brain. Consistent with published evidence the dominant ADCY10 protein detected in sperm lysates was ~48 kda [1]. (g) A proximity ligation assay was also employed to confirm that ODF2 is a substrate for tyrosine phosphorylation during the capacitation of crocodile spermatozoa. This assay produces punctate red fluorescent signals when target antigens of interest (ODF2 and phosphotyrosine residues) reside within a maximum of 40 nm from each other [2]. All experiments were replicated on independent samples from 5 different crocodiles and representative immunolabelling and PLA labelling patterns are shown. In all experiments, cells were counterstained with DAPI (blue). Scale bar = 5µm. 1. Jaiswal B.S., Conti M Identification and functional analaysis of splice variants of the germ cell soluble adenylyl cyclase. J Biol Chem 276(34), (doi: /jbc.m ) 2. Soderberg O., Gullberg M., Jarvius M., Ridderstrale K., Leuchowius K.J., Jarvius J., Wester K., Hydbring P., Bahram F., Larsson L.G., et al Direct observation of individual endogenous protein complexes in situ by proximity ligation. Nat Methods 3(12), (doi: /nmeth947).