The protamines of the noble false widow spider Steatoda nobilis provide an example of liquid-liquid phase separation chromatin transitions during spermiogenesis

Abstract

While there is extensive information about sperm nuclear basic proteins (SNBP) in vertebrates, there is very little information about Arthropoda by comparison. This paper aims to contribute to filling this gap by analyzing these proteins in the sperm of the noble false widow spider Steatoda nobilis (Order Araneae, Family Theridiidae). To this end, we have developed a protein extraction method that allows the extraction of cysteine-containing protamines suitable for the preparation and analysis of SNBPs from samples where the amount of starting tissue material is limited. We carried out top-down mass spectrometry sequencing and molecular phylogenetic analyses to characterize the protamines of S. nobilis and other spiders. We also used electron microscopy to analyze the chromatin organization of the sperm, and we found it to exhibit liquid-liquid phase spinodal decomposition during the late stages of spermiogenesis. These studies further our knowledge of the distribution of SNBPs within the animal kingdom and provide additional support for a proposed evolutionary origin of many protamines from a histone H1 (H5) replication-independent precursor.

Keywords: Sperm nuclear basic proteins (SNBPs); liquid-liquid phase separation; mass spectrometry; phylogeny; protamines; spider.

Fig. 1.. SNBPs of S. nobilis in comparison to other groups of arthropods.

(A) Acid acetic urea PAGE of S. nobilis SNBPs (SN) in relation to the chromosomal proteins of other organisms used as protein markers. CM: chicken erythrocyte nuclear histones. MC: Mytilus californianus (California mussel) SNBPs indicating its major PL-II (Mr = 15.6 kDa) (Carlos et al., 1993b); PL-III (11.3 kDa) (Rocchini et al., 1995a) and PL-IV (6.5 kDa) components (Carlos et al., 1993a) SL: salmine protamine (4.0–4.3 kDa) (Ando, 1973) from the salmon Onchorhyncus keta. (B) Simplified phylogeny of Arthropods adapted from Giribet and Edgecombe (2019) (Giribet and Edgecombe, 2019). (C) Electrophoretic analysis of the SNBPs of several Arthropod representative species. LP: Limulus polyphemus (horse shoe crab) (Munoz Guerra et al., 1982); SN: S. nobilis; CP: Cancer pagurus (brown crab) (Kurtz et al., 2008); AM: Apis mellifera (honey bee) (unpublished) in comparison to salmine. H: Histones; P: Protamines.

Fig. 2.. Protein sequences of spider protamines.

(A) Partial amino acid sequences of the P1 (~6359Da) and P2 (~6396Da) protamines from S. nobilis determined by LC-MS. (B) 1. Amino acid sequence alignment of chicken histone H5 (AAA48798.1) and A. ventricosus H1-H5 like protein (GBL99906). 2. The protamine region (red) is part of the C-terminal region of a histone H5-lke protein (blue). (C) Sequence alignments of the histone H5-like proteins from different spiders: Cyclosa laticauda (IAWK01030102.1); Neoscona scylla (ICBS01001118.1); Eriophora transmarina (IBPW01032809.1); Eriovixia pseudocentrodes (ICEC01024091.1); Larinoides cornutus (IBUI01019351.1) and Larinia phthisica (IAJV01034118.1). In these alignments, (*) indicates that the amino acid is the same for all the sequences at that position and (:) indicates that some of the sequences have different amino acids at that position, but that the chemical properties of the different amino acids are similar. (D) Consensus sequence of spider protamines.

Fig. 3.. Chromatin transitions during Steatoda grossa spermiogenesis.

(1) Early spermatids with beginning of chromatin condensation; (2 a-d) Cross-sections of late spermatids at different stages of chromatin condensation undergoing spinodal decomposition (SD) (2 a-b) and nucleation (2 c-d). (3) Longitudinal section of a late spermatid. (4) Mature sperm. AxB, axonemal basis; Gly, glycogen; N, Nucleus; MM, manchette of microtubules.

Fig. 4.. Chromatin transitions during S. nobilis spermatogenesis.

(A-1) Fig. 3–2b showing spermatid chromatin undergoing spinodal decomposition. (A-2) Three D schematic representation of the cube section indicated in (A-1). (A-3) Representation of the dynamic lamellae in (A-2). (B) Cartoon representation of the major chromatin changes during S. nobilis spermatogenesis. During post-meiotic spermiogenesis, histones of the nucleosomal chromatin fiber become hyperacetylated (yellow flags) in preparation for histone eviction and replacement by protamine precursors. This is a highly dynamic complex process that involves histone degradation and several precursor protamine post-translational modifications (cleavage and phosphorylation) presumably assisted by chromatin remodeling complexes and chaperone proteins that takes place at the interchromatin compartment (Cremer et al., 2020). By the late stages of spermiogenesis, protamines replace most of the germ cell progenitors.