Polymorphism in abalone fertilization proteins is consistent with the neutral evolution of the egg's receptor for lysin (VERL) and positive darwinian selection of sperm lysin

The evolution of species-specific fertilization in free-spawning marine invertebrates is important for reproductive isolation and may contribute to speciation. The biochemistry and evolution of proteins mediating species-specific fertilization have been extensively studied in the abalone (genus Haliotis). The nonenzymatic sperm protein lysin creates a hole in the egg vitelline envelope by species-specifically binding to its egg receptor, VERL. The divergence of lysin is promoted by positive Darwinian selection. In contrast, the evolution of VERL does not depart from neutrality. Here, we cloned a novel nonrepetitive region of VERL and performed an intraspecific polymorphism survey for red (Haliotis rufescens) and pink (Haliotis corrugata) abalones to explore the evolutionary forces affecting VERL. Six statistical tests showed that the evolution of VERL did not depart from neutrality. Interestingly, there was a subdivision in the VERL sequences in the pink abalone and a lack of heterozygous individuals between groups, suggesting that the evolution of assortative mating may be in progress. These results are consistent with a model which posits that egg VERL is neutrally evolving, perhaps due to its repetitive structure, while sperm lysin is subjected to positive Darwinian selection to maintain efficient interaction of the two proteins during sperm competition.     

Full-length sequence of VERL,the egg vitelline envelope receptor for abalone sperm lysin

Abalone sperm use 16 kDa lysin to create a hole in the egg vitelline envelope (VE) by a species-specific, nonenzymatic mechanism. To create the hole, lysin binds tightly to VERL (the VE receptor for lysin), a giant, unbranched glycoprotein comprising 30% of the VE. Binding of lysin to VERL causes the VERL molecules to lose cohesion and splay apart creating the hole. Lysin and VERL represent a cognate pair of gamete recognition proteins, one male the other female, which mediate fertilization. The coevolution of such cognate pairs may underlie the establishment of species-specific fertilization which could be a component of the mechanism to achieve reproductive isolation and hence new species. Here we present the full-length cDNA sequence (11,166 bp) of VERL from the red abalone (Haliotis rufescens). There are 42 amino acids from the start Met residue to the beginning of the first 'VERL repeat'. Most of VERL (9981 bp; 89.4%) consists of 22 tandem repeats of a approximately 153 amino acid sequence that is predicted to be beta-sheet. The last VERL repeat is followed by 353 non-repeat amino acid residues containing a furin cleavage site (RTRR), a ZP domain and a hydrophobic COOH-terminus with a 3' UTR of only 10 nucleotides. VERL repeats 3-22 have been subjected to concerted evolution and consequently have almost identical sequences. Curiously, comparisons of repeats from other species shows that repeats 1 and 2 of red abalone VERL have not been subjected to concerted evolution since the divergence of the red species from the other six California species.     

The Tegula tango: a coevolutionary dance of interacting, positively selected sperm and egg proteins

Reproductive proteins commonly show signs of rapid divergence driven by positive selection. The mechanisms driving these changes have remained ambiguous in part because interacting male and female proteins have rarely been examined. We isolate an egg protein the vitelline envelope receptor for lysin (VERL) from Tegula, a genus of free-spawning marine snails. Like VERL from abalone, Tegula VERL is a major component of the VE surrounding the egg, includes a conserved zona pellucida (ZP) domain at its C-terminus, and possesses a unique, negatively charged domain of about 150 amino acids implicated in interactions with the positively charged lysin. Unlike for abalone VERL, where this unique VERL domain occurs in a tandem array of 22 repeats, Tegula VERL has just one such domain. Interspecific comparisons show that both lysin and the VERL domain diverge via positive selection, whereas the ZP domain evolves neutrally. Rates of nonsynonymous substitution are correlated between lysin and the VERL domain, consistent with sexual antagonism, although lineage-specific effects, perhaps owing to different ecologies, may alter the relative evolutionary rates of sperm- and egg-borne proteins.     

鲍配子识别蛋白的研究

配子相互作用的生化机制对于进一步阐明生殖过程具有重要作用,它是深入了解细胞内识别的理想体系。精卵细胞相互作用包括一系列的步骤,开始于精子与卵细胞外被的接触,终止于两性细胞的融合及精子核进入卵细胞质中,而精卵细胞的识别具有建立于各自性细胞表面成分基础上的种的特异性,鲍则是研究精卵识别的好材料。鲍精子在发生顶体反应时释放出两种蛋白质——细胞溶素(1ysin)和18ku糖蛋白(spl8),其中的细胞溶素与其卵黄膜上的受体紧密结合,并利用非酶反应在卵黄膜上穿一个小孔,整个精子则从此孔穿过卵黄膜与卵细胞融合;spl8释放后则覆盖到精子细胞膜表面,起到溶解卵细胞脂质体的作用,即spl8介导精、卵细胞膜的融合。鲍卵细胞膜上存在细胞溶素受体,它是大的不分支的糖蛋白分子,占据了卵黄膜30％的组分,可以专一性地与细胞溶素相结合。这些配子识别蛋白共同进化且速度很快,其中细胞溶素和18ku糖蛋白通过正向选择进化,而细胞溶素受体进行协同进化。     

Coevolution of Interacting Fertilization Proteins

Reproductive proteins are among the fastest evolving in the proteome, often due to the consequences of positive selection, and their rapid evolution is frequently attributed to a coevolutionary process between interacting female and male proteins. Such a process could leave characteristic signatures at coevolving genes. One signature of coevolution, predicted by sexual selection theory, is an association of alleles between the two genes. Another predicted signature is a correlation of evolutionary rates during divergence due to compensatory evolution. We studied female–male coevolution in the abalone by resequencing sperm lysin and its interacting egg coat protein, VERL, in populations of two species. As predicted, we found intergenic linkage disequilibrium between lysin and VERL, despite our demonstration that they are not physically linked. This finding supports a central prediction of sexual selection using actual genotypes, that of an association between a male trait and its female preference locus. We also created a novel likelihood method to show that lysin and VERL have experienced correlated rates of evolution. These two signatures of coevolution can provide statistical rigor to hypotheses of coevolution and could be exploited for identifying coevolving proteins a priori. We also present polymorphism-based evidence for positive selection and implicate recent selective events at the specific structural regions of lysin and VERL responsible for their species-specific interaction. Finally, we observed deep subdivision between VERL alleles in one species, which matches a theoretical prediction of sexual conflict. Thus, abalone fertilization proteins illustrate how coevolution can lead to reproductive barriers and potentially drive speciation.     

Speciation genes in free-spawning marine invertebrates

Research on speciation of marine organisms has lagged behind that of terrestrial ones, but the study of the evolution of molecules involved in the adhesion of gametes in free-spawning invertebrates is an exception. Here I review the function, species-specificity, and molecular variation of loci coding for bindin in sea urchins, lysin in abalone and their egg receptors, in an effort to assess the degree to which they contribute to the emergence of reproductive isolation during the speciation process. Bindin is a protein that mediates binding of the sperm to the vitelline envelope (VE) of the egg and the fusion of the gametes' membranes, whereas lysin is a protein involved only in binding to the VE. Both of these molecules are important in species recognition by the gametes, but they rarely constitute absolute blocks to interspecific hybridization. Intraspecific polymorphism is high in bindin, but low in lysin. Polymorphism in bindin is maintained by frequency-dependent selection due to sexual conflict arising from the danger of polyspermy under high densities of sperm. Monomorphism in lysin is the result of purifying selection arising from the need for species recognition. Interspecific divergence in lysin is due to strong positive selection, and the same is true for bindin of four out of seven genera of sea urchins studied to date. The differences between the sea urchin genera in the strength of selection can only partially be explained by the hypothesis of reinforcement. The egg receptor for lysin (VERL) is a glycoprotein with 22 repeats, 20 of which have evolved neutrally and homogenized by concerted evolution, whereas the first two repeats are under positive selection. Selection on lysin has been generated by the need to track changes in VERL, permitted by the redundant structure of this molecule. Both lysin and bindin are important in reproductive isolation, probably had a role in speciation, but it is hard to determine whether they meet the strictest criteria of "speciation loci," defined as genes whose differentiation has caused speciation.     

Seasonal variations in biochemical composition during the reproductive cycle of the veined rapa whelk Rapana venosa (Valenciennes, 1846) from the northern coast of China

Seasonal variations in the biochemical composition of Rapana venosa in relation to reproductive cycle and environment on the northern coast of China were investigated from March 2012 to February 2013. The results indicated that R. venosa has an annual reproductive cycle with synchronized gonad development in both females and males. Gametogenesis was initiated in September and gametes developed slowly during the winter, followed by rapid gonad development during spring and summer. Most individuals from this study were sexually mature between May and June, and gamete release occurred mainly between May and August. The peak of spawning was found in July and the recovery of the gonad was observed between August and November. The key biochemical components including glycogen, protein and lipid were analysed in four tissues, specifically the gonad, digestive gland, mantle and foot. The declining glycogen content in the gonad, digestive gland and mantle during maturation suggested that glycogen was consumed during the development of the gonad. Lipids and protein can be stored in the digestive gland and used during the winter in a period of food shortage. The protein and lipid contents in the ovaries increased during gonad development, which suggested that the protein and lipid had been accumulated as vitellin in oocytes.     

Annual changes in testicular development and plasma sex steroids in the captive male dojo loach <Emphasis Type="Italic">Misgurnus anguillicaudatus</Emphasis>

Testicular development in the captive male dojo loach Misgurnus anguillicaudatus was examined monthly in relation to the levels of plasma sex steroids [testosterone (T), 11-ketotestostrone (11-KT), and 17,20β-dihydroxy-4-pregnen-3-one (DHP)]. On the basis of testicular histology, the annual gonadal cycle was found to be divisible into 3 periods: the recovery and proliferation period, which mainly consists of early spermatogenic testis from August to November (reproductive phase I); the preparation period for the next spawning period, which mainly consists of late spermatogenic testis from December to April (reproductive phase II); and the mature period, characterized by a high proportion of mature testis from May to July (reproductive phase III). Individual variability in testicular development was high, and continuous spermatogenesis was observed throughout the year. High levels of plasma T, 11-KT, and DHP were observed during reproductive phase III. 11-KT began to increase in February, while T was present at low levels in reproductive phase II. These results suggest that the physiologically active season of testis development for breeding in the dojo loach is from May to July, although spermatogenesis occurs throughout the year.     

Reproductive cycles in a reared strain of the mummichog, a daily spawner

Annual, lunar, and diel samplings were taken from a strain of mummichog (Arasaki strain) reared in outdoor tanks under natural conditions, to examine gonadal maturity. Gonads of yearling fish were quite immature in September. During late autumn and winter, a gradual increase in the GSI of both sexes was observed, and the growth of cortical alveolus phase oocytes in females and basal spermatogenesis in males progressed. In late February, a rapid increase in the GSI of both sexes, vitellogenesis in females, and active spermatogenesis in males, occurred. The spawning period of the yearling fish was from late March to August judging from the presence of milt-producing males and ovulated females. The spawning period of the underyearling fish started in the same month as the yearlings, but terminated 1 month earlier. Plasma levels of oestradiol-17 β (E₂) in females and testosterone in males were high during the spawning period in the yearlings. In the underyearlings, however, E₂ levels peaked in early spring, and declined in the latter part of the spawning period. Neither a lunar nor semilunar cycle was evident in the reproductive activity of this fish, which proved to be a typical daily spawner. Females showed an apparent daily reproductive cycle; oocyte maturation commenced at about 1200 hours, germinal vesicle breakdown (GVBD) occurred at about 2400 hours, and ovulation was completed by 2400 hours, 24 h after GVBD. Such clear annual and daily reproductive cycles make this strain of mummichog a suitable model for the study of environmental and endocrine regulation of reproductive cycles in marine and estuarine teleosts.     

Variations in the levels of progesterone in Mytilus edulis during the annual reproductive cycle

• 1.1. Progesterone levels in Mytilus edulis males and females during the annual reproductive cycle were analysed in the whole animal and in the gonads using gas-liquid chromatography and radioimmunoassays.
• 2.2. The high hormone levels in the whole animal were observed in July and October, coincident with the main spawning seasons.
• 3.3. The levels of progesterone in gonad extracts also show a maximum in summer (July).
• 4.4. The patterns of the progesterone levels in males and females throughout the annual reproductive cycle are similar.
• 5.5. These data are discussed in relation to the role of progesterone in the regulation of sex-specific processes, particularly gametogenesis.

     

Purification and immunocytochemical localization of the vitelline coat lysin of abalone spermatozoa

Spermatozoa of the abalone Haliotis discus were treated with high-calcium seawater to induce the acrosome reaction. The soluble components released from the sperm acrosomal vesicles showed potent lytic activity on the egg vitelline coat. A vitelline coat lysin was purified by salting-in, preparative polyacrylamide gel electrophoresis, and high-performance liquid chromatography. Its molecular weight was 15,500 and its isoelectric point 9.6. These properties were similar to those of other molluskan vitelline coat lysins. The lysin was immunocytochemically localized using a protein A-gold technique, in the posterior half of the acrosomal vesicle.     

The gene fl(2)d is needed for the sex-specific splicing of transformer pre-mRNA but not for double-sex pre-mRNA in Drosophila melanogaster

In Drosophila melanogaster, regulation of the sex determination genes throughout development occurs by sex-specific splicing of their products. The first gene is Sex-lethal(Sxl). The downstream target of Sxl is the gene transformer (tra): the Sxl protein controls the female-specific splicing of the Tra pre-mRNA. The downstream target of the gene tra is the gene double-sex (dsx): the Tra protein of females, controls the female-specific splicing of the Dsx pre-mRNA. We have identified a gene, female-lethal-2-d fl(2)d, whose function is required for the female-specific splicing of Sxl pre-mRNA. In this report we analyze whether the gene fl(2)d is also required for the sex-specific splicing of both Tra and Dsx pre-mRNAs. We found that the Sxl protein is not sufficient for the female-specific splicing of Tra pre-mRNA, the fl(2)d function also being necessary. This gene, however, is not required for the female-specific splicing of Dsx pre-mRNA.     

The first sex-specific molecular marker discovered in the moss Pseudocalliergon trifarium

Most dioecious plants do not exhibit discernible sexual dimorphism before sexual maturity. Therefore, it is impossible to address any sex-related questions during the prereproductive phase unless a genetic sex marker is available for gender determination. The aim of the present study was to develop a genetic sex marker for the moss Pseudocalliergon trifarium to allow gender and sex ratio determination at any stage in the life cycle. A high proportion of P. trifarium populations do not express sex. The screening of genomic DNA with inter simple sequence repeat (ISSR) primers was used to discover sex-specific polymerase chain reaction (PCR) amplification products. A presumably female-specific band was found, excised from the gel, cloned, and sequenced. A sequence-walking method was used to characterize the same region in males. A primer pair was designed to allow the amplification of a 159-bp portion of the female-specific DNA region. All tested material, up to 16-year-old herbarium specimens, provided unambiguous amplification products. This study successfully provides, for the first time in a moss, a sex-specific DNA marker. It allows reliable determination of gender and sex ratios. The short length of the amplification product is an advantage as satisfactory PCR products are more likely when the targeted sequence is short. The amount of variation in the DNA region shared by both sexes was relatively high. If the male sequence can be better characterized, the sex-specific regions could possibly be used to evaluate sex-specific phylogeographic patterns.     

The regulation of the yolk protein genes,a family of sex differentiation genes in Drosophila melanogaster

There are many obvious morphological and behavioural differences between male and female Drosophila, whose differing phenotypes are produced by a hierarchy of sex determination genes. These genes have been well characterised at the genetic and molecular level. Similarly, a number of sex-specific differentiation genes have been characterised, such as the chorion and vitelline membrane genes in females and the sex peptide and other accessory gland proteins in males. Despite the depth of these parallel studies, there is only one example of a direct link between the sex determination pathway and the downstream sex differentiation genes, namely the regulation of the female-specific yolk protein genes. The yolk proteins are synthesised in the fat body and ovarian follicle cells of the adult female and are subsequently transported to the oocyte where they are stored for utilization during embrygenesis. The expression of the yolk protein genes is not entirely controlled by the sex determination hierarchy, as several different regulatory pathways must interact to direct their correct sexual, temporal and spatial regulation during development.     

Relative growth and reproductive cycle in two populations of <Emphasis Type="Italic">Bolinus brandaris</Emphasis> (Gastropoda: Muricidae) from northern Tunisia (Bizerta Lagoon and small Gulf of Tunis)

A study on the relative growth and reproductive cycle of the purple dye murex (Bolinus brandaris) was performed from March 2007 to February 2008 in two populations with contrasting imposex levels (Carthage Byrsa — CB in the small Gulf of Tunis and Menzel Abderrahmane — MA in the Bizerta Lagoon). Both populations presented balanced sex ratios. Comparison of allometric relationships established between linear and ponderal variables (sexes confounded) revealed higher growth in CB than in MA. Mature individuals were found throughout the year, except for September in MA. Spawning periods occurred from March–April to May and from June to August in both sites. Spawning was associated to an increase in seawater temperature. Besides increasing the current knowledge on the biology of B. brandaris from the southern Mediterranean, the information gathered in the present study constitutes a useful baseline for the sustainable management of local wild stocks, namely by prohibiting collection of snails during the spawning season.     

Screening and identification of female-specific DNA sequences in octaploid sturgeon using comparative genomics with high-throughput sequencing

In this study, six candidate female-specific DNA sequences of octaploid Amur sturgeon (Acipenser schrenckii) were identified using comparative genomic approaches with high-throughput sequencing data. Their specificity was confirmed by traditional PCR. Two of these sex-specific sequences were also validated as female-specific in other eight sturgeon species and two hybrid sturgeons. The identified female-specific DNA fragments suggest that the family Acipenseridae has a ZZ/ZW sex-determining system. However, one of the two DNA sequences has been deleted in some sturgeons such as Sterlet sturgeon (Acipenser ruthenus), Beluga (Huso huso) and Kaluga (H. dauricus). The difference of sex-specific sequences among sturgeons indicates that there are different sex-specific regions among species of sturgeon. This study not only provided the sex-specific DNA sequences for management, conservation and studies of sex-determination mechanisms in sturgeons, but also confirmed the capability of the workflow to identify sex-specific DNA sequences in the polyploid species with complex genomes.