Ovarian fluid plays an essential role in attachment of Eurasian perch, Perca fluviatilis eggs

Eggs of the Eurasian perch, Perca fluviatilis, are strongly attached to each other to form 0.5-5.5 m long and 1-6 cm wide floating jelly strands or ribbons. The processes involved in the initiation of egg attachment were investigated in the present study. Eggs are released together with small amounts of ovarian fluid. When the eggs come in contact with water, a fibrous jelly-like structure is formed by interactions between proteins of the ovarian fluid and proteins of the zona radiata externa (ZRE) leading to the egg attachment with each other. This mechanism is unique in teleost fish and is described for the first time in the present paper.     

Adhesion mechanisms in European whitefish Coregonus lavaretus eggs: is this a survival mechanism for high‐energy spawning grounds?

This study examined the potential biochemical and mechanical structures that may contribute to egg adhesion in European whitefish Coregonus lavaretus. Experiments showed that eggs from a population of C. lavaretus from Loch Eck remained non‐adhesive in a solution chemically similar to ovarian fluid but became adhesive seconds after contact with water. Examination of the ultrastructure of the chorion showed that the morphology changed significantly after contact with water, with nodule‐like protuberances attached to connective filaments on the surface present in water‐hardened but not non‐water hardened eggs. Biochemical analysis showed the presence of Chain A, RNase ZF‐3e proteins in the chorion of water‐hardened but not non‐water hardened eggs. Histochemical staining of the chorion of C. lavaretus eggs showed that the externa, but not the interna, stained positively for the presence of glycoproteins. From these results, it was concluded that C. lavaretus from Loch Eck possess both anatomical and biochemical adhesive mechanisms that have been undocumented in this species so far.     

Amino acid cues emanating from Pacific salmon eggs and ovarian fluid

The eggs of salmonid fishes are an important food source for many aquatic predators that detect eggs using olfaction. Moreover, chemicals from eggs and ovarian fluid aid sperm cells in detecting and locating eggs for fertilization, and ovarian fluid is attractive to conspecific males. Thus chemicals from eggs and ovarian fluid may facilitate reproduction but may also attract egg predators. The authors sampled mature females of three Pacific salmon species – Chinook (Oncorhynchus tshawytscha), coho (Oncorhynchus kisutch) and sockeye (Oncorhynchus nerka) – and determined the proportional representation of amino acids, potent fish odorants, from their eggs and ovarian fluid (Chinook and coho salmon only). They then tested juvenile coho salmon, an egg predator, for responses to ovarian fluid and egg odours using the electro-olfactogram (EOG) recording technique. The amino acid compositions of the salmon species were significantly and positively correlated with each other, and the interspecific differences were comparable to those between individuals of the same species. The egg water samples were, on average, dominated by lysine, alanine and glutamine (12.6%, 12.4% and 10.9%, respectively). The ovarian fluid samples were dominated by lysine (20.5%), followed by threonine (9.7%), glycine (9.2%) and arginine (8.8%). EOG recordings demonstrated the ability of juvenile coho salmon to detect the chemical traces of eggs and ovarian fluid. It is concluded that salmon eggs are a potent source of odours for potential predators but likely not highly differentiated among salmon species.     

The cytochemistry of Limulus eggs.

Cytochemical studies on uninseminated mature eggs of Limulus demonstrate the presence of carbohydrates, lipids and proteins in the egg envelopes and yolk. The vitelline envelope, cortical region and yolk are rich in 1,2-glycols, with the vitelline envelope, containing fewer reactive 1,2-glycol groups than other components of the egg. Neutral mucopolysaccharides are found in the cortical region and yolk, but only the cortical region of the eggs demonstrate the presence of sulfated mucosubstances (which are in part glycoprotein in nature) and glucose-6-phosphatase. Protein is evident in all egg components. Biochemical analysis demonstrate the protein in the egg envelopes of uninseminated eggs is composed of sixteen amino acids while that of developing eggs contain seventeen amino acid residues. Electrovalent linkages and non-S-S- covalent linkages between protein chains are shown to be instrumental in maintaining the stuctural integrity of Limulus egg envelopes. Neutral lipids, unsaturated lipids, phospholipids and fatty acids are demonstrated in yolk bodies and lipoproteins, unsaturated lipids and fatty acids constitute part of the egg envelopes. DNA is concentrated in the cortical region and the yolk bodies     

A rapid and sensitive method for the analysis of carbohydrate components in glycoproteins using gas-liquid chromatography

A rapid and simple gas-liquid chromatographic method for the determination of subnanomolar amounts of carbohydrates derived from glycoproteins is described. The procedure involves methanolysis in the presence of methyl acetate followed by removal of hydrogen chloride by coevaporation with t-butyl alcohol and trimethylsilylation. The method is also applicable to samples containing uronic acids and lipids.     

Gamete Interactions in Xenopus laevis: Identification of Sperm Binding Glycoproteins in the Egg Vitelline Envelope

A quantitative assay was developed to study the interaction of Xenopus laevis sperm and eggs. Using this assay it was found that sperm bound in approximately equal numbers to the surface of both hemispheres of the unfertilized egg, but not to the surface of the fertilized egg. To understand the molecular basis of sperm binding to the egg vitelline envelope (VE), a competition assay was used and it was found that solubilized total VE proteins inhibited sperm-egg binding in a concentration-dependent manner. Individual VE proteins were then isolated and tested for their ability to inhibit sperm binding. Of the seven proteins in the VE, two related glycoproteins, gp69 and gp64, inhibited sperm-egg binding. Polyclonal antibody was prepared that specifically recognized gp69 and gp64. This gp69/64 specific antibody bound to the VE surface and blocked sperm binding, as well as fertilization. Moreover, agarose beads coated with gp69/64 showed high sperm binding activity, while beads coated with other VE proteins bound few sperm. Treatment of unfertilized eggs with crude collagenase resulted in proteolytic modification of only the gp69/64 components of the VE, and this modification abolished sperm-egg binding. Small glycopeptides generated by Pronase digestion of gp69/64 also inhibited sperm-egg binding and this inhibition was abolished by treatment of the glycopeptides with periodate. Based on these observations, we conclude that the gp69/64 glycoproteins in the egg vitelline envelope mediate sperm-egg binding, an initial step in Xenopus fertilization, and that the oligosaccharide chains of these glycoproteins may play a critical role in this process.     

The testicular main ducts and the spermatic ducts in some cyprinid fishes—II. Composition of the seminal fluid

The composition of the organic compounds of the seminal fluid, pH values and osmolalities were investigated in three cyprinid species, the bleak ( Alburnus alburnus ), the chub ( Leuciscus cephalus ) and the zaehrte ( Vimba vimba ). The seminal fluid contains monosaccharides (glucose, fructose, galactose, xylose), lipids (cholesterol, fatty acids, phosphatidylcholine, glycolipids) and proteins, and exhibits activities of acid phosphatase, β-glucuronidase, proteases and to some extent of alkaline phosphatase. The composition of free amino acids reveals species specific differences.     

Carp ovarian cystatin binds and agglutinates spermatozoa via electrostatic interaction

A sperm-agglutinating factor was purified from ovulated carp eggs and the conditioned medium (CM) of cortical-reacted eggs. It was identified to be the carp ovarian cystatin. Three cystatin isoforms were found. The cystatin isolated from the CM had a higher sperm-agglutinating activity than that isolated from eggs, although the cystatins have identical N-terminal amino acid sequences, masses, and positive charges. Differences in sperm-agglutinating activity between the cystatins of the CM and eggs may be caused by the different conformations because they differed in circular dichroism spectrum and tryptic map. Cystatin was discharged from cortical granules to the perivitelline space after fertilization and is abundant in the perivitelline fluid (PVF) of early stage embryos. Cystatin rapidly agglutinated spermatozoa via an electrostatic interaction. Other basic proteins also agglutinated carp spermatozoa. Their activities were inhibited by salt and high pH. Cystatin bound to the entire surface of carp spermatozoa. The PVF of early embryos agglutinated carp spermatozoa. The activity was related to the cystatin content and influenced by ionic strength and pH. Therefore, cystatin is the major sperm-agglutinating factor of PVF. Owing to the rapid action of cystatin on spermatozoa agglutination and the presence of a high concentration of cystatin in PVF, cystatin is considered important for preventing polyspermy in carp eggs.     

雌核发育银鲫与两性生殖彩鲫卵壳蛋白组分的比较分析及其受精前后的变化

以雌核发育银鲫和两性生殖彩鲫的成熟卵为材料,分离卵壳,经处理得到卵壳可溶性蛋白组分。SDS－PAGE梯度凝胶电泳分析在雌核发育银鲫中揭示出3条较明显的差异蛋白带。同时,采用相同处理方法对受精前后卵壳蛋白组分进行比较分析后发现,这些差异蛋白带在受精后发生了变化,其带纹表现为减弱或消失,表明这些差异蛋白可能与受精过程相关。 Abstracts:Egg chorions were isolated from unfertilized and fertilized eggs of gynogenetic silver crucian carp and gonochoristic color crucian carp by homogenization and further purification techniques.Then,soluble proteins were extracted from the isolated egg chorions,and were analyzed by gradient SDS-PAGE.Three differential protein bands were revealed between the gynogenetic silver crucian carp and gonochoristic color crucian carp.Furthermore,these differential proteins were demonstrated to undergo obvious changes during fertilization.It was suggested that these differential proteins should be related to the special fertilization process in gynogenetic silver crucian carp.     

Biochemical composition of cuttlefish (Sepia esculenta) eggs during embryonic development

Changes in egg volume, water content, proteins, carbohydrates, lipids, amino acids and fatty acids in cuttlefish (Sepia esculenta) were determined during embryogenesis to understand the nutritional requirements in the early life phase. The egg volume and the water content decreased significantly (P<0.05) during early embryonic development, and then increased abruptly after the beginning of organ differentiation. During embryonic development, protein was the major content in the yolk and the per cent composition varied to a large extent (55.19–78.45%) with carbohydrates (9.86–15.56%) and lipids (1.62–2.97%). Proteins and lipids decreased 23.3% and 0.4%, respectively, in dry weight, while carbohydrates increased 0.46%. Total amino acids and essential amino acids (EAAs) were stable during the early embryonic developmental stage, but decreased significantly until the eggs hatched (P<0.05). The largest utilisation of the yolk protein possibly occurred with respect to EAAs (25.7%) because of a decrease in methionine (70.3%), valine (63.0%) and phenylalanine (58.5%). The most important fatty acids were saturated fatty acid (SFA) C16:0 and polyunsaturated fatty acids (PUFAs) C22:6 and C20:5. Unsaturated fatty acids (UFAs) and SFA decreased at a similar rate during embryonic development (5.69% and 6.15%, respectively). For UFAs, monounsaturated fatty acids were consumed at a greater rate than PUFAs (29.6% and 0.05%, respectively).     

Osmotic changes during the development of eggs and larvae of the lumpsucker, Cydopterus lumpus L.

The demersal eggs of Cyclopterus appear to osmoregulate like the pelagic eggs of cod and plaice. Unfertilized eggs in ovarian fluid exhibited ovoplasm osmolarities similar to those of adult blood and ovarian fluid (356–359 mosmol kg⁻¹). Yolk osmolarities remained virtually constant from fertilization and during development (356–366 mosmol), with a slight decrease near hatching (to 332 mosmol). Yolk and body fluids of larvae (338 mosmol) had osmoconcentrations similar to egg yolk values near hatching. Yolk osmoconcentration of unfertilized eggs remained unchanged during the first 12 h in sea water, with a slow increase thereafter. Fertilized eggs of bad quality cultures exhibited higher yolk osmoconcentrations than eggs of good quality. Cyclopterus eggs were found to develop normally and survive in 20–34%o salinity, larvae seemed to have the same salinity range.     

Homology model and molecular dynamics simulation of carp ovum cystatin

In this study, a homology model of carp ovum cystatin was constructed based on the crystal structure of chicken egg white cystatin. The results of amino acid sequence alignment indicate that these two proteins exhibit 36.11% of sequence identity. The resultant homology model reveals that carp ovum cystatin shares similar folds as chicken egg white cystatin, particularly in the conserved regions of Q48-V49-G52 and P98-W99 and the locations of two disulfide bonds, C67-C76 and C90-C110. However, the results of 1 ns molecular dynamics simulations show that carp ovum cystatin exhibits less structural integrity than chicken egg white cystatin in explicit water at 300 K. The relatively hydrophilic Met62 of carp ovum cystatin, corresponding to the hydrophobic Leu68 of human cystatin C and Ile66 of chicken egg white cystatin, may destabilize the hydrophobic core and form a dimeric structure more easily through domain swapping. A total of 16 positively charged residues are equally distributed on the surface of carp ovum cystatin, resulting in agglutination with the negatively charged spermatozoa via electrostatic interaction. Thus, carp ovum cystatin is considered to be important in preventing carp eggs from polyspermy.     

Fibroin-like substance is a major component of the outer layer of fertilization envelope via which carp egg adheres to the substratum

Seven cDNA encoding silkworm fibroin homologues were cloned from a carp ovarian cDNA library. The encoded proteins are denoted as carp ovarian fibroin-like substances (FLS). FLS contain a repetitive domain consisting of tandem repeats of dipeptide of Gly-X, where X may be any amino acid. Each FLS has its own unique repeating sequence, such as GQGAGQGS, GQGMGQGM, GRGQGEGHGS, and GFGFGQGS, indicating a family of FLS genes exists in carp. FLS is exclusively expressed in oocytes and is stored in cortical granules. During cortical reaction, FLS is exocytosed to perivitelline space and then gradually added to the outer layer of the fertilization envelope (FEo). The FLS of fertilization envelope is conjugated with cystatin and cathepsin-like substance (CLS) and appears in multiple bands of molecular weights ranging from 40 to 205 kDa. After fertilization or artificial activation, carp eggs adhere firmly to the substratum via FEo. FLS is a major component of FEo. The presence of transglutaminase inhibitor, cadaverine or ethylene diaminetetraacetic acid, in the cortical reaction medium can impair or block the recruitment of FLS and other substances to FEo. As a consequence, FEo is not formed or is greatly reduced, resulting in a great reduction of egg adhesion.     

Structural and functional relationships between mouse and hamster zona pellucida glycoproteins

The hamster egg's extracellular coat, or zona pellucida, consists of three glycoproteins, designated hZP1, hZP2, and hZP3, that exhibit extensive heterogeneity on SDS-PAGE. hZP1 is a relatively minor component of hamster zonae pellucidae, as compared with hZP2 and hZP3. In the presence of reducing agents, hZP1, 200,000 apparent Mr, migrates on SDS-PAGE with an apparent Mr of 103,000. This suggests that hZP1, like mouse ZP1, is composed of two polypeptides held together by intermolecular disulfides. When purified hamster ZP glycoproteins were tested at relatively low concentrations in an in vitro competition assay, employing either hamster or mouse gametes, only hZP3 (56,000 apparent Mr) exhibited sperm receptor activity (i.e., inhibited binding of sperm to eggs). Thus, apparently hZP3 is the hamster counterpart of mouse ZP3, the mouse egg receptor for sperm. Furthermore, at relatively high concentrations, solubilized hamster egg ZP preparations induced both hamster and mouse sperm to undergo the acrosome reaction in vitro. hZP3 is encoded by a relatively abundant ovarian mRNA that is detected by a mouse ZP3 cDNA probe and is the same size, about 1.5 kb, as mRNA encoding the mouse sperm receptor, ZP3 (83,000 apparent Mr). Like mouse ZP2, hZP2 undergoes limited proteolysis following artificial activation of hamster eggs in vitro. Results of in vitro assays employing intact eggs and isolated zonae pellucidae demonstrate that hamster eggs possess a ZP2-proteinase which has a substrate specificity similar to that of the mouse enzyme. These observations are discussed in terms of structural and functional relationships that may exist between hamster and mouse zona pellucida glycoproteins.     

Differences in carbohydrate profiles in batch culture grown planktonic and biofilm cells of Amphora rostrata Wm. Sm

Diatoms are abundant in biofilms developed on surfaces immersed in sunlit waters. In both the planktonic and the biofilm mode of growth, diatoms produce carbohydrate polymers which perform several functions including motility, protection, production of macro-aggregates and detoxification. However, little is known about the differences, if any, in the production and characterization at the molecular level of carbohydrates in planktonic and biofilm cells. In order to identify the differences in these two modes of growth, the concentration of total carbohydrates, carbohydrate fractions, neutral carbohydrates, uronic acids and amino sugars in planktonic and biofilm cells of Amphora rostrata were measured. The results showed that the distribution of carbohydrate fractions, uronic acids and amino sugars was different in biofilm and planktonic cells. Cell normalized concentrations of these components were two to five times greater in planktonic cells compared with biofilm cells. The concentrations of glucose and glucosamine decreased, whereas fucose increased in planktonic cells over the period of cultivation. Conversely, the concentrations of glucose and glucosamine increased while that of fucose decreased in attached cells. The study suggests that marked differences exist between the carbohydrates of the planktonic and the biofilm cells of A. rostrata.     

Lectin affinity chromatography of proteins bearing O-linked oligosaccharides: application of jacalin-agarose.

The lectin jacalin immobilized on agarose was found to bind a variety of glycoproteins known to contain typical O-linked oligosaccharides, including human IgA, C1 inhibitor, chorionic gonadotropin, plasminogen, bovine protein Z, bovine coagulation factor X, and fetuin. These proteins were eluted from columns of jacalin-agarose specifically by alpha-galactopyranosides such as melibiose and alpha-methylgalactopyranoside but not by lactose or other sugars. Treatment of asialofetuin with endo--alpha--N--acetylgalactosaminidase eliminated its affinity for the lectin column, and other proteins known to contain only N-linked oligosaccharides such as ovalbumin, transferrin, and alpha 1-acid glycoprotein were not retained by the lectin. Binding of proteins with O-linked oligosaccharides to the lectin column did not require divalent cations and was affected little by changes in pH and ionic strength over a wide range. Virtually all of the glycosidically linked oligosaccharides of fetuin, chorionic gonadotropin, and plasminogen are known to be sialated. Thus, binding of these glycoproteins to jacalin, which is known to have affinity for the core disaccharide, 1-beta-galactopyranosyl-3-(alpha-2-acetamido-2-deoxygalactopyranoside ), in O-linked oligosaccharides of these proteins, was not prevented by the presence of sialic acids. Affinity of oligosaccharides for jacalin did appear to be reduced by occurrence of sialic acids as it was found that higher concentrations of melibiose were required to elute asialofetuin than fetuin from jacalin-agarose. Results of the present study indicate that affinity chromatography using this lectin is a widely applicable technique for identifying and purifying proteins bearing O-linked oligosaccharides.     

A proteome analysis of the subcutaneous gel in avian hatchlings

An appropriate level of water loss from eggs is critical to successful hatching. This water may be lost from the egg by evaporation, but where water loss is suboptimal, it is commonly observed that the hatchlings contain substantial amounts of a subcutaneous gel-like fluid. To characterize this fluid, we have analyzed the proteins that are contained within it. The protein complement comprised a small number of proteins in high concentrations. Proteomics analysis of the constituent proteins identified virtually all of these abundant proteins and confirmed that the subcutaneous gel was very similar in protein composition to plasma. However, the subcutaneous gel was substantially depleted of fibrinogen. It is possible that activation of the final stages of the coagulation process might account for the enhanced viscosity, creating a gel-like material that is relatively immobile in the subcutaneous space. This gel may function as a water volume that is partitioned during embryonic development in order to mitigate the effects of high water content of the egg caused by low mass loss during incubation and in some instances might also function as a water reserve to support the hatchling in the first few hours of life free of the shell.     

CRYPTIC CHOICE OF CONSPECIFIC SPERM CONTROLLED BY THE IMPACT OF OVARIAN FLUID ON SPERM SWIMMING BEHAVIOR

Despite evidence that variation in male–female reproductive compatibility exists in many fertilization systems, identifying mechanisms of cryptic female choice at the gamete level has been a challenge. Here, under risks of genetic incompatibility through hybridization, we show how salmon and trout eggs promote fertilization by conspecific sperm. Using in vitro fertilization experiments that replicate the gametic microenvironment, we find complete interfertility between both species. However, if either species’ ova were presented with equivalent numbers of both sperm types, conspecific sperm gained fertilization precedence. Surprisingly, the species’ identity of the eggs did not explain this cryptic female choice, which instead was primarily controlled by conspecific ovarian fluid, a semiviscous, protein‐rich solution that bathes the eggs and is released at spawning. Video analyses revealed that ovarian fluid doubled sperm motile life span and straightened swimming trajectory, behaviors allowing chemoattraction up a concentration gradient. To confirm chemoattraction, cell migration tests through membranes containing pores that approximated to the egg micropyle showed that conspecific ovarian fluid attracted many more spermatozoa through the membrane, compared with heterospecific fluid or water. These combined findings together identify how cryptic female choice can evolve at the gamete level and promote reproductive isolation, mediated by a specific chemoattractive influence of ovarian fluid on sperm swimming behavior.     

Proteomic Analysis of Chinook Salmon (Oncorhynchus tshawytscha) Ovarian Fluid

The ovarian, or coelomic, fluid that is released with the egg mass of many fishes is increasingly found to play an important role in several biological processes crucial for reproductive success. These include maintenance of oocyte fertility and developmental competence, prolonging of sperm motility, and enhancing sperm swimming speed. Here we examined if and how the proteome of chinook salmon (Oncorhynchus tshawytscha) ovarian fluid varied among females and then sought to examine the composition of this fluid. Ovarian fluid in chinook salmon was analyzed using 1D SDS PAGE and LC-MS/MS tryptic digest screened against Mascot and Sequest databases. We found marked differences in the number and concentrations of proteins in salmon ovarian fluid across different females. A total of 174 proteins were identified in ovarian fluid, 47 of which were represented by six or more peptides, belonging to one of six Gene Ontology pathways. The response to chemical stimulus and response to hypoxia pathways were best represented, accounting for 26 of the 174 proteins. The current data set provides a resource that furthers our understanding of those factors that influence successful egg production and fertilisation in salmonids and other species.     

Acquisition of Salmonella flora by turtle hatchlings on commercial turtle farms

A commercial turtle pond in South Louisiana was studied to identify the mechanism by which turtle hatchlings acquire Salmonella flora. The visceral organs and mature eggs removed from 31 adult gravid female turtles over the course of two egg-laying seasons and from 37 adult females during one winter dormant period were examined bacteriologically for Salmonella. Pond water, egg nest soil, and hatchlings produced by eggs removed from the oviducts and nest soil were also tested. Eighty-eight turtles hatched from eggs removed from the oviducts of 15 turtles at necropsy did not excrete or harbor systemically Salmonella, nor were these pathogens isolated from ovarian tissue or immature eggs. The findings suggest transovarian transmission of these pathogens does not occur frequently. Turtles hatched from eggs retrieved from soil nests 1 to 2 h after deposition harbor and excrete these organisms. This result coupled with the isolation of these pathogens from the cloaca, colon contents, and bursal fluid from 18 females captured in the act of egg laying supports the cloaca to egg and nest soil to egg mode for salmonellae infection in the resultant hatchling. Salmonella arizonae and Salmonella serogroups B, C2, and E1 were isolated from the cloaca, colon contents, pond water, and nest soil, and were excreted by hatchlings produced from eggs removed from the soil nests. These same serogroups were isolated from the colon contents of 19 of 37 females tested during the dormant period, suggesting the salmonellae persist in the pond environment in the adult throughout the year.