cDNA cloning and sequence analysis of the Xenopus laevis egg envelope glycoprotein gp43

The glycoproteins of the Xenopus laevis egg envelope function in fertilization and development. As the unfertilizable coelomic egg transits the pars recta region of the oviduct, it is converted to a fertilizable egg by limited proteolysis of the envelope glycoprotein gp43 to gp41. This conversion is caused by an oviductally secreted serine active site protease, oviductin. We cloned a cDNA for gp43 from an oocyte cDNA library. The cDNA encoded a 454 amino acid protein homologous to the ZPC family of glycoproteins previously shown to be present in mammalian and fish egg envelopes. Conserved ZPC domains and motifs present in the Xenopus sequence included a signal peptide sequence, an N-linked glycosylation site, and 12 aligned Cys residues. In mammalian and Xenopus sequences, a furin-like (convertase) site and a C-terminal transmembrane domain were present reflecting the biosynthesis of ZPC in these species via the secretory glycoprotein pathway. However, fish envelope glycoproteins lack these sequences since they are synthesized via a different route (in the liver, transported to the ovary, and assembled into the egg envelope surrounding the oocyte). Consensus amino acid residues were identified by sequence comparisons of seven ZPC family members; 19% of the amino acid residues were invariant and 48% of the residues were identical in at least four of the seven sequences. The consensus sequence was used to make structure-fertilization function predictions for this phylogenetically conserved family of glycoproteins.     

Cloning and characterization of vitellogenin cDNA from the common Japanese conger (Conger myriaster) and vitellogenin gene expression during ovarian development

The major yolk protein precursor, vitellogenin (VTG) was detected in plasma from vitellogenic females and estradiol-17β (E₂)-treated immature females, but not in males and immature females by Western blotting in common Japanese conger Conger myriaster. Its molecular mass was approximately 180 kDa under denaturing and reducing conditions. The common Japanese conger VTG cDNA was cloned from the liver of vitellogenic female. It contains 5110 nucleotides including an open reading frame that encodes 1663 amino acids. The deduced amino acid sequence of the common Japanese conger VTG shares 80% identity with that of eel Anguilla japonica VTG-1, and 45–55%, 32–34% and 27–29% identity with the deduced amino acid sequences of other fish, amphibian and avian VTG with polyserin domain, respectively. In female common Japanese conger, VTG gene was highly expressed in the liver of this species similar with other oviparous vertebrates. The expression levels of VTG gene in the liver increased from the oil droplet stage to the tertiary yolk globule stage and were maintained until the migratory nucleus stage.     

Pheromone-Induced Spawning of Pacific Herring

A spawning pheromone in the milt (semen) and testes of the Pacific herring,Clupea harengus pallasi,is thought to facilitate school spawning of this species. We found that responsiveness to the spawning pheromone was variable among ripe fish (milt-producing or ovulated). Measurement of five principle reproductive steroids in the free form and five steroids in conjugated forms in the plasma of male fish early in the spawning season (newly ripe fish) showed that elevated plasma levels of 3α,17α-dihydroxy-5β-pregnan-20-one and 17α-hydroxyprogesterone coincided with responsiveness to the spawning pheromone in these fish; levels of other steroids did not differ. In contrast, responsiveness to the pheromone by female fish later in the spawning season (ripe-and-holding fish) coincided with lower levels of glucuronated 17α,20β-dihydroxyprogesterone and a lower gonadosomatic index. We suggest that these differences indicate a more advanced mature reproductive state in the responsive individuals among both the newly ripe male and the ripe-and-holding female fish. We found no differences in the level of cortisol in the blood of the herring that could be correlated with differences in pheromonal responsiveness. We conclude that differences in responsiveness to the spawning pheromone coincide to some extent with levels of reproductive maturation but probably not with recent stress.     

The third egg envelope subunit in fish: cDNA cloning and analysis, and gene expression

The inner layer of the egg envelope of a teleost fish, the medaka, Oryzias latipes, consists of two major subunit groups, Zl-1,2 and Zl-3. On SDS-PAGE, the Zl-1,2 group presents three glycoprotein bands that were considered to be composed of a common polypeptide moiety derived from their precursor, choriogenin H (Chg H). Zl-3 is a single glycoprotein derived from the precursor, choriogenin L (Chg L). In the present study, a fraction of a novel subunit protein was found in the V8 protease digest of Zl-1,2 that was partially purified from oocyte envelopes. This protein fraction was not present in the purified precursor, Chg H. By RT-PCR employing the primers based on the amino acid sequence of this fraction, a cDNA for the novel subunit was amplified, and a full-length clone of the cDNA was obtained by screening a cDNA library constructed from the spawning female liver. The clone consisted of 2025 b.p. and contained an open reading frame encoding the novel protein of 634 amino acids. This protein included Pro-X-Y repeat sequences in two-fifths of the whole length from its N-terminus. Northern blot analysis revealed that the gene expression for this protein occurred in the liver but not in the ovary of spawning female fish. This protein is considered as the third major subunit of the inner layer of the egg envelope of medaka.     

Electric signalling and reproductive behaviour in a mormyrid fish, the bulldog Marcusenius macrolepidotus (South African form)

Bulldog fish (Marcusenius macrolepidotus) generate short (<1 ms) electric-organ discharges (EODs), separated by much longer and highly variable interdischarge intervals (IDIs). We observed overt behaviour and electrical activity during reproductive behaviour in a male and in a female bulldog, and identified IDI patterns with putative signal functions. In contrast to Pollimyrus adspersus and Pollimyrus isidori, in which an elaborate and extended courtship precedes spawning proper, our fish started spawning almost immediately when we allowed the female to enter the males territory. The male did not construct a nest, and neither parent provided parental care. The male showed very little aggression towards the intruding female. Fish spawned in bouts near the males hiding place, and eggs were scattered by the females vigorous tail flips as she left the spawning site, only to return shortly thereafter. During spawning bouts, both fish generated highly stereotyped IDI patterns: the male generated a series of IDIs gradually decreasing from about 200 ms to about 55 ms that was abruptly terminated by a long IDI. The female generated a series of relatively regular IDIs (about 54 ms) that was followed by a marked increase in IDI duration (the probable time of spawning). Finally, a sharp decrease in IDIs to about 20 ms accompanied the females sudden escape from the spawning site. In between spawning bouts, both fish generated series of very short IDIs (high discharge rate, HD) that alternated abruptly with very low-rate inter-HD activity (especially in the male). IDIs as short as 9 ms (male) or 11 ms (female) occurred during HD displays. No visible aggression, in fact very little overt behaviour, occurred during these HD displays in both fish. Agonistic interactions between male and female, outside a reproductive context, were similar to those previously described in male pairs, including overt behavioural patterns such as parallel swimming, antiparallel display and attack, as well as HD displays. When not interacting, fish did not generate HD displays. We suggest the HD display is a communication signal in both reproductive and agonistic contexts.     

Differential expression of ZPC in the bovine ovary,oocyte, and embryo

Using nonradioactive in situ hybridization (ISH), the mRNA encoding the zona glycoprotein bZPC was localized in bovine ovaries, oocytes, and embryos. In the ovary, the distribution of the mRNA was correlated with the developmental stage of the follicle. Whereas in primordial and primary follicles the mRNA was predominantly seen in the oocyte, it was found in both the oocyte and the follicle cells of secondary and tertiary follicles. In 2-day-old embryos produced by in vitro fertilization (IVF), no mRNA encoding ZPC could be demonstrated. Immunoblotting using monospecific polyclonal antibodies against porcine ZPC revealed a distinct band at a molecular weight of 47 kD in the ovarian cortex of cows, calves, and fetuses as well as in bovine follicle cells. Immunohistochemistry using the ZPC antibody displayed a strong signal in the zona pellucida of bovine oocytes and 2- to 6-day-old embryos as well as in the follicle cells. Our results show that during follicular development bovine ZPC is synthesized by the oocyte of the primary follicle and by both the oocyte and the follicle cells of the secondary and tertiary follicle. After fertilization, the synthesis of the zona protein is finished. Mol. Reprod. Dev. 49:435–443, 1998. © 1998 Wiley-Liss, Inc.     

Chemical signals in gametogenesis, spawning, and larval settlement and defense of the soft coral Sinularia polydactyla

 Mass spawning strategies of hard and soft corals on the Great Barrier Reef, Australia have been described in recent years. Nonetheless, the applicability of those studies to corals on other reef systems has not been well documented. Here we describe the mass spawning behavior of the soft coral Sinularia polydactyla on coral reefs surrounding Guam; specifically we describe the events in an annual gametogenic cycle including steroidogenesis, spawning, settlement and early life history defense. The gametogenic cycle of female colonies lasted 12 months while male colonies produced viable sperm within 9 months. Sinularia polydactyla exhibited a split spawn between March and June that correlated with a significant reduction in tissue concentrations of progesterone and testosterone. Estradiol was released into the water column, apparently by female colonies, just prior to spawning. There was a trend for preferential larval settlement in the presence of the crustose coralline algae Hydrolithon reinboldii rather than coral rubble, a natural biofilm, or filtered seawater. The defensive compounds pukalide and 11β-acetoxypukalide were found in eggs and larvae at adult level and three-fold lower than adult-level concentrations, respectively. These compounds provided some predator deterrent and antimicrobial protection against an ecologically relevant omnivorous fish Canthigaster solandri and a sympatric microbe Vibrio sp. Accepted: 10 September 1998     

Mating,male Ixodes scapularis express several genes including those with sequence similarity to immunoglobulin-binding proteins and metalloproteases

We created a cDNA library from mating, male Ixodes scapularis ticks and screened the library with a subtracted probe to eliminate genes common to feeding female and mating male I. scapularis ticks. A total of seven unique cDNAs were identified in this screen. One cDNA had sequence similarity to the IGBP-MC gene from Rhipicephalus appendiculatus, and another cDNA potentially encodes a protein with similarity to metalloproteases. RT-PCR, using RNA isolated from male and female I. scapularis ticks, confirmed that these genes are expressed in male, but not female ticks. The remaining five cDNAs did not match any sequences in the GenBank database. This revised version was published online in July 2006 with corrections to the Cover Date.     

Interannual variability in reproductive traits of the Patagonian toothfish Dissostichus eleginoides around the sub‐Antarctic island of South Georgia

Commercial fisheries data, collected as part of an observer programme and covering the period 1997–2014, were utilized in order to define key reproductive traits and spawning dynamics of the Patagonian toothfish Dissostichus eleginoides at South Georgia. Multi‐year spawning site fidelity of D. eleginoides was revealed through the identification of previously unknown spawning hotspots. Timing of female spawning was shown to have shifted later, leading to a shorter spawning duration. A decrease in length and mass of female and male spawning fish and a reduced number of large spawning fish was found, evidence of a change in size structure of spawning D. eleginoides. During the study period fewer later maturity stage females (including spawning stage) were observed in conjunction with increased proportions of early stage female D. eleginoides. The findings are discussed in the context of reproductive success, with consideration of the possible effects such spawning characteristics and behaviours may have on egg and larval survival. This work presents the first long‐term assessment of D. eleginoides spawning dynamics at South Georgia and provides valuable knowledge for both the ecology of the species and for future fisheries management of this commercially important species.     

Environmental cues for natural reproduction of the Chinese sturgeon,Acipenser sinensis Gray, 1835, in the Yangtze River,China

For effective conservation, it is important to explore the environmental cues initiating the spawning activities of a fish species. Based on monitoring data gathered between 1998 and 2011, the relationships between spawning activities of the Chinese sturgeon, Acipenser sinensis, and several environmental cues were analyzed using the rare events logistic regression ‘Relogit’ method, which indicated that water temperature, 1‐day ?‐discharge, and atmospheric pressure were among the key spawning cues for A. sinensis (P < 0.05). It is suggested that Chinese sturgeon might have an optimal environment window of 17–20°C water temperature, high day‐to‐day discharge increase, and low atmospheric pressure for spawning. In support of Chinese sturgeon reproduction, suggested modifications to the operational procedures for the Three Gorges Reservoir (TGR) to trigger spawning are: lowering the downstream water temperature to below 20°C before mid‐October and expanding the period with water temperatures of between 17 and 20°C; to create a day‐to‐day intermittent increase in the discharge to an optimal spawning water temperature; and to regulate flow at nights with a low atmospheric pressure.     

A Test for Male Parental Care in a Fundulid, the Bluefin Killifish, Lucania goodei

Under field conditions, breeding male bluefin killifish, Lucania goodei, have been observed aggressively defending territories from other breeding males, non-breeding females, and minnows (mainly Notropis harperi). We performed an aquarium experiment to test whether male aggression serves to protect newly deposited eggs from predation. We allowed a male and a female to spawn in a yarn mop, removed the female, and exposed the eggs to one of four treatments (spawning male present, two minnows present, spawning mal+two minnows present, no adult fish present). Mops were censused daily for seven days. Egg predation rates were highest in the male+minnows and male only treatments. Egg predation rates in the male+minnows treatment did not differ from the predicted predation rate (sum of male only and minnows only treatments). Hence, there is no evidence for male parental care in L. goodei. In addition, we compared the egg predation rates (filial cannibalism) between males of 3 different color morphs and found no evidence for differential egg cannibalism.     

Molecular cloning and expression analysis of Fshr and Lhr in relation to Fshb and Lhb subunits during the period of temperature‐dependent sex determination in pejerrey Odontesthes bonariensis

In this study, we cloned and characterized the follicle stimulating hormone receptor (Fshr) and luteinizing hormone receptor (Lhr) cDNAs of pejerrey Odontesthes bonariensis, a species with temperature‐dependent sex determination (TSD), and analyzed their expression in relation to Fshb and Lhb subunits during gonadogenesis at temperatures producing only females (17°C, FPT), both sexes (25°C, MixPT), and only males (29°C, MPT). The pejerrey Fshr cDNA had 3,069 bp for a mature protein of 694 amino acids (aa) and a signal peptide of 22 aa; the Lhr cDNA had 2,936 bp for a mature protein of 676 aa and a signal peptide of 25 aa. With the exception of Lhr in fish at the MPT, all genes showed significant increases and/or peaks of expression before histological differentiation of the gonads regardless of temperature. Larvae at the FPT had lower Fshb and Lhb but higher Lhr expression during the TSD period than those at the MPT; a clear pattern could not be ascertained for Fshr. At the MixPT, Fshb, Lhb, and Lhr mRNA increased in approximately half of the fish during TSD and sex differentiation and the sex ratio was 55.2% male. Based on the above results, it is suggested that animals with high Fshb and Lhb and low Lhr values represent putative males. These evidences, together with other studies, suggest that temperature may signal through the pituitary (differential expression of Fshb and Lhb) down to the gonads (differential expression of Lhr), probably affecting the regulation of steroidogenesis during the TSD process of pejerrey. Mol. Reprod. Dev. 77: 521–532, 2010. © 2010 Wiley‐Liss, Inc.     

Estradiol-17β effects on lipid and carbohydrate metabolism and on the induction of a yolk precursor in goldfish, CarassiusAuratus

The effects of estradiol-17β treatment on plasma lipid levels, liver lipid and glycogen reserves were examined during different phases of the reproductive cycle in goldfish, $\text{Carassius}$$\text{auratus}$. Estrogen therapy resulted in increased plasma and hepatic lipid levels except during the spawning season. Hepatic glycogen deposits were depleted by estradiol injections during all seasons. Treatment of fish with the estrogen antagonist, CI-628, during the spawning season caused a reduction in plasma and liver lipid levels. Electrophoretic studies conducted during the post-spawning season showed that estrogen induces the appearance of a specific lipoprotein, probably a yolk precursor, in the serum and liver of goldfish.     

Polygynous Mating System of Trimma okinawae (Pisces: Gobiidae) at Kagoshima,Japan with a Note on Sex Change

Mating system, social structure and reproductive behaviour of the gobiid fish Trimma okinawae were studied at Akamizu, Kagoshima, Japan. Mating system of this species is polygyny. There were two types of habitat-related distribution patterns of male and female home ranges: females have home ranges outside a small territory of the male; female home ranges are covered by male territory. Spawnings take place with a cycle of four to five days during the spawning season of June to Sept. In the morning of the day of spawning, males stay at the spawning sites. Females visit the site and spawn with the male. On days between spawnings males exhibit courtship displays at female home ranges. Higher reproductive success is not connected with frequency of such courting of the male, but with male size. The largest female of a social unit changes into a male when the dominant male is removed, which shows that T. okinawae is a hermaphrodite.     

Collapse of the reproductive process of two migratory fish (Prochilodus argenteus and Prochilodus costatus) in the Três Marias Reservoir,São Francisco River,Brazil

Generally, migratory fish can complete vitellogenesis even when the migration process is interrupted; however, final oocyte maturation does not occur and therefore there is no spawning. To evaluate the reproductive process of migratory fish inside the reservoir of the Três Marias Dam (a lentic environment), 90 female and 81 male Prochilodus argenteus and 172 female and 115 male Prochilodus costatus were captured in this environment. Males and females from both species in the resting stage were pre‐dominant, and the highest frequency of specimens in advanced maturation stages occurred in November, December and January. According to histological analysis, ovarian regression was frequent and vitellogenic oocytes undergoing atresia were intensely observed, moreover, no fish with ovaries and testes with characteristics of spawning and spermiation, respectively, were captured. This indicated that both species were preparing for reproduction and reaching advanced gonad maturation, but were unable to complete the process of reproduction in this environment. The data from this study indicate that the modification of portions of rivers to lentic environments can have a negative impact on the reproductive process of migratory fish in freshwater.