The hagfish oocyte at late stages of oogenesis: structural and metabolic events at the micropylar region

The hagfishes (Class Agnatha), primitive vertebrates and massive secretors of mucus, occupy a unique niche in a marine benthic environment and exists in abundance despite an infrequent ovulation of a low number (12-45) of notably large, prolate ellipsoidal (~8 mm x 28 mm. width x length, respectively) eggs. To establish factors that might contribute to survivability of one species (Eptatretus stouti), we examined three groups of mature females and document structural events characteristic of oocytes at late developmental stages: attached to the gonad, after ovulation into the body cavity, and after deposition on aquaria substrata. Quantitative autoradiographic analyses following administration in vivo of [(3)H]-leucine was useful to confirm post-vitellogenic stages and late metabolic events at the micropylar region located at one end of each egg. After egg disassembly, a combination of light microscopy and scanning electron microscopy was used to delineate structural features and 3-D relationships in juxtaposition to the micropylar cup and canal. A thick (~200mum) complex tripartite chorion surrounds a large micropylar cup (~300mum, width at base) consisting of fused polygonal (mainly hexagonal) substructural units (3-4 mum, width) and collectively serves to stabilize the micropylar canal (of size to prevent polyspermy) that penetrates the ooplasm near the germinal vesicle. A tuft composed of numerous chorionic appendages ('anchor filaments', ~2-3 mm length) is enmeshed in a gel matrix and surrounds the cup. When the eggs are deposited, intermediate filament aggregates (IFA) from slime gland mucus become interspersed in the tuft/gel complex. A similar IFA/tuft/gel region found on the vegetal pole-end facilitates firm egg-end attachments and possibly assists localization of egg clutches to a favorable substratum. Although the mode of fertilization is as yet unknown, the collective structural characteristies of the hagfish egg arc indicative of physical strength and are markedly different from eggs of the other group of cyclostomes (lampreys) and from tcleosts. The hagfish produces fewer but larger eggs which are prolate ellipsoidal rather than spherical and have a thicker chorion, a greater chorion to egg width ratio, a much larger micropylar cup with a unique substructure, larger and more localized chorionic appendages, and appendage tufts interspersed with adhesive substances derived not only from the follicular epithelium but also from the slime glands.     

Mature males of Arctic charr,Salvelinus alpinus,release F-type prostaglandins to attract conspecific mature females and stimulate their spawning behaviour

Synopsis We examined the behavioural and biological functions of prostaglandins (PG) of F-type as a chemosensory signal during spawning in Arctic charr. Immunoassay revealed F-type PGs in water inhabited by ripe charr of both sexes performing spawning-related acts. High concentrations of immunoreactive PGFs seemed to coincide with male spawning activity. Ripe isolated males were more odorous to other charr (as assayed by EOG) than either females or immature charr. When these males received chemical cues from ovulated females, they increased their release of odorants, including F-type PGs, into the water. PGF_2α at concentrations of 0.1 nM attracted ovulated females and ripe males. It also stimulated digging behaviour in mature females. It is concluded that F-type PGs or their derivatives are released by ripe males on the spawning grounds to attract females and elicit their spawning behaviour.     

Reproductive biology of the endangered Oxleyan pygmy perch Nannoperca oxleyana Whitley

The reproductive biology of the Oxleyan pygmy perch Nannoperca oxleyana is described from simultaneous studies of wild populations in north-eastern New South Wales and mature fish held in aquaria. In the wild, 50% of males and females matured at total lengths of 24·0–25·9 and 28·0–29·9 mm, respectively. The species displays sexual dichromatism during the spawning season, with males developing more intense red and brown fin and body colouration, and black pelvic fins. Captive male broodfish displayed territoriality during the breeding season, closely guarding sites within artificial, plant-like substrata in which pairs of fish spawned adhesive eggs. Protracted serial spawning of wild and captive fish occurred from September to April and May at mean water temperatures ≥16·6° C and day length ≥10·7 h. Captive broodfish spawned on an average of 57% of days during the 256 day spawning period. Gonado-somatic indices averaged 0·7% for all ripe males and 4·1–4·2% for all ripe females collected. Mean total and batch fecundities of captive females were 1323 eggs per fish and 7·8 eggs per fish per day, respectively, and relative fecundity was 587 eggs g⁻¹ of body mass. Batch fecundity of wild females was estimated at 7·8 eggs per fish. The adaptive significance of this reproductive strategy in a harsh, variable environment is discussed.     

Mating‐induced ovulation in loggerhead sea turtles,Caretta caretta

Mating‐induced ovulation is common in mammals, but has been rarely described in other taxa. Observations of several mature female loggerhead sea turtles, Caretta caretta, held in captivity seemed to indicate that ovulation did not occur in the absence of a male. This study was designed to determine whether this was an effect of captivity or an effect of the absence of a male. Two mature female loggerheads were followed over six annual reproductive cycles. Ultrasound exams were performed approximately every 2 weeks to follow the development of follicles in the ovaries. During the first two seasons, no male was present, in the next two seasons, a mature male was present without mating, and in the final two seasons a mature male was present, mating with one or both females. When no male was present or when present without mating, ovarian follicles developed to full size, but ovulation did not occur and the follicles gradually began to decrease in diameter and undergo changes evident on ultrasound. In the fifth season, only one of the females mated, dropping two eggs after 7 days, and continued to oviposit throughout the following months (total 275 eggs). The unmated female did not ovulate, showing the same pattern as earlier seasons. In the final season, both females mated and ovulated, dropping eggs for the next four months (total 539 eggs). The following year, no males were present and neither female ovulated. This study provides clear evidence that ovulation in loggerhead sea turtles is induced by mating. Zoo Biol 27:213–225, 2008. © 2008 Wiley‐Liss, Inc.     

Reproductive regulation in the female Japanese minnow, Pseudorasbora parva (Cyprinidae)

The Japanese minnow, Pseudorasbora parva, is a multiple spawner that lays eggs repeatedly during the spawning season. In laboratory aquaria, 19 of 63 females did not spawn, whereas 44 other females laid 167 to 6285 eggs in 1 to 14 mating sequences. Neither fish density nor sex ratio (number of males per female) affected fecundity, but the increase in fish density significantly decreased the growth rate of females. The increase in male length significantly increased fecundity in aquaria which contained only a single male, whereas it decreased female growth rate. These relationships were not evident in aquaria housing three or ten males. Since larger males were more dominant and had larger testes than smaller males, the correlation between fecundity and male size strongly suggests that individual females regulated fecundity to increase their reproductive success.     

Pattern of sea bass oocyte development after ovarian stimulation by LHRHa

The cyclic pattern of oocyte development in the sea bass, Dicentrarchus labrax L., was studied after induction of spawning by two injections, 24 h apart, of a luteinizing hormone releasing-hormone analog (LHRHa) administered at the end of vitellogenesis. The first difference in the developmental stage of the ovary and in the size-frequency distribution of oocytes between the LHRHa treated group and the control group, was detected 32 h after the first injection, the LHRHa group showing a higher proportion of the 900 μm diameter oocyte class (maturing oocytes) ( P <0.01). At 48 h LHRHa-treated females showed an increase in the 1000 and 1100 μm classes (maturing oocyte and ovulated eggs) ( P <0.01) and at 72 h these females exhibited a bimodal pattern, reaching the highest proportions in the 1100 (27.4%) and the 600 (14.7%) μm classes (ovulated eggs and advanced vitellogenic oocytes, respectively). Bimodal distributions were present in 80% of the LHRHa-treated females. Once oocyte final maturation was triggered by LHRHa the time needed for ovulation was about 48 h and the interval between consecutive ovulations and spawnings seemed to be 48–72 h.     

Abnormal zonae pellucidae in mice lacking ZP1 result in early embryonic loss.

All vertebrates have an egg shell that surrounds ovulated eggs and plays critical roles in gamete recognition. This extracellular matrix is known as the zona pellucida in eutherian mammals and consists of three glycoproteins, ZP1, ZP2 and ZP3 in the mouse. To investigate the role of ZP1 in fertilization and early development, we have used targeted mutagenesis in embryonic stem cells to create mouse lines (Zp1(tm/tm)) lacking ZP1. Although a zona pellucida composed of ZP2 and ZP3 was formed around growing Zp1(tm/tm) oocytes, the matrix was more loosely organized than zonae around normal oocytes. In some Zp1 null follicles, this structural abnormality resulted in ectopic clusters of granulosa cells, lodged between the zona matrix and the oolemma, that perturbed normal folliculogenesis. Comparable numbers of eggs were ovulated from Zp1 null females and normal females following hormonal stimulation. However, after mating with males, fewer two-cell embryos were recovered from Zp1 null females, and their litters were significantly smaller than those produced by normal mice. Therefore, although mouse ZP1 is not essential for sperm binding or fertilization, it is required for the structural integrity of the zona pellucida to minimize precocious hatching and reduced fecundity.     

Oviposition preference in the oligophagous tomato fruit fly, Neoceratitis cyanescens

We assessed the role of visual and olfactory cues on oviposition preference in the oligophagous tomato fruit fly, Neoceratitis cyanescens (Bezzi) (Diptera: Tephritidae). In a field survey, we evaluated the stage of susceptibility of field‐grown tomatoes by monitoring N. cyanescens infestations from fruit‐setting up to harvest, in relation to post‐flowering time, size, and visual properties of fruit. In two‐choice laboratory experiments, we tested the degree to which females use visual and olfactory cues to select their host plant for oviposition. In addition, we investigated the ability of flies to avoid fruit already infested by conspecific eggs or larvae, and the influence of natal host fruit on oviposition preference. Neoceratitis cyanescens females preferentially lay their eggs in small yellow‐green unripe fruit (2–3.5 cm diameter, 10–21 days post‐flowering). Damage to fruit was significantly affected by brightness and size properties. In laboratory experiments, females chose to lay their eggs in bright orange rather than yellow domes. On the sole basis of olfactory stimuli, females showed a significant preference for unripe vs. ripe host fruit, for unripe fruit vs. flowers or leaves, and for host vs. non‐host fruit (or control). However, colour interacted with odour as females dispatched their eggs equally between the yellow dome and the bright orange dome when unripe fruit of tomato was placed under the yellow dome vs. ripe fruit under the bright orange dome. When offered real ripe and unripe tomatoes, females preferred unripe tomatoes. Females significantly chose to lay eggs in non‐infested fruit when they were given the choice between these or fruit infested with larvae. In contrast, recent stings containing eggs did not deter females from laying eggs. Rather, they could have an attractive effect when deposited within <1 h. Regardless of their natal host plant, tomato or bugweed, N. cyanescens females laid significantly more eggs in a dome containing bugweed fruit. However, 15% of females originating from tomato laid eggs exclusively in the dome with tomato, against 3% of females originating from bugweed.     

Oviposition deterrence associated with larval frass of the Japanese pine sawyer,Monochamus alternatus (Coleoptera: Cerambycidae)

To determine the effect of larval frass of Monochamus alternatus on oviposition preference of the female adults, three tests were performed in the laboratory. Individual females were provided with a frass-coated, Pinus densiflora bolt and an untreated bolt simultaneously and were allowed to oviposit for 24 h. They deposited a significantly smaller number of eggs on frass-coated bolts. The females supplied with frass-coated bolts deposited no eggs on them, indicating that the larval frass deterred the females from oviposition. When individual females were provided simultaneously with a pine bolt applied with a methanol extract of larval frass and another bolt applied with methanol alone, they deposited a significantly smaller number of eggs on frass extract-applied bolts for 24 h. These results suggest the presence of a putative oviposition deterrent in larval frass of M. alternatus. Each test was replicated 19-20 times using different females.     

Male-female Parental Roles in Sarotherodon galilaeus (Pisces: Cichlidae)

Sarotherodon galilaeus is a predominantly biparental, sexually monomorphic mouthbrooder. 15 spawnings of pairs in the weight range 150–450 g were observed in aquaria. Only clutches of females larger than 320 g were reared jointly; with those of smaller females, the first fish to pick up eggs brooded alone, while the comparatively few eggs left for the second fish were invariably eaten. The latency after which eggs were picked up increased with the duration of spawning and indirectly with fish size. This increase was larger in males than in females, so large males started to collect eggs later than their mates. In small pairs (female weight < 200 g), by contrast, the male would pick up eggs first and carry them alone. This is the case in a smaller congeneric species, S. melanotheron, indicating that the apportioning of broodcare is based on a unitary, size-dependent mechanism among Sarotherodon. Yet picking-up order was not entirely determined by these equations. Some fish did not show any reaction towards the eggs and occasionally males became care-givers despite expected longer latency, seemingly because of aggressive dominance.     

Influence of sperm and phytoplankton on spawning in the echinoid Lytechinus variegatus

The cues triggering large-scale broadcast-spawning events in marine invertebrates are not fully understood. Using the sea urchin Lytechinus variegatus, we tested the effectiveness of a variety of potential spawning cues in eliciting a spawning response. In the laboratory, during two consecutive spawning seasons, about 400 isolated sea urchins were exposed to phytoplankton, sperm, or eggs, singly or in combination. The likelihood of spawning, time to spawning, and spawning behavior were recorded for both sexes. Sperm was most successful at inducing spawning. No response to eggs was noted. Phytoplankton alone did not trigger spawning, but when a phytoplankton cue was followed by the addition of sperm, spawning behavior was induced, the time between addition of sperm and spawning was reduced, and the variance among individuals in the time of spawning initiation was reduced. Males spawned sooner in response to cues than females and rarely spawned spontaneously in phytoplankton or control treatments. A semilunar pattern in the sensitivity to spawning cues was noted. During time periods when sea urchins were less ripe, the ratio of spawning males to spawning females increased. Our results indicate that seasonal and lunar cycles, together with the presence of phytoplankton, increase the sensitivity of these sea urchins to spawning cues and the precision of their responses to conspecific sperm.     

Sperm storage and motility in the ovary of the marine sculpin Alcichthys alcicornis (Teleostei: Scorpaeniformes), with internal gametic association.

Elkhorn sculpin, Alcichthys alcicornis, is a marine teleost with a unique reproductive mode called "internal gametic association," in which sperm introduced into the ovary by copulation enter the micropylar canal of ovulated eggs in the ovarian cavity, but actual sperm-egg fusion does not occur until the eggs have been released into sea water. It is also known that this fish is a multiple spawner, which spawns at intervals of a few days for one month, and the sperm introduced into the ovary at the beginning of the spawning season retain their fertilizability for the entire period. To clarify how the fertilizability of sperm is maintained internally, the ultrastructure of sperm, the morphological characteristics related to sperm storage in the ovary, and the characteristics of sperm motility were investigated. Mature sperm generally have the normal form of teleost sperm, devoid of acrosomal structures. However, it was found that the midpiece is comparatively elongated and has a compact aggregation of many small-size mitochondria. The intraovarian sperm remained floating in the ovarian fluid throughout the spawning season. The sperm showed high motility in isotonic and weak alkaline solution, containing sodium ions, which was similar to the ovarian fluid of this fish. Sperm continued to move in artificial ovarian fluid for 7-14 days. Considering these results together, it is thought that the intraovarian sperm move throughout the spawning season due to the plentiful energy generated by the many mitochondria.     

The reproductive biology of Gerres in Natal estuaries

An investigation of the reproductive state and gonad development of Gerreidae in Natal showed that the fry of Gerres rappi, G. acinaces and G. filamentosus entered estuaries at 10 mm (s.l.). They stayed in the estuary until attainment of maturity, which was between 70 and 110 mm according to species. Gonad activity was initiated in estuaries but all females left before their eggs were ripe. Testes ripened while the fish were still in the estuary but no ripe running individuals were found. Since few large and no spent individuals occurred in the estuaries sampled, it appears that most fish do not return from the sea. Sequence of oocyte maturation and testes development were determined histologically. The estuarine area may be of importance to the fry as a safe nursery ground and to maturing individuals as an environment in which gonad maturity is reached for the first time.     

Envelope surface ultrastructure and specific gravity of artificially fertilized Pacific cod Gadus macrocephalus eggs

The envelope surface ultrastructure and specific gravity of artificially fertilized eggs of the Pacific cod Gadus macrocephalus were examined. The unfertilized, demersal and slightly adhesive eggs of G. macrocephalus were almost spherical and had no oil globules. Wrinkled envelope surface with elaborated hexagonal reticulated patterns and type I micropyle were observed under a scanning electron microscope. The adhesiveness of the eggs was lost at the blastodermal‐cap stage after fertilization. The micropylar canal was sealed by secretion of the perivitelline fluid, and the entire surface became rough. Numerous bacilli were deposited at the micropyle and the outer envelope surface at the late germ‐ring stage and at the embryo five‐eighths around the yolk stage. The micropyle was completely deformed at the embryo seven‐eighths around the yolk stage. The specific gravity of the fertilized G. macrocephalus eggs ranged from c. 1·0316 to 1·0454. These values, however, sharply decreased towards the end stages of egg development to produce pelagic larvae. The ultrastructural changes in the micropyle and envelope surface of the G. macrocephalus eggs protected the embryo from microorganism infections and mechanical stress during the long incubation period. The adhesiveness and specific gravity of the eggs influenced their dispersion potential.     

Ovulatory rhythms and over-ripening of eggs in cultivated turbot, Scophthalmus maximus L.

The aim of this study was to investigate ageing of ovulated eggs retained in the ovarian lumen of captive turbot, Scophthalmus maximus L., prior to handstripping. The ovulation times of egg-batches were determined by catheterization, handstripping and plotting the percentage fertilizations and hatches of resulting eggs against time. The catheterization experiments showed that eggs age and change appearance rapidly after ovulation. The stripping method demonstrated that freshly-ovulated eggs showed greater than 90% fertilization and up to 97% hatch, but ovulated eggs retained in the ovary lumen for 1 day before stripping gave 0% hatch. The turbot showed precise ovulatory rhythms. The time between ovulations varied between individuals but was constant for any one female, making it possible to predict future ovulation times. Three of the females studied had ovulatory periods of alternating length.     

Egg concealment in ascidians by females of the Japanese tubesnout, Aulichthys japonicus (Gasterosteiformes), and its subsequent copulation

The reproductive behaviors of the Japanese tubesnout, Aulichthys japonicus, the only fish among gasterosteiform fish that conceals eggs in ascidians, were observed in aquaria. Yellow nuptial-colored males attending at ascidian courted gravid females. The nuptial color could be an advertisement to females for depositing sites, and courtships seemed to facilitate female spawning. The females spawned eggs above the ascidian and immediately placed their eggs with their mouths into the peribranchial cavity. Males never ejaculated on eggs after spawning, but attempted to copulate with females during a period of several minutes, and one or a few males with the remarkable nuptial color frequently copulated using their urogenital papilla. An evolutionary scenario of unique egg concealment and subsequent copulation is proposed.     

Clutch production in the darter Etheostoma lynceum and its implications for life-history study

We tested two hypotheses that describe previously untested assumptions about the appropriate stage(s) for use in determining clutch sizes in darters. Three sequential stages of clutch/ovarian development are recognized: mature oocyte/ovary, ripening oocytc/ovary, and ripe oocyte/ovary. Mean ratios of clutch size to female length (relative clutch size, RCS) were significantly smaller and variances of RCS were significantly greater in ripe females when compared to mature and ripening females. Correlation coefficients for the relationship between clutch size and standard length (S.L.) were significantly greater for mature and ripening females than for ripe females. Mean clutch size, adjusted for the S.L. covariate, was significantly greater in mature and ripening females than in ripe females. Thus, we conclude that counts of eggs from ripe females yield fecundity estimates that are lower and more variable than estimates from counts of oocytes from mature and ripening females collected at the same time. Based on this conclusion, we discuss methods for developing fecundity estimates in darters. Our results may be broadly applicable to other taxa of fishes with group-synchronous ovum development.     

Deterred oviposition response of Monochamus alternatus (Coleoptera: Cerambycidae) to oviposition scars occupied by eggs

1 The oviposition behaviour and response of Monochamus alternatus females to oviposition scars were investigated in the laboratory. 2 Prior to oviposition, females gnawed at the bark surface of Pinus densiflora bolts to make a wound. Then females turned their bodies 180° to position their ovipositors over the wounds and inserted them under the bark through the wounds. After an oviposition, a jelly was deposited while the ovipositor was still inserted. The females then withdrew their ovipositors and rubbed the oviposition scars with the tips of their abdomens. 3 When searching females encountered oviposition scars, they stopped walking and drummed the surface and inside of the oviposition scars with their maxillary and labial palpi. 4 Eighty-six percent of females left oviposition scars containing single eggs after the palpation. By contrast, when females encountered oviposition scars containing no eggs, 76% of them began to gnaw at the scars and 64% deposited single eggs. The response to artificial oviposition scars was similar to that to vacant oviposition scars made by the females. 5 The results of various observations and experiments showed that the females could recognize oviposition scars and discriminate the scars occupied by single eggs from vacant ones, and suggested that the palpation of oviposition scars was the critical discrimination behaviour, indicating mediation by chemical cues.     

Reproductive Behavior, Captive Breeding, and Restoration Ecology of Endangered Fishes

Species recovery efforts generally focus on in situ actions such as habitat protection. However, captive breeding can also provide critical life history information, as well as helping supplement existing or restoring extirpated populations. We have successfully propagated nine species in captivity, including blackside dace, spotfin chubs, bloodfin darters, and boulder darters. Threatened blackside dace, Phoxinus cumberlandensis, were induced to spawn in laboratory aquaria by exposing them to milt from a reproductively mature male stoneroller, Campostoma anomalum or river chub, Nocomis micropogon. The latter are nest-building minnows, with which Phoxinus may spawn in nature. Eggs are broadcast among gravel and pebbles. Blackside dace individuals reared in captivity were used for translocation. Threatened spotfin chubs, Cyprinella monacha, fractional crevice spawners, deposited eggs in laboratory aquaria in the spaces created between stacks of ceramic tiles. Captively produced spotfin chubs were used as part of a larger stream restoration and fish reintroduction project in the Great Smoky Mountains National Park. The bloodfin darter, Etheostoma sanguifluum, was first used as a surrogate to develop techniques for spawning a closely related species, the endangered boulder darter, E. wapiti. Both darter species mated in a wedge created between two ceramic tiles. Our efforts have had variable but generally high success, with survival rates of 50–90% of eggs deposited. Captive production of nongame fishes can aid recovery of rare species or populations, aid in watershed restoration, and can help to refine water quality standards. In addition, captive breeding allows discovery of important behavioral or life history characteristics that may constrain reproduction of rare species in altered natural habitats.     

Induced spawning of the striped mullet Mugil cephatus L.

The response of adult mullet to artificial hypophysalion, immediately before the onset of the spawning migration was studied. Both males and females received intraperitoneal injec-tions of mullet or salmon pituitary homogenates wth ACTH or Synahorin (APE+HCG) three times per week for 12 days. At the outset, conlrol females were in the tertiary yolkstage with a low mean GSI of 7.6, while males gave thick, non-dispersing milt. Two of fourfemales receiving one salmon pituitary plus 25 RU Synahorin per injection spawnedspontaneously in laboratory aquaria. Released eggs were buoyant, well formed and had a mean diameter of 570μm. Egg fertility could not be ascertained due to protracted contact with sea water. Spawned females extruded a meinbranous tissue 'plug' from the cloaca1 region which was subsequently retracted. The remaining experimental females were refractory to treatment and histological examination of the ovaries revealed a predominance of atretic oocytes. All males were refractory to treatment and milt hydration was not achieved. It is suggested that induction of early spawning is possible especially with higher doses of pituitary homogenates and Synahorin. [Oceanic lnstitute Contribution No, 62 Sea Grant Program, GH-76].