Twelve novel polymorphic microsatellite loci for the Yellow grouper (Epinephelus awoara) and cross-species amplifications

Yellow grouper (Epinephelus awoara) is a commercially important marine fish species. A dinucleotide-enriched genomic library of E. awoara was constructed using the method of FIASCO. Twelve loci were polymorphic in a test population with alleles per locus ranging from two to eight, and observed and expected heterozygosities per locus from 0.13 to 1.00 and from 0.20 to 0.86, respectively. Five loci significantly deviated from Hardy–Weinberg equilibrium and no significant linkage disequilibrium was found between pairs of loci. Cross-species amplification of these polymorphic microsatellite loci was performed in additional two related species. These polymorphic microsatellite loci would be useful for investigating genetic diversity of E. awoara and related species. L. Zhao and C. Shao have contributed equally to this work.     

De novo assembly of a chromosome‐level reference genome of red‐spotted grouper (Epinephelus akaara) using nanopore sequencing and Hi‐C

The red‐spotted grouper Epinephelus akaara (E. akaara) is one of the most economically important marine fish in China, Japan and South‐East Asia and is a threatened species. The species is also considered a good model for studies of sex inversion, development, genetic diversity and immunity. Despite its importance, molecular resources for E. akaara remain limited and no reference genome has been published to date. In this study, we constructed a chromosome‐level reference genome of E. akaara by taking advantage of long‐read single‐molecule sequencing and de novo assembly by Oxford Nanopore Technology (ONT) and Hi‐C. A red‐spotted grouper genome of 1.135 Gb was assembled from a total of 106.29 Gb polished Nanopore sequence (GridION, ONT), equivalent to 96‐fold genome coverage. The assembled genome represents 96.8% completeness (BUSCO) with a contig N50 length of 5.25 Mb and a longest contig of 25.75 Mb. The contigs were clustered and ordered onto 24 pseudochromosomes covering approximately 95.55% of the genome assembly with Hi‐C data, with a scaffold N50 length of 46.03 Mb. The genome contained 43.02% repeat sequences and 5,480 noncoding RNAs. Furthermore, combined with several RNA‐seq data sets, 23,808 (99.5%) genes were functionally annotated from a total of 23,923 predicted protein‐coding sequences. The high‐quality chromosome‐level reference genome of E. akaara was assembled for the first time and will be a valuable resource for molecular breeding and functional genomics studies of red‐spotted grouper in the future.     

Early gonadal development and primary males in the protogynous epinepheline, Cephalopholis boenak

Early gonadal development of the protogynous epinepheline, Cephalopholis boenak, was examined histologically in 289 specimens with standard length (L_S) of 42–130 mm, collected from May 2000 to April 2002 in Hong Kong waters, to determine male developmental pathways and establish its sexual pattern. All juvenile gonads developed an ovarian lumen with primary‐growth stage oocytes and scattered spermatogenic tissue prior to sexual differentiation and first sexual maturation. From this bisexual phase containing both female and male tissues, some gonads differentiated as ovaries with further oocyte growth to cortical‐alveolus and vitellogenic stages, the rest differentiated as testes with the proliferation of spermatogenic tissue and the formation of a sperm sinus. All testes retained the lumen and primary‐growth stage oocytes, and sperm sinuses ran within the gonad wall. Unlike most protogynous species, among functional males it was impossible to distinguish those resulting from juveniles through sexual differentiation (i.e. primary male) from those resulting from functional females through sex change (i.e. secondary male) based solely on testicular morphology. A proportion‐spermatogenic‐tissue index (I_ST) was, therefore, developed and determined to be a reliable quantitative indicator for distinguishing differentiating, primary males before a sperm sinus was evident, from differentiating females during sexual differentiation. Since sexually transitional specimens with the concominant appearance of degenerating vitellogenic, or later, stage oocytes and spermatogenic tissue in the gonads were previously noted from Hong Kong, diandric, protogynous hermaphroditism is confirmed in C. boenak. For species, such as this and other epinephelines, in which all males have the same testicular morphology, a complete analysis of a wide range of body sizes from juveniles to adults is necessary for understanding male developmental pathways, and determining sexual pattern.     

Comparison of reproductive development in triploid and diploid female rainbow trout Oncorhynchus mykiss

The diploid rainbow trout Oncorhynchus mykiss reached sexual maturity 3 years after hatching and its oogenesis underwent four stages, which were oogonia, primary oocyte, secondary oocyte and egg. Reproductive development and hormone changes of 4 to 35 month‐old female O. mykiss were investigated using histological and radioimmunoassay methods in order to provide a theoretical and practical basis for the use of triploid female O. mykiss. The oogonium of the triploid female could develop into the oocytes of the prophase with abortion occurring later; the oogonium was surrounded by stroma cells to form the oogonium cluster and the gonads showed a virilescent tendency when the oogonium clusters were gradually replaced by spermatogenic‐like cytocysts. After 13 months, amounts of gonadotropic hormone (GtH‐I, GtH‐II) and oestradiol (17β‐E₂) in triploid females were lower than in diploid fish at corresponding time periods, but the amounts of testosterone (T) increased consistently after 21 months and were more than in diploid fish in the corresponding time periods (P > 0·05). The infertility of triploid females resulted from meiosis failure, which caused developmental abortion of oocytes and oogonium formed cytocysts before the prophase oocytes. The cytocyst formation was due to the lack of the normal interaction of ovum and follicular cells, the development of follicular cells producing steroids were inhibited, the arylate path from T to 17β‐E₂ was interrupted, concentration of 17β‐E₂ decreased and concentration of T increased in the blood, the content of vitellogenin (Vg) decreased in the liver with a low 17β‐E₂ and high T caused to ovaries to show a tendency to be virilescent.     

The influence of social factors on juvenile sexual differentiation in a diandric,protogynous grouper <Emphasis Type="Italic">Epinephelus coioides</Emphasis>

The influence of social factors on juvenile sexual differentiation was explored in a diandric, protogynous grouper Epinephelus coioides. Experimental social units were established as singles, pairs and quartets using sexually undifferentiated juveniles at an age of 22 weeks post-hatching (WPH); all gonads were examined histologically at 130 WPH, the age of first sexual maturation. The percentage of primary males was about 39% at the end of the experiment, higher than the <5% obtained under mariculture conditions or from wild stocks for similar age or body size. This study demonstrated for the first time that social factors can significantly influence juvenile sexual differentiation in E. coioides.     

Reproductive features of the non-native Siganus luridus (Teleostei, Siganidae) during early colonization at Linosa Island (Sicily Strait, Mediterranean Sea)

In July 2003, the finding of a newly settled population of Siganus luridus at Linosa Island (Sicily Strait, Mediterranean Sea) gave us the unusual opportunity to examine the reproductive condition of a Lessepsian migrant during early phases of colonization. Aspects of gonad morphology, fecundity, atresia and oocyte dynamics were investigated by using 43 pioneer specimens collected in concomitance with their first record in the Pelagie Islands. Ovarian development was consistent with the group‐synchronous type, and testicular organization was of the unrestricted spermatogonial testis type, with cystic spermatogenesis. Both males and females had reached final stages of gonad maturation. The rates of follicular atresia were moderate: out of 17 adult females, 10 individuals did not present atretic oocytes; six exhibited <15.1% of secondary growth phase (SGP) oocytes in α‐atresia, while one female presented 45.7% of SGP in α‐atresia. Fecundity estimates did not diverge from what was observed in a reference population along the Lebanese coast. Absolute fecundity ranged from 115 739 to 740 433 oocytes per female (16.5–24.5 cm L_T). Relative fecundity ranged from 1239 to 3162 oocytes g⁻¹, with a mean of 1885 ± 868 oocytes g⁻¹. Our observations indicated that these early settled siganids are reproductively active at Linosa and suggested the forthcoming of self‐maintaining populations across the central Mediterranean area.     

Life‐history strategies of the rock hind grouper Epinephelus adscensionis at Ascension Island

Epinephelus adscensionis sampled from Ascension Island, South Atlantic Ocean, exhibits distinct life‐history traits, including larger maximum size and size at sexual maturity than previous studies have demonstrated for this species in other locations. Otolith analysis yielded a maximum estimated age of 25 years, with calculated von Bertalanffy growth parameters of: L_∞ = 55·14, K = 0·19, t₀ = ?0·88. Monthly gonad staging and analysis of gonad‐somatic index (I_G) provide evidence for spawning from July to November with an I_G peak in August (austral winter), during which time somatic growth is also suppressed. Observed patterns of sexual development were supportive of protogyny, although further work is needed to confirm this. Mean size at sexual maturity for females was 28·9 cm total length (L_T; 95% C.I. 27·1–30·7 cm) and no females were found >12 years and 48·0 cm L_T, whereas all confirmed males sampled were mature, >35·1 cm L_T with an age range from 3 to 18 years. The modelled size at which 50% of individuals were male was 41·8 cm (95% C.I. 40·4–43·2 cm). As far as is known, this study represents the first comprehensive investigation into the growth and reproduction of E. adscensionis at its type locality of Ascension Island and suggests that the population may be affected less by fisheries than elsewhere in its range. Nevertheless, improved regulation of the recreational fishery and sustained monitoring of abundance, length frequencies and life‐history parameters are needed to inform long‐term management measures, which could include the creation of marine reserves, size or temporal catch limits and stricter export controls.     

A redescription of Pseudorhabdosynochus epinepheli (Yamaguti, 1938), the type-species of Pseudorhabdosynochus Yamaguti, 1958 (Monogenea: Diplectanidae), and the description of P. satyui n. sp. from Epinephelus akaara off Japan

Pseudorhabdosynochus epinepheli (Yamaguti, 1938) has been recorded from a variety of hosts, mainly groupers. All type-specimens of Diplectanum epinepheli Yamaguti, 1938, Pseudorhabdosynochus epinepheli Yamaguti, 1958 and Cycloplectanum hongkongensis Beverley-Burton & Suriano, 1981 are figured: it is concluded, as did Kritsky & Beverley-Burton (1986), that the three species are synonymous. In addition, numerous monogenean specimens from a deep-sea grouper, Epinephelus chlorostigma, collected off New Caledonia, South Pacific, were prepared using various methods and described. These specimens are also conspecific with P. epinepheli (Yamaguti, 1938) and represent a new geographical record. This species has a sclerotised vagina with a very characteristic primary chamber. The simultaneous presence of P. lantauensis (Beverley-Burton & Suriano, 1981) and P. epinepheli was noted in both type-slides of D. epinepheli from Japan (host: E. akaara) and C. hongkongensis from Hong Kong (host: E. bruneus). Several causes are suspected for the alleged ‘generalist’ character of P. epinepheli, including the misidentification of either fish or monogeneans and the accidental exchanges of monogeneans between fishes of different species kept alive in the same tank. Finally, the confirmed list of hosts of P. epinepheli includes E. akaara, E. awoara and E. chlorostigma; it is suggested that the latter, a widespread deep-sea fish, serves as a reservoir for the infection of the other species, which are associated with shallow waters. P. satyui n. sp. is described from two specimens found on slides from E. akaara (from the Inland Sea of Japan) deposited by Yamaguti; the new species has a sclerotised vagina with characteristic spherical chambers.

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Résumé   Pseudorhabdosynochus epinepheli (Yamaguti, 1938) a été mentionné chez différents h?tes, surtout des mérous. Tous les spécimens-types de Diplectanum epinepheli Yamaguti, 1938, Pseudorhabdosynochus epinepheli Yamaguti, 1958 et Cycloplectanum hongkongensis Beverley-Burton & Suriano, 1981 sont figurés, et on conclut, comme Kritsky & Beverley-Burton (1986), que les trois espèces sont synonymes. De plus, de nombreux spécimens de monogènes collectés chez un mérou de profondeur, Epinephelus chlorostigma, de Nouvelle-Calédonie, Pacifique Sud, ont été préparés avec des méthodes variées. Ces spécimens sont aussi conspécifiques de P. epinepheli (Yamaguti, 1938) et la Nouvelle-Calédonie représente une nouvelle mention géographique. L’espèce a une vagin sclérifié avec une chambre primaire très caractéristique. La présence simultanée de P. lantauensis (Beverley-Burton & Suriano, 1981) et P. epinepheli a été notée dans les lames-types de D. epinepheli du Japon (h?te, E. akaara) et de C. hongkongensis de Hong-Kong (h?te, E. bruneus). Plusieurs causes sont soup?onnées pour le caractère prétendument ‘généraliste’ de P. epinepheli, dont les mauvaises identifications de poissons, des monogènes, et l’échange accidentel de monogènes chez des poissons gardés vivants ensemble dans un même contenant. Finalement, la liste des h?tes confirmés de P. epinepheli comprend E. akaara, E. awoara et E. chlorostigma. On fait l’hypothèse que E. chlorostigma, espèce de profondeur, a servi de réservoir pour l’infestation des autres espèces, plus associées aux eaux peu profondes. P. satyui n. sp. est décrit de deux spécimens trouvés dans des lames de parasites de E. akaara (de la mer intérieure du Japon) déposées par Yamaguti. La nouvelle espèce a un vagin sclérifié avec des chambres sphériques caractéristiques.

     

Reproductive biology and implications for management of the orange-spotted grouper Epinephelus coioides in the southern Arabian Gulf

The reproductive biology of Epinephelus coioides was determined from the examination of 1455 individuals collected between July 2005 and June 2007 in the southern Arabian Gulf. Histological preparations of gonads indicated that males were either derived from a juvenile phase or the transition of postspawning females, confirming a diandric protogynous sexual pattern. The spawning season was well defined, occurring once a year during April and early May. Peaks in spawning occurred after the full and new moons and was completed within a single lunar cycle. The presence of mature males over the entire size and age range and the absence of inactive mature females during the spawning season suggested that the population was not constrained by sperm limitation. While specimens undergoing sexual transition were only observed in size and age ranges of 335–685 mm total length ( L _T) and 5–6 years, patterns in the proportion of males in size and age classes suggested that sex change occurred at a relatively constant rate after female maturation up to the maximum size (1002 mm L _T) and age (11 years). Relationships between reproductive output and capacity with size and age indicated that conventional regulations that equate the mean size at first capture to sexual maturation are unsuitable for the management of E. coioides . The maximum age, small size and young age at sexual maturation ( L _min= 320 mm L _T, 2 years, for females and 242 mm L _T, 1 year, for males) conflict with the general pattern for large epinepheline groupers and may be a direct result of the intensive demersal fishery in the southern Arabian Gulf.     

Socially controlled male-to-female sex reversal in the protogynous orange-spotted grouper,Epinephelus coioides

Socially controlled sex change in teleosts is a dramatic example of adaptive reproductive plasticity. In many cases, the occurrence of sex change is triggered by a change in the social context, such as the disappearance of the dominant individual. The orange-spotted grouper Epinephelus coioides is a typical protogynous hermaphrodite fish that changes sex from female to male and remains male throughout its life span. In this study, male-to-female sex reversal in male Epinephelus coioides was successfully induced by social isolation. The body length and mass, gonadal change, serum sex steroid hormone levels and sex-related gene expression patterns during the process of socially controlled male-to-female sex reversal in E. coioides were systematically examined. This report investigates the physiological mechanisms of the socially controlled male-to-female sex reversal process in a protogynous hermaphrodite grouper species. The results enable us to study the physiological control of sex change, not only from female to male, but also from male to female.     

Gustatory adaptation affects sexual maturation in male German cockroaches,Blattella germanica

Adaptations to hazardous environmental factors are essential for survival, although they may be maladaptive in conditions where the hazard is absent. In German cockroach (Blattella germanica L.) populations, glucose aversion has evolved rapidly in response to glucose‐containing insecticidal baits, but little is known about the consequences of this behaviour in the absence of bait. In the present study, glucose‐averse (GA) and wild‐type (WT) male German cockroaches are restricted to a range of nutritionally defined diets containing either glucose or fructose as the sole carbohydrate source, and time to first expression of courtship is measured by stimulating the male antennae daily with isolated antennae from receptive, 6‐day‐old females. Glucose‐averse males that are restricted to glucose‐containing diets mature their courtship responses significantly later than GA males restricted to fructose‐containing diets, whereas there is no difference in maturation of courtship responses between GA males restricted to fructose‐containing diets and WT males restricted to diets containing either sugar type. Glucose‐averse males furthermore respond later to GA female antennae than to WT female antennae, all from 6‐day‐old females. This suggests that GA females are less sexually stimulating, and the results are also consistent with earlier findings showing that GA females contain less developed oocytes than WT females at this age. These findings demonstrate that an adaptive gustatory mutation conferring protection from a toxin may have comparatively detrimental effects under conditions where the toxin has vanished, both by delaying female sexual maturation and signalling and by delaying male sexual maturation and courtship under conditions where glucose is a major energy source.     

Reproductive biology of black seabream Acanthopagrus schlegelii,threadfin porgy Evynnis cardinalis and red pargo Pagrus major in the northern South China Sea with consideration of fishery status and management needs

The reproductive biology of three commercially significant seabream species (family: Sparidae) Acanthopagrus schlegelii, Evynnis cardinalis and Pagrus major, taken from Hong Kong and adjacent northern South China Sea (SCS) waters, were investigated for their sexual patterns, spawning seasons, length at maturity and exploitation in relation to their conservation and management status. Histological analysis showed E. cardinalis and P. major to be functionally gonochoristic, the latter having a bisexual juvenile stage and being a rudimentary hermaphrodite. Acanthopagrus schlegelii is a protandric hermaphrodite. Standard length (L_S) at 50% sex change for A. schlegelii is 291 mm. L_S at 50% female maturity for E. cardinalis and P. major are 117 and 332 mm, respectively. For all three species, the spawning period falls between November and March. The study highlights geographical differences in reproductive biology among the species and a paucity of fishery or other population‐related data. While heavy fishing pressure, life‐history characteristics and absence of effective management throughout the geographic ranges of these species make them susceptible to overfishing, they nonetheless appear to be generally more resilient than many other taxa that comprise the multi‐species fisheries of the region, possibly due to their relatively rapid sexual maturation and spatial movement patterns. Overall, however, little is known of the biology, fishing history and current fishery status of sparids in general in the northern SCS and the current study is one of the first to examine such aspects of this family in the region and to consider appropriate management options.     

Comparisons of body sizes at sexual maturity and at sex change in the parrotfishes of Hawaii: input needed for management regulations and stock assessments

First estimates of sex allocation patterns and body size‐at‐sexual maturity and at protogynous sex change are presented for the five major (including one endemic) species of parrotfishes of Hawaii. Median body size at initial maturation as a female (L_M50) and at protogynous sex change from adult female to adult male (L_Δ50) varied greatly among the five species. Estimates of L_M50 were about 14, 17, 24, 34 and 35 cm fork length (L_F) in palenose Scarus psittacus, Pacific bullethead Chlorurus spilurus, stareye Calotomus carolinus, spectacled Chlorurus perspicillatus and redlip parrotfish Scarus rubroviolaceus. Values of L_Δ50 were c. 23, 27, 37, 46 and 47 cm L_F in the respective species. Length at female maturation was proportional to maximum body size (L_max) of the respective species, ranging from 50 to 72% and averaging 62% of L_max across species. L_Δ50 was also proportional to L_max, ranging from 82 to 97% and averaging 92%. Males of both pairs of Scarus and Chlorurus spp. reported here are diandric. Only one of the five major species (C. carolinus) is functionally monandric, with either all or nearly all males secondarily derived from adult females. The broadly differing absolute body sizes at sexual maturation and at sex change among the five species have important implications for improving regulatory size limits for parrotfishes in the State of Hawaii, where parrotfish species have historically been managed based on a single minimum size limit of 30·5 cm L_F. This study provides a model demonstration of why catch data for parrotfishes, and other size‐structured reef‐fish populations, should be recorded either by species or by functional size‐groups of species that allow setting more meaningful minimum size limits.     

Effects of exploitation on reproductive capacity of blackspot snapper, Lutjanus fulviflamma (Pisces: Lutjanidae) in Mafia Island, Tanzania

There is paucity of information on the effects of exploitation on reproductive characteristics of blackspot snapper, Lutjanus fulviflamma (Forsskål 1775) in Tanzanian coastal waters. We compared size at first sexual maturity (L_M50), sex ratio, fecundity, and breeding season of L. fulviflamma in least fished Mafia Island Marine Park (MIMP) and intensively fished areas (IFA) between May 1999 and April 2001. Fish in MIMP matured at significantly smaller size (female: L_M50 = 206.3 mm; male: L_M50 = 195.5 mm) than in IFA (female: L_M50 = 216.7 mm; male: L_M50 =212.1 mm) total body length. Sex ratio was balanced at 1.03 : 1 (female : male) in MIMP, but it was skewed 0.9 : 1 (female : male) in IFA. Size‐related differences in sex ratio were observed with males predominating in the smaller sizes and females in the larger sizes. Total fecundity of fish in MIMP was determined at 45,200–430,200 oocytes in females of between 207 and 293 mm total length. Lutjanus fulviflamma in MIMP has a prolonged spawning season lasting from September to March peaking in December. None of the fish from IFA were in breeding state, suggesting recruitment overfishing is an added matter of concern for the long‐term sustainability of the fishery at the current exploitation level.     

Growth and reproduction of the highfin grouper Epinephelus maculatus

Highfin grouper Epinephelus maculatus sampled in Chuuk, Micronesia, exhibited a moderate growth rate and a relatively short life span compared to other epinephelids of a similar size. Combined gonad and otolith analysis provide preliminary evidence that the species conforms to a protogynous sexual pattern. Mean total length at maturity for females was 308 mm with first age at maturity 2·8 years for females and 4 years for males, which differs from other regional studies. Based on the gonado‐somatic index and microscopic analysis of gonads, E. maculatus in Chuuk have a 4 month spawning season (January to April) that corresponds with seasonal lows in sea surface water temperature and overlaps with that of other aggregating epinephelids. The estimated von Bertalanffy growth factor (K) was 0·51 year^?1, while total mortality was 0·34 year^?1. Current management for E. maculatus in Chuuk includes a January to May catch, sale and export ban, which overlaps with its reproductive season. The effectiveness of these arrangements will require on‐going monitoring to determine whether alternative management strategies are required to ensure population persistence.     

Neuropeptides trigger oocyte maturation and subsequent spawning in the hydrozoan jellyfish Cytaeis uchidae

Oocyte maturation and subsequent spawning in hydrozoan jellyfish are generally triggered by light‐dark cycles. To examine if the initiation of the maturation process after light stimulus is mediated by neurotransmitters, neuropeptides isolated originally from Hydra magnipapillata were applied to sexually mature female medusae of the hydrozoan jellyfish Cytaeis uchidae. Among the Hydra neuropeptides tested, Hym‐53 (NPYPGLW‐NH₂), as well as a nonphysiological peptide, CGLWamide (CGLW‐NH₂), were most effective in inducing oocyte maturation and spawning. Hym‐355 (FPQSFLPRG‐NH₂) also triggered these events, but the stimulatory effect was weaker. Since Hym‐53‐OH (NPYPGLW) and Hym‐355‐OH (FPQSFLPRG) had no effect, amidation at the C‐terminus may be critical for the stimulatory activities of the peptides. Exposure to Hym‐53 for 2 min was sufficient to trigger of oocyte maturation, and the spawned eggs were able to be fertilized and to develop normally. Transmission electron microscopy confirmed that bundles of axon‐like structures that contain dense‐core synaptic vesicles and microtubules are present in the ovarian ectodermal epithelium overlying the oocytes. In addition, immunohistological analyses revealed that some of the neurons in the ectodermal epithelium are GLWamide‐ and PRGamide‐positive. These results suggest that a neuropeptide signal transduction pathway is involved in mediating the induction of oocyte maturation and spawning in this jellyfish. Mol. Reprod. Dev. 80: 223–232, 2013. © 2013 Wiley Periodicals, Inc.     

The reproductive biology of the two-banded sea bream (Diplodus vulgaris) from the southwest coast of Portugal

The study of the reproduction of Diplodus vulgaris (Geoff.) as part of a base‐line study of the fishery resources of the south‐west coast of Portugal, was based on the analysis of the spawning season, gonad maturation, size at maturity, fecundity and hermaphroditism. The spawning season is relatively long, from December to March, with peaks in January and February. No significant differences were found either in the sex ratio (M : F = 1.01) over the year or by size. The size at first maturity (L₅₀) for all sexes and undetermined individuals combined was 18.33 cm total length (TL), with no significant differences between males and females. The estimated L₅₀ is considerably greater than the minimum legal size in Portugal of 15.0 cm. Mean absolute fecundity (F_a) was 131 127 oocytes, ranging from 31 523 to 250 608. The relationship between absolute fecundity and total length (TL) (F_a = 25 398 TL – 484 426) and somatic weight (SW) (F_a = 878.8SW – 71 416) was of the linear type. The mean number of oocytes per gram of somatic weight was 526, ranging from 194 to 887. The reproductive strategy of this species is characterized by a rudimentary hermaphroditism with possible protandry, as evidenced by the existence of individuals in sexual transition.     

Timecourse of oocyte development in saithe Pollachius virens

Wild caught North Sea saithe Pollachius virens were monitored for growth, sex steroid profiles and oocyte development pre‐spawning and measured for egg size and group fecundity during the spawning season in the laboratory. Vitellogenesis commenced in late October–early November, at a leading cohort size (C_L) of c. 250 µm, after which oocytes grew rapidly in size until spawning started in February. Notably, a distinct cortical alveoli stage was virtually absent with yolk granules observed in developing oocytes at the very beginning of vitellogenesis. Little atresia was observed pre‐spawning, but atretic re‐absorption of remnant oocytes containing yolk granules was found in all females immediately post‐spawning. As expected, concentrations of sex steroids, oestradiol‐17β (females), testosterone (both sexes) and 11‐ketotestosterone (both sexes), increased pre‐spawning before dropping post‐spawning. The present experiment provides the first validation of sex steroid levels in P. virens. Post‐ovulatory follicles were visible in histological sections from female gonads 9–11 months post‐spawning, but then disappeared. Spawning commenced around a C_L of c. 750 µm (700–800 µm). Hydrated oocytes (eggs) measured between 1·04 and 1·31 mm (mean = 1·18 mm) with decreasing sizes towards the end of spawning. The average estimated realized fecundity was c. 0·84 million eggs (median female total length, L_T = 60 cm). Spawning lasted from 13 February to 29 March.     

Metabolism of glucose,pyruvate, and glutamine during the maturation of oocytes derived from pre‐pubertal and adult cows

The aim of the study was to compare the energy metabolism of oocytes from pre‐pubertal (2 to 3 months) and adult cows during maturation, to identify the cause of poor developmental potential in many pre‐pubertal oocytes. The metabolism of [5‐³H] glucose, [2‐¹⁴C] pyruvate, and [G‐³H] glutamine was measured at 0 hr, 12 hr, and 24 hr maturation. Oxidative metabolism was important during maturation of oocytes from both pre‐pubertal and adult cows, with pyruvate metabolism peaking at 12 hr and glutamine metabolism increasing linearly and peaking at 24 hr. Peak oxidative metabolism was significantly lower in oocytes from pre‐pubertal animals, for both pyruvate and glutamine (P < 0.05). Glucose metabolism increased significantly during oocyte maturation in both groups (0hr to 24 hr). Glucose metabolism was significantly lower in oocytes from pre‐pubertal cows at 12 hr (P < 0.05). Oocytes from pre‐pubertal animals were significantly smaller than oocytes from adult cows at 0 hr, 12 hr, and 24 hr maturation (P < 0.05). When metabolic rates were corrected for oocyte volume, there were no significant differences in substrate metabolism between oocytes from pre‐pubertal and adult cows. There was however, a delay in the increase in glucose metabolism in pre‐pubertal oocytes 0 hr to 12 hr maturation. Germinal vesicle breakdown was slower in oocytes from pre‐pubertal animals with more oocytes still at the germinal vesicle stage approximately 5 hr post‐aspiration, compared to oocytes from adult cows (P < 0.05). By 24 hr, development to metaphase II was equivalent for pre‐pubertal and adult oocytes. This study identified differences in energy metabolism, oocyte size, and meiotic progression between the oocytes from pre‐pubertal and adult cows that may account for the poor developmental potential of many pre‐pubertal oocytes. Mol. Reprod. Dev. 54:92–101, 1999. © 1999 Wiley‐Liss, Inc.     

BAPTA‐AM dramatically improves maturation and development of bovine oocytes from grade‐3 cumulus‐oocyte complexes

Intracellular free calcium ([Ca²⁺]_i) is essential for oocyte maturation and early embryonic development. Here, we investigated the role of [Ca²⁺]_i in oocytes from cumulus‐oocyte complexes (COCs) with respect to maturation and early embryonic development, using the calcium‐buffering agent BAPTA‐AM (1,2‐bis[2‐aminophenoxy]ethane‐N,N,N′,N′‐tetraacetic acid tetrakis [acetoxymethyl ester]). COCs were graded based on compactness of the cumulus mass and appearance of the cytoplasm, with Grade 1 indicating higher quality and developmental potential than Grade 3. Results showed that: (i) [Ca²⁺]_i in metaphase‐II (MII) oocytes from Grade‐3 COCs was significantly higher than those from Grade‐1 COCs, and was significantly reduced by BAPTA‐AM; (ii) nuclear maturation of oocytes from Grade‐3 COCs treated with BAPTA‐AM was enhanced compared to untreated COCs; (iii) protein abundance of Cyclin B and oocyte‐specific Histone 1 (H1FOO) was improved in MII oocytes from Grade‐3 COCs treated with BAPTA‐AM; (iv) Ca²⁺ transients were triggered in each group upon fertilization, and the amplitude of [Ca²⁺]_i oscillations increased in the Grade‐3 group upon treatment with BAPTA‐AM, with the magnitude approaching that of the Grade‐1 group; and (v) cleavage rates and blastocyst‐formation rates were improved in the Grade‐3 group treated with BAPTA‐AM compared to untreated controls following in vitro fertilization and parthenogenetic activation. Therefore, BAPTA‐AM dramatically improved oocyte maturation, oocyte quality, and embryonic development of oocytes from Grade‐3 COCs.