Histological characterization of the spawning process in the catarina scallop,Argopecten ventricosus (Sowerby II, 1842) induced by thermal shock and serotonin injection

Summary

The histology of the spawning catarina scallop Argopecten ventricosus was studied to characterize cellular and anatomical changes occurring along the gonad-kidney pathway. Broodstock were conditioned for 25–30 days and induced to spawn with two methods: thermal shock and serotonin by intracardiac injection. Samples of the kidney-gonad-digestive gland complex were fixed at the sperm and oocyte stages of gamete emission and at the end of spawning. Both induction methods elicited gametes, and no particular difference in the microscopic anatomy of the acini-kidney pathway was detected. However, serotonin injection, apart from producing a rough valve opening, sporadic movement (opening and closure of valve), and pronounced foot extension, induced gamete emission in less time than thermal shock. Primary gonad ducts opened to acini to permit sperm emission; at the same time, germinal vesicles broke down in female acini and meiosis restarted. Shortly afterward, oocytes gradually changed form from typically poly-hedral to a more malleable form. This probably contributed to oocyte transport through ciliary activity along the gonad and kidney ducts. Nucleoli disintegration, chromosome condensation, and appearance of meiotic figures were observed along the acini-kidney pathway. Although sperm and oocytes were sometimes observed in gonad or kidney ducts, self-fertilization was rarely observed.     

Gamete number and size correlate with adult size in the egg parasitoid Trichogramma euproctidis

In parasitoids, the size of the adult is influenced by the size and quality of the host in which it develops. Body size is generally positively correlated with several adult fitness proxies (fecundity, longevity, and mating capacity). The initial resources available to an individual can influence gamete production (sperm and oocytes), and the number and quality of gametes produced directly influence the expected fitness of both males and females. Gamete production in relation to adult body size was quantified in Trichogramma euproctidis (Girault) (Hymenoptera: Trichogrammatidae), a short‐lived egg parasitoid of lepidopteran species. To avoid host quality variation, male and female parasitoids of different body sizes were produced using superparasitism by allowing mated and virgin female parasitoids to oviposit on Trichoplusia ni Hübner (Lepidoptera: Noctuidae) eggs. Seminal vesicles and ovaries of their offspring were dissected to count oocytes and to measure sperm length and oocytes volume. Tibia length was also measured to estimate body size. The number of oocytes, volume of oocytes, maternal investment index [= (number of oocytes × mean volume of oocytes)/10 000] and sperm length were all significantly positively correlated to body size. These results show that initial resources acquired during larval stage induce phenotypic plasticity in gamete production in both male and female T. euproctidis. Whereas number of sperm and oocytes can influence the fitness of males and females through increased mating capacity and fecundity, variation in gamete size (sperm length and oocyte volume) could also affect the fitness of an individual through sperm and larval competition.     

Phenotypic plasticity in the reproductive traits of a parasitoid

Organisms show phenotypic plasticity - the capacity for a given genotype to express different phenotypes - in response to changes in the environment. Among the several factors that can cause phenotypic plasticity, nutritional constraints during development can affect the size of organisms and, consequently, affect most life-history traits, including reproductive traits. As their larvae are restricted by the amount of food contained in their host, parasitoids are a good model to study phenotypic plasticity related to size. The phenotypic plasticity of reproductive traits was investigated in the egg parasitoid Trichogramma euproctidis (Hymenoptera: Trichogrammatidae) by using host species of different sizes. Adult size, sperm storage organs (seminal vesicles and spermatheca), number of sperm stored and gamete size (sperm and oocyte) are all influenced by the host species; larger individuals have larger organs which contain more sperm, and both sperm and oocytes are correlated with adult size. However, while females become larger than males and mature larger oocytes in larger hosts, increase in sperm length stops after a given threshold.     

Reproductive strategy of the Patagonian catfish Hatcheria macraei

This study describes the reproductive strategy of the stream‐dwelling catfish Hatcheria macraei in the Pichileufu River, Argentina. Gonad maturity phases, classified on the basis of histological analysis, stages of gamete development and the frequency distribution of oocyte size, were correlated with macroscopic features of the gonads. Hatcheria macraei has a cystovarian ovary, asynchronous oocyte development and lobular testes. Five oocyte and four spermatogenic stages were identified and related to macroscopic gonad characteristics, making it possible to divide gonad development into five phases for females and males. Mature oocyte diameter ranged from 922 to 1935 µm. Absolute fecundity in mature females varied from 115 to 480 oocytes. Hatcheria macraei has multiple spawning during a protracted reproductive season that extends from December to April. This, together with its small size, is characteristic of an opportunistic reproductive strategy, commonly found in species that inhabit adverse and unpredictable environments, such as the low‐order rivers of Patagonia.     

Gamete rescue in the African black rhinoceros (Diceros bicornis)

Mortality rates are high among captive African black rhinoceroses (Diceros bicornis), due to increased susceptibility to disease. The ability to rescue genetic material from individuals that die unexpectedly represents a practical approach to assist ex situ conservation efforts. The objectives of the present study were to attempt postmortem oocyte recovery from ovaries of African black rhinoceroses (N = 6) and to test the efficacy of equine protocols for rhinoceros oocyte IVM and IVF using cryopreserved rhinoceros sperm. The interval from ovary removal to oocyte recovery was 25.3 ± 13.9 h (mean ± SD). Ovaries were transported at 4 °C or 22 °C and effects of temperature on postmortem oocyte competence was evaluated. Numbers of oocytes collected per female averaged 15.8 ± 6.9. In total, 95 oocytes were recovered. Of these, 85 were inseminated using homologous sperm and 10 were inseminated using heterologous sperm. Overall, substantial numbers of viable oocytes were retrieved from African black rhinoceros ovaries 1 to 2 days postmortem from ovaries stored at ambient temperature. A proportion of these oocytes matured and underwent penetration and fertilization by heterologous or homologous frozen-thawed rhinoceros sperm. The reproductive competence of postmortem oocytes was further demonstrated by development of a single two-cell embryo. Despite the need for further refinements, gamete rescue in the rhinoceros has promise for producing rhinoceros embryos, as well as testing sperm functions in vitro.     

Evidence of daily spawning in natural populations of the New Zealand snapper Pagrus auratus (Sparidae)

Synopsis New Zealand snapper, Pagrus auratus, were captured by trawling from NE New Zealand over two successive spawning seasons, and examined for acute temporal changes in gonad condition. Fish with oocytes completing final oocyte maturation predominated during the morning, with a peak in ovulated fish occurring just after midday. Afternoon catches were dominated by fish in which the most advanced oocytes had yet to begin final maturation. This suggests that ovulation is synchronised to occur soon after midday, and the high proportion (up to 100% of the catch) of fish with particular gonad stages captured at any one time indicates that daily spawning involves most of the population. Diurnal changes in oocyte diameter support a daily spawning rhythm, with numbers of large hydrated oocytes peaking in the late morning, followed by the disappearance of these oocyte stages in the afternoon. Snapper captured alive by longlining were returned to the laboratory to examine the relationship between ovulation and probable time of spawning. Unovulated fish generally ovulated close to midday on the day of capture (morning captures), or the following day (afternoon captures). The viability of ovulated eggs (proportion undergoing division after fertilization) decreased markedly after oviduct residence times of over 8 hours. This suggests that natural spawning occurs before the late evening. The results of this study are consistent with anecdotal evidence suggesting that spawning occurs every day during the late afternoon or early evening, and is similar to the reproductive patterns displayed by a number of closely related sparids. Department of Zoology, University of Otago, PO Box 56, Dunedin, New Zealand     

Disruption of O‐GlcNAc homeostasis during mammalian oocyte meiotic maturation impacts fertilization

Meiotic maturation and fertilization are metabolically demanding processes, and thus the mammalian oocyte is highly susceptible to changes in nutrient availability. O‐GlcNAcylation—the addition of a single sugar residue (O‐linked β‐N‐acetylglucosamine) on proteins—is a posttranslational modification that acts as a cellular nutrient sensor and likely modulates the function of oocyte proteins. O‐GlcNAcylation is mediated by O‐GlcNAc transferase (OGT), which adds O‐GlcNAc onto proteins, and O‐GlcNAcase (OGA), which removes it. Here we investigated O‐GlcNAcylation dynamics in bovine and human oocytes during meiosis and determined the developmental sequelae of its perturbation. OGA, OGT, and multiple O‐GlcNAcylated proteins were expressed in bovine cumulus oocyte complexes (COCs), and they were localized throughout the gamete but were also enriched at specific subcellular sites. O‐GlcNAcylated proteins were concentrated at the nuclear envelope at prophase I, OGA at the cortex throughout meiosis, and OGT at the meiotic spindles. These expression patterns were evolutionarily conserved in human oocytes. To examine O‐GlcNAc function, we disrupted O‐GlcNAc cycling during meiotic maturation in bovine COCs using Thiamet‐G (TMG), a highly selective OGA inhibitor. Although TMG resulted in a dramatic increase in O‐GlcNAcylated substrates in both cumulus cells and the oocyte, there was no effect on cumulus expansion or meiotic progression. However, zygote development was significantly compromised following in vitro fertilization of COCs matured in TMG due to the effects on sperm penetration, sperm head decondensation, and pronuclear formation. Thus, proper O‐GlcNAc homeostasis during meiotic maturation is important for fertilization and pronuclear stage development.     

Maturity and gonad changes of Oreochromis (Nyasalapia) karongae raised in fish ponds in Malawi

The reproductive biology of pond-raised Oreochromis (Nyasalapia) karongae was investigated. Gonad histology and gonadosomatic indices (GSIs) indicated a potential for multiple spawning in a season. Several peaks of oocyte-size distribution and several maturation stages occurred in the same gonad. GSIs of 2.5% and 1.4% were recorded in female and male fish, respectively. Three stages of oocyte maturation (primary growth, formation of yolk vesicles, vitellogenesis) and three stages of sperm development (spermatogonia, spermatid, spermatozoa) were observed. With the exception of the final maturation stage, all development phases seemed to proceed satisfactorily. The final stage of maturation was attained at oocyte size of 2.70 ± 0.54 mm and was selectively impaired in some female fish by a lack of deposition of vitellogenin. This abnormal condition led to atrophic oocytes lacking yolk granules and vesicles. Sexual maturation was attained at a relatively large size of 16.0 cm (114 g) compared with other tilapia of the mossambicoid group (i.e. Oreochromis mossambicus and Oreochromis shiranus ). A combination of gravimetric and histological techniques was successful in charting gonad changes and calibrating external against internal gonad features.     

The histology of mature gonads of Oreochromis (Nyasalapia) karongae (Trewavas)

Histology of gonads of Oreochromis karongae was undertaken to study internal cell characteristics during maturation. This study was necessitated by low spawning output of the fish species. Several oocyte stages, ranging from primary forms to vitellogenesis, suggest that the maturation was generally succesfully attained in the fish ponds. Pre‐vitellogenesis oocytes (oogonia to perinuclear stage) and more advanced vitellogenesis (primary vesicle to tertiary yolk vesicle) oocyte stages were all found in the same gonads. However, there were some discontinuities observed during stages 3 and 4, suggesting selective maturation. Failure of gonads to mature normally is attributed to an ecological crunch that was in a previous study associated with environmental factors. Atretic oocytes were also recorded in the same gonads, a sign that some oocytes failed to mature normally. This indicates insufficient stimuli for normal gonad development. Several stages of spermatogenesis (spermatocytes, spermatid and spermatozoa) were also found in the same gonads. Selective recrudescence was more pronounced in O. karongae because generally less oocytes attained final maturation stages compared to Oreochromis niloticus and other tilapias. This could be the main reason for low natural breeding that has been observed in both wild and captive stocks, and led to the abandonment of its use in aquaculture. This study corroborates findings of previous studies that depended solely on external gonad characteristics. Histology provides conclusive evidence from internal cell characteristics that other techniques are unable to show.     

Reproductive patterns of the Pacific oyster Crassostrea gigas in France

Summary

In France, national management programs focus research on understanding reproductive factors in Crassostrea gigas to confront problems of the oyster industry. However, little information has been documented in which reproductive patterns include sexual changes. The reproductive cycle of oysters at three sites of the Atlantic coast of France was examined from 1996 to 1998, and the seasonal variations in oocyte size-frequencies, and sex ratio were described. The results showed a synchronism within the population concerning reproductive behavior. Young oocytes are generated after spawning and show no apparent changes during winter. Growth of oocytes begins in spring and cells reach maturity in April-May and are ready for a single spawning season in June-July. Oocytes that were not released during spawning are reabsorbed within the gonad. The significant difference between sites is that spawning occurred 1 month later in the southern area. A modal analysis showed that oocyte populations in the sample individuals are primordially bimodal, but with polymodal occurrences in June-July, in some cases. Irregular alternative sexuality was detected at all sites, and hermaphrodites appear to be a transition phase that allows changes from male to female during early spring. Previous observations, together with the study of the development of oocyte cohorts over time, permit a hypothetical model concerning the kinetics of gametogenesis in C. gigas. The model suggests that primary oocytes are generated from energy supplied from degenerating, as well as young oocytes that do not reach the mature stage within the gonad during autumn-winter. It seems that, during vitellogenesis, there is disintegration of smaller cells coupled with transfer of energy to the larger oocytes, which continue to grow and mature.     

Incorporation and fate of specific sperm plasma membrane components following insemination as revealed by ultrastructural immunocytochemistry

Studies have been carried out to 1) further characterize sperm specific plasma membrane polypeptides (33 and 35 kDa) that are recognized by a monoclonal antibody previously described (Longo, 1989) and 2) follow the incorporation and dispersal of these proteins within plasmalemmae of monospermic and polyspermic sea urchin (Arbacia punctuluta) eggs and oocytes utilizing immunocytochemical methods at the ultrastructural level of observation. Only sperm labeled when incubated with monoclonal antibody to the 33 and 35 kDa proteins followed by colloidal gold-tagged second antibody. Colloidal gold label was observed on the egg plasma membrane immediately after gamete membrane fusion; the amount and extent of label, i.e., the distance from the site of sperm incorporation, increased with time postinsemination. By 20 min postinsemination approximately one hemisphere of the inseminated egg/oocyte was associated with label. The expanding distribution of colloidal gold label on inseminated eggs and oocytes vs. time reflects the free diffusion of 33 and 35 kDa sperm surface proteins among egg/oocyte plasma membrane components. Label was also found in forming endocytotic vesicles, suggesting that sperm plasma membrane proteins may be internalized.     

Large P body-like RNPs form in C. elegans oocytes in response to arrested ovulation, heat shock, osmotic stress, and anoxia and are regulated by the major sperm protein pathway

As Caenorhabditis elegans hermaphrodites age, sperm become depleted, ovulation arrests, and oocytes accumulate in the gonad arm. Large ribonucleoprotein (RNP) foci form in these arrested oocytes that contain RNA-binding proteins and translationally masked maternal mRNAs. Within 65 min of mating, the RNP foci dissociate and fertilization proceeds. The majority of arrested oocytes with foci result in viable embryos upon fertilization, suggesting that foci are not deleterious to oocyte function. We have determined that foci formation is not strictly a function of aging, and the somatic, ceh-18, branch of the major sperm protein pathway regulates the formation and dissociation of oocyte foci. Our hypothesis for the function of oocyte RNP foci is similar to the RNA-related functions of processing bodies (P bodies) and stress granules; here, we show three orthologs of P body proteins, DCP-2, CAR-1 and CGH-1, and two markers of stress granules, poly (A) binding protein (PABP) and TIA-1, appear to be present in the oocyte RNP foci. Our results are the first in vivo demonstration linking components of P bodies and stress granules in the germ line of a metazoan. Furthermore, our data demonstrate that formation of oocyte RNP foci is inducible in non-arrested oocytes by heat shock, osmotic stress, or anoxia, similar to the induction of stress granules in mammalian cells and P bodies in yeast. These data suggest commonalities between oocytes undergoing delayed fertilization and cells that are stressed environmentally, as to how they modulate mRNAs and regulate translation.     

Gamete membrane fusion in hamster spermatozoa with reacted equatorial segment

Mammalian spermatozoa must undergo many changes to be able to fertilize the oocyte. One of these changes, the acrosome reaction, has been established as a requisite for gamete membrane fusion to occur; it consists of the fusion and vesiculation of the sperm plasma membrane with the outer acrosomal membrane of the principal segment of the acrosome. Reaction of the equatorial segment has occasionally been observed. The objective of the present work was to determine whether the presence of the sperm plasma membrane over the equatorial segment is necessary for gamete membrane fusion to occur. Golden hamster spermatozoa were capacitated in vitro in TAPL 10K, and the maximum possible percentage of acrosome reaction was determined at 82.79% + 1.69% SD (P = 0.27; r = 0.21). Ultrastructural studies showed that 93.6% of the reacted spermatozoa in this population had their principal and equatorial segments reacted. The fertilizing ability of these spermatozoa was assayed using zona-free hamster oocytes. The percentage of fertilized ova obtained was 98.8% (308/312). Ultrastructural studies snowed the presence of spermatozoa with reacted equatorial segment inside the cytoplasm of immature oocytes. The evidence presented in this work demonstrates that the plasma membrane of spermatozoa with reacted equatorial segment retains its ability to fuse with the oocyte.     

Morphological development of the gonads in zebrafish

Gonadogenesis of zebrafish Danio rerio was investigated by means of light microscopy to test the suitability of gonad histology as an endpoint in hazard assessment of endocrine‐active compounds. At age 2 weeks post‐fertilization (pf), primordial germ cells were found in a dorsocaudal position in the body cavity. At 4 weeks pf, the majority of the fish (86%) possessed paired gonads with meiotic germ cells; these gonads represented presumptive ovaries. At week 5 pf, 87% of the fish examined had ovaries with perinucleolar oocytes. Further development of the gonads in female zebrafish up to week 11 pf was characterized by an increase in gonad size as well as in the number and size of perinucleolar oocytes. Starting with week 5, some fish showed alterations of gonad morphology, including a decrease in the number and size of the oocytes, an enhanced basophilia and irregular shape of the oocytes, and finally their degeneration into residual bodies. With the decline in oocyte number, stromal cells became more numerous and they infiltrated the gonadal matrix. In several 7 week‐old zebrafish with altered gonadal morphology, enhanced numbers of gonial cells arranged in cyst‐like groups appeared. These gonads were interpreted as presumptive testes. In one fish out of 32 individuals examined, spermatocytes were detected, in addition to the gonial cells. During the subsequent weeks, the percentage of fish showing early testes with spermatogonia, spermatocytes and spermatids increased and reached 40% at 11 weeks pf. The sequence of gonadal alterations taking place in some of the individuals from week 5 pf onwards was interpreted to reflect the transition of protogynic ovaries into testes. The developmental pattern described identifies zebrafish to be a juvenile hermaphrodite. The results of this study are of relevance for the use of gonadal histopathology as endpoint in endocrine disruption testing, particularly in order to avoid false diagnoses of ‘intersex gonads’ in zebrafish.     

Phagocytosis of sperm heads lacking the acrosomal process by unfertilized starfish oocytes

In sperm of the starfish Asterina pectinifera, the acrosomal process and the flagellum were mechanically separated from the sperm head with a disperser. The sperm head fraction was then used to examine the direct interaction between the sperm head and the egg surface. Sperm heads lacking the acrosomal process and the flagellum did not fertilize oocytes, even after removal of the vitelline coat. Transmission electron microscopy showed that each denuded oocyte engulfed the sperm head without gamete membrane fusion. The sperm-engulfing response, similar to phagocytosis, was induced without the mediation of the acrosomal process. The present results suggest that the process of sperm incorporation consists of two independent events, acrosomal process-egg surface fusion and the phagocytotic movement of the egg surface.     

Reproductive traits and relative gonad expenditure of the sexes of the free spawning Chiton articulatus (Mollusca: Polyplacophora)

ABSTRACT

Reproductive studies of an intertidal free-spawning population of Chiton articulatus (Mollusca: Polyplacophora) from Puerto Angel, Oaxaca, Mexico were undertaken during 2011. We used gonad histology and gonadal indices to assess the relative gonad expenditure of the sexes (RGES) and other reproductive traits, accounting for individual and seasonal variation within this population. At this location, C. articulatus is gonochoric, without sexual dimorphism, except internally by gonad colour (testis is ‘salmon’ coloured and ovary olive green). Annual and monthly sex ratios (m/f) do not differ significantly from 1:1. Highest population-level gonadosomatic index (GSI) corresponded to maximum (peak) ripe stage (i.e. maximum gonad investment), with a first peak in May with a high value (8.4 ± 0.5) and a second peak during August-September with a lower value (4.7 ± 0.3). GSI fluctuated throughout year implying that gonad expenditure may be seasonally constrained, but with overall synchrony between sexes of ripe and spawning stages. July to December was the main reproductive season with some facultative spawning occurring off-season. Ripe and spawning RGES did not differ between sexes, suggesting that either sperm competition is intense and/or that sperm limitation is high. Early spawning individuals may quickly replenish their gametes for a second phase of gamete release later.

Abbreviations: RGES: relative gonad expenditure of the sexes; GSI: gonadosomatic index; GDS: gonad developmental stages; MiMI: microscopic maturity index; SST: sea-surface temperature.     

The effects of temperature and chorionic gonadotropin on the functional properties of lactate dehydrogenase from oocytes of loach Misgurnus fossilis

Acclimation of loach to relatively low and high temperatures gives rise to changes in the properties of LDH from oocytes. The minimum of apparent K_m values for pyruvate of LDH from immature oocytes of fish acclimated to low temperature was registered at low assay temperatures, whereas adaptation to high temperatures leads to the enzyme showing a minimum at high temperatures. In thermal acclimation of fish the K_m minimum value drifts during 15 days. During oocyte maturation, the K_m minimum drifts during 40 hr. The differences in the kinetic features of LDH from oocytes both at thermal acclimation and during oocyte maturation are not associated with the appearance of new isozymes.     

Fast polyspermy block and activation potential : Electrophysiological bases for their changes during oocyte maturation of a starfish

In the starfish oocyte, the activation potential (AVP) upon fertilization establishes a fast polyspermy block. In the present paper, factors affecting the peak level of the AVP were analyzed by comparing current-voltage relations of the oocyte membrane just before and after insemination at various maturation stages. These factors were: (1) conductance of the oocyte membrane before insemination; (2) magnitude of the conductance increase induced by sperm; and (3) equilibrium potential of the AVP. Mature oocytes showed extremely low membrane conductance before insemination, and this provided a more positive-going AVP establishing monospermy. Overmature oocytes before fertilization showed higher conductance than mature oocytes and usually became polyspermic upon insemination. Application of Ba²⁺ ions reduced the conductance of the unfertilized, overmature oocyte to a state similar to that of the mature oocyte. Correspondingly, Ba²⁺ reduced the probability of polyspermy in overmature oocytes. Old oocytes showed even higher conductance before insemination than overmature oocytes and, in addition, apparently showed a large negative value for the equilibrium potential of the AVP. In old oocytes, these two factors account for the small amplitude of the AVP, which allows multiple sperm entries. Furthermore, the magnitude of the conductance increase induced by sperm seemed to change during the maturation process.     

Ovarian activity and plasma sex steroid levels of Distichodus antonii in relation to environmental conditions in the upper basin of the Congo River

Distichodus antonii is an endemic fish species of the Congo River basin in which the stocks of wild populations are threatened by overfishing pressure. Knowledge of its reproductive biology would be useful in consideration of conservation and management options for the species. Therefore, this study investigated changes in ovarian activity and levels of steroid profiles in wild populations in relation to variation in temperature and rainfall. Adult females (n = 101, body weight of 3 183 ± 14.75 g, SE) were captured monthly over one year (2013–2014). Apart from evaluation of oocyte diameters and gonad developmental stages, gonado-, hepato-, lipososomatic indices (GSI, HSI, LSI) and plasma levels of sex steroids (testosterone-T, estradiol-17β-E2) were determined. The results suggested a synchronous development of oocytes with two annual reproductive seasons over the one-year study. Plasma T and E2 levels peaked during spawning periods likely reflecting active oogenesis. The highest values of morphosomatic indices were observed during the longest rainfall period in September, and were associated with high steroidogenic activity evidenced by increased E2 production. In addition, more vitellogenic oocytes (September and October) were observed during the latter season than during the short rainy season (in May).