Germinal Vesicle Migration Research Articles

Real-Time Observation of Germinal Vesicle Migration During Oocyte Meiotic Cell Division Using Ovarian Fluorescent Transgenic Zebrafish.

The transgenic (TG) zebrafish allows researchers to bio-image specific biological phenomena in cells and tissues in vivo. We established TG lines to monitor changes in the ovaries of live fish. The original TG line with ovarian fluorescence was occasionally established. Although the cDNA integrated into the line was constructed for the expression of enhanced green fluorescent protein (EGFP) driven by the medaka β-actin promoter, the expression of EGFP is restricted to the oocytes and gills in adult fish. Furthermore, we found that germinal vesicles (GVs) in oocytes of the established line can be observed by relatively strong fluorescence around the GV. In this study, we tried to capture the dynamic processes of germinal vesicle breakdown (GVBD) during meiotic cell division using the GV fluorescent oocytes. As a result, GV migration and GVBD could be monitored in real time. We also succeeded in observing actin filaments involved in the migration of GV to the animal pole. This strain can be used for education in the process of oocyte meiotic cell division.

Effects of IGF1 and IGF2 on In Vitro Ovarian Follicle Maturation in Rainbow Trout, Oncorhynchus mykiss

Ovarian follicle maturation (OFM), including the resumption of meiosis in the oocyte, is under hormonal regulation. Insulin-like growth factors (IGF) have been shown to participate in the regulation of OFM with species-specific actions in teleost fishes. In the present study, in vitro treatment of rainbow trout ovarian follicles with recombinant human insulin-like growth factor-1 (rhIGF1) or rhIGF2 did not induce germinal vesicle breakdown (GVBD), a marker for the resumption of meiosis, in the oocytes. Co-incubation of follicle-enclosed oocytes with rhIGF1 and the maturation-inducing steroid (MIS) in rainbow trout, 17α,20β-dihydroxy-4-pregnen-3-one (17,20βP), also did not induce GVBD in follicles from rainbow trout that were not able to respond to the MIS alone suggesting IGFs cannot induce oocyte maturational competence (OMC), which is the ability of the oocyte to respond to the MIS. Nevertheless, the addition of rhIGF1 with the MIS increased the proportion of oocytes completing GVBD compared with MIS alone, although this potentiation was small and varied greatly among clutches of follicles from fish with oocytes at different stages of germinal vesicle migration. Collectively, these observations suggest IGFs may have synergistic actions with the MIS but cannot induce resumption of meiosis directly at the oocyte and are not potent inducers of OMC in rainbow trout. Rainbow trout are the first teleost fish in which IGFs were found to induce neither OMC nor resumption of meiosis in vitro.

Reproductive biology of wahoo (Acanthocybium solandri) in the Canary Islands (eastern-central Atlantic)

The reproductive status of wahoo (Acanthocybium solandri) in the eastern-central Atlantic was investigated to better understand their migration and breeding patterns. The waters around the Canary Islands are considered a breeding area for wahoo; and this study documents the first described breeding activity found in the eastern Atlantic. The presence of many mature and spawning capable wahoo reported in this study confirms its spawning activity in this region. Off the Canary Islands, only mature individuals spawn in the region where no immature individuals were observed to date. Most of the specimens captured in Canary Islands waters exceed 100 cm TL (about 2 years old). Values of the gonadosomatic index (GSI) of males were much lower than those estimated for females. Testes showed little changes in size throughout gametogenesis, while ovaries increased significantly (p<0.05). The low values make it inadvisable to use GSI in males to determine spawning activity. In the Canary Islands, wahoo females spawn (fishes with tertiary vitellogenic oocytes, postovulatory follicles or germinal vesicle migration, yolk coalescence, germinal vesicle breakdown and hydrated oocyte) mainly during a 4-month period in summer. Both females of spawning capable outside the main summer breeding period and females in regressing phase during the spawning period have been observed in the Canary Islands. Males were found in the spawning capable phase throughout the year, with greater prevalence between May and October.

Effects of 3,3’,4,4’,5-pentachlorobiphenyl on in vitro oocyte maturation in dusky tripletooth goby, Tridentiger obscurus: an implication of estrogenic potency

Polychlorinated biphenyls (PCBs) are endocrine-disrupting chemicals that mimic estrogen in fish. Among the various PCBs, 3,3’,4,4’,5-pentachlorobiphenyl (PCB126), considered an estrogen antagonist, has been reported to elicit estrogenic activity in fish. We investigated the estrogenic potency of PCB126 in in vitro oocyte maturation in dusky tripletooth goby, Tridentiger obscurus. In this study, we quantified steroid metabolites following exposure to PCB126 and estradiol-17β (E2). Vitellogenic ovarian follicles were incubated, in vitro, with 10 and 100 ng/mL PCB126 or E2 with [3H]17α-hydroxyprogesterone as a precursor. Testosterone (T) and E2, were identified using thin-layer chromatography, high-performance liquid chromatography, and gas chromatography-mass spectrometry. Both PCB126 and E2 increased T and E2 metabolite production. Further, vitellogenic ovarian follicles were exposed to PCB126 (1, 10 and 100 ng/mL) or E2 (0.01, 0.1 and 1 ng/mL) in vitro, and T and E2 from the incubation media were measured. PCB126 (100 ng/mL) inhibited T production and increased E2 production. Furthermore, we investigated the effects of PCB126 on the final oocyte maturation process. Germinal vesicle migration ovarian follicles were in vitro incubated with 0.1, 1, 10 and 100 ng/mL of PCB126 or E2. High doses of PCB126 (10 and 100 ng/mL) inhibited germinal vesicle breakdown. These results suggest that PCB126 has an estrogenic potency in oocyte maturation in T. obscurus.

Reproductive development of female wallago catfish (Wallago attu) in captivity

The wallago catfish (Wallago attu) is a new potential fish for aquaculture in Vietnam. Data related to the reproductive cycle of W. attu in captivity are, however, not available. To provide reliable indicators for oocyte maturation (OM) and the spawning season of the captive W. attu, this study investigated the temporal variation in hepatosomatic and gonadosomatic indices, oocyte diameter and color (greenish vs yellowish), germinal vesicle migration, and plasma concentrations of estradiol-17β (E2) and vitellogenin (Vtg) in female broodstock in association with changes in light density, temperature and amount of rainfall during the reproductive cycle. The results of this study displayed a clear seasonality in all the investigated parameters. The highest concentration of E2 (2.6 ± 3.5 ng/mL) was found in April, followed by a peak of Vtg (543 ± 43 ng/mL) in June. Meanwhile, the largest mean oocyte diameter (1.70 ± 0.02 mm) was observed in June. The shortest distance between the germinal vesicle and the edge of the oocyte (0.20 ± 0.01 mm) was recorded in July. Correspondingly, the amount of rainfall increased remarkably in July from 43.9 mm to over 200 mm in August. Taken together, we conclude that OM and the onset of the spawning season of captive W. attu occur in July and August, respectively. The percentage of greenish oocytes increased significantly over sampling time points. The changes in the color of oocytes combined with oocyte diameter could, therefore, be considered as promising indicators to predict the OM and spawning season of captive W. attu.

Transcriptomic analysis provides insights into the growth and maturation of ovarian follicles in the ricefield eel (Monopterus albus)

The ricefield eel is becoming an important aquaculture species in China. However, the regulation of oocyte growth and maturation in ricefield eels is poorly understood. In this study, the development of ricefield eel ovarian follicles was classified into nine stages, namely the primary growth/previtellogenic (PG/PV), early vitellogenic (EV), mid-vitellogenic (MV), late vitellogenic (LV), full-grown (GV0), germinal vesicle migration 1 (GV1), germinal vesicle migration 2 (GV2), germinal vesicle migration 3 (GV3), and germinal vesicle breakdown (GVBD) stages, which are based on follicle morphologies and sizes, vitellogenic states, and the positions of germinal vesicles. Comparative transcriptomic analysis was performed on isolated ovarian follicles from the MV to the GVBD stages. A total of 438,410 high-quality reads were obtained after filtering from six cDNA libraries of ovarian follicles at the MV, LV, GV0, GV1, GV2, and GVBD stages, 92.09% of which were perfectly mapped to the ricefield eel genome. Real-time PCR analysis showed that the expression profiles of 20 representative genes related to oocyte growth and maturation largely paralleled with those inferred from transcriptome data, which indicated the reliability of RNA-Seq data for gene expression analysis in the present study. The estradiol biosynthesis pathway was highly activated in ovarian follicles at the LV stage, which may be related to the stimulation of vitellogenin synthesis and/or meiotic arrest. The high expression of the Vtg receptor gene vldlr in ovarian follicles at MV and LV is consistent with active vitellogenesis at these stages. Thirteen cathepsin-encoding genes were highly expressed in ovarian follicles with different expression profiles during development from MV to GVBD, which implied their differential roles in vitellogenin hydrolysis during oocyte growth and maturation. Many cell cycle-related genes showed the lowest expression at GV0 and then increased expression at GV1 and GV2, which indicated that the resumption of meiosis may be initiated at GV0. The highest expression of the adenyl cyclase genes adcy3a, adcy5, adcy6, adcy6-like, and adcy7 at MV and/or LV, while that of phosphodiesterase genes pde1c, pde4b, pde4b-like, pde4c, pde7a, pde8b, pde10, and of the cyclic AMP-dependent transcription factor atf-3 gene observed at GV0 indicated that cAMP signals may play important roles in oocyte meiotic arrest and resumption in ricefield eels. Parallel expression profiles of urok, mmp2, mmp9, mmp11, mmp14a, mmp14b, mmp17a, mmp19, and mmp25 suggest their potential roles in tissue remodeling during oocyte maturation and ovulation in ricefield eels. Collectively, the data of the present study will aid in further understanding the molecular mechanisms underlying oocyte growth and maturation in ricefield eels as well as in other teleosts.

Ovarian dynamics, batch fecundity and spawning phenology of the lessepsian migrant Etrumeus golanii DiBattista, Randall &amp; Bowen, 2012 (Clupeidae: Dussumieriinae)

The Golani’s round herring Etrumeus golanii is an Erythraean small pelagic fish (lessepsian migrant) that entered into the Mediterranean Sea through the Suez Canal. It has expanded its distribution from the east to the western Mediterranean with well-established local populations. We investigated basic aspects of its reproductive biology off the island of Crete (eastern Mediterranean) using ovarian histology and analysis of oocyte size-frequency distributions. The species exhibited a protracted breeding period (winter to early summer), with all ovaries examined during the main spawning season having markers of recent (postovulatory follicles, POFs) or imminent spawning (advanced oocyte batch in germinal vesicle migration or hydration). The advanced batch (AB) increased rapidly in size and was fully separated from the remainder, less developed oocytes in 95% of females with “old” POFs (POFs with signs of degeneration) and all females in final maturation. The growth of the subsequent batch (SB) was arrested at sizes &lt;630 µm until full maturation of the AB. Mean diameter of hydrated oocytes ranged from 1181 to 1325 µm and relative batch fecundity was low ranging from 56 to 157 eggs g-1. The simulation of a coupled hydrodynamic/biogeochemical model (POM/ERSEM) provided evidence that E. golanii takes advantage of the seasonal cycle of planktonic production to reproduce and exhibits monthly changes in batch fecundity that appear to be closely related with the seasonal cycle of mesozooplankton concentration.

Gonadal recrudescence and annual reproductive hormone pattern of captive female Spotted Scats (Scatophagus argus)

The Spotted Scat (Scatophagus argus), an important euryhaline fish inhabiting mangrove and coastal regions of Indo-Pacific waters, is both an ornamental and food fish in India. Detailed insight into maturation of Spotted Scat when maintained in aquaculture systems, therefore, needs to be elucidated. Lack of information on annual maturation dynamics of female scat collected from their natural habitat and reared in earthen ponds is the basis of this study. Oocytes were classified into five developmental stages: pre-vitellogenic, vitellogenic, late-vitellogenic, ripe, and follicular atresia. Ovarian maturity stages were subsequently categorized as immature (Stage 1), vitellogenesis (Stage 2), maturing (Stage 3), mature (Stage 4), and spent (Stage 5). In oocytes in primary, secondary and tertiary yolk stages, there are greater concentrations of E2 in vitellogenic females between March and June. Significant increases of E2, T, and 17-OHP paralleled the increase of diameter of late–vitellogenic oocytes in maturing females during July. The completion of vitellogenesis and initiation of germinal vesicle migration in the cytoplasm were evident in mature females (Stage 4) with a decreasing trend of sex steroids in and subsequent to the month of August. There were 50 % of oocytes in the final oocyte maturation stage (FOM) (490–620 μm) until completion of Stage 4 in September. The results of this study indicate there is complete ovarian maturation in female scats captured in their natural habitat and maintained in an earthen pond, which may be important information for hatchery management for induction of spawning of Spotted Scat in aquaculture systems.

Reproductive physiology of bonefishes (Albula spp.) across the Northwest Bahamas.

Bonefishes (Albula spp.) are classified within the superorder Elopomorpha, which is comprised of over 1000 species that share a unique leptocephalus larval stage. Bonefishes have a circum-tropical distribution, inhabiting inshore shallow water flats and gathering in presumptive nearshore pre-spawn aggregations (PSA) during spawning months. These fishes support economically important recreational fisheries and subsistence fisheries throughout their ranges, yet little is known regarding their reproductive biology. Analysis of oocyte development and nutrient composition, and sex and gonadotrophic hormone levels, was conducted on females sampled in Grand Bahama, Central Andros, and South Andros, The Bahamas, to assess their reproductive state. Fish collected from the flats habitats along all three islands exhibited four major reproductive phases (immature, developing, spawning capable, and regressing). In contrast, all females captured at presumptive PSA sites had eggs in the final stage of oocyte maturation, significantly higher levels of all reproductive hormones (17β-estradiol, testosterone, and LH), larger vitellogenic oocytes, and oocytes exhibiting germinal vesicle migration and germinal vesicle breakdown. In addition, monthly variability in hormone levels and spawning readiness between Grand Bahama and Andros PSAs suggest that peak spawning times may differ among regions. Fatty acid and free amino acid composition and profiles, with high proportions of docosahexaenoic acid, histidine, and taurine, suggest that these nutrients are not only relevant as energy reserves, but also help achieve buoyancy and osmoregulation of oocytes. This study expands upon our understanding of fish reproductive and developmental physiology, and indicates potential factors influencing the survival and recruitment of bonefishes.

Oogenesis of Lithodes santolla: histological and histochemical characterization

Although Lithodes santolla is a resource with high commercial importance that has aroused interest in controlled reproduction in captivity, currently, little knowledge is available about basic reproductive aspects of this species. This research study describes detailed oogenesis stages at the histological level in adult females of the southern king crab of the Magellan and Chilean Antarctic Region. Oogenesis was quantitatively analyzed from the proliferation of 10-24 μm oogonia and during oocyte growth. Five stages of ovary development were identified: multiplication (0), previtellogenesis (I), vitellogenesis (II), maturity (III) and spawning (IV). Furthermore, 11 oocyte substages were distinguished: oogonia, chromatin nucleolus, early perinucleolus, late perinucleolus, oil globule, early vitellin globule, late vitellin globule, postvitelogenic, germinal vesicle migration and germinal vesicle breakdown. Primary vitellogenesis occurred in oocytes 180-185 μm, containing acidophilic globules and surrounded by a layer of thick follicle cells (20-40 μm). Secondary vitellogenesis was evident in oocytes at 315-321 μm with numerous acidophilic granules in the cytoplasm and surrounded by thin follicle cells; then, cortical crypts appeared, indicating the prematuration stage and preparation for ovulation. This study allows establishing gonadic changes that occur during a reproductive cycle of female L. santolla and help to strengthen aquaculture initiatives and management in the Magellan and Chilean Antarctic Region.

Induction of fertilizable eggs by conspecific vitellogenin implantation in captive female walking catfish,Clarias batrachus(Linn.)

The synthesis of vitellogenin (Vg) is induced by conspecific Vg (Vg1 and Vg2) and estradiol-17β (E2) as demonstrated by the pattern of 3H-serine incorporation in the liver and plasma proteins. The incorporation studies indicated that the label was first incorporated into the liver after which it appeared in the blood in both E2- and Vg-treated male catfish. Since Vg was capable of inducing its own synthesis, experiments were conducted in females during preparatory–prespawning period (March–May) to make them gravid by implanting Vg pellets. Two implantations of 4 mg Vg1 pellets into female catfish with an interval of 15 days, followed by laboratory maintenance for 45 days of initial implantation showed a significant increment in ovarian weight with concomitant formation of yolky oocytes through synthesis and incorporation of Vg, whereas Vg2 implantation was not effective in this regard. Histological observation of yolky oocytes in Vg1-treated group showed the peripheral migration of germinal vesicle (eccentric germinal vesicle), which indicates the onset of maturation. On 45th day, third implantation with 2 mg Vg pellets was performed and after 15 days, fish were hormonally induced with a single injection of hCG (2,000 IU/kg fish). Six groups were considered such as initial control, BSA-implanted control, Vg1-implanted, Vg2-implanted, catfish collected from the field on the last day of the experiment and catfish collected during spawning period in this experiment with 3–7 fish in each group. Each of the experimental fish was sexually mature and the body weight was between 100 and 125 g. The percentage of ovulation and fertilization in the eggs of Vg1-implanted group was 91% and 78%, respectively, which was almost similar to that of gravid female catfish collected during breeding period (July). The breeding performance in BSA- and Vg2-treated females was very poor. The fertilized eggs were hatched in the laboratory conditions. Thus, in the female catfish, Vg1 not only induces vitellogenesis but also makes the oocytes viable for fertilization.

Preovulatory follicle growth dynamics indicates daily spawning synchronicity in wild-caught Atlantic horse mackerel off Portugal

The Atlantic horse mackerel, Trachurus trachurus, is a commercially important species whose daily spawning dynamics are not yet fully understood. Existence of spawning synchronicity would facilitate application of the daily egg production method for estimating the spawning biomass of the southern stock of the species in the Northeast Atlantic. The intent of this work was to study preovulatory follicle (PREF) growth dynamics in wild Atlantic horse mackerel to determine the existence of daily spawning synchronicity in the population. The size and translucency of hydrated oocytes increased significantly between early morning and late afternoon; this pattern was the same for two study years. This morpho-developmental shift led to the appearance of previously indistinguishable oil drops in ovarian whole mounts which is an easily observed microscopic descriptor of PREF growth. The results clearly suggested that PREF growth was well synchronized between Atlantic horse mackerel that were capable of spawning and provided evidence for the existence of daily spawning synchronicity in the population. Apart from sampling time of day, the size of hydrated oocytes was shown to be affected by body size: a 10 cm difference in maternal length corresponded to a 0.030 mm difference in the diameter of hydrated oocytes. In contrast, the size of early germinal vesicle migration oocytes was correlated neither to sampling time nor to maternal size which suggests that the abrupt increase in PREF size only commences after the onset of the actively spawning sub-phase.

Effect of human chorionic gonadotropin on sexual maturation, sex steroids and thyroid hormone levels in Caspian lamprey (Caspiomyzon wagneri Kessler, 1870).

The objective of this study was to determine the effect of human chorionic gonadotropin (hCG) on sexual maturation, plasma sex steroids (17β-estradiol, E2 and 17α-hydroxy progesterone (17α OHP)) and thyroid hormones (triiodothyronine, T3 and thyroxin, T4) levels in upstream - migrating Caspian lamprey. During the experiment, 36 fish (24 females and 12 males) in spring 2013 and 36 fish (24 females and 12 males) in fall 2013 were collected from the Shirud River estuary in Mazandaran Province, the Southern Caspian Sea during their upstream migration to the freshwater. All fish were injected with hCG at the doses of 1000, 1500 and 2000 IU.kg BW-1. The injection was a two - step process (50% of hormone in each step) by 12 h interval. After the first injection, fish were retained in the cages in the river beds and 24 hours after the second injection, fish were checked for egg and sperm release after mild abdomen pressure. Blood samples were taken for determining sex steroid levels. Results showed that hCG hormone injections caused increase in migration of germinal vesicle in the oocyte of female and sperm release in males. Significant differences were found in the serum E2 and 17α-OHP levels in hCG - injected fish compared to the control. However, no significant differences were found in serum T3 and T4 levels in the hCG - injected fish. According to the results, the appropriate hCG dosage to induce the reproduction acceleration in Caspian lamprey is 1500-2000 IU.kg BW-1.

Simultaneous Germinal Vesicle Migration and Oil Droplet Coalescence Predict Final Oocyte Maturation in the Leopard Grouper

AbstractThe size of oocytes during germinal vesicle migration (GVM) and germinal vesicle breakdown (GVBD) was revised for the Leopard Grouper Mycteroperca rosacea. Histology of ovaries, plasma levels of estradiol‐17ß (E2), and testosterone (T) were analyzed in Leopard Grouper at 0, 12, 16, and 24 h after a priming dose of 1,000 IU/kg body weight of human chorionic gonadotropin (hCG) and were compared with control females injected with saline solution. Histological observations were verified in hormonally induced females by observing in vivo intraovarian biopsies after using the same priming dose and a second resolving dose of 500 UI/kg body weight of hCG 16 or 24 h later, which served to analyze spawning performance. Histological and in vivo intraovarian biopsies showed that in full‐yolk oocytes (~455 μm diameter) GVM and GVBD were concomitant with coalescence of a single oil droplet 16 and 24 h after the first injection. The oil droplet became a morphological marker as well as a predicting indicator of the competence and synchronization of maximum vitellogenic oocytes and the effectiveness to respond to hormonal therapy. In the experimental fish group E2 and T reached peaks at the same time, which were significantly higher than those in the controls. Mean fecundity was 168,000 eggs/kg, mean fertilization was 88%, and mean viability was 71% (114,000 eggs/kg), all of which were within the range of other cultivated groupers. These results can be used to improve the induction of final maturation in the Leopard Grouper in captivity and may be used in other species of Mycteroperca that show the same marker.

Spawning cycle and fecundity of a multiple spawner round herring Etrumeus teres off southern Japan: Oocyte growth and maturation analysis

The spawning characteristics of round herring Etrumeus teres remain unclear despite its commercial and ecological importance. The oocyte growth and maturation during the spawning cycle of round herring were examined based on the samples collected at different hours of the day off southern Japan during their 2015 spawning season. The ovarian histological analysis showed that germinal vesicle migration (GVM) and subsequent germinal vesicle breakdown occurred from the afternoon to the next morning. The appearance of hydrated oocytes indicated that round herring spawned between midday and evening. Degeneration of post-ovulatory follicles and the ovarian developmental stage suggested that individual round herring did not spawn on consecutive days but rather spawned at an interval of at least 2days. The oocyte diameters of the second clutch (subsequent spawning batch) increased gradually when the first clutch (spawning batch) underwent GVM and hydrated, but oocytes in the second clutch increased significantly in size immediately after spawning, suggesting that vitellogenesis of the second clutch may be accelerated for the next spawning. Size at maturity in all females was estimated to 17.6cm in body length (BL), and batch fecundity was 1409–21,023 oocytes (15.7–24.0cm in BL). Our findings provide a foundation for conducting stock assessment studies on round herring using an egg production method.

Interactive effects of dietary composition and hormonal treatment on reproductive development of cultured female European eel, Anguilla anguilla

Farmed female eels were fed two experimental diets with similar proximate composition but different n-3 polyunsaturated fatty acid (PUFA) levels. Both diets had similar levels of arachidonic acid (ARA), while levels of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) in one diet were approximately 4.5 and 2.6 times higher compared to the other diet, respectively. After the feeding period, each diet group was divided into two and each half received one of two hormonal treatments using salmon pituitary extract (SPE) for 13 weeks: i) a constant hormone dose of 18.75mg SPE/kg initial body weight (BW) and ii) a variable hormone dosage that increased from 12.5mg SPE/kg initial BW to 25mg SPE/kg initial BW. Results showed a significant interaction between diets and hormonal treatments on gonadosomatic index (GSI), indicating that the effect of broodstock diets on ovarian development depends on both nutritional status and hormonal regime. Females fed with higher levels of n-3 series PUFAs and stimulated with the constant hormonal treatment reached higher GSIs than those receiving the variable hormonal treatment. However, when females were fed lower levels of n-3 series PUFAs there was no difference in the effect of hormonal treatments on GSI. We also found that, independent of hormonal treatment, the diet with higher levels of n-3 series PUFAs led to the most advanced stages of oocyte development, such as germinal vesicle migration. Concentration of sex steroids (E2, T, and 11-KT) in the plasma did not differ between diets and hormonal treatments, but was significantly correlated with ovarian developmental stage. In conclusion, increasing dietary levels of n-3 PUFAs seemed to promote oocyte growth, leading to a more rapid progression of ovarian development in European eel subjected to hormonal treatment.

Stereological comparison of oocyte recruitment and batch fecundity estimates from paraffin and resin sections using spawning albacore (Thunnus alalunga) ovaries as a case study

Traditional histological protocols in marine fish reproductive laboratories using paraffin as the embedding medium are now increasingly being replaced with protocols using resin instead. These procedures entail different degrees of tissue shrinkage complicating direct comparisons of measurement results across laboratories or articles. In this work we selected ovaries of spawning Mediterranean albacore (Thunnus alalunga) as the subject of our study to address the issue of structural changes, by contrasting values on oocyte recruitment and final batch fecundity given from the same tissue samples in both paraffin and resin. A modern stereological method, the oocyte packing density (OPD) theory, was used supported by initial studies on ovarian tissue sampling and measurement design. Examples of differences in the volume fraction of oocyte stages, free space and connective tissue were found between the embedding media. Mean oocyte diameters were smaller in paraffin than in resin with differences ranging between 0.5% in primary growth and 24.3% in hydration (HYD) stage oocytes. Fresh oocyte measurements showed that oocytes shrank as a consequence of the embedding process, reaching the maximal degree of shrinkage for oocytes in the HYD stage (45.8% in paraffin and 26.5% in resin). In order to assess the effect of oocyte shrinkage on the OPD result, and thereby on relative batch fecundity (Fr), oocyte diameters corrected and uncorrected for shrinkage, were used for estimations. Statistical significant differences were found (P<0.05) between these two approaches in both embedding media. The average Fr was numerically smaller in paraffin compared to resin (86±61 vs. 106±54 oocytes per gram of body mass (mean±SD)). For both embedding media statistical significant differences (P<0.05) were seen between Fr results based on either oocytes in the germinal vesicle migration stage or HYD stage. As a valuable adjunct, the present use of the OPD theory made it possible to document that the oocyte recruitment of spawning ovaries of Mediterranean albacore followed the typical pattern of an asynchronous oocyte development and indeterminate fecundity.

Effect of thiamine injection on growth performance, hematology and germinal vesicle migration in sterlet sturgeon Acipenser ruthenus L.

The present study aimed to investigate the effect of thiamine on growth, hematological, egg thiamine content and oocyte nucleus migration indices in sterlet sturgeon (Acipenser ruthenus). A total of 45 female fish (698.6 ± 8.9 g) were distributed in nine fiberglass tanks with three treatments (each in three replicates). Experimental fish were fed once a day with practical diet supplemented with 1 g kg−1 amprolium hydrochloride (as the antithiamine) for 5 months before spawning. Thiamine hydrochloride was injected to fish with one of three doses at 0, 5 and 50 mg kg−1 body weight (BW) at three different stages. At the end of each month, fish were weighed and growth parameters such as weight gain, specific growth rate, feed efficiency and final weight were determined. At the end of the 5-month period, hematological parameters and egg thiamine content were measured. The results showed no significant differences in terms of growth performance. Hemoglobin, number of red blood cells and white blood cells were not significantly different among the treatments, but hematocrit was significantly higher in fish injected with 5 mg kg−1 BW thiamine. The mean corpuscular volume and mean corpuscular hemoglobin were not significantly different, but lower value of mean corpuscular hemoglobin concentration was observed in fish injected with 5 mg thiamine. Percentage of lymphocytes, neutrophils, eosinophils and monocytes were not significantly different among treatments. The results of the germinal vesicle migration index at different stages showed no significant differences in different groups. Results showed that free thiamine, thiamine pyrophosphate and total thiamine in eggs significantly increased at 50 mg kg−1 injection dose compared with the control, but no significant different was found in thiamine monophosphate. The results revealed that thiamine injection to sterlet broodstocks has positive accumulation in egg thiamine content and some hematological parameters but have no effect on growth and ovarian maturation. We conclude that thiamine can affect some physiological features of broodstock, which needs to be specified in further studies.

Determining Ovarian Maturity in Farmed Sturgeon 1 (Acipenser transmontanus) for Caviar Production Using Fourier Transform Infrared Spectroscopy (FT-IR)

Ovarian maturity of white sturgeon (Acipenser transmontanus, Acipenseridae) farmed in California (Sterling Caviar, LLC) (N=400) and Idaho (Fish Breeders and Blind Canyon Aqua Ranch) (N=143) was determined by correlating blood plasma spectral features [(Fourier transform infrared (FT-IR, 4000-400 cm-1) spectroscopy] with oocyte polarization index (PI), an index of germinal vesicle migration. A total of ~20,000 spectra were collected over a period of four years (2007, 2008, 2009 and 2010). Mathematical models could predict maturity in fish at a later year of harvest (i.e., 2010) and at either the California or Idaho production sites. PI values of 0.10, 0.15, and 0.20 were selected for segregating fish into subgroups based on ovarian maturity. Spectral features for specific proteins, lipids, carbohydrates and nucleic acids were related to fish maturity stages. Mathematical models could predict the actual PI values based on plasma spectral features in fish from 2010 based on models developed and validated from fish harvested in 2007-2009. These models worked equally well whether the fish were raised in California or Idaho. This research indicates that infrared spectroscopy provides a rapid and less invasive method to segregate sturgeon females according to maturity levels and has the potential to substitute for the traditional surgical biopsy to determine stage of maturity.

Transcriptional signature of progesterone in the fathead minnow ovary (Pimephales promelas)

A growing number of studies have examined transcriptional responses to sex steroids along the hypothalamic–pituitary–gonadal axis in teleost fishes. However, data are lacking on the molecular cascades that underlie progesterone signaling. The objective of this study was to characterize the transcriptional response in the ovary of fathead minnows (Pimephales promelas) in response to progesterone (P4). Fathead minnow ovaries were exposed in vitro to 500ng P4/L. Germinal vesicle migration and breakdown (GVBD) was observed and microarrays were used to identify gene cascades affected by P4. Microarray analysis identified 1702 differentially expressed transcripts after P4 treatment. Functional enrichment analysis revealed that transcripts involved in the molecular functions of protein serine/threonine kinase activity, ATP binding, and activity of calcium channels were increased after P4 treatment. There was an overwhelming decrease in levels of transcripts of genes that are structural constituents of ribosomes with P4 treatment. There was also evidence for gene expression changes in steroid and maturation-related transcripts. Pathway analyses identified cell cycle regulation, insulin action, hedgehog, and B cell activation as pathways containing an over-representation of highly regulated transcripts. Significant regulatory sub-networks of P4-mediated transcripts included genes regulated by tumor protein p53 and E2F transcription factor 1. These data provide novel insight into the molecular signaling cascades that underlie P4-signaling in the ovary and identify genes and processes that may indicate premature GVBD due to environmental pollutants that mimic progestins.