Stages Of Vitellogenesis Research Articles

The annual ovarian cycle in Northern pit vipers (Bothrops atrox) using serum steroids and ultrasound of the female reproductive tract

Knowledge on hormonal regulation of reproductive cycles in viperid snakes is still incipient, especially when it comes to females and tropical species. There is an urgent need to understand the reproduction of venomous snakes to improve assisted reproduction techniques and optimize the maintenance of these animals in captivity. With this in mind, we monitored Northern pit viper females year-round throughout different seasons via serum levels of progesterone (P4) and estradiol (E2) in conjunction with ultrasound examinations. Ovarian follicles were classified according to their size and stage of vitellogenesis in F-I and F-II (non-vitellogenic phase) or in F-III and F-IV (vitellogenic phase). During autumn and winter, five adult males were rotated among these females for reproductive pairing, which resulted in 17 copulations and 2 pregnancies in the first year and 12 copulations and 5 pregnancies in the second year. Then, we assessed changes in P4 and E2 levels according to seasons, predominant ovarian structures and the presence of embryos or eggs in the oviduct. Our findings showed high levels of E2 when a greater number of vitellogenic follicles were detected, indicating a possible influence of E2 on vitellogenesis and higher levels of P4 whenever eggs and embryos were visualized in the oviduct, implying its role in maintaining pregnancy. Descriptive analysis of the vipers’ ovarian cycles revealed a greater number of vitellogenic follicles during winter, probably as a result of increases in E2; whereas pregnancies occurred predominantly in spring, under the influence of P4. The use of ultrasound images, as a minimally invasive methodology, associated with serum steroid levels has proven to be an efficient approach in the reproductive monitoring of Northern pit vipers in vivo. In addition, these data suggest that female pit vipers under human care display a seasonal reproductive cycle, despite earlier studies involving captive males of the species indicating a lack of seasonality in sperm production and quality.

Deepening our understanding of the environmental influences on the reproductive dynamics of the main fishery stock of Argentine hake, Merluccius hubbsi, in the southwestern Atlantic Ocean

AbstractThis study analyzed the inter‐annual variability in the reproductive dynamics of the Argentine hake from Patagonian waters during the last 20 years concerning the environmental conditions and the nutritional status of females. Macroscopic and histological information on gonadal maturity, female condition factor (K), and oceanographic data at different sampling sites throughout the studied region were obtained from research surveys carried out during the main spawning season of this species. Four stages, according to the maturity ovarian phases composition, were assigned annually at each sampling site to assess reproductive dynamics: vitellogenesis, ovulation, regression, and resting. The spatial pattern of inter‐annual variability in reproductive dynamics was described, and the influence of temperature and female condition on the probability of ovulation and regression was assessed using generalized linear models. The area with the lowest inter‐annual variability in the hake reproductive dynamics was observed between 43 and 45°S (50 to 80 m depth) in relation to ovulation events. The vitellogenesis stage was more frequently found adjacent to the reproductive area, whereas the regression was mainly observed in deeper waters. Both bottom and surface temperatures explained the ovulation and regression stages, showing a positive and negative relationship, respectively. Lower values of the K factor, associated with low food availability, were further linked to the occurrence of regression. Finally, it is worth noting that the relationship observed between the ovulation/spawning dynamics of Argentine hake and water temperature suggests that the reproductive success of this species could be affected in the future scenario of climate change.

Integrated transcriptomic and metabolomic analyses identify key factors in the vitellogenesis of juvenile Sichuan bream (Sinibrama taeniatus)

Vitellogenesis is the most important stage of ovarian maturation in fish, involving the synthesis and transport of essential yolk substances and their complex mechanisms and coordination process. The liver is the main tissue involved in the vitellogenesis of oviparous animals, but studies of vitellogenesis in fish rarely include the liver and ovary as a whole. The aim of this study was to explore the molecular mechanism and associated regulatory factors behind vitellogenesis in Sichuan bream (Sinibrama taeniatus). The different stages of oogenesis were first identified by successive histological observations. Then, ovary and liver tissues that developed to 115 days (stage II, previtellogenesis stage), 165 days (stage III, vitellogenesis stage) and 185 days (stage IV, late-vitellogenesis stage) were collected for transcriptomic and metabolomic analyses, and serum testosterone (T), 17β-estradiol (E2), vitellogenin (Vtg), triiodothyronine (T3), and thyroxine (T4) levels were measured at the corresponding stages. We found that energy redistribution during vitellogenesis is mainly regulated through glycolysis, fatty acid biosynthesis and the citrate cycle pathway. In the liver, energy metabolism was promoted by activating glucolipid metabolic pathways to provide sufficient ATP, but at the same time, the ovary tends to retain nutrients rather than decompose them to produce energy. In addition, we have identified several key factors involved in the metabolism of neutral lipids, polar lipids, amino acids and vitamins, which are involved in the assembly and transport of important yolk nutrients. The initiation of vitellogenesis was found to be associated with a surge in serum E2 levels, but the sustained increase in Vtg levels in the late stage may be due more to upregulation of the estrogen receptor. These results provide valuable information about the regulation of ovarian development in cultured fish.

Histological analysis of oogenesis and ovarian development of the pharaoh cuttlefish, Sepia pharaonis

AbstractThe pharaoh cuttlefish, Sepia pharaonis, has become a commercially cultured cephalopod in coastal southeast China. However, information on the reproductive histology of this species remains limited. To describe its reproductive development, this study investigated the ovarian structure and oogenesis of S. pharaonis from hatchlings to the postbreeding stage using anatomical methods and histological techniques. The results showed that oogenesis in the ovary was asynchronous and morphologically variable, with immature oocytes attached to the gonadal cord and encircling the ovarian stroma in sequence. After maturation, the egg was free in the ovarian cavity and was discharged through the fallopian tube to combine with the sperm. Furthermore, serial histological dissections of the ovaries demonstrated that oocyte development was asynchronous. Based on egg size and morphology, follicular cell morphology, and yolk formation, oogenesis in S. pharaonis was divided into five distinct stages: The oogonia stage, protoplasm growth stage, follicular penetration stage, vitellogenesis stage, and resorbing stage. Moreover, based on the appearance of follicular cells in the protoplasm growth stage and their disintegration and disappearance in the vitellogenesis stage, it can be inferred that follicular cells secrete yolk substances and participate in the formation of egg membranes. Through the dynamic observation and description of the ovary development and oogenesis, these results provide an important foundation for studies of the regulatory mechanisms of oogenesis in this species, enriching the theory of cephalopod reproductive biology and improving artificial reproduction technology.

Marine pollution effects on the reproduction process of Perinereis cultrifera (Annelida, Polychaeta) in Algeria

The Mediterranean Sea is one of the busiest areas worldwide in terms of maritime activity and faces considerable anthropogenic disturbances, such as pollution by hydrocarbons and heavy metals. This study evaluated the environmental status of three sites on the Algerian coast using the annelid polychaete Perinereis cultrifera as a biomonitoring sentinel species. It included different reproductive parameters such as oocyte diameter, frequency distributions, sexual maturity index, biochemical quantitative parameters represented by the vitellogenins and vitellins, and a histological study of the coelome during the reproductive period. The results showed that the females collected from El-Kala (healthy site) have the highest diameter 304.1 ± 24.3 μm during April but the values were increased in Annaba 290.0 ± 21.0 μm and Skikda 245.3 ± 26.7 μm. Significant differences were observed between worms from the three study sites during the three months of the study. Moreover, the size-frequency indicated that a relatively high proportion of females containing mature oocytes was found in April in females collected at El-Kala 52.4%, 46.4% in females collected at Annaba and 36.7% in females collected at Skikda. In addition, regarding the sexual maturity index, the highest values were recorded in April (3.8, 2.9, and 1.9) at the El-Kala, Annaba, and Skikda study sites. However, the biochemical analysis demontrated that the highest mean concentration of vitellogenin was in females of El-Kala (5.3 ± 0.3 µg/mg of coelomic fluid) in April, and the lowest mean concentration was measured in females of Skikda (1.5 ± 0.2 µg/mg of coelomic fluid). Therefore, the highest mean concentration of vitellin was measured in females of El-Kala (4.4 ± 0.2 µg/mg of oocytes) in April, while the lowest mean concentration of vitellin was measured in females of Skikda (1.02 ± 0.2 µg/mg of oocytes) in May. Significant differences were observed between worms from the three sites during the three months of the study. Furthermore, histological observations revealed that oogenesis was asynchronous, with oocytes at different stages of vitellogenesis simultaneously present. The structure of the oocytes of females collected at El-Kala showed intense vitellogenic activity due to the presence of very dense yolk cells. In contrast, the oocytes of the females collected from the two polluted sites (Annaba and Skikda) were small, vitellogenesis presented low intensity, and yolk cells were less frequent at the periphery and less uniform at the cytoplasmic mass; the nucleus was smaller, indicating a slower vitellogenic activity. These observations confirm the previously obtained results.

DNA methylation mediates gonadal development via regulating the expression levels of cyp19a1a in loach Misgurnus anguillicaudatus

DNA methylation-mediated transcriptional regulation has been considered to significantly impact some steroidogenic enzyme genes expression. To uncover the roles of DNA methylation on the regulation of aromatase gene expression during gametogenesis in Misgurnus anguillicaudatus, the expression profiles and cellular localization of cyp19a1a and cyp19a1b were analyzed, and the landscape of DNA methylation dynamics was investigated. We found that cyp19a1a was predominantly expressed in granulosa cells of oocytes, while cyp19a1b expression was enriched in radial glial cells of the forebrain. In ovary, cyp19a1a was highly expressed until the vitellogenesis stage. The average methylation levels, especially for two CpG sites within the cAMP response element, were negatively correlated with cyp19a1a expression levels, indicating that methylation could regulate cyp19a1a transcriptional activity by modulating the binding efficiency of cAMP to its response elements. Compared with in ovary, cyp19a1a showed lower expression in testis but was hypermethylated. Cyp19a1b in female brain weakly expressed before the vitellogenesis stage, but significantly elevated at the maturation stage. In both sexes, it maintained high methylation levels in brain despite the obvious fluctuation of the cyp19a1b expression. This study revealed that DNA methylation plays a key role in establishing cyp19a1a spatiotemporal expression patterns and thus mediates gonadal development in teleosts.

The vitellogenesis-inhibiting hormone (SpVIH) of the crab Scylla paramamosain is not likely to have a vitellogenesis-inhibiting function in the ovary

The function and molecular characterization of the previously reported crab (Scylla paramamosain) vitellogenesis inhibiting hormone SpVIH is still unclear. In this experiment, SpVIH’s bioinformatic and functional characterizations were analyzed. Sequence analysis showed SpVIH was clustered with several type II MIH/GIHs of the Brachyura. The expression of SpVIH in the eyestalk was low in the early ovarian developmental stage I and increased with the advancement of ovary maturation. Interestingly, the SpVIH transcript was still at a high level in the ovary of females at the middle to late maturation stage. SpVIH’s function was studied according to the expression of vitellogenin (Vg) and vitellogenin receptor (VgR) in the ovary and hepatopancreas after incubation with SpVIH-dsRNA and recombinant SpVIH protein (rSpVIH). In vitro assay results indicated that rSpVIH has a significant stimulative effect on the expression of Vg in the hepatopancreas in the middle stage of vitellogenesis and in the ovary of each vitellogenesis stage. However, SpVIH-dsRNA has no significant effect on the expression of Vg when SpVIH-dsRNA is introduced to the ovary and hepatopancreas at the early, middle, and late stages of vitellogenesis. Furthermore, the co-incubation experiment result indicated that the eyestalk consists of factor(s) that can significantly up-regulate the expression of Vg in the ovary, with a further combination of dsVIH and eyestalk under incubation, the significant increase of Vg-mRNA expression levels were also found in the ovary of four ovarian development stages of S. paramamosain. Contrary to the previous reports, SpVIH does not possess a vitellogenesis-inhibiting function but can promote vitellogenesis in the ovary and hepatopancreas.

Resveratrol Alleviates Inflammation and ER Stress Through SIRT1/NRF2 to Delay Ovarian Aging in a Short-Lived Fish.

Aging is a complex process in which the structure and function of various tissues and organs gradually decline with age, and ovarian aging affects the reproductive capacity of females and induces age-related diseases. Resveratrol, a natural polyphenol compound, extends the life span and has a protective effect on the ovaries of vertebrates. However, the effects and underlying mechanisms of resveratrol delaying ovarian aging are unclear. In this study, using an annual fish Nothobranchius guentheri, we demonstrated that senescence-associated-beta-galactosidase (SA-β-gal) activity and lipofuscin accumulation increased with age in the ovaries, and resveratrol reversed this phenomenon. Resveratrol increased proliferating cell nuclear antigen (PCNA) expression and the oocyte proportions of the primary growth stage, cortical alveolus stage and vitellogenesis stage, and decreased the number of atretic follicles in the ovaries of 6-, 9-, and 12-month-old fish. Moreover, the expression of SIRT1 and NRF2 decreased and the levels of NF-κB, pro-inflammatory cytokines IL-1β, TNF-α, and IL-8 and endoplasmic reticulum (ER) stress markers GRP78 and CHOP increased with aging, while resveratrol up-regulated SIRT1 and NRF2 expression and down-regulated NF-κB, IL-1β, TNF-α, IL-8, GRP78, and CHOP levels in the ovaries of 6- and 9-month-old fish. In HEK293T cells, knockdown SIRT1 decreased NRF2 and increased NF-κB p65, pro-inflammatory cytokines (IL-1β and TNF-α), and ER stress marker GRP78 expression markedly. Silencing SIRT1 and then treating the cells with resveratrol significantly reversed the phenomenon. Collectively, resveratrol might activate SIRT1/NRF2 to reduce inflammation and ER stress, and finally delay ovarian aging in a short-lived fish. This study highlights the protective effect and mechanism of resveratrol on ovarian aging.

Expression and cellular localization of insulin-like growth factor 3 in gonads of the seasonal breeding teleost silver pomfret (Pampus argenteus).

Insulin-like growth factor 3 plays an important role in gonad development in teleost fish. Previous studies found that igf3 was specifically expressed in gonads of silver pomfret (Pampus argenteus). Unlike in other fish, IGF3 is a membrane protein in silver pomfret, and its specific role in gonads is unclear. Herein, we explored the importance of IGF3 in oogenesis and spermatogenesis in silver pomfret by analyzing gene expression and cellular localization. During follicular development, igf3 was detected in ovaries at both mRNA and protein levels during the critical stages of vitellogenesis (IV-VI). Localization analysis detected igf3 mRNA and protein in somatic cells, including theca and granulosa cells around oocytes. Similar to cathepsin L and cathepsin K, igf3 was consistently expressed in ovaries during vitellogenesis, suggesting that it might play a key role in vitellogenesis of oocytes. During spermatogenesis, igf3 mRNA and protein levels were high in stages II, IV, and V, similar to sycp3 and dmc1, and the highest igf3 mRNA and protein levels were reached in stage VI. Furthermore, igf3 mRNA and protein were detected in spermatogonia, spermatocytes, spermatids, and surrounding Sertoli cells, but not in spermatozoon, indicating that IGF3 might be involved in differentiation and meiosis of spermatogonia.

The insulin-like growth factor 1 stimulates ovarian steroidogenesis and oocyte maturation in spotted steed Hemibarbus maculatus

The insulin-like growth factor 1 (IGF1) plays important roles in fish reproduction. To understand the role of IGF1 in steroidogenesis and oocyte maturation, we investigated expression pattern and functions of Igf1 during in vitro maturation of spotted steed (Hemibarbus maculatus) oocytes. Igf1 expression quantified by qPCR and western blotting revealed that both Igf1 mRNA and protein was significantly increased in mature follicles. The immunofluorescence analyses showed IGF1 protein signal was detected in the theca cells of ovarian follicles during vitellogenesis stage, and in both theca cells and granulosa cells of matured oocytes. Incubation of ovarian follicles with recombinant IGF1 (rIGF1) significantly enhanced the rate of spotted steed oocyte germinal vesicle breakdown (GVBD), a marker of oocyte maturation. qPCR analysis showed that the transcription of steroidogenesis-related genes (3β-hsd, 20β-hsd, Cyp17a and Cyp19a1a) were influenced after IGF1 treatment. In addition, the expression profiles of receptors of Igf1, Fsh and Lh indicated that a cross talk response to IGF1 induced oocyte maturation. Furthermore, IGF1 treatment did not affect ERK protein expression but significantly upregulated CyclinB and AKT protein expression. All together, these findings help us to understand the molecular mechanism of IGF1 in regulation ovarian steroidogenesis and oocyte maturation of spotted steed.

Molecular Characteristics of Scylla paramamosain Ubiquitin-Conjugating Enzyme E2 and Expression Analysis During Oogenesis

Ubiquitination is an essential post-translational modification of proteins and involved in many physiological processes, including sperm differentiation, oocyte maturation, and embryonic development. Among them, the ubiquitin-conjugating enzyme E2 plays a crucial role in gametogenesis. However, the participation of E2 in the regulation of the gonad development of crustaceans is still very limited, especially for oogenesis. In this research, we isolated and identified full-length complementary DNA of ubiquitin-conjugating enzyme E2 from mud crab (Scylla paramamosain) (Sp-Ubc2), detected distribution in different tissues by real-time quantitative PCR, and described expression patterns during oogenesis by in situ hybridization. The results demonstrated that the deduced proteins with a ubiquitin-conjugating (UBC) catalytic domain was clustered with other arthropods. In addition, the Sp-Ubc2 was widely distributed in different tissues of female individuals and kept the highest expression level in the ovary. Moreover, the expression level of Sp-Ubc2 changed significantly during oogenesis and reached peak in secondary and tertiary vitellogenesis stages. At the same time, the Sp-Ubc2 signal in the ovary gradually concentrated in nuclei. These results indicated that Sp-Ubc2 might play important roles in the cell cycle of oogenesis. This study would provide insights into the regulatory mechanism of gonad development in S. paramamosain, and it also enriched the theoretical basis of ubiquitin family genes involved in crustacean gonad development.

Growth, sexual transition, and maturation of blacktip grouper Epinephelus fasciatus under long-term artificial rearing: Puberty and its associated physiological and endocrine changes

The blacktip grouper Epinephelus fasciatus is considered a valuable marine fish for aquaculture. This study was conducted to obtain information on its age of puberty, growth performance, gonadal development, sexual transition pattern, and plasma sex steroid levels from juvenile stage to maturity in specimens reared in captivity. The stages of gonadal development were identified by histological analysis, and plasma sex steroid levels were measured using an enzyme-linked immunosorbent assay. Our results show that maturation and spawning in blacktip groupers occurred at almost 3 years of age; although some females matured at 2 years. Plasma 17β-estradiol (E2) levels increased rapidly to high levels during puberty at 3 years of age, especially during the late stage of vitellogenesis at the tertiary yolk stage, and reduced at the ripe and ovulated stages prior to spawning. The gonadal somatic index also showed an increase consistent with the rise in plasma E2 levels. Plasma 11-ketotestosterone (11-KT) levels increased dramatically during sex change and in males. The significant decrease in plasma E2 levels and remarkable increase in plasma 11-KT were the key factors for the progress of sex change in these fish. Interestingly, there were the concomitant appearances of spermatocytes in ovaries of immature females at 1 year 3 months of age. The earliest appearance of sex-transitioning and male fish in blackfish groupers was observed at 2 years, although no mature females were observed below this age. This indicated that the sex change and male transformation in these fish took place without them undergoing the mature female stage. In conclusion, this species had the ability to reach puberty at 2 years of age, but most individuals matured and were functional at 3 years old. Puberty might be affected by age rather than size. In addition, although this species had a protogynous hermaphrodite pattern, when individuals born in the same year were reared together, some individuals were capable of directly becoming males without experiencing a mature female stage. This was due to the lack of socialization in captivity wherein individuals of various ages do not coexist, unlike that in the wild.

Steroid Profiles of Wild Terror Green (Andinoacara rivulatus) Associated with Gonadal Histology in the Baba River, Ecuador

Native cichlids in Ecuador are represented by Andionoacara rivulatus and Cichlasoma festae mainly. These aquatic species are a fundamental part of the food chain of rural communities in the western subtropical region of Ecuador. For the first time, a detailed study on the gonadal development in A. rivulatus males and females was carried out, and the characterization of sex hormones present in their blood plasma from May 2019 to January 2020. Fish samples were captured in the multipurpose Baba River dam upstream of the Guayas River, Ecuador. Gonad and blood samples were collected for histological analysis and assaying of steroidal hormones such as 17β-estradiol and 11-ketotestosterone. The sex steroids were quantified by enzyme immunoassay (ELISA). As a result, the fish samples have asynchronous characteristics and show all development stages in testes and ovaries. The serological fluctuations show statistical differences (p <0.05) between the development stages, with low concentrations of estradiol and 11-KT. These results are somewhat related to the hydrological period of capture, where the temperature, luminosity, and rainfall play a fundamental role in the oogenesis and spermatogenesis in the captured fishes. The highest concentration of E2 is detected in female vitellogenesis stages and the highest concentration of 11-KT in the male spermatogenesis stage. These results confirm the fundamental role of these hormones in the key periods of gonadal development. In future studies, it is important to monitor the maturational hormone 17α, 20β-DHP, and the vitellogenin concentrations and thus understand the reproductive physiology of A. rivulatus.

New insights into reproductive physiology in Antarctic fish: a trial in Lepidonotothen nudifrons

The spatio-temporal delimitation of fish reproduction is essential for the appropriate management and conservation strategies in populations. Assessing this feature in Antarctic Ichthyology is particularly difficult because harsh environmental conditions limit sample collection. This study shows how physiology can contribute compelling evidence to understand reproduction in Antarctic fish using the notothenioid Lepidonotothen nudifrons as a model species. Sampling included 121 specimens caught at Potter Cove (PC), South Shetland Islands (SSI), from November to late March of 2016–2018. Gonadal macroscopic and histologic features are reported. Oocyte growth and change in testosterone and estradiol plasma levels throughout the ovarian growth of L. nudifrons adult females is provided. In March, females (n = 17) attained gonado-somatic index of 13–20% (16.73 ± 4.20), total fecundity of 2196–4652 oocytes/female, the leading clutch oocytes measured 1.7–2.1 mm, and males (n = 5) showed spermatozoids in their testicles. The leading clutch growth was significantly associated with photoperiod, with no diameter variation until the summer solstice, when it began to grow linearly with an estimated rate of 0.01 mm/day. Testosterone and estradiol increased together with the oocyte growing throughout the analysed months, with a higher rate of increase during March. The reproductive effort data, and especially the significant plasma level increase in both sex steroids observed in March, suggest that (1) females were at a late vitellogenesis stage, just prior to the oocyte final maturation, and thus L. nudifrons spawning period might begin in March at SSI; (2) PC is likely a spawning site for L. nudifrons, which indicates that nearshore areas are spawning grounds for some notothenioids.

Structural and ultrastructural characters of the reproductive tract in females of the mint leaf beetle Chrysolina herbacea (Duftschmid 1825) (Coleoptera: Chrysomelidae)

AbstractIn this study, Chrysolina herbacea female reproductive system, differentiating stages of vitellogenesis, the egg chorion are examined with light and scanning electron microscopies. C. herbacea is chosen for this study because it is considered to be one of the most harmful insects that destroy mint plants, and it is an agricultural pest. Female reproductive system consists of a pair of ovaries, a pair of lateral oviducts, a common oviduct, spermathecae and a genital chamber. The ovary has 18 meroistic telotrophic ovariole. Each ovariole is divided into four regions: terminal filament, germarium, vitellarium and pedicel. Terminal filament is a long tubular structure. Germarium contains trophocytes (nurse cells), young oocytes and prefollicular cells. Vitellarium consists differentiating oogenic stages including developmental periods of previtellogenesis, vitellogenesis and choriogenesis. The ovarioles open into lateral oviduct by pedicel. The single spermatheca is located near the base of the common oviduct. Spermathecae consist of spermathecae reservoir for sperm storage and a short spermathecae duct. C. herbacea eggs are yellow and elongated. The structure of female reproductive system, the egg chorion and larva of C. herbacea are important to understand the outbreak of this insect pest and to biological control this harmful species.

Molecular characterization of gonadotropin subunits (fshβ, lhβ and cgα) of largemouth bass ( micropterus salmoides ) and their expression in response to luteinizing hormone‐releasing hormone analogue and dopamine antagonists

Gonadotropins (GtHs), including follicle-stimulating hormone (FSH) and luteinizing hormone (LH), are important hormones involved in gametogenesis, gonadal steroidogenesis, and maturation. In this study, GtH subunits (FSHβ, LHβ and CGα) of largemouth bass (Micropterus salmoides) were cloned and characterized, and their regulation by luteinizing hormone-releasing hormone analog (LHRHa2) and dopamine antagonist (DOM) treatment in vivo was investigated. The full-length cDNA sequences of FSHβ, LHβ, and CGα were 683, 576, and 685bp, encoding 120, 152, and 132 amino acids, respectively. The deduced amino acid sequence analysis revealed that GtH subunits contain conserved cysteine residues and potential N-linked glycosylation sites and showed high homology with the corresponding subunit sequences from other Perciformes by phylogenetic analysis. In the primary growth cortical alveoli stage of largemouth bass, the three GtH subunits were highly expressed in the pituitary, brain and ovary, but weakly in other tissues. After LHRHa2 and DOM injection, mRNA levels of FSHβ, LHβ, and CGα in the pituitary were significantly increased at 12 or 24 hr (p <.05), but significantly decreased at 48 hr; plasma FSH and LH levels showed a consistent trend. Histological analysis classified ovaries from LHRHa2- and DOM-treated fish into the early oocyte maturation stage and late vitellogenesis stage, while those from the control group fish were classified into the early vitellogenesis stage. These results suggested that both LHRHa2 and DOM stimulated GtH subunit expression, increased plasma FSH and LH and accelerated ovary development of largemouth bass, providing a framework for a better understanding of the mechanisms of hormone-mediated reproduction control in teleosts.

Molecular cloning, characterization, and expression analysis of a sex-biased transcriptional factor sox9 gene of mud crab Scylla paramamosain.

Sox9 gene, a crucial member of the Sox gene family, is present in various organisms and involved in many physiological processes, especially in sex determination and gonad development. In this study, we reported a sox9 gene (designated as Spsox9) from Scylla paramamosain through analyzing published gonad transcriptome data. Meanwhile, the accuracy was validated by PCR technology, and the 3' sequences were cloned with 3' RACE technology. The full-length cDNA of Spsox9 is 2843bp, consisting of a 243bp 5' UTR, an 1124bp 3' UTR, and a 1476bp ORF encoding 491 amino acids. Furthermore, to better understand its conservation among crustacean species, the sox9 gene ortholog was identified in several other crustaceans species with their published transcriptome data, respectively. All of the Sox9 proteins identified in the current study had the common feature of Sox proteins (HMG domain) and were highly conserved among analyzed crustacean species. In all examined tissues, the Spsox9 was mainly expressed in the gonad (testis and ovary), eyestalk, and cerebral ganglion. During embryo development, Spsox9 was highly expressed in 5 pairs of appendages, 7 pairs of appendages, and eye-pigment formation stage. During ovary development, the expression level of Spsox9 remained stable in the first 4 stages (O1-O4) and decreased in the tertiary vitellogenesis (O5) stage. During testis development, the expression level of Spsox9 was highest in the spermatid stage (T2) and was significantly different from that in the spermatocyte stage (T1) and mature sperm stage (T3) (p<0.05). In addition, Spsox9 exhibited a sex-biased expression pattern in T1 and O1. These present results indicated that the Spsox9 gene might play crucial roles in the gonad and embryo development of mud crab.

Reproductive Characters of the Ricefield Eel (Monopterus albus Zuieuw) in Babakan Village, Karang Lewas District, Banyumas, Central Java

One of the villages in Banyumas Regency which provides a high potential habitat for ricefield eel (Monopterus albus Zuieuw), a protogynous hermaphrodite, is Babakan. However, information concerning their reproduction cycles is lacking. This research aimed to understand the reproductive characters including Gonad Maturity Level (GML), Gonad Maturity Index (GMI), fecundity, egg diameter, and to examine the correlation of body length with both GML and GMI of the fish. We applied the survey method with purposive sampling design. The sampling was done once in a month for four times from November 2017 to March 2018. There were 30 samples for each sampling time (total of 120 eels). The results showed that the ricefield eels underwent intersex phase at a body length of 30.5-30.7 cm. The fish less than 30.5 cm were females, and those more than 30.7 cm was a male. The ricefield eel sex based on stages of gonad development was GML I (there were oocytes equipped with nucleus and cytoplasm), GML II (the presence of cortical alveoli stage), GML III (the stage of vitellogenesis), and GML IV (mature stage). The presence of degenerated oocytes and testicular lobes characterized their intersex stage. A testicular lobe characterized the male stage. The dominant GML during November, December, January, and March were GML I and II, GML I and II, GML III, and GML IV, respectively. Fecundity of the ricefield eels during the research was between 207 and 370 eggs, with an average of 252 eggs. The egg diameter of 0.22-2.5 mm indicated that the ricefield eel was on the batch spawner. The highest GMI of the eels occurred in January between 00017 and 0.0242 during the entire month of sampling. The body length of the fish did not correlate to their GML. On the other hand, their body length correlated to their GMI.

Cyclin B protein undergoes increased expression and nuclear relocation during oocyte meiotic maturation of the freshwater prawn Macrobrachium rosenbergii and the Chinese mitten crab Eriocheir sinensis.

Cyclin B functions as a regulatory protein through association with its catalytic partner Cdc2 kinase forming M-phase promoting factor (MPF), which plays a central role in the meiotic maturation of oocyte. To gain insight into the molecular events, we here cloned a cyclin B cDNA from the ovary of the prawn Macrobrachium rosenbergii and compared its spatial-temporal expression patterns during oocyte maturation with those of crab Eriocheir sinensis. The prawn cyclin B cDNA encodes a 398 amino acid protein with predicted molecular weight of 45.16kDa. Immunodetection of cyclin B protein by Western blot showed that a target band of approximately 53kDa protein in the prawn ovaries at both late vitellogenesis (lVt) and germinal vesicle breakdown (GVBD) stages, whereas a 41kDa band was present in the crab ovaries. Cyclin B protein expression changes indicating that the newly synthesis of cyclin B proteins could be required for GVBD in both prawn and crab. Immunohistochemical analysis revealed that both the prawn and crab cyclin B proteins, were localized in the ooplasm of previtellogenic oocytes, then relocated into germinal vesicle at vitellogenesis stage and localized on meiotic spindle at M phase. These similar behaviors suggested that the prawn and the crab cyclin B proteins associated with Cdc2 kinase have conserved roles in inducing GVBD and regulating the formation of meiotic spindle. The similar expression patterns of the cyclin B proteins during oocyte maturation implicated that the molecular mechanisms for MPF activation could be identical between the prawn and the crab.

Functional expression patterns of four ecdysteroid receptor isoforms indicate their different functions during vitellogenesis of Chinese mitten crab, Eriocheir sinensis

In arthropods, alternative splicing of ecdysteroid receptor gene (EcR) leads to multiple functions of different EcR isoforms during metamorphosis, growth and ovarian development via ecdysteroid signaling pathway. This study was conducted to investigate the expression patterns of four EcRs of Eriocheir sinensis (EsEcRs) and the changes of haemolymph ecdysteroid titer during the ovarian development. The results showed that four EsEcR isoforms had the tissue-specific expression among 12 examined tissues, and the highest transcript levels of the four EsEcR isoforms were detected in Y-organ or sinus gland. During the ovarian development, EsEcR1 showed the highest transcript abundance of the four EsEcR isoforms. The expression profiles of all the EsEcR isoforms were similar in the hepatopancreas during the ovarian maturation cycle of E. sinensis with a trend of “high-low-high-low”. In ovary, the highest expression levels of EsEcR1 and EsEcR4 were both found at stage V ovary, while the peaks of EsEcR2 and EsEcR3 were found on stage III ovary and stage IV ovary, respectively. Meanwhile, the ecdysteroid titer in haemolymph decreased gradually during ovarian maturation cycle. Further regression analysis revealed significant negative correlations were found between the ovarian EsEcR3/ EsEcR4 expression levels and haemolymph ecdysteroid titer during part or whole ovarian development cycle. These results together indicated that four EsEcR isoforms may have different functions during ovary maturation of E. sinensis. All EcR isoforms and ecdysteroid seemed to have important roles in the hepatopancreas during early ovarian development stages, while EsEcR3 and EsEcR4 were closely related to the mid-late vitellogenesis stages.