Ovarian Lumen Research Articles

Evaluation of Female Recipient Infertility and Donor Spermatogonial Purification for Germ Cell Transplantation in Paralichthys olivaceus.

Since the advent of germ cell transplantation (GCT), it has been widely used in shortening the fish breeding cycle, sex-controlled breeding and the protection of rare and endangered fish. In this study, the effectiveness of female sterile recipient preparation and donor stem cell isolation and purification were comprehensively evaluated for spermatogonial stem cell transplantation (SSCT) in Paralichthys olivaceus. The best way to prepare sterile recipients was found to be giving three-year-old fish four intraovarian injections of busulfan (20 mg/kg body weight) combined with exposure to a high temperature (28 °C) after the spawning season compared with the two other ways, which induced apoptosis of most of the endogenous germ cells, resulting in shrinkage of the spawning plate and enlargement of the ovarian lumen. Further analysis showed that both the gonadosomatic index and germ-cell-specific vasa expression were significantly lower than those of the natural-temperature group before treatment (p < 0.05). A high percentage (>60.00%) of spermatogonial stem cells (SSCs) were obtained after isolation and purification and were transplanted into the prepared recipients. After three weeks of SSCT, the numbers of PKH26-labeled SSCs were increased in the ovaries of the recipients. These findings provide a basis for the establishment of an ideal SSCT technique using P. olivaceus females as the recipients, ultimately contributing to the efficient conservation of male germplasm resources and effective breeding.

Membrane molecule bouncer regulates sperm binding activity in immature oocytes in the viviparous teleost species Poecilia reticulata (guppy).

Generally, in vertebrates, the first step toward fertilization is the ovulation of mature oocytes, followed by their binding to sperm cells outside of the ovary. Exceptionally, the oocytes of poeciliid fish are fertilized by sperm cells within the follicle, and the developmental embryo is subsequently released into the ovarian lumen before delivery. In the present study, we aimed to identify the factor(s) responsible for intrafollicular fertilization in a viviparous teleost species, Poecilia reticulata (guppy). Sperm tracking analysis in this regard indicated that in this species, sperm cells reached immature oocytes including the germinal vesicle, and the insemination assay indicated that the immature oocytes robustly adhered to the sperm cells; similar binding was not observed in Danio rerio (zebrafish) and Oryzias latipes (medaka). We also identified the Ly6/uPAR protein bouncer as the factor responsible for the observed sperm binding activity of the immature oocytes in this species. The recombinant bouncer peptide acted as an inhibitory decoy for the sperm-oocyte binding in guppy. On the other hand, ectopic expression of guppy bouncer in zebrafish oocytes resulted in interspecific sperm-oocyte binding. These results argue that bouncer is responsible for sperm-immature oocyte binding. Our findings highlight the unique reproductive strategies of guppy fish and enhance our understanding of the diverse reproductive mechanisms in vertebrates.

Toxic effect of Callistemon viminalis essential oil on the ovary of engorged Rhipicephalus microplus female ticks (Acari: Ixodidae).

Rhipicephalus microplus is the main ectoparasite of cattle and its parasitism can reduce weight gain of hosts, in addition to causing anemia, increasing the risk of myiasis, and resulting in the transmission of Babesia bovis and Anaplasma marginale, among other pathogens. The use of synthetic chemicals plays an important role in controlling these ticks. However, its frequent and indiscriminate use has contributed to the selection of resistant strains, resulting in greater interest in the search for natural-origin products. The shrub Callistemon viminalis (Myrtaceae), also known as weeping bottlebrush, is known for its antibacterial, acaricidal, repellent, and antifungal activities, but there are no reports in the literature about its effects on the internal morphology of ticks. This study aimed to extract and characterize the essential oil obtained from the leaves of C. viminalis. Additionally, its effects on the ovary morphology of engorged R. microplus were assessed through histological, histochemical, and morphometric techniques. Exposure to C. viminalis caused dose-dependent morphological changes, such as cellular alterations in the epithelial layer lining the ovary lumen and the pedicel, irregularity of the chorion and oocyte shape, changes in protein and carbohydrate content, decrease in oocyte size, reduction in the size of the nucleus as well as cytoplasmic and nucleolar vacuolation. Thus, C. viminalis essential oil exhibited a toxic effect on the reproductive system of R. microplus, which may result in the reproductive impairment of this tick species.

Envelope-Fusion-Syncytium Formation in Microplitis bicoloratus bracovirus Maturation.

The viral envelope is essential for virus maturation. Virus-mediated syncytium formations are induced by viral envelope proteins that cause membrane fusion of the infected cells. Polydnaviridae (Polydnavirus) are enveloped viruses with multiple nucleocapsids, and virions mature in symbiotic parasitoid wasp ovaries. However, the mechanism governing the envelope packaging of multiple nucleocapsids remains unclear. In this study, we used transmission electron microscopy to examine the process whereby multiple nucleocapsids of Microplitis bicoloratus bracovirus are packaged into an envelope and observed envelope-fusion-syncytium formation in symbiotic wasp calyx cells during virus maturation. The virus maturation process in calyx cells comprised four stages: pre-virogenic stroma, virogenic stroma, assembly, and fusion. Each virus contained a single envelope with one nucleocapsid in the assembly stage; multiple envelopes then fused to form a viral envelope with multiple nucleocapsids (i.e., the envelope-fusion-syncytium) around the envelope fusion core in the fusion stage. The envelope-fusion-syncytium then stabilized the virions that were released into the lumen of the ovary across the calyx epithelial layer. The phagocytic calyx epithelial cells on the border of the calyx and ovary lumen cleared the majority of non-enveloped nucleocapsids. In contrast, non-phagocytic calyx epithelial cells with microvilli and a cuticular line between the ovary wall and the lumen remained intact in the ovary lumen. These results indicate that envelope-fusion-syncytium formation is important for packaging multiple nucleocapsids in bracovirus maturation.

Expression and antimicrobial activity of liver-expressed antimicrobial peptides in the ovaries of the viviparous teleost Xenotoca eiseni

The mechanism via which the mothers of viviparous animals regulate the internal environment of pregnancy-associated organs for maintaining offspring growth is poorly understood. Environmental niches in organs contain fluid components for supporting embryonic growth; however, they may serve as nutrients for microbes. Therefore, microbial control is essential in viviparous animals to reduce the risk of infection in the ovarian lumen. Its importance may be higher than that in the case of oviparous animals. In this study, we investigated the antimicrobial factors in a viviparous teleost, Xenotoca eiseni. Four transcripts of the liver-expressed antimicrobial peptide (LEAP) were identified via RNA-Seq analysis. Some of the genes were expressed in the ovaries or intraovarian embryos of the fish. In particular, high expression of leap1a was detected in the ovaries of both pregnant and non-pregnant fish. Moreover, the ovary extracts from X. eiseni and transformed leap genes exhibited antimicrobial activity against Escherichia coli. Our results suggest that viviparous teleosts utilize antimicrobial peptides to reduce the risk of infection in the ovarian lumen.

Morphological basis for maternal nutrient provision to embryos in the viviparous fish Ataeniobius toweri (Teleostei: Goodeidae).

In viviparous Mexican fishes of the family Goodeidae, embryos develop in the maternal ovarian lumen. They typically absorb maternal nutrients during gestation by means of "trophotaeniae," that is, specialized, elongated extensions of the hindgut that are exposed to the fluids, which occupy the ovarian lumen. The sole exception is Ataeniobius toweri, whose embryos lack trophotaeniae but are nevertheless matrotrophic. Thus, how its embryos obtain maternal nutrients is unclear. We studied a series of non-pregnant and pregnant ovaries of A. toweri using histology to identify the mechanism of maternal-embryo nutrient transfer. By early-gestation, embryos have depleted their yolk supplies. Yolks are released into the ovarian lumen and are ingested by the developing embryos, as shown by yolk material in their digestive tracts. The embryonic gut is lined by an epithelium consisting of columnar cells with apical microvilli, providing a means for nutrient absorption. Contrary to statements in the literature, embryos develop minuscule trophotaenial rudiments that extend slightly into the ovarian lumen. These structures are formed of an absorptive epithelium that overlies a vascular stroma, similar to the trophotaeniae of other goodeids. Through late gestation, vitellogenic follicles form and oocytes are discharged into the ovarian lumen, contributing to embryonic nutrition. Thus, histological evidence suggests that embryos chiefly obtain nutrients from ingestion of yolk and maternal secretions released into the ovarian lumen. This function possibly is supplemented by uptake via the small hindgut protrusions and other absorptive surfaces (e.g., the skin and the gill epithelium). Our observations are consistent with two evolutionary interpretations of the hindgut protrusions: (a) that they are rudimentary, evolutionary precursors of trophotaeniae formed by exteriorized hindgut; and (b) that they are vestigial remnants of trophotaeniae that were lost during a switch to a form of matrotrophy involving nutrient ingestion.

Ontogeny and Dynamics of the Gonadal Development, Embryogenesis, and Gestation in Xenotoca eiseni (Cyprinodontiformes, Goodeidae)

We characterized the ontogeny and dynamics of gonadal development, embryogenesis, and gestation in captive stocks of the viviparous redtail splitfin, Xenotoca eiseni. Using histology, we showed that gonads were fully differentiated at the time of birth with a male:female sex ratio of 1:1 in the captive stock. External secondary sex features included a modified anal fin and a distinctive orange tail coloration. These features first appeared at 4 weeks after birth and were discriminative for males thereafter. There was no sex-related dichotomy in body size, and X. eiseni reached sexual maturity at approximately 12 weeks of age. We found no evidence for sperm storage in females. Gestation normally took 6 weeks, and there was a positive correlation between female body size and the number of offspring produced, with up to 27 offspring for a single pregnancy. Yolk is the main food source for developing embryos for the period up to 2 weeks, and thereafter, trophotaeniae in embryos act as nutrient exchange surfaces in the ovarian lumen, which subsequently undergo complete regression within 2 weeks of birth. In our final analysis, we discuss the great potential of X. eiseni as a model for studying the effects of chemicals on sexual development.

Shift of the Vegetal Pole Area of Full-Grown Oocytes Toward the Ovulatory Site of the Ovary in the Medaka Fish, Oryzias latipes (Beloniformes: Adrianichthyidae).

This study presents novel findings on the dynamics of growing oocytes in the ovary of the medaka fish, Oryzias latipes. In the ovary of mature females, all follicles are anchored tightly to the abdominal ovarian rete via fibrous follicular stalks on the follicle surface. The follicular stalks lie at the end of the follicle opposite the site of its attachment to the ovarian wall. Various lengths of the follicular stalks reflect the spatial arrangement of follicles in the ovary. Herein, a line that connects the follicular stalk to the opposite side of the follicle toward or attaching to the ovarian wall is called the follicle axis. The animal-vegetal axis in late stage III oocytes is already recognizable as a line that connects the center of the oocyte nucleus and the vegetal pole area; this is ascertainable by the morphological landmark of a compact distribution of granulosa cells or rudimentary attachment filaments. In growing oocytes later than stage V, the beginning of vitellogenesis, the tufted attachment filaments are located on a discrete region of the vegetal pole area. Our observations reveal that during growth, oocytes are arranged randomly between the animal-vegetal axis and the follicle axis, whereas the vegetal pole area of full-grown oocytes in preovulatory follicles turns close to the inner surface of the ovarian wall, from which mature oocytes subsequently ovulate into the ovarian lumen. It is suggested that in the O. latipes ovary, mature oocytes always transpose the vegetal pole area to the ovulatory site of the ovarian wall prior to ovulation. The expulsion of mature oocytes from the vegetal pole appears to be the regular mode of ovulation in O. latipes.

Structures Associated with Oogenesis and Embryonic Development during Intraovarian Gestation in Viviparous Teleosts (Poeciliidae)

Viviparity in teleosts involves, invariably, the ovary in a gestational role. This type of viviparity is due to the combination of unique aspects, different from those found in the rest of vertebrates. These aspects are: The ovary has a saccular structure; the germinal epithelium lines the ovarian lumen; the absence of oviducts; and the intraovarian insemination, fertilization, and gestation. The communication of the germinal zone of the ovary to the exterior is via the caudal zone of the ovary—the gonoduct. The germinal epithelium is composed of oogonia and oocytes scattered individually or in cell nests among somatic epithelial cells. In the ovarian stroma the follicles are included which are formed by the oocyte, which is surrounded by follicular cells and the vascularized theca. The oogenesis comprises three stages: chromatin-nucleolus, previtellogenesis, and vitellogenesis. There is no ovulation, as the oocyte is retained in the follicle. During the insemination, the spermatozoa enter into the ovarian lumen and the intrafollicular fertilization occurs, followed by intrafollicular gestation. The intraovarian gestation of poeciliids involves morphological characteristics associated with the intrafollicular embryogenesis and types of nutrition, such as lecithotrophy and matrotrophy. In lecithotrophy, the nutrients come from the yolk reserves stored during oogenesis, whereas in matrotrophy the nutrients are provided by supplies from maternal tissues to the embryo during gestation. The maternal–embryonic metabolic interchanges converge through the development of the association of maternal and embryonic blood vessels, establishing a follicular placenta.

Meiotic Chromosomes, Synaptonemal Complexes in a Female Viviparous Lizard (Zootoca vivipara) in Prophase I of Meiosis

In the females of the viviparous lizard Zootoca vivipara (Lichtenstein, 1823) (family Lacertidae) from Northwest Russia (2n = 35: 32A (acrocentric autosomes) + Z1Z2W sex chromosomes), the ovarian lumen germinal vesicles (oocytes), as well as germinal lamina cells, were examined. Chromosome preparations were obtained using the direct method and the method of total oocyte nuclei spreading developed by Dresser and Moses. Chromosome preparations were stained with Giemsa; for visualization of synaptonemal complexes (SCs), total preparations of oocyte nuclei were stained with silver nitrate and DAPI. It was demonstrated that, during oogenesis in the female, primary follicles enter the early stages of the meiotic prophase I (stages from leptotene to diplotene, lampbrush chromosomes are formed). Here, for the first time, total oocyte spreads were obtained and studied. On the basis of light microscopic analysis of the oocyte SCs and taking into account their length in a female with 2n = 35, the SC karyotype is presented, consisting of 19 SC elements, among which 16 SC autosomal bivalents are distinguished. The remaining three SC elements, according to the authors, can be univalents of Z1Z2W sex chromosomes or one WZ1 bivalent and Z2 and B chromosome univalents. As in the SC karyotype of Z. vivipara males, in a female, specific features in the morphology of SC elements of chromosomes 5 and 6 were observed.

Change of lecithotrophic to matrotrophic nutrition during gestation in the viviparous teleost Xenotoca eiseni (Goodeidae).

Teleosts possess unique features of the female reproductive system compared with the rest of vertebrates, features that define the characteristics of their viviparity. Viviparity involves new maternal-embryonic relationships detailing the most diverse structures during gestation that include embryonic nutrition. In order to analyze the morphological features of the complex nutrition in viviparous teleosts during intraovarian gestation, this study utilizes the goodeid Xenotoca eiseni as a model. Ovarian gestation in X. eiseni, as in all goodeids, is intraluminal; the early embryo moves from the follicle to the ovarian lumen where gestation continues. The scarce yolk in the oocytes implies that the initial lecithotrophy is replaced by matrotrophy, with nutrients provided via maternal tissues. The nutrients are absorbed by the embryo mainly by trophotaenia, extensions of the embryonic intestine into the ovarian lumen. This histological study analyses the structures involved in these two types of nutrition and when they occur during gestation in X. eiseni. The morphology displayed in this study demonstrated the extended simultaneity of lecithotrophy and matrotrophy during gestation with the progressive reduction of lecithotrophy and increase of matrotrophy. Similarly, it describes the development of complex embryonic structures for metabolic exchange with the maternal tissues associated with matrotrophy; specifically the branchial placenta and mainly the trophotaenia.

Persistent expression of Cotesia plutellae bracovirus genes in parasitized host, Plutella xylostella.

Cotesia plutellae (= vestalis) bracovirus (CpBV) is symbiotic to an endoparasitoid wasp, C. plutellae, and plays crucial roles in parasitism against the diamondback moth, Plutella xylostella. CpBV virion genome consists of 35 circular DNAs encoding 157 putative open reading frames (ORFs). This study re-annotated 157 ORFs with update genome database and analyzed their gene expressions at early and late parasitic stages. Re-annotation has established 15 different viral gene families, to which 83 ORFs are assigned with remaining 74 hypothetical genes. Among 157 ORFs, 147 genes were expressed at early or late parasitic stages, among which 141 genes were expressed in both parasitic stages, indicating persistent nature of gene expression. Relative frequencies of different viral circles present in the ovarian lumen did not explain the expression variation of the viral ORFs. Furthermore, expression level of each viral gene was varied during parasitism along with host development. Highly up-regulated CpBV genes at early parasitic stage included BEN (BANP, E5R and NAC1), ELP (EP1-like protein), IkB (inhibitor kB), P494 (protein 494 kDa) family genes, while those at late stage were mostly hypothetical genes. Along with the viral gene expression, 362 host genes exhibited more than two fold changes in expression levels at early parasitic stage compared to nonparasitized host. At late stage, more number (1,858) of host genes was regulated. These results suggest that persistent expression of most CpBV genes may be necessary to regulate host physiological processes during C. plutellae parasitism.

Insemination, intrafollicular fertilization and development of the fertilization plug during gestation in Heterandria formosa (Poeciliidae).

The viviparous teleost Heterandria formosa is a remarkable species for its reproductive characters including: (a) the smallest oocyte in viviparous fish species; (b) a high level of matrotrophy with a complex placenta; and (c) the highest level of superfetation. Superfetation involves (d) the continuous development of oocytes and fertilization at the same time with embryos in gestation. The sequential fertilization of oocytes requires (e) storage of spermatozoa in the ovary. Among these characteristics, fertilization is of fundamental interest, specifically the intrafollicular fertilization of poeciliids, species that do not present micropyle, and the consequent formation of the fertilization plug, a structure developed at the periphery of the follicle where the entrance of spermatozoa occurs. Both processes intrafollicular fertilization and formation of the fertilization plug have been rarely described. There is only one study illustrating, the fertilization plug of H. formosa with a drawing. In the context of reproductive aspects of H. formosa, the goal of this study is to describe the morphology of the ovary during insemination, intrafollicular fertilization and development of the fertilization plug. After insemination, spermatozoa enter the ovary and occupy folds of the lamella near follicles of all stages of oogenesis, the delle, where the germinal epithelium establishes contact with the follicular epithelium. The results of the present study provide evidence that both epithelia open at the distal end of the delle, this morphological change allow that the spermatozoa to make contact with the zona pellucida of the oocyte. After fertilization, the delle becomes blocked by proliferation of cells of the germinal epithelium, to form the fertilization plug that persists throughout gestation. Abundant reticular fibers and blood vessels are seen around the fertilization plug. Persistence of the fertilization plug suggests that it could be the site where the juvenile will gain entrance to the ovarian lumen during birth.

Germ Cell Migration, Proliferation and Differentiation during Gonadal Morphogenesis in All-Female Japanese Flounder (Paralichthys Olivaceus).

In this study, all genetic female (XX) broods of Japanese flounder were produced artificially by mating the females with sex-reversed males. The proliferation and migration of primordial germ cells (PGCs), formation of ovary and oogenesis were described in detail. After hatching, around 20 individual PGCs migrated from the lateral to the dorsal of trunk region. At 15 days posthatching (dph), a part of PGCs were covered by a single layer somatic cells and formed the genital ridge. By 22 dph, the elongated gonadal primordia appeared under the ventral kidney, where the PGCs were totally enclosed by somatic cells. During the process of migration, PGCs were presumed to be mitotically inactive. From 63 to 73 dph, somatic cells rearrangement resulted in the formation of a narrow crevice, which became deeper and formed ovarian lumen. However, at 52 dph, dramatic mitotic proliferation of germ cell occurred and germline nest formed before the appearance of ovarian lumen. The onset of intensive germ cell proliferation and appearance of cell nests could be accepted as a criterion of initial ovarian differentiation. Then germ cells and somatic epithelial cells were gradually delimited by basement membrane and formed the germinal epithelium. In this period, results from in situ hybridization revealed that the early forkhead box L2 (pofoxl2) was expressed in somatic cells and oocytes in primary growth, which indicated the prefollicle cells formed. Then oogonia or oocytes, follicle cells, basement membrane, and theca cells composed a follicle complex. Finally, oocytes underwent meiosis and developed into to mature eggs. Anat Rec, 301:727-741, 2018. © 2017 Wiley Periodicals, Inc.

Ovarian structure and oogenesis of the extremophile viviparous teleost Poecilia mexicana (Poeciliidae) from an active sulfur spring cave in Southern Mexico.

The structure of the ovary and oogenesis of Poecilia mexicana from an active sulfur spring cave is documented. Poecilia mexicana is the only poeciliid adapted to a subterranean environment with high hydrogen sulfide levels and extreme hypoxic conditions. Twenty females were captured throughout one year at Cueva del Azufre, located in the State of Tabasco in Southern Mexico. Ovaries were processed with histological techniques. P. mexicana has a single, ovoid ovary with ovigerous lamella that project to the ovarian lumen. The ovarian wall presents abundant loose connective tissue, numerous melanomacrophage centers and large blood vessels, possibly associated with hypoxic conditions. The germinal epithelium bordering the ovarian lumen contains somatic and germ cells forming cell nests projecting into the stroma. P. mexicana stores sperm in ovarian folds associated with follicles at different developmental phases. Oogenesis in P. mexicana consisted of the following stages: (i) oogonial proliferation, (ii) chromatin nucleolus, (iii) primary growth, subdivided into: (a) one nucleolus, (b) multiple nucleoli, (c) droplet oils-cortical alveoli steps; (iv) secondary growth, subdivided in: (a) early secondary growth, (b) late secondary growth, and (c) full grown. Follicular atresia was present in all stages of follicular development; it was characterized by oocyte degeneration, where follicle cells hypertrophy and differentiate in phagocytes. The ovary and oogenesis are similar to these seen in other poeciliids, but we found frequent atretic follicles, melanomacrophage centers, reduced fecundity and increased of offspring size.

Histological and ultrastructural investigation of the female reproductive system of Argulus bengalensis Ramakrishna, 1951 (Crustacea: Branchiura).

In order to understand branchiuran reproductive biology, it is imperative to know the sites of oogenesis and oocyte maturation, locate the accessory reproductive glands, and identify the fertilization site with the present knowledge of the sperm transfer mechanism of the genus Argulus. With these objectives, we attempted to describe the female reproductive system of Argulus bengalensis using serial histological sections through the ovaries and associated ducts in the transverse, longitudinal, and sagittal planes. The reproductive organs include a median ovary, one pair of ovarian lumina, a median oviduct, and a pair of collateral accessory glands. A duct from each of the collateral accessory glands leads into the proximal part of the median oviduct, which opens to the exterior through a genital opening at the distal end. The glandular secretion presumably contributes to the jelly coat of the egg. The ovary is bound with a tunica propria which extends further diametrically inside the ovary forming the paired lumina. The lumina are confluent into the median oviduct. Two distinct areas, the germarium and differentiating zones, are clearly distinguishable within the ovary. The tunica propria itself houses the oogonia within a matrix, serving as the germarium. Transmission electron micrograph reveals that the matrix is made of collagen. The collagen matrix confers elasticity to the tunica propria to accommodate the postvitellogenic oocytes within the ovarian lumen. The differentiating zone is situated in between the germarium: dorsally it is covered with a chromatophore layer. The ovary is ensheathed by a circum ovarian striated muscle. The presence of spermatophores in the ovarian lumen indicates the fertilization site. J. Morphol. 277:707-716, 2016. © 2016 Wiley Periodicals, Inc.

Primary male development of two sequentially hermaphroditic groupers, Epinephelus akaara and Epinephelus awoara (Perciformes: Epinephelidae).

Gonad ontogeny of the Hong Kong grouper Epinephelus akaara (a bi-directional sex changer) and the yellow grouper Epinephelus awoara (a protogynous hermaphrodite) was examined for the first time from post-larval phase until first sexual maturation, by histology. Approximately 20 specimens of each species were collected randomly every 2-7 weeks from rearing tanks with natural sea water and temperature between June 2013 and June 2014. The paired gonadal primordia (GP) were observed at 6 weeks after hatching (wah) for both species; however, gonia were first observed in GP at 16 wah for E. akaara and at 8 wah for E. awoara. The timings for the appearance of primary-growth stage oocytes (O1) and the completion of ovarian lumen (OL) varied; both at 27 wah for E. akaara, and at 18 and 23 wah for E. awoara respectively. A bisexual-phase gonad with an OL, O1 and scattered spermatogenic cysts (SC) was observed at 27-29 wah for both E. akaara and E. awoara. Sexual differentiation was subsequently observed from the bisexual-phase gonad at 34 wah for E. akaara, and 41 wah for E. awoara, with the appearance of cortical-alveolus stage oocytes (O2) for developing female and the proliferation of SC for developing primary male (i.e. from juvenile directly). Ovaries of mature females contained the vitellogenic stage oocytes (O3) and scattered SC; testes of mature primary males had sperm in sperm sinuses within the gonadal wall and remained O1. Minimum age of first sexual maturation for both female and primary male of E. akaara was at 41 wah; minimum total length (LT ) of female (143 mm) was larger than that of primary male (137 mm L(T)). Minimum age and size of first sexual maturation for female of E. awoara (47 wah and 149 mm L(T), respectively) were larger than those of E. akaara. Developing primary males of E. awoara were found at 41-58 wah, however, mature males were not observed, indicating inconsistency in first sexual maturation for E. awoara. This study provided strong evidences of primary male pathway in E. akaara and E. awoara; the latter is confirmed to be diandric.

Wnt4 in protogynous hermaphroditic orange-spotted grouper (Epinephelus coioides): identification and expression.

Wnt4 (Wingless-type MMTV integration site family member 4) has been demonstrated to play critical roles in ovarian development in mammals, but its function in fish reproduction is still unclear. In the present study, two full-length wnt4 cDNA sequences (named wnt4a and wnt4b) were cloned from the orange-spotted grouper (Epinephelus coioides). Amino acid alignment analysis showed that both orange-spotted grouper Wnt4s proteins had the typical characteristics of the Wnt family. RT-PCR revealed that both wnt4a and wnt4b were highly expressed in the ovaries of the orange-spotted grouper. Temporal expression profiles of both wnt4 genes during embryonic and ovarian development were examined. The expressions of wnt4a and wnt4b genes were first detected at the embryonic morula stage, but the gens showed different expression patterns. During ovarian development, high expression of wnt4a was observed in the ovarian lumen formation and gonium proliferation stage, while wnt4b exhibited strong expression in the early developmental stage of oocytes. Taken together, the present study indicates that the two wnt4 genes are involved in the regulation of ovarian development in the orange-spotted grouper.

Reproductive characteristics of the humbug damselfish, Dascyllus aruanus, in Chuuk Lagoon, Micronesia

This study was conducted to investigate the reproductive characteristics of the humbug damselfish, Dascyllus aruanus, through the measurement of gonadosomatic indices (GSI) and histological examination of the gonads. Fish were collected from Chuuk Lagoon, Micronesia (7°27′N; 151°53′E), between August 2009 and July 2010. Overall, the functional sex ratio was approximately 1:1; however, there was a female bias in the smaller size range (35–40 mm standard length [SL]) and male bias in the larger size range (45–60 mm in SL). The process of oocyte development exhibited a group synchronous pattern, from the vitellogenic phase oocytes in the gonads following the two clutches of oocytes, as the primary growth stage and yolk vesicle stage. The testis with an ovarian lumen exists as a central slit, and the sperm ducts extend into the medial hilar region of the gonads, indicating that males of D. aruanus have a secondary testis of protogynous species. Monthly variations in the GSI and evolution of gonad status indicated that reproductive activity in this species occurs throughout the year in Chuuk Lagoon, Micronesia.

Development of the germinal epithelium and early folliculogenesis during ovarian morphogenesis in the cichlid fishCichlasoma dimerus(Teleostei, Perciformes)

Formation of the germinal epithelium and folliculogenesis during ovarian development in Cichlasoma dimerus were described at the light- and electron-microscopic levels. Prior to gonadal differentiation, germ cells and enveloping support cells reside within an inpocketing of the coelomic epithelium. Separation of the germinal and interstitial compartments of the gonad by a basement membrane is apparent from early gonadal development. Upon ovarian differentiation, oogonia undergo cyst-forming divisions leading to the formation of clusters of interconnected cystocytes that synchronously enter meiosis, becoming oocytes. At the pachytene step, each oocyte becomes individualized by cytoplasmic extensions of prefollicle cells, thereby developing as an ovarian follicle. Subsequent somatic reorganization leads to the formation of the ovarian lumen in a cephalo-caudal gradient. As a result, the germinal epithelium becomes internalized and lines the ovarian lumen. As defined by its origin from the germinal epithelium, the ovarian follicle is composed of an oocyte and the surrounding follicle cells. Thecal cells derived from the stroma encompass the basement membrane outside the follicle, thus forming a follicle complex. A common basement membrane is shared by the germinal epithelium and the follicle complex along a small portion of its surface. This point of attachment represents the site at which the oocyte would be released to the ovarian lumen during ovulation.