Gonadal development and non-functional protogyny in a coral-reef damselfish, Dascyllus albisella Gill

All gonads of the Hawaiian dascyllus Dascyllus albisella , irrespective of the final sex of individuals, developed an ovarian lumen and primary-growth-stage oocytes after an initially undifferentiated state. From this ovarian state or from more differentiated ovaries, some gonads redifferentiated into testes. None of 117 individuals examined had a gonad containing degenerating vitellogenic oocytes and proliferating spermatogenic tissue. Eleven individuals had gonads containing degenerating cortical-alveolus-stage oocytes and developing spermatogenic tissue. The size of these individuals overlapped with the female size range in which the majority of the females were still in the middle of the maturation process. They were absent from the larger size range where the majority of females had vitellogenic oocytes. This indicated that the transition toward maleness is likely to have occurred after the onset of cortical-alveolus stage, but before final oocyte maturation and spawning as females. Therefore the protogynous pattern of gonadal development was non-functional. There was no dimorphism in the sperm duct configuration, and all the testes were secondary testes reported for diandric, protogynous species with undelimited gonads. Very early development of an ovarian lumen appeared to have resulted in a secondary-male configuration in all testes, although redifferentiation into males appeared to have occurred before sexual maturity and spawning as females.     

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 (L_T) 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.     

Gonadal development and an indication of functional protogyny in the Indian damselfish (Dascyllus carneus)

The objective of this study was to determine the sexual pattern of the Indian dascyllus Dascyllus carneus . After an initially undifferentiated state, gonads of D. carneus developed an ovarian lumen and primary growth stage oocytes, and subsequently cortical-alveolus stage oocytes. From ovaries with cortical-alveolus stage oocytes and from more developed ovaries, some gonads redifferentiated into testes. From a sample of 163 individuals, two had a gonad containing degenerating vitellogenic oocytes and proliferating spermatogenic tissue, nine had a gonad containing degenerating cortical-alveolus stage oocytes and spermatogenic tissue, and five had a gonad with degenerating primary growth stage oocytes and spermatogenic tissue. The size of these individuals overlapped greatly with the size range of mature females, suggesting that at least in some individuals, redifferentiation toward a testis occurred after spawning as females. This indicates that D. carneus is a functional, diandric protogynous hermaphrodite. Removal of a dominant male(s) did not induce a sex change in any of the ranking females in the laboratory and field groups. There was no difference in the number of chases and signal jumps performed by the ranking female between control and experimental field groups, or before and after removal of the male. However, the sizes of the ranking females were at or beyond the size range of individuals with a mixed-stage gonad, suggesting that the developmental window for female-to-male sex change may not be open ended. In 41 of 43 field groups, in which sex of fish was determined histologically or by the shape of the urogenital papilla, one to several highest size ranks were occupied by males, followed by one to several females. Mature males, however, were not limited to the highest ranks and occurred at various lower size ranks within groups. Individuals with a mixed-stage gonad also occupied various size ranks within groups.     

Gonadal development and diandric protogyny in two populations of Dascyllus reticulatus from Madang, Papua New Guinea

Two Dascyllus reticulatus populations from Madang, Papua New Guinea exhibited diandric protogyny. In both populations, gonads began as undifferentiated, and then developed oocytes in the primary growth stage and an ovarian lumen. From this ovarian state or from more developed ovaries containing oocytes beyond the primary‐growth stage, some gonads developed into testes. The first sign of testicular development was degeneration of oocytes, degeneration of oocytes in the primary growth stage in ovarian gonads and degeneration of oocytes of all growth stages present including the primary growth stage in ovaries, which was then followed by development of spermatogenic tissue. In both populations, most of the fish that had gonads with degenerating oocytes were smaller than the smallest mature females, indicating that development towards testes was mostly initiated in immature gonads containing only pre‐vitellogenic oocytes. On some occasions, however, females as large as other mature females also had gonads with degenerating oocytes, suggesting that development towards testes may have occurred in mature ovaries as well. This latter notion is further strengthened by the discovery of a fish having a gonad that contained both degenerating vitellogenic oocytes and developing spermatogenic tissue. Taken together, these results suggest that D. reticulatus can exhibit diandric protogyny, because testes in D. reticulatus developed from juvenile gonads as well as from mature ovaries.     

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

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

Patterns of structural change in gonads of the divine dwarfgoby Eviota epiphanes as they sexually transition

This study documents changes in gonadal structure for the serial hermaphrodite (or bidirectional sex changer) divine dwarfgoby Eviota epiphanes (family Gobiidae) as individuals transition in both directions. To evaluate transitional gonad morphology, individuals actively producing the same gamete type (oocytes or sperm) were set up into pairs and euthanised over a period of 14 days to get a time series of morphological changes during gonad transformation. Results from this study show that rapid changes in the gonad take place at a structural level as individuals change their reproductive function and gamete production. Changing from oocyte production (o-phase) to sperm production (s-phase) starts with the breakdown of vitellogenic oocytes (i.e., atresia) followed by the appearance and proliferation of spermatogenic tissue which, in most cases, was not previously visible. Changing from sperm production to oocyte production included the cessation of sperm production, a reduction in size and number of seminiferous lobules and the maturation of previtellogenic oocytes already present in the gonads. Experimental fish changed from oocyte production to sperm production more readily than from sperm production to oocyte production. The hypothesis that shifts in sexual function among serially hermaphroditic fish species have a similar cost in either direction is not supported in E. epiphanes.     

Histological and ultrastructural evidence for the role of gonadal steroid hormones in sex change in the protogynous wrasse Thalassoma duperrey

Synopsis The process of sex change in the protogynous wrasse, Thalassoma duperrey, was investigated through histological and ultrastructural observations on the gonads of females changing sex to male. Changes in plasma steroid levels concomitant with structural changes were measured by radioimmunoassay. The process of sex change from ovary to testis was divided into six stages on the basis of changes in the structure of the germinal and somatic elements. Ovaries of females were filled with vitellogenic oocytes during the breeding season, but contained no spermatogenic tissue (Stage 1). At the commencement of sex change (Stage 2), vitellogenic oocytes began to degenerate, and were ingested by macrophagous cells. This stage was accompanied by a rapid drop in plasma levels of estradiol-17. Thereafter, previtellogenic oocytes (Stage 3) also began to degenerate, and aggregations of stromal tissue, and loose connective tissue were observed in the central region of the lamellae. Steroid producing cells (Leydig cells), developed at the border of this loose connective tissue. Presumed spermatogonia proliferated on the periphery of the lamellae, and Leydig cells increased in size and number (Stage 4). Spermatogonia formed cysts, and underwent spermatogenesis (Stage 5). Finally, sex change to male was considered complete, with the beginning of active spermatogenesis and spermiation (Stage 6). Plasma levels of testosterone remained low throughout the sex change, but a second androgen, 11-ketotestosterone increased gradually in parallel to the increased numbers of Leydig cells and spermatogonia. Preliminary in vitro incubation of gonads with salmon gonadotropin, revealed that sex-changed males had higher levels of 11-ketotestosterone production than did females, while females had higher levels of estradiol-17 production than did males. Production of both these steroids increased in a dose-related fashion with increasing doses of gonadotropin.     

Developmental temperature affects the expression of ejaculatory traits and the outcome of sperm competition in Callosobruchus maculatus

The outcome of post‐copulatory sexual selection is determined by a complex set of interactions between the primary reproductive traits of two or more males and their interactions with the reproductive traits of the female. Recently, a number of studies have shown the primary reproductive traits of both males and females express phenotypic plasticity in response to the thermal environment experienced during ontogeny. However, how plasticity in these traits affects the dynamics of sperm competition remains largely unknown. Here, we demonstrate plasticity in testes size, sperm size and sperm number in response to developmental temperature in the bruchid beetle Callosobruchus maculatus. Males reared at the highest temperature eclosed at the smallest body size and had the smallest absolute and relative testes size. Males reared at both the high‐ and low‐temperature extremes produced both fewer and smaller sperm than males reared at intermediate temperatures. In the absence of sperm competition, developmental temperature had no effect on male fertility. However, under conditions of sperm competition, males reared at either temperature extreme were less competitive in terms of sperm offence (P₂), whereas those reared at the lowest temperature were less competitive in terms of sperm defence (P₁). This suggests the developmental pathways that regulate the phenotypic expression of these ejaculatory traits are subject to both natural and sexual selection: natural selection in the pre‐ejaculatory environment and sexual selection in the post‐ejaculatory environment. In nature, thermal heterogeneity during development is commonplace. Therefore, we suggest the interplay between ecology and development represents an important, yet hitherto underestimated component of male fitness via post‐copulatory sexual selection.     

Habitat Association and Social Structure of the Chocolate Hind, <Emphasis Type="BoldItalic">Cephalopholis boenak</Emphasis> (Pisces: Serranidae: Epinephelinae), at Ping Chau Island,Northeastern Hong Kong Waters

Synopsis The chocolate hind, Cephalopholis boenak, is the smallest and last remaining grouper of any abundance in Hong Kong waters. We investigated its habitat association and social structure to understand its sexual pattern and seek possible approaches to protect this species. Juveniles and adults are distributed in the same habitat and strongly associated with corals, in particular, Pavona decussata when available, for settlement and residence. Social groups of C. boenak are relatively small with a single male, one or two smaller females, and varying numbers of sexually inactive individuals (which are mature but sexually inactive, or juvenile). Males defend their boundaries by chasing neighbouring males and have significantly larger home ranges than females. Males visit females and sexually inactive individuals at one or more specific locations, consistently following similar paths. There was a positive linear correlation between body size and home range in individuals ≥90 mm TL. We observed male courtship in the reproductive season, within his home range during the late afternoon. Spawning behaviour is not associated with any particular moon phase and may occur more than once within the reproductive season. Although C. boenak is a diandric, protogynous hermaphrodite, primary and secondary males could not be distinguished by behavioural patterns in the field. Population management of this species should include habitat protection.     

Seasonal changes in sex ratio and size‐related sex reversal of the protogynous hermaphroditic bluespotted seabass Cephalopholis taeniops (Valenciennes, 1828)

Seasonal changes in sex ratio and size‐related sex reversal of the protogynous hermaphroditic Cephalopholis taeniops were studied from histological and population data of 218 individuals captured by hook and line, July 2009–November 2012, in Cape Verde archipelago. This study showed that C. taeniops have a diandric protogynous hermaphrodite sexual model, with young individuals undergoing bisexual development and hermaphrodites above 28 cm. All gonads had a bisexual immature stage with primary and secondary males. Primary males possibly originate from immature bisexual individuals, whereas secondary males likely result from females that have already reproduced and changed sex.     

中国大鲵幼体的性别分子鉴定与性腺形态

为探明中国大鲵(Andrias davidianus)雌雄幼体的性腺发育特征,确定适合的性别分子鉴定方法,对15尾5月龄和17尾17月龄养殖个体进行形态测量、解剖观察、性腺组织切片及PCR扩增雌性特异DNA片段。结果发现,引物adf225和adf340的扩增效果好,判定5月龄个体8雌7雄;17月龄个体8雌9雄,与依据性腺形态结构区分的结果一致。体视显微镜下5月龄幼体中肾腹侧有两条半透明细条状的原始生殖嵴;组织切片显示生殖细胞形态分化不明显。17月龄卵巢波浪状弯曲,有颗粒感,精巢呈光滑的白条状,形态分化明显;组织切片显示,卵巢分化出体积较大的卵母细胞,同时保留原始卵泡,精巢分化出生精小叶和精原细胞、支持细胞。外形测量显示,5月龄与17月龄性二型不明显,不能根据外形判断性别。本研究确定了大鲵幼体性别分子鉴定的最佳引物,可用于养殖过程中雌雄选配,以节约资源。     

Anatomical changes to the gonad during protogynous sex change in the half-moon grouper Epinephelus rivulatus (Valenciennes)

Anatomical changes to the gonad during sex change in the protogynous grouper Epinephelus rivulatus are described from histological observations. A decrease in ovarian mass occurred soon after the onset of sex change as oocytes atrophied and were removed from the gonad. Blood supply and abundance of unidentified somatic cells increased at this time as proliferation of sperm tissue commenced. As the gonad was cleared of ovarian tissue, the rate of spermatogenesis increased and the lamellae soon became dominated by sperm and connective tissue. Putative Leydig cells, the probable sites of male steroid production, appeared in transitional gonads and were most abundant in the testes of immature males. Peripheral sperm sinuses subsequently formed within basal tissue layers of the tunica and expanded as they filled with spermatids. The process of sex change, occurring as a result of experimental manipulation of wild populations at the start of the spawning season, took c. 3 weeks. This appears rapid compared to other hermaphroditic species and may reduce the impacts of fishing on reproductive output by E. rivulatus populations.     

Morphological evidence for limited sperm production in the enigmatic Tasmanian cave spider Hickmania troglodytes (Austrochilidae,Araneae)

The male reproductive systems of spiders are highly diverse in structure across all major spider taxa. Little is known about this organ system in basal araneomorph spiders, especially Austrochiloidea; such knowledge is necessary for a more complete understanding of the evolutionary morphology of the male reproductive system in spiders. In the present study, we describe the male reproductive system of an austrochilid spider, the enigmatic troglophilic Tasmanian cave spider Hickmania troglodytes, using light and electron microscopic techniques. The male reproductive system consists of tubular testes leading into convoluted deferent ducts, which are fused close to the genital opening to an unpaired ejaculatory duct. Spermatogenesis occurs only in the subadult testes, whereas adult testes showed neither spermatogenic stages nor any generative tissue in all investigated specimens. The testes of adult males are drastically reduced in size compared with those of subadult males, but the deferent ducts are filled with large numbers of mature spermatozoa. Thus, our data suggest that males of H. troglodytes are sperm‐limited, but not necessarily sperm‐depleted as described for certain orb‐weaving spiders. Due to the absence of generative tissue, limited sperm production is permanent (PSL) and probably has an influence on the reproductive strategies in this species. As nearly no data are available on the life history of H. troglodytes, and in particular information on the phenology of males is lacking, implications of the evolution of PSL in this species are unclear. Nonetheless, our data on other representatives of Austrochilidae (Austrochilus forsteri, Thaida chepu) and Gradungulidae (Progradungula otwayensis) suggest that PSL evolved within Austrochiliodea only in H. troglodytes and might be an adaptation to its troglophilic lifestyle.     

Evolutionary change in testes tissue composition among experimental populations of house mice

Theory assumes that postcopulatory sexual selection favors increased investment in testes size because greater numbers of sperm within the ejaculate increase the chance of success in sperm competition, and larger testes are able to produce more sperm. However, changes in the organization of the testes tissue may also affect sperm production rates. Indeed, recent comparative analyses suggest that sperm competition selects for greater proportions of sperm‐producing tissue within the testes. Here, we explicitly test this hypothesis using the powerful technique of experimental evolution. We allowed house mice (Mus domesticus) to evolve via monogamy or polygamy in six replicate populations across 24 generations. We then used histology and image analysis to quantify the proportion of sperm‐producing tissue (seminiferous tubules) within the testes of males. Our results show that males that had evolved with sperm competition had testes with a higher proportion of seminiferous tubules compared with males that had evolved under monogamy. Previously, it had been shown that males from the polygamous populations produced greater numbers of sperm in the absence of changes in testes size. We thus provide evidence that sperm competition selects for an increase in the density of sperm‐producing tissue, and consequently increased testicular efficiency.     

Development of the deep‐sea viviparous quill worm Leptoecia vivipara (Hyalinoeciinae,Onuphidae, Annelida)

Development of Leptoecia vivipara, a brooding deep‐sea onuphid polychaete with a circum‐Antarctic distribution, was studied using light microscopy, scanning and transmission electron microscopy, and histology. All specimens examined were brooding viviparous females or juveniles; no male gametes were detected. Anterior segments of juvenile and adult worms bore paired compact ovaries with clusters of vitellogenic oocytes. In adults, the mid‐body region formed a chamber containing up to 12 offspring at different stages of development, from oocyte to 13 chaetigers. Mature oocytes freely floated in the coelomic fluid, while embryos and juveniles were enclosed in peritoneal envelopes. Chaetal replacement in juveniles and the morphology of the provisional maxillae are described. Leptoecia vivipara is argued to be a progenetic species with juvenile‐like external morphology and accelerated sexual maturation. These traits may have arisen as adaptations to epibenthic life in a high‐latitude deep‐sea environment affected by seasonal pulses of organic matter.     

The reproductive biology of Plagioscion squamosissimus (Heckel, 1840) in the Pará River estuary (Amazon Estuary)

This study describes the reproductive biology of the South American silver croaker Plagioscion squamosissimus (Heckel, 1840) in the Pará River estuary. Data were collected bimonthly from June 2007 to May 2008. In all, 334 specimens were examined. The gonads were processed with histological techniques, and the ovaries were subjected to morphometric analysis. The length at first maturity (L₅₀) values were 16.14 and 21.43 cm total length for females and males, respectively. The sex ratio based on the total number of individuals favoured the females (1.11 females: one male). Macroscopically, the gonads were classified as immature (A), maturing (B), mature (C) and spent (D). Microscopically, four germ cell types were distinguished based on the development of the ovaries. Chromatin nucleolus (I), perinucleolar (II), cortical alveoli (III) and full‐vitellogenic (IV) oocytes were observed in all ovarian stages, except that of the mature individuals which showed the largest number of full‐vitellogenic oocytes (IV), with a mean diameter of 565.4 μm (±147.4). Substantial percentages of individual P. squamosissimus were found to be in a reproductive condition. Reproductive activity was recorded throughout the year in the Pará River estuary, primarily in February/March and August/September.     

Histological and ultrastructural investigation of early gonad development and sex differentiation in Adriatic sturgeon (Acipenser naccarii,Acipenseriformes, Chondrostei)

Gonad development and sex differentiation from embryos to 594‐day‐old individuals were investigated in farmed Acipenser naccarii using light and transmission electron microscopy. The migrating primordial germ cells first appear along the dorsal wall of the body cavity in embryos 1.5 days before hatching. The gonadal ridge, containing a few primary primordial germ cells (PGC‐1) surrounded by enveloping cells, appears in 16‐day‐old larvae. At 60 days, the undifferentiated gonad is lamellar and PGC‐1 multiply, producing PGC‐2. In 105‐day‐old juveniles, a distinct germinal area with advanced PGC‐2 appears on the lateral side near the mesogonium and the first blood vessels are visible. At 180 days, putative ovaries with a notched gonadal epithelium and putative testes with a smooth one appear, together with adipose tissue on the distal side. In 210‐day‐old juveniles, active proliferation of germ cells begins in the putative ovaries, whereas putative testes still contain only a few germ cells. The onset of meiosis and reorganization of stromal tissue occurs in ovaries of 292‐day‐old individuals. Ovaries with developed lamellae enclosing early oocyte clusters and follicles with perinucleolar oocytes occur at 594 days. Meiotic stages are never found, even in anastomozing tubular testes of 594‐day‐old individuals. Steroid producing cells are detected in the undifferentiated gonad and in the differentiated ones of both sexes. Anatomical differentiation of the gonad precedes cytological differentiation and female differentiation largely precedes that of the male. Gonad development and differentiation are also associated with structural changes of connective tissue, viz. collagen‐rich areas are massive in developing testes and reduced in ovaries. J. Morphol., 2008. © 2008 Wiley‐Liss, Inc.     

Protogynous hermaphroditism in the parrotfish,Calotomus japonicus

Sexual patterns of the parrotfish,Calotomus japonicus, were studied using fifteen specimens collected in February and April, 1990, near Makurazaki, Kagoshima Prefecture. The males ranged from 265 to 345 mm SL, and were larger than the females on average. Most females ranged from 202 to 260 mm SL, but a female of 305 mm SL was obtained. Sexual dichromatism was conspicuous. Histological examination of the gonads and low values of gonado-somatic indices of both males and females indicated that they were not in spawning season. It was concluded that C.japonicus is a protogynous hermaphrodite, because all male specimens had secondary testes. It was pointed out that the present species has a weak size-sex relationship. Because the specimens examined did not include primary males, it is suggested that primary males are relatively rare, if they occur at all.     

A null mutation for tissue inhibitor of metalloproteinases-3 (Timp-3) impairs murine bronchiole branching morphogenesis

Juvenile zebrafish are hermaphroditic; undifferentiated gonads first develop into ovary-like tissues, which then either become ovaries and produce oocytes (female) or degenerate and develop into testes (male). In order to fully capture the dynamic processes of germ cells' proliferation and juvenile hermaphroditism in zebrafish, we established transgenic lines TG(beta-actin:EGFP), harboring an enhanced green fluorescent protein (EGFP) gene driven by a medaka beta-actin promoter. In TG(beta-actin:EGFP), proliferating germ cells and female gonads strongly expressed EGFP, but fluorescence was only dimly detected in male gonads. Based on the fluorescent (+) or nonfluorescent (-) appearance of germ cells seen in living animals, three distinct groups were evident among TG(beta-actin:EGFP). Transgenics in ++ group (44%) were females, had fluorescent germ cells as juveniles, and female gonads continuously fluoresced throughout sexual maturation. Transgenics in +- (23%) and -- (33%) groups were males. Fluorescent germ cells were transiently detected in +- transgenics from 14 to 34 days postfertilization (dpf), but were not detected in -- transgenics throughout their life span. Histological analyses showed that 26-dpf-old transgenics in ++, +-, and -- groups all developed ovary-like tissues: Germ cells in -- group juveniles arrested at the gonocyte stage and accumulated low quantities of EGFP, while those in ++ group juveniles highly proliferated into diplotene to perinucleolar stages and accumulated high quantities of EGFP. In +- group juveniles, degenerating oocytes, gonocytes, and spermatogonia were coexistent in transiently fluorescent gonads. Therefore, the fluorescent appearance of gonads in this study was synchronous with the differentiation of ovary-like tissues. Thus, TG(beta-actin:EGFP) can be used to visualize germ cells' proliferation and juvenile hermaphroditism in living zebrafish for the first time.