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.     

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.     

Early gonadal development and primary males in the protogynous epinepheline, Cephalopholis boenak

Early gonadal development of the protogynous epinepheline, Cephalopholis boenak, was examined histologically in 289 specimens with standard length (L_S) of 42–130 mm, collected from May 2000 to April 2002 in Hong Kong waters, to determine male developmental pathways and establish its sexual pattern. All juvenile gonads developed an ovarian lumen with primary‐growth stage oocytes and scattered spermatogenic tissue prior to sexual differentiation and first sexual maturation. From this bisexual phase containing both female and male tissues, some gonads differentiated as ovaries with further oocyte growth to cortical‐alveolus and vitellogenic stages, the rest differentiated as testes with the proliferation of spermatogenic tissue and the formation of a sperm sinus. All testes retained the lumen and primary‐growth stage oocytes, and sperm sinuses ran within the gonad wall. Unlike most protogynous species, among functional males it was impossible to distinguish those resulting from juveniles through sexual differentiation (i.e. primary male) from those resulting from functional females through sex change (i.e. secondary male) based solely on testicular morphology. A proportion‐spermatogenic‐tissue index (I_ST) was, therefore, developed and determined to be a reliable quantitative indicator for distinguishing differentiating, primary males before a sperm sinus was evident, from differentiating females during sexual differentiation. Since sexually transitional specimens with the concominant appearance of degenerating vitellogenic, or later, stage oocytes and spermatogenic tissue in the gonads were previously noted from Hong Kong, diandric, protogynous hermaphroditism is confirmed in C. boenak. For species, such as this and other epinephelines, in which all males have the same testicular morphology, a complete analysis of a wide range of body sizes from juveniles to adults is necessary for understanding male developmental pathways, and determining sexual pattern.     

Cytomorphology of female and male gonads in Antarctic toothfish <Emphasis Type="Italic">Dissosctichus mawsoni</Emphasis> (Nototheniidae) from the Ross Sea in the summer period

Results of histological analysis of gonads of female and male Antarctic toothfish Dissostichus mawsoni caught in the Ross Sea of the Pacific sector in the period of Antarctic summer (December–February) 2004–2005 are presented. Morphological indices and index of gonad maturity are described and ecological criteria of assessment of maturity stages of ovaries, cytological indices of oocytes, and the type of toothfish oogenesis are determined. It was established that in the period of fishing Antarctic toothfish, females and males with gonads at maturity stage III dominate. Ovaries contain two groups of oocytes of the period of trophoplasmatic growth and large oocytes of the nearest spawning season that comprise the smallest proportion of total sex cells. In the testes of two male toothfish, the primary fusion of renal and generative tissues was revealed. It is suggested that the termination of gonad maturity of toothfish takes place from March to April, and spawning takes place from June to August.     

Historical aspects of protandrous sex change in the anemonefish Amphiprion melanopus (Pomacentridae, Teleostei)

Gonadal structure and cellular composition were examined in juveniles, males and females of the protandric hermaphrodite, Amphiprion melanopus. Functional sex change was experimentally induced in the field and gonad structure was histologically examined both qualitatively and quantitatively at 10, 20, 30 and 45 days after its initiation. Juvenile gonads consist primarily of immature ovarian tissue. Functional male gonads are ovotestes with co-existing mature spermatogenic tissue and immature ovarian tissue, while females possess only ovarian tissue. The initiation of sex change is marked by a rapid maturation of spermatogenic tissue and proliferation of putative oogonia. Gonads were essentially female by 20 days into sex change, but evidence of mature female function (marked by the initiation of vitellogenesis) was not observed until 45 days. Considerable variation between individuals was seen in quantitative measures of gonadal change in the early stages of sex change, but not in later stages. Progress in sex change as indicated by histological indicators was, however, consistent within stages. Duet systems for gamete transport changed from the male to the female form after all male tissue had been replaced.     

Gonad development and evidence of protogyny in the red-throat emperor on the Great Barrier Reef

The gonad development in the red-throat emperor Lethrinus miniatus is described and the first detailed evidence for protogyny in this species provided. The identification of transitional individuals, bimodal sex-specific size-frequency distributions and female biased sex ratios suggest that L. miniatus is most likely a protogynous hermaphrodite. Transitional L. miniatus gonads were characterized by the concurrent degeneration of all oocytes and the proliferation of spermatocysts near the edge of the lamellae, an increase in blood vessels along strands of stromal tissue within the lamellae and the formation of multiple sperm sinuses. The sites of oocyte degeneration and proliferation of spermatocysts were spatially segregated. An increase in blood vessels along strands of stromal tissue within the lamellae of transitional phase gonads is likely to assist in the breakdown of oocytes and the proliferation of spermatocysts. Most mature resting females containing spermatocysts occurred within the transitional size-frequency distribution, suggesting that the presence of spermatocysts in these females may be an early sign of sex change. Oocytes within female gonads were interrupted by filamentous strands of stromal tissue within the lamellae. The testis contained a remanent ovarian lumen but no residual oocytes. Three characteristics of transitional L. miniatus gonads were found to be unusual and described for few other species of coral reef fishes. These included the absence of oocytes within testes, increased numbers of blood vessels, and the presence of strands of stromal tissue within the lamellae.     

Morphological development of the gonads in zebrafish

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

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.     

Differentiation of ambisexual gonads and immunohistochemical localization of P450 cholesterol side-chain cleavage enzyme during gonadal sex differentiation in the protandrous anemonefish, Amphiprion clarkii

To clarify the relationship between steroid hormones and sex differentiation of the protandrous anemonefish Amphiprion clarkii, we histologically examined its gonadal differentiation. From hatching to 30 days post hatching (dph), all of the gonads surveyed were sexually undifferentiated. The gonads of all fish first differentiated into ovaries at 60 dph, and the oocytes gradually developed and increased in number as the ovaries grew up until 183 dph. Some cysts of differentiated spermatogenic germ cells appeared in the ovaries at 214 dph, and ambisexual gonads with both ovarian and testicular tissues formed by 273 dph. Using immunohistochemistry, we then investigated the expression of cytochrome P450 cholesterol side-chain cleavage enzyme (P450scc), during gonadal sex differentiation. P450scc-immunopositive reactions first appeared in sexually undifferentiated gonads at 30 dph. Beginning at 60 dph, the number of strongly positive cells increased throughout the differentiation of the ovaries and continued to increase during the testicular differentiation until 210 dph. Immunopositive cells were observed more frequently in ovarian tissue than in testicular tissue in the ambisexual gonads at 270 dph. These results suggest that endogenous steroid hormones are important for the sex differentiation, including the primary sex differentiation and subsequent testicular differentiation, of the anemonefish.     

The gonads of Aegla platensis Schmitt (Decapoda, Anomura, Aeglidae): a macroscopic and histological perspective

The aim of this study is to characterize the gonads of Aegla platensis, relating aspects of colour and size of ovaries, testes and vasa deferentia (VD) to histological observations. The ovaries are H‐shaped, extending from behind the stomach and ending at the 1st or 2nd abdominal somite. The male has a pair of testes from which the VD issue and extend over the pereon. For males and females, macroscopic categories of gonad development are defined. Stages of the male gonad are defined as types 1, 2 and 3. Type 1 includes the less developed gonads, and type 3 includes extremely developed testes and completely differentiated VD. The ovaries are classified into four stages according to their colour: I (white), II (yellow), III (orange) and IV (red), beginning with the least developed gonads. The number and size of oogonia and oocytes depend on the stage of the ovary and coincide with the different degrees of development as indicated by ovary colour. In males, the presence or absence of spermatozoa in the testes and VD is determined only for the more developed gonads. Neither spermathecae (female) nor spermatophores (male) were found. The lack of spermatophore in the present species is a rare characteristic among the anomurans.     

Juvenile bisexuality in the red sea bream,Pagrus major

The histology of the gonad of the red sea bream,Pagrus major, was examined in order to study the early gonadal development, sexual maturation and sex ratio in a natural population. A total of 1,117 fish between the ages of 4 months and 8 years were examined. Gonads of 4-month-old fish were either sexually undifferentiated with a central cavity, or ovarian in form. Gonads of 12- and 18-month-old fish were ovaries or bisexual gonads, while those of 2-year-old fish were ovaries, bisexual gonads or testes. Fish aged between 3 and 8 years had ovaries or testes, except for a few bisexual gonads found in 3- and 4-year-old fish. The chronological appearance of females, hermaphrodites and males in that order, and histological evidence, suggested that the testis originates from the ovary via a bisexual gonad in the juvenile stage. The sex ratio of females to males at the age of 2 years and over was about 1:1, suggesting that hermaphroditic red sea bream appear in about 50% of the juvenile population. The sexual pattern in this species, therefore, is concluded to be gonochorism with a bisexual juvenile stage.     

Seasonal cycles of gonadal development and plasma sex steroid levels in Epinephelus morio, a protogynous grouper in the eastern Gulf of Mexico

In a field population of the protogynous red grouper Epinephelus morio in the eastern Gulf of Mexico, females with oocytes at all stages of development were collected during the spawning season suggesting that several batches of oocytes may be released over the spawning period. Plasma oestradiol (E₂) levels were highest in ripe females whose gonads contained both cortical alveoli and vitellogenic oocytes during the breeding season. Males were still spermiating as late as August, although levels of androgens 11-ketotestosterone (11-KT) and testosterone (T) had declined from their peaks in March. A few red grouper with either perinucleolar or cortical alveoli stage oocytes were undergoing sex change both during and after the spawning period. Low levels of E₂, T and 11-KT were detected in transitionals. Proliferation of male tissue was not restricted to any specific area of the gonad but occurred in pockets within the ovarian lumen. The sequence of an increase in gonial cells along the periphery of the lamellae, increase in interstitial tissue, degradation of female elements, and formation of a sperm duct seemed to be concurrent with spermatocyte proliferation and the process of preparing the gonad to function as a testis.     

Histological study of the development and sex differentiation of the gonad in the European eel

The histological structure of the gonads was studied in yellow eels sampled from a coastal lagoon and from stocks reared in an aquaculture plant showing different sex ratios. Gonad development related to body size rather than to age and underwent an intermediate stage characterized by a structure of an early testis but containing oogonia and oocytes. This gonad was called the Syrski organ and the stage juvenile ambisexual. Ovaries were found in eels from 22–30 cm in length, possibly derived from undifferentiated gonads or from Syrski organs. Fully differentiated testes were found in eels >35 cm, derived from Syrski organs. These observations support the results of previous research. From elvers and in eels up to 15–16 cm in length, growth of the gonadal primordium is due to primordial germ cell migration. In eels > 15 cm multiplication of primordial cells begins. Oogonial clones were found in eels > 18 cm in length, whilespermatogonium B clones were observed in eels >30 cm in length. The dynamics of sex differentiation was different among stocks with different ultimate sex ratios: ovaries were found in shorter eels in stocks with a prevalence of females, in longer eels in stocks with a prevalence of males. This result supports the hypothesis of a metagametic (environmental) sex determination. The somatic cells in contact with germ cells and those in the interstitium appeared early during gonad development and preceded germ cell differentiation. This suggests that somatic cells are the targets of the environmental factors influencing sex differentiation.     

Sexual maturation in female triploid plaice, Pleuronectes platessa, and plaice × flounder, Platichthys flesus, hybrids

The gonads of immature female triploid plaice ( Pleuronectes plafcssa ) and plaice × flounder ( Platichthys flesus ) hybrids produced from cold-shocked eggs were examined. They were less than half the size of those in diploids of similar age and contained only small numbers of developing oocytes. These were similar both in size and cytological appearance to those in the controls. The bulk of the ovarian tissue was composed of nests of small undifferentiated cells resembling the oogonia in the ovaries of diploid fish. Mature, 5 year old triploid hybrids which had not produced eggs over the previous two spawning seasons were killed in January when the diploid controls were nearing spawning condition. The ovaries of these fish were smaller than those of the controls and contained degenerating oocytes at maturation stage V. The advantage of sterile female triploid fish for fish-cultivation is discussed.     

Protogynous hermaphroditism in the half-banded sea perch, Hypoplectrodes maccullochi (Serranidae)

The half-banded sea perch, Hypoplectrodes maccullochi (Serranidae) exhibits protogynous hermaphroditism, based on the presence of transitional individuals collected from rocky reefs along the coast of New South Wales, Australia. Males dominated the larger size and age classes, whereas females predominated the smaller size and age groups. Histological preparations suggested that sex change occurs between 55–85 mm s.l. and 1 + to 4+ years of age. Three transitional phases were identified which varied according to the proportion of gametogenic tissue and remnant oocytes present within their gonads. All individuals greater than 85 mm s.l. were functional males containing well defined crypts of spermatids and spermatozoa. Remnant oocytes within these testes indicated prior female function. H. maccullochi were sexually monochromatic. The occurrence of some small males derived from pre-spawning females suggested pre-maturational sex change occurred in some individuals. Sex changing individuals were found in all the months of the year that gonads were examined in detail (January, February, April and June 1989). Fish were ripe in August and November. H. maccullochi conforms with the pattern of reproduction evident within the genus Hypopleclrodes .     

Human chorionic gonadotropin (hCG) induces gonad reversal in a protogynous fish, the bluehead wrasse, Thalassoma bifasciatum (Teleostei, Labridae)

Female to male successive hermaphroditism (protogyny) is common in several groups of marine fish. Thalassoma bifasciatum, the bluehead wrasse (Labridae), found in the reefs of the Caribbean normally undergoes sex reversal after receiving behavioral cues. This report deals with the successful use of human chorionic gonadotropin (hCG) in inducing gonad reversal in this species. Eighty percent (n = 40) of the treated fish showed signs of reversal in 1-6 weeks; only 11% (n = 54) of the control (nontreated) group showed signs of reversal during the same period. The number of fish undergoing reversal increased with the length of the treatment period, 55% after 1 week, 100% after 6 weeks. A bluehead color pattern, typical of the terminal male phase, also appeared more frequently in the longer treated groups. To determine the efficacy of hCG in inducing gonad reversal, morphological criteria for reversal had to be established. For the majority of treated fish, the presence of both spermatogenic cysts and degenerating oocytes was sufficient to label these gonads unambiguously as undergoing reversal. However, at the extremes of this process, i.e., onset (early) and endpoint (late) stages, ambiguity could arise in identifying a gonad as undergoing reversal, and, therefore, key criteria were established for these stages. In our hands, the most consistent and reliable criterion for the early stage reversal was appearance of male germ line cells, clusters of "spermatogonial-like" cells. For the late stage, recognizable remnants of late-state oocytes had to be present. Some details of the histological changes that occur during early, middle, and late stages of reversal are also described. It is suggested that these results with hCG shed new light on the endocrine control of gonad reversal in T. bifasciatum.     

Induction of meiosis in fetal mouse testis in vitro

Fetal mouse testes and ovaries with their urogenital connections were cultured singly or in pairs on Nuclepore filters. When a testis in which the sex was not yet morphologically detectable was cultured together with older ovaries containing germ cells which were progressing through the meiotic prophase, the male germ cells were triggered to enter meiosis. When older fetal testes in which the testicular cords have developed were cultured together with ovaries of the same age with germ cells in meiosis, the oocytes were prevented from reaching diplotene stage. It was concluded that the fetal male and female gonads secrete diffusable substances which influence germ cell differentiation. The male gonad secretes a "meiosis-preventing substance" (MPS) which can arrest the female germ cells within the meiotic prophase. The female gonad secretes a "meiosis-inducing substance" (MIS) which can trigger the nondifferentiated male germ cells to enter meiosis.     

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.     

Gonadal structure of the serial-sex changing gobiid fish Trimma okinawae

In order to obtain basic information about the role played by endogenous sex hormones in bringing about sex changes in the serial-sex changing gobiid fish Trimma okinawae, the gonadal structure of male and female phases were observed histologically. Steroid-producing cells (SPC; Leydig cells in a testis) were observed ultrastructurally in the ovaries and testes of both female-phase and male-phase fish. In addition, gonadal expression of P450 cholesterol side-chain-cleavage (scc) was examined immunohistochemically. Gonads of fish in female and male phases were observed to have both ovaries and testes simultaneously. Female-phase fish had matured with many developed vitellogenic oocytes, while male-phase individuals had immature ovaries with many numbers of previtellogenic oocytes at the perinucleolus stage. Testes of fish in different sexual phases had active spermatogenic germ cells. Organellae of SPC in the ovaries of female-phase fish had active structures of steroid production. In contrast, SPC in the ovaries of male-phase fish did not show active structures of steroid production. Immunopositive reactions against the scc antibody in the ovaries of female-phase fish were very strong, but immunoreactions in the ovaries of male-phase fish were very weak. In the testis, moderate immunopositive signals were obtained from dual-phase male/females.