Differentiation of steroid-producing cells and folliculogenesis in the developing ovary of the Nile tilapia Oreochromis niloticus

Differentiation and development of steroid-producing cells (SPCs) and folliculogenesis during ovarian differentiation in the Nile tilapia Oreochromis niloticus were immunohistochemically and ultrastructurally examined. Clusters of immunopositive cells (IPCs) against antibodies (ABs) of cholesterol side-chain cleavage cytochrome P450 (P450scc), 3β-hydroxysteroid dehydrogenase (3βHSD), and cytochrome P450aromatase (P450arom) only appeared in the area near blood vessels in the fish ovaries at 50-60 days after hatching (dah). Ultrastructural results showed that differentiation and development of SPCs from undifferentiated to maturation occurred in the area near blood vessels, indicating that it would be the original site of SPCs. At 70-80 dah, IPC clusters invaded the interstices among oocytes at the perinucleolar stage from the area near the blood vessels. IPCs increased in number in the interstices among the previtellogenic oocytes, and some clusters began to enclose the outer thecal layer of the previtellogenic oocytes at 90 dah. The process of folliculogenesis was ultrastructurally observed. SPCs enclosed by fibroblastic cells invaded the interstitial areas among oocytes and some reached the surfaces of oocytes. The upper portions of these elongations opened and began to enclose the outer surfaces of developed oocytes to become thecal layer. Later, newly migrated SPCs reach the thecal layer to become thecal cells. These results indicate that steroid-producing thecal cells originate from the SPCs in the area near blood vessels. After thecal layer formation, an immunopositive reaction against P450arom AB, but not against P450scc or 3β-HSD ABs, appeared first in the granulosa cells enclosing the vitellogenic oocytes at 100 dah. At this time, estrogen production in serum levels rapidly increased. Thus, folliculogenesis could be essential for active production of estrogen in the ovary.     

Evidence of steroidogenesis in postovulatory follicles of the tilapia,Oreochromis mossambicus

Summary Postovulatory follicles of the tilapia, Oreochromis mossambicus, were examined with electron microscopy and enzyme histochemistry for evidence of steroid-hormone production. Light microscopy was also used to examine changes in the ovary with time after spawning. Electron microscopy detected the presence of smooth endoplasmic reticulum, lipid droplets, and mitochondria with tubular cristae in certain cells of the theca interna. These structures are suggestive of cells that synthesize steroid hormones. Granulosa cells also contained some smooth endoplasmic reticulum, along with an augmentation of Golgi complexes, vesicles, microvilli, and microfilaments within 5–7 days after spawning. Enzyme histochemistry demonstrated an intense reaction of 5, 3-hydroxysteroid dehydrogenase (3-HSD) in variably placed thecal cells up to 7 days after spawning. At this time, the thecal cells of vitellogenic oocyte follicles also began to show strong 3-HSD activity. During the first 7 days after spawning, there was an increase in young primary oocytes and recruitment of some of these to vitellogenic oocytes. By 10 days after spawning, certain thecal cells in the follicles of these vitellogenic oocytes showed an intense 3-HSD reaction, while the postovulatory follicular tissue demonstrated a weak reaction. This arrangement continued for the lifespan of the postovulatory follicular tissue. Postovulatory follicles had a lifespan of up to 25 days after spawning in females that continued to hold the developing fry inside their mouths, i.e., mouthbrooders. At 25 days after spawning, the postovulatory follicular tissue showed signs of degeneration with the presence of vacuoles and lysosomes. In females that ate the zygotes, therefore exhibiting no parental behavior, the postovulatory follicular tissue showed signs of degeneration at l0days after spawning. In these females, the next clutch of eggs also developed at a higher rate than in mouthbrooders.     

Steroid Producing Cells during Ovarian Differentiation of the Tilapia, Sarotherodon niloticus

In order to examine the initial appearance and development of the steroid producing cells (SPCs) during the process of ovarian differentiation, histology and ultrastructure of tilapia ( Sarotherodon niloticus ) ovaries were investigated from 10 to 50 days after hatching. In gonads of fry at 23–26 days after hatching, initial ovarian differentiation was confirmed by the differentiation of stromal aggregations in the proximal and distal region of the gonad on the side facing the lateral wall. This represents the initial formation of the ovarian cavity. At the same time as ovarian differentiation, a few large cells appeared initially in the vicinity of blood vessels. They have some of the ultrastructural features characteristic of SPCs such as a moderate number of mitochondria with tubular cristae, a large amount of smooth endoplasmic reticulum and many free ribosomes. Based on these ultrastructural criteria, together with the present finding that these cells further differentiated into the typical SPCs at older stages, these cells were identified as SPCs. Thereafter, by 30–50 days, SPCs increased gradually in number in the area enclosing the blood vessels of ovaries. The increase in SPCs coincided with the development of germ cells, including the multiplication of oogonia and the transformation from oogonia to oocytes.     

Aromatase immunoreactivity and the role of enzymes in steroid pathways for inducing sex change in the hermaphrodite gobiid fish Trimma okinawae

The role of aromatase (Arom) in the process of bi-directional sex change in the gobiid fish Trimma okinawae was investigated by immunohistochemical methods. Irrespective of sexual phase, gonads comprised both ovarian and testicular tissues. In each sexual phase of females, the 2nd (2DF-M) and 4th (4DF-M) days after initiation of sex change to male, males, and the 2nd (2DM-F), 4th (4DM-F) and 6th (6DM-F) days after the initiation of reversion from male to female, ovarian and testicular histological observations were made. During the female, 2DF-M, 4DF-M and 6DM-F phases, the ovary contained vitellogenic and previtellogenic oocytes, compared with previtellogenic oocytes in the other phases. Although sperm was found in the testis in every phase, sperm ducts were apparent in the male phase, but not the female phase. Arom immunoreactivity was detected in the interstitial cells between the oocytes in all phases. On the other hand, it was localized in the thecal and granulosa cells of the follicular layer enclosing the oocytes in the female, 2DF-M, 4DF-M and 6DM-F phases. Activity of Arom in the thecal and granulosa cells is thought to be important for the development of oocytes and subsequent sex change.     

Immunohistological localization of 17β-estradiol and testosterone in the ovary of the rainbow trout (Salmo gairdneri Richardson) during the preovulatory period

Summary Antisera (AS) raised in rabbits against 17-estradiol (E) and testosterone (T) were tested for their suitability to localize E and T on deparaffinized, rehydrated sections of preovulatory trout ovaries, using the unlabeled antibody technique.Conventional control experiments demonstrated the specificity of the staining reactions. Furthermore, no staining was observed after the removal of T-specific antibodies by affinity chromatography, or following gonadectomy when non-gonadal tissue sections of male trout were incubated with T-AS. Antiserum, raised against 11-oxotestosterone and devoid of antibodies cross-reacting with T, did not stain ovarian sections.The loci at which E and T are detected in the somatic compartment are consistent with the two-cell concept of estrogen synthesis, where aromatizable androgens are produced in the thecal/interstitial layer and serve as substrates for estrogen synthesis in granulosa cells.Both steroids were detected in yolk vesicles from the stage of endogenous vitellogenesis. T-AS showed affinity for nuclei of vitellogenic oocytes. Nucleoli were not stained.     

Oogenesis in the Polychaete Dipolydora commensalis

Oocytes of the polychaete Dipolydora commensalis develop in the gonad, in close contact with the wall of the genital blood vessel, up to the late stages of vitellogenesis. At the blood vessel wall, between the neighboring vitellogenic oocytes, and sometimes on the apical surface of the oocytes, there are flattened follicular cells. However, no continuous, well-expressed gonad envelope is found. Oogenesis is asynchronous. Gametes at all developmental stages, from oogonia to late vitellogenic oocytes, occur in the gonad. Dividing oogonia vary from 6 to 10 m in diameter. RNA, proteins, glycogen, and lipids accumulate in the oocytes during vitellogenesis. The breakdown of the oocyte germ vesicle occurs in the gonad. Before spawning, gametes accumulate in the coelom and reach 80–90 m in diameter, at which point a new generation appears in the gonad.     

Varicose axons bearing “synaptic” vesicles on the basal lamina of the human seminiferous tubules

Summary Normal (infant and adult) and pathological testes were examined by electron microscopy in order to study testicular innervation. Nerves composed of non-myelinated fibres were abundant in the tunica vasculosa of the tunica albuginea. These nerves penetrated into the testicular septa reaching the interstitial tissue. This showed numerous non-myelinated nerve fibres running among the Leydig cells and blood vessels. Single axons or small groups of them, partially surrounded by Schwann cells, approached: 1) the Leydig cells, 2) the interstitial blood vessels, and 3) the seminiferous tubules. Single naked axons were also observed primarily in the proximity of the seminiferous tubules. These axons showed varicosities containing both small and large synaptic vesicles. The latter were less numerous and contained a central dense core. Small vesicles were agranular. Some varicose axons ran across the myofibroblast layer of the tunica propria reaching the basal lamina of the seminiferous tubules at the level of the Sertoli cells but not at the level of the spermatogonia. The intercellular space between Sertoli cell and axon membrane was about 150–200 nm.Profesor Agregado de Histología y EmbriologíaProfesor Adjunto de Citología e HistologíaProfesor Adjunto de Histología y Embriología     

Ovarian development rates in the Old World screw-worm fly, Chrysomya bezziana

Rates of ovarian development in relation to temperature were determined for autogenous females of the screw-worm fly, Chrysomya bezziana. Percentage durations of the different ovarian stages (scaled 2–10) were estimated on the basis of observed lengths of the developing oocytes. Mean durations (h) of each ovarian stage was determined at 20, 25, 28 and 35°C. A model of ovarian development rate (%/d) in relation to temperature (T) is presented, the fitted curve being give by R(T)=EXP (–2.73+0.362T–0.0055T²).     

The gas bladder of Argentina silus L., with special reference to the ultrastructure of the gas gland cells and the countercurrent vascular bundles

Summary The orifice between the two chambers of the gas bladder in Argentina silus is surrounded by a sphincter muscle. Gas analyses of the gas bladder contents of fish from 400 meters depth give 0–1% carbon dioxide and 9–72% oxygen. Micro-retia mirabilia form a countercurrent vascular system, and the arterial component has peripherally a sphincter mechanism. The function of the glandular layer of the anterior chamber remains uncertain, but the structure indicates secretion into blood capillaries. The lining epithelium of the anterior chamber may secrete some substance into blood or directly into the lumen, which may be involved in a secretory mechanism. This conclusion is not supported by our histochemical tests. The posterior chamber has no micro-retia and the blood vessels have a different origin from those of the anterior chamber. The blood vessels form a plexus of capillaries or sinuses in contact with the flat lining epithelium, thus allowing gases to pass freely by diffusion. — The muscular layers of both chambers are innervated by catecholamine-containing nerve fibres.The investigation was supported by grants from the Swedish Natural Research Council (No. 99-35) and by the Faculty of Mathematics and Science, University of Lund.     

Sexual Maturation, Annual Reproductive Cycle, and Spawning Periodicity of the Shore Scorpionfish, Scorpaenodes littoralis

The ovarian structure, sexual maturation, annual reproductive cycle, and spawning periodicity of the shore scorpionfish, Scorpaenodes littoralis, in Uchiura Bay, central Japan, were examined using specimens collected between May 1995 and March 1998 and fishes reared in laboratory. The ovarian stroma and blood vessels run longitudinally through the center of each ovarian lobe. The ovarian peduncles radiate from the central stroma. During the spawning season, gelatinous material is secreted from the epithelia of both the ovarian peduncle and ovarian wall, and the epithelia show morphological changes accompanying the ovarian maturation cycle. The minimum standard length at maturity was 55.2mm for males and 40.2mm for females. Males with mature testes were collected from March to November. Females in the mature or post spawning stages were collected between May and October, when the mean gonadosomatic indices were also high. This indicates that the spawning season of this species occurs between May and October. Four successive types of oocytes were grouped in the mature ovary, comprised of mature, late and early vitellogenic and previtellogenic oocytes respectively, suggesting that this species is a multiple spawner. Four captive females spawned repeatedly at intervals of 2–8 days over a prolonged period (4–8 months); a 2-day spawning interval was the most common for all females. This suggests that female S. littoralis have a 48-h spawning cycle in captivity.     

Previtellogenic and vitellogenic oocytes in ovarian follicles of cultured siberian sturgeon Acipenser baerii (Chondrostei,Acipenseriformes)

Previtellogenic and vitellogenic oocytes in ovarian follicles from cultured Siberian sturgeon Acipenser baerii were examined. In previtellogenic oocytes, granular and homogeneous zones in the cytoplasm (the ooplasm) are distinguished. Material of nuclear origin, rough endoplasmic reticulum, Golgi complexes, complexes of mitochondria with cement and round bodies are numerous in the granular ooplasm. In vitellogenic oocytes, the ooplasm comprises three zones: perinuclear area, endoplasm and periplasm. The endoplasm contains yolk platelets, lipid droplets, and aggregations of mitochondria and granules immersed in amorphous material. In the nucleoplasm, lampbrush chromosomes, nucleoli, and two types of nuclear bodies are present. The first type of nuclear bodies is initially composed of fibrillar threads only. Their ultrastructure subsequently changes and they contain threads and medium electron dense material. The second type of nuclear bodies is only composed of electron dense particles. All nuclear bodies impregnate with silver, stain with propidium iodide, and are DAPI‐negative. Their possible role is discussed. All oocytes are surrounded by follicular cells and a basal lamina which is covered by thecal cells. Egg envelopes are not present in previtellogenic oocytes. In vitellogenic oocytes, the plasma membrane (the oolemma) is covered by three envelopes: vitelline envelope, chorion, and extrachorion. Vitelline envelope comprises four sublayers: filamentous layer, trabecular layer 2 (t2), homogeneous layer, and trabecular layer 1 (t1). In the chorion, porous layer 1 and porous layer 2 are distinguished in most voluminous examined oocytes. Three micropylar cells that are necessary for the formation of micropyles are present between follicular cells at the animal hemisphere. J. Morphol. 278:50–61, 2017. ©© 2016 Wiley Periodicals,Inc.     

Possible sites of ultrafiltration in Tubifex tubifex Müller (Annelida,Oligochaeta)

Summary The endothelia of Tubifex tubifex Müller consist of myoendothelial cells, chloragocytes, or podocytes. The latter seem to occur only as windows on the ventral vessel which has an endothelium of myoendothelial cells elsewhere. The podocytes are large cells, with several processes on the inner side which ramify into several pedicels. These are aligned upon the outside of the basement membrane which lines the inside of the endothelium. The gaps between adjacent pedicels are about 40 nm wide. In capillaries fenestrated endothelia occur with irregular spacings measuring up to 0.4–1 m. A diaphragm in podocytes or capillary fenestrations do not seem to exist. The basement membrane is the only continuous layer lining the blood vessels and capillaries of Tubifex with a rather uniform diameter in the range of 50 nm. It is the only permeability barrier between blood and coelomic fluid.     

Effect of monosodium glutamate on apple leaf consumption by codling moth larvae

We have demonstrated that larvae of codling moth, Cydia pomonella (L) can successfully complete their first instar when fed apple leaves instead of fruit. Larvae fed leaves after hatching maximized their feeding intensity (about 320 g/larva/day) on day 2. Weight gain revealed a stereotypic sigmoid pattern that peaked on day 3. Although the maximum body weight of larvae fed leaves was 70–85% less than for larvae maintained on apples or on artificial diet, 100% of larvae fed leaves molted to the second instar 3–5 days after hatching. Our investigation revealed a diurnal pattern of leaf ingestion, and neonates' feeding intensity decreased significantly during the scotophase. We also demonstrated that monosodium glutamate (MSG) increased feeding on leaves by codling moth larvae. Depending on the duration of the bioassay, and larval age at time of initial exposure, 0.05 mg/ml and 0.1 mg/ml MSG increased apple leaf consumption by 25–60% over leaves alone. The effect of monosodium glutamate was best demonstrated during the first day following hatching. Exposure to MSG also accelerated molting to the second instar. Larvae exposed to MSG initiated consumption of leaf tissue significantly earlier than control neonates. The feeding stimulatory effect of MSG was not observed if exposure to this chemical was delayed until 3–4 h after hatch.The addition of feeding stimulants to pesticides that act via the alimentary tract may reduce the amount of active ingredients needed to maintain the efficacy of these formulations. Here, we postulate that first instar codling moth larvae are potential targets for treatment with pesticide formulations enhanced with monosodium glutamate.     

Effects of pituitary glycoprotein hormones and thyroid hormones on in-vitro vitellogenin incorporation into organ-cultured oocytes in the Japanese eel, Anguilla japonica

The objectives of the present study were to establish a long-term culture system for previtellogenic ovarian fragments of the Japanese eel (Anguilla japonica) and to identify the effects of salmon pituitary glycoprotein fraction (SPG), thyroxine (T4), and 3,5,3'-triiodothyronine (T3) on the uptake of vitellogenin (VTG) by cultured ovarian fragments evidenced by the appearance of yolk globules (YGs) within the oocytes. Yolk globules first appeared in the oocytes incubated in media containing only VTG (VTG-only group) after 9 days, whereas YGs began to accumulate in the oocytes of ovarian fragments cultured in media containing VTG+SPG (SPG group) following only 3 days of incubation. Furthermore, the occurrence of vitellogenic oocytes (%VO) and proportion of YGs within oocytes (%YG area) were significantly higher in follicles cultured in 30 ng/ml SPG throughout the culture period. No such stimulatory effects of T4 on VTG uptake were observed. Incubation of ovarian fragments with VTG and T3 (T3 group; 50 ng/ml) resulted in an increased %VO compared to follicles in the VTG group by day 9 of culture, and from day 10 onwards, both %VO and %YG area became significantly higher in follicles of the T3 group. Interestingly, SPG stimulated VTG incorporation and YG accumulation even in small oocytes (approximately 150 microm), whereas T3 showed these effects only in larger sized oocytes (> 180 microm). These results suggest that both SPG and T3 can accelerate VTG incorporation, but the mechanisms whereby this is achieved may differ between these hormone preparations.     

Susceptibility of the cattle tick Boophilus microplus (Acari: Ixodidae) to isolates of the fungus Beauveria bassiana

The susceptibility of the tick Boophilus microplus to Beauveria bassiana was evaluated by inoculating eggs, larvae and engorged females of the tick with five fungal isolates at concentrations of 10⁶, 10⁷ and 10⁸ conidia/ml. Tick eggs (0.25 g) were immersed in 1 ml of a suspension of the different conidial concentrations for 1 min. Similar exposure was performed by immersion of 2000 larvae and homogeneous groups of nine engorged females in 2 and 20 ml of conidial suspension, respectively. Treated eggs, larvae and adults were placed in an incubator at 27 ± 1 °C and relative humidity above 80% for evaluation of the fungal action. All fungal isolates applied at all conidial concentrations reduced the hatching rate of larvae from treated eggs by 1.36–65.58% and increased the mortality rate of inoculated larvae by 0.8–70.49%. In the bioassay with engorged females, oviposition period was reduced by 9.69–47.80%, egg mass weight by 4.71–53.87%, estimated reproduction by 8.3–60.62%, egg production index by 5.03–54.20%, percent larval hatching by 0.27–13.96%, and the mortality rate of treated females was increased by 96.60–100%. The reduction of the estimated reproduction obtained for the treated groups ranged from 8.37 to 64.52%. The sporulation of the pathogen on dead females ranged from 3.70 to 88.88% depending on the isolate and concentration used. Isolates AM 09, CB 7 and JAB 07 were the most effective and effectiveness increased with increasing concentrations of conidia in the suspensions.     

Prostaglandins E and F in uterine venous plasma in relation to peripheral plasma levels of progesterone and 20α-hydroxyprogesterone in the rat throughout pregnancy and parturition

Prostaglandins E and F in uterine venous plasma and progesterone (P) and 20α-hydroxyprogesterone (20α-OH-P) in peripheral plasma were measured by radioimmunoassays throughout pregnancy and parturition in the rat. E Prostaglandins are low (approx. 2 ng/ml) and maintain a more or less constant level throughout most of the pregnancy except just before parturition when they rise to 3.8 ng/ml on day 20. F Prostaglandin levels are always higher than E prostaglandins and show distinct peaks around day 5 (5 ng/ml), day 11 (7 ng/ml), and before parturition (8.4 ng/ml).Progesterone levels are higher than 20α-OH-P levels throughout most of the pregnancy (day 6–20); however, during early pregnancy (day 1–5) and before parturition more 20α-OH-P than P is present in peripheral blood.The possible role of uterine venous prostaglandin levels in altering the 20α-OH-P/P ratio during pregnancy and parturition is discussed.     

Population growth rate of the parasitic rotifer Proales gigantea,and susceptibility to parasitization in the snail Lymnaea acuminata at different stages of embryonic development

Developing eggs of the host snail Lymnaea acuminata were experimentally parasitized with the parasitic rotifer Proales gigantea to study the population growth rate of the parasite within the snail egg capsule and the susceptibility of the host eggs at different stages of embryonic development. The population growth rate of P. gigantea was 0.46 ± 0.07 individual^–1 day^–1 at the ambient temperature of 18–22 °C. Snail eggs were most susceptible to rotifer attack during the initial stages of development, becoming progressively more resistant after the hippo stage. Yet, regardless of the stage of development, the host embryo was doomed to die without hatching even if one individual rotifer gained entry inside the egg capsule. The presence of P. gigantea within the parasitized egg capsules or in the mucilage had no effect on the developmental rates and hatching success of non-parasitized eggs within the same egg mass.     

Barrier membranes at the outer surface of the brain of an elasmobranch,Raja erinacea

Summary This report gives the results of the first electron-microscopic examination of the cell layers covering the outer brain surface and the inner surface of the cartilaginous skull in the skate, Raja erinacea. The perivascular glial blood-brain barrier — a characteristic of elasmobranchs — extends to the outer surface of the brain. This outer barrier layer is surrounded, in turn, by a subarachnoid compartment (depth: 30–40 m), containing loose connective tissue and blood vessels; by an arachnoid-like epithelium (10–15 cell layers), impermeable to horseradish peroxidase; and, by perimeningeal fluid, a fluid with a slow turnover rate and a protein composition different from plasma. The inside of the skull, facing the perimeningeal fluid, is covered by a multilayered (10–15 layers) cuboidal epithelium, also impermeable to horseradish peroxidase. Closely apposed cells in the luminal layer of this epithelium have apical microvilli and numerous vesicular profiles, containing material of moderate electron density. These observations may explain, in terms of structure, the regulated protein content of perimeningeal fluid and the restricted exchange of solutes between brain and perimeningeal fluid in elasmobranchs.     

Gonadal structure and P450scc and 3β-HSD immunoreactivity in the gobiid fish Trimma okinawae during bidirectional sex change

The process of sex change in the gobiid fish Trimma okinawae was investigated by gonad histology and immunohistochemistry of two steroidogenic enzymes, P450 cholesterol-side-chain-cleavage (P450scc) and 3-hydroxysteroid dehydrogenase (3-HSD). Irrespective of sexual phase, gonads comprised both ovarian and testicular tissues. Females changed sex to male within 7 days, reverting again to female over an 11-day period. In each sexual phase of the females, the 2nd (2DF-M) and 4th (4DF-M) day after the initiation of sex change to male, the males, and 2nd (2DM-F), 4th (4DM-F), and 6th (6DM-F) days after the initiation of reversion from male to female, histological observations were made. In the ovary during the female, 2DF-M, 4DF-M, and 6DM-F phases, both vitellogenic and previtellogenic oocytes were present, but only previtellogenic oocytes were found in the other phases. The testis contained sperm in all phases, but sperm ducts were not visible in the female phase. In the ovary, P450scc immunoreactivity of interstitial cells was strongly or moderately detected, although weak in the male phase. In contrast, P450scc immunoreactivity in thecal cells was found in all but the male and 2DM-F phases. 3-HSD immunoreactive interstitial cells were detected in all phases, but only weakly so in the male and 2DM-F phases. 3-HSD immunoreactive thecal cells were observed in all stages without the male and 2DM-F and 4DM-F phases. In the testis, moderate P450scc and 3-HSD immunoreactivity was regularly found in the Leydig cells in all the phases. These results suggest that functional steroids including testosterone are produced in any sexual phases.     

Determination of trans-resveratrol in human plasma by high-performance liquid chromatography

The first method using high-performance liquid chromatography (HPLC) has been developed for the determination of trans-resveratrol in human plasma. The method involves a liquid–liquid extraction followed by reversed-phase HPLC with UV detection. The detection limit of trans-resveratrol in human plasma was 5.0 ng/ml. Standard curves are linear over the concentration range of 5.0–5000.0 ng/ml. Intra-assay variability ranged from 1.9 to 3.7% and inter-assay variability ranged from 2.5 to 4.0% at the concentration range of 15.0–4000.0 ng/ml.