Ultrastructural and cytochemical studies of the female gonad of Prorhynchus sp. (Platyhelminthes,Lecithoepitheliata)

The female gonad of Prorhynchus is heterocellular (neoophoran organization) and consists of an unpaired, elongate germovitellarium enveloped by a finely granular extracellular lamina. It is composed of a posterior germinative area where early oocytes are randomly associated with differentiating vitellocytes and a growth area with follicular organization. In each follicle a single oocyte is surrounded by a layer of vitellocytes. By electron microscopy, the oocytes showed features typical of non-vitellogenic germ cells; they had chromatoid bodies, annulate lamellae, lipid droplets and R.E.R. and Golgi complexes producing small granules with a multilamellar pattern. Vitellocytes showed features typical of secretory cells with the R.E.R. and Golgi complex developed to a great extent and involved in the production of type A and type B globules, respectively. We speculate that type A globules are shell-globules and type B globules are yolk. The structure, composition and role of vitellocyte globules of Prorhynchus are compared with those of homologous inclusions from other Platyhelminthes.Abbreviations A type A globule - B type B globule - ECL extracellular lamina - GC Golgi complex - L lipid - RER rough endoplasmic reticulum - O oocyte - V vitellocyte     

Gonadal dysgenesis induced by prenatal exposure to ethinyl estradiol in mice

Daily oral administration of ethinyl estradiol (0.02, 0.2, or 2.0 mg/kg of body weight) to pregnant Jc1:ICR mice resulted in ovotestis and intra-abdominal testis with persistent Müllerian duct and Wolffian duct in male fetuses and ovarian hypoplasia in female fetuses when it was given from day 11 through day 17 of gestation (before gonadal differentiation in the fetus). The ovotestis consisted of testicular and ovarian portions. In the testicular portion, a few solid seminiferous tubules containing spermatogonia, some with pachytene nuclei with Sertoli cells and compact interstitial tissue including Leydig cells, were seen. In the ovarian portion, pachytene nuclei were seen. The intra-abdominal testis was smaller and contained more spermatogonia per tubule in cross section than the control testis. These findings suggest that in male fetuses ethinyl estradiol affects Sertoli cell differentiation resulting in suppression of Müllerian inhibiting factor. On the other hand, in the ovarian hypoplasia, the primordial follicles and follicular cells in a primordial follicle were significantly decreased in number, and the number of the degenerated primordial follicles was significantly increased. It seems likely that ethinyl estradiol affects the intimate contact between follicular cells and oocytes to cause degeneration of primordial follicles.     

Ultrastructural observations on the oogenesis of Triops cancriformis (Crustacea,Notostraca)

Summary Each ovarian follicle of Triops cancriformis is four-celled; these cells (one oocyte and three nurse cells) are interconnected by cytoplasmic bridges. In the course of differentiation, the nurse cells are early recognizable; they increase in size more than the oocyte and their nuclei contain many nucleoli. For the first time in Arthropoda, yolk globules are reported to be present in nurse cell cytoplasm; these globules arise from the smooth endoplasmic reticulum. The functional significance of the intercellular bridges and the trophic role of the nurse cells are discussed.The authors are grateful to Dr. Bruno Sabelli for his support and to Mr. Francesco Monte for his technical assistance     

Morphology of female reproductive system of Mediterranean flatheaded peachborer,Capnodis tenebrionis (Linnaeus, 1761) (Coleoptera: Buprestidae)

Capnodis tenebrionis causes damage in many species of Rosaceae. The present study investigates on the morphology of the female reproductive system of C. tenebrionis. The female reproductive system of C. tenebrionis has a pair of ovaries, lateral oviducts, a common oviduct, spermatheca, and bursa copulatrix. Each ovary in C. tenebrionis consists of approximately 24 telotrophic meroistic type ovarioles. The ovarioles of C. tenebrionis have four regions (terminal filament, tropharium, vitellarium, and pedicel). Tropharium have trophocytes, young oocytes, and prefollicular cells. Vitellarium consists of previtellogenic, vitellogenic, and choriogenic oocytes. Previtellogenic oocyte is surrounded by cylindrical epithelial cells. Its ooplasm is homogeneous and basophilic. In vitellogenic oocyte, there are intercellular spaces between monolayered follicle cells. Its ooplasm has yolk granules and lipid droplets. Choriogenic oocyte are surrounded by chorion and single-layered cylindrical cells. There are yolk granules and lipid droplets in its ooplasm which is asidophilic. In C. tenebrionis female, spermatheca and bursa copulatrix wall is surrounded by thin cuticular intima, monolayer epithelial, glandular cells, and muscle layer. Spermatheca lumen contains a large number of spermatozoa. Bursa copulatrix lumen is filled with secretory material. This study may be useful in terms of the morphology of mature female reproductive organs of Buprestidae and other coleopteran species.     

Seasonal histological changes in the gonads of Sebastodes paucispinis ayres,an ovoviviparous teleost (family scorpaenidae)

The elongate paired testes of Sebastodes paucispinis consist of tubules which radiate from a single longitudinal sperm duct and terminate blindly at the periphery of the testis. They are lined by an epithelium consisting of columnar cells with distinct elliptical nuclei. During fall and winter, germ cells migrate inward from the fibrous capsule of the testis and become lodged among the tubule-boundary cells of the seminiferous tubules where they mature into primary spermatogonia. Each of these undergoes several mitotic divisions to produce large cysts of secondary spermatogonia. Subsequent spermatogenic divisions within these cysts produce large sperm-filled cysts which rupture, releasing the spermatozoa into the lumina of the seminiferous tubules. Seasonal cycles of cholesterol and carbohydrate production by the tubule-boundary cells suggest that they perform the same functions as the Leydig cells (androgen production) and Sertoli cells (nutrition) of other vertebrates. The paired fusiform ovaries consist of spongy tissue surrounded by thin-walled muscular ovisacs that converge posteriorly to form a genital duct. The spongy tissue is arranged in transverse lamellae composed of fibrovascular trunks which support epithelial and ovigerous tissue. A series of oocytes (up to 150 μ in diameter) is produced continually from oogonial nests distributed throughout each lamella. Vitellogenesis begins in July and continues throughout the summer. The follicle surrounding the mature oocyte consists of a bilaminar striated vitelline membrane, two epithelial layers (granulosa and theca), and a profuse capillary network. Spermatozoa appear within the ovaries from October to March. Ovulation probably precedes fertilization since spermatozoa were never found within pre-ovulatory or post-ovulatory follicles. The follicular epithelium regresses after ovulation but the capillary beds remain intact, thus providing a mechanism for fetal-maternal exchange of gases and nitrogenous wastes.     

Ultrastructural observations on oogenesis in Drosophila

The ultrastructure of the follicle cells and oocyte periplasm is described during the stages of oogenesis immediately prior to, during, and immediately subsequent to, vitellogenesis. A number of features have not been described previously in Drosophila. Some yolk appears prior to pinocytosis of blood proteins. However, most of the protein yolk forms while the periplasm is filled with micropinocytotic invaginations and tubules derived from the oolemma. These tubules retain the internal layer of material characteristic of coated vesicles and are found to fuse with yolk spheres. No accumulation of electron-dense material in the endoplasmic reticulum or Golgi of the oocyte is found. Both trypan blue and ferritin are accumulated by the oocyte. The follicle cells have an elaborate endoplasmic reticulum during the period of maximum yolk accumulation. Adjacent cells are joined at their base by a zonula adhaerens, forming a band around the cells, and by plaques of gap junctions. Gap junctions are also present between nurse cells and follicle cells. During chorion formation, septate junctions also appear between follicle cells, adjacent to the zonula adhaerens.     

Effect of follicle cells on in vitro fertilization of pig follicular oocytes

We investigated the effect of cumulus and granulosa cells (follicle cells) on in vitro fertilization of pig follicular oocytes matured in vitro. Oocytes surrounded by cumulus and connected with a piece of parietal granulosa cells (complexes) were matured in vitro for 46hours and were then divided into 4 groups: Group I oocytes were surrounded by expanded cumulus and granulosa cells; Group II oocytes were surrounded by expanded cumulus cells; Group III were denuded oocytes; and Group IV were denuded oocytes with cumulus cells from other complexes. After incubation for 4 hours and 40 minutes with frozen, thawed and preincubated pig epididymal spermatozoa, the oocytes were cultured for 5 hours and 20 minutes. When oocytes were inseminated in the presence of cumulus cells, the penetration rates were higher (92.5% for Group II and 89.5% for Group IV) than when cumulus cells were not used for insemination (Group III, 66.8%) or when oocytes with follicle cells were inseminated (Group I, 72.3%). Denudation of follicle cells before insemination (Group III) decreased the percentage of male pronuclear formation (50.8%) compared with that of oocytes surrounded by follicle cells (66.7% for Group II and 80.2% for Group I). These results support the ability of a moderate number of follicle cells to facilitate sperm penetration of pig follicular oocytes and male pronuclear formation.     

Microtubule organization and nucleation in the differentiating ovarian follicle of the lizard Podarcis sicula

We analyzed the organization of the microtubular cytoskeleton and the distribution of centrosomes at the different stages of differentiation of the ovarian follicle of the lizard Podarcis sicula by examining immunolabeled α‐ and γ‐tubulins using confocal microscopy. We observed that in the follicular epithelium the differentiation of the nurse pyriform cells is accompanied by a reorganization of the microtubules in the oocyte cortex, changing from a reticular to a radial pattern. Furthermore, these cortical microtubules extend in the cytoplasm of the connected follicle cells through intercellular bridges. Radially oriented microtubules were still more marked in the oocyte cortex during the final stages of oogenesis, when the yolk proteins were incorporated by endocytosis. The nucleation centres of the microtubules (centrosomes) were clearly detectable as γ‐tubulin immunolabeled spots in the somatic stromal cells of the germinal bed. A diffuse cytoplasmic immunolabeling together with multiple labeled foci, resembling the desegregation of the centrosomes in early oogenesis of vertebrates and invertebrates, was revealed in the prediplotenic germ cells. In the cytoplasm of growing oocytes, a diffuse labeling of the γ‐tubulin antibody was always detectable. In the growing ovarian follicles, immunolabeled spots were detected in the mono‐layered follicle cells which surrounded the early oocytes. In follicles with a polymorphic follicular epithelium, only the small follicle cells showed labeled spots. A weak and diffuse labeling was observed in the pyriform cells while in the enlarging intermediate cells the centrosomes degenerated like in the early oocytes. Our observations confirm that in P. sicula most of the oocyte growth is supported by the structural and functional integration of the developing oocyte with the pyriform nurse cells and suggest that their fusion with the oocyte results in an acquirement by these somatic cells of characteristics typical of the germ cells. J. Morphol. 2012. © 2012 Wiley Periodicals, Inc.     

Histological, histochemical and immunohistochemical study on the growing oocytes of the abyssal teleost Hoplostethus mediterraneus (V)

The oocytes of the abyssal Teleost, Hoplostethus mediterraneus were studied. Four stages of growth were observed and the oocytes of all the stages were surrounded by follicular cells and had several nucleoli in the nucleus. In the oocytes of the II degrees stage, vacuoles without contents, in oocytes of the III degrees stage several vacuoles with a basophilic contents and small yolk globules were identified. General and basic proteins, ribonucleoproteins, acid proteoglycans with -COOH groups were recognized in the cytoplasm, in the nucleoli of oocytes in the II degrees stage and in the vacuolar contents of oocytes in the III degrees stage. In the follicular cells, in the pellucid zone, in the yolk globules, from their beginning, glycoproteins were present. Positivity, for all lectins used, was revealed in the follicular cells and in the four stages of oocytes growth. alpha-D-glucose and alpha-D-mannose binding sites were in the pellucid zone and in the initial yolk globules. In the lather galactose and beta-N-acetyl glucosamine were present too. nNOS and VIP immunopositivity revealed at the periphery of the cytoplasm and at network of nerve fibres between oocytes, suggests NO is involved in a mechanism of regulation of the gametogenesis and of the spawning.     

Ovarian Structure in Milnesium tardigradum (Tardigrada, Milnesiidae) during Early Vitellogenesis

The ultrastructure of the ovary of Milnesium tardigradum during early vitellogenesis is described. Within the ovary, there were large multinuclear cells surrounded by many mononuclear oocytes. Observation of serial sections revealed four multinuclear cells that were connected to each other by cytoplasmic bridges. Each peripheral oocyte was connected to the multinuclear cell. An enormous ER-like structure was conspicuous in the centre of the multinuclear cell. The presence of large numbers of lipid droplets and yolk granules in both multinuclear cells and many mononuclear oocytes suggested a role as nurse cells. A small number of these oocytes grow to be eggs. The structural features of the multinuclear nurse cell were compared with other known examples.     

Yolk formation in Isohypsibius (Eutardigrada)

Summary In Isohypsibius granulifer, yolk is autosynthesized. The Golgi apparatus is mainly responsible for the formation of yolk, which consists of irregular platelets with heterogeneous contents and a diameter of about 1 m. Dense globules, 300 nm in diameter, are visible among yolk platelets. These develop in the vesicles of the rough endoplasmic reticulum. The genesis of these vesicles is associated with the outer membrane of the nuclear envelope, which forms blebs intensively during previtellogenesis and early vitellogenesis. The developing oocytes are assisted by nurse cells, to which they are jointed by cytoplasmic bridges. For every oocyte, there are a number nurse cells, which are sister cells of the oocyte. In addition to rRNA, nurse cells transfer to the oocyte lipids, platelets of yolk formed in their cytoplasm, mitochondria and cortical granules.     

Spatial and Temporal Changes in Testis Morphology and Sperm Ultrastructure of the Sportfish Sauger (Sander canadensis)

The objective of this study was to assess testicular morphology and spermatozoal structure spatially within the reproductive tract and temporally among seasons in the sauger (Sander canadensis). The testis exists as two separate lobes joined at the urogenital pore and were characterised as unrestricted lobular with seminiferous tubules terminating at the ventral periphery and coalescing dorsally on the main sperm duct. Differences were observed between the pre-breeding season (November) and breeding season (March), with every stage of spermatogenesis occurring in spermatocysts in pre-breeding season in contrast to only spermatozoa being present in the tubules and main duct during the breeding season. Longitudinal folds in the main duct epithelium increased in number with increasing proximity to the urogenital pore, greatly increasing epithelial height regardless of season. Sauger spermatozoa consisted of an ovoid head, a midpiece containing 2 – 4 mitochondria incorporated into the head and a single flagellum containing an asymmetrical lateral ribbon. Motile spermatozoa were found throughout the testis during the breeding season. A decrease in sperm concentration was quantified moving proximally, suggesting a hydration effect by the main duct epithelium during the breeding season. These observations fill an important knowledge gap regarding reproductive biology of this impactful recreational fish species.     

A case report of ovotesticular disorder of sex development (OT‐DSD) in a baboon (Papio spp.) and a brief review of the non‐human primate literature

Disorders of sexual development are rare in non‐human primates. We report a case of true hermaphroditism in a 19‐year‐old, nulliparous, female baboon (Papio spp.). At necropsy, the animal was obese with adequate muscle mass and hydration. Reproductive organs appeared normal with the exception of 2 firm nodular structures in the myometrium (1‐1.5 cm diameter) and a thickened, dark endocervical mucosa. Histologically, both gonads were ovotestes and contained discrete areas of ovarian and testicular tissue. There were follicles in various stages of development surrounded by ovarian stroma. Other areas contained hypoplastic seminiferous tubules lined by Sertoli cells, but lacked germ cells and spermatozoa. The uterine lesions were consistent with adenomyosis and cystic endometrial hyperplasia. Cervical lesions were consistent with atypical glandular hyperplasia and squamous metaplasia with dysplasia. We report the first case of ovotesticular disorder of sexual development (OT‐DSD), or true hermaphroditism in a baboon.     

Annual reproductive cycle based on histological changes in the ovary of the female mosquitofish,Gambusia affinis, in central Japan

To clarify the annual reproductive cycle of wild female mosquitofish,Gambusia affinis, in Mie Prefecture, central Japan, changes in ovarian histology were investigated. Female mosquitofish kept in aquaria under constant temperature (25°C) and photoperiod (16L: 8D) conditions produced successive broods at intervals of 22.1±0.46 days (n=7). Between days 0–3 following parturition, females began active vitellogenesis. Between days 3–5, fully grown oocytes matured and were fertilized, and embryonic development began in the follicles. By day 10, as fertilized eggs continued embryonic development, some oocytes at the oil-droplet stage had begun to accumulate yolk globules for the next gestation. Thus, vitellogenesis of the succeeding batch of oocytes overlaps with gestation during reproduction in the mosquitofish. A rearing experiment showed the annual reproductive cycle of mosquitofish breeding in Nagashima to be as follows. Although oocytes had not at that point developed to the yolk globule stage, copulation occurred in February. Females began vitellogenesis in early May, the first pregnancy of the year commencing in mid-May. From mid-May to August, females repeated the gestation cycle (vitellogenesis, maturation, fertilization, pregnancy and parturition) at around one month intervals. In September, oocyte recruitment from the oil-droplet to the yolk globule stage ceased. After the final parturition, the ovaries contained only non-vitellogenic oocytes. Spermatozoa in the ovarian cavity were scare from November to January.     

The histology and histochemistry of oogenesis in the water strider, Gerris remigis Say

In each ovariole of Gerris remigis, nurse cells arise by mitotic divisions at the anterior end of the germarium. These cells enlarge as they move posteriorly. This size increase is possibly caused by fusion of cells, but probably by endopolyploidy as well. The nurse cells then establish connections with a central trophic core, which receives the products of subsequent nurse cell degradation. Two possible pathways of nuclear degradation are suggested: one involves the condensation of chromatin within the nucleus; the other, the release of DNA as fine granules into the cytoplasm. Cytoplasmic areas containing such DNA are also rich in proteinaceous granules, but have a meager content of RNA. The remainder of the cytoplasm of the mature nurse cells contains a high concentration of RNA, as do the nucleoli. Posteriorly the trophic core connects via nutritive cords with each developing oocyte in the prefollicular region and in the anterior vitellarium. RNA is apparently contributed to the ooplasm via the trophic stream. Patches of cytoplasmic DNA are present in the young oocytes; the origin and fate of this DNA is uncertain. During early oocyte maturation chromosomal stainability decreases, and the nucleolus enlarges. In previtellogenic stages, numerous proteinaceous bodies appear in association with the nucleolus-chromosome complex. These bodies, like the nucleolus, have only a low RNA content. They may pass to the cytoplasm, but cannot be traced with certainty. During the latter part of this period a complex population of small proteinaceous and lipid preyolk bodies accumulates peripherally in the oocyte. Definitive protein and lipid yolk are probably derived by the enlargement and inward migration of these bodies. The oocytes are each surrounded by a layer of follicle cells proliferated in the prefollicular region. These become binucleate and enlarge as the enclosed oocytes grow and elongate. RNA also increases in the nucleoli and cytoplasm of the follicle cells as they move posteriorly in the vitellarium. There is no evidence of transfer of nucleic acids or protein from the follicle cells to the oocyte. The nurse cells are therefore implicated as the major source of nucleic acids for the maturing oocyte.     

Structure of the ovotestis and evidence for heterosynthetic incorporation of yolk precursors in the oocytes of the nudibranch mollusc,Spurilla neapolitana

The ovotestis of Spurilla neapolitana consists of a series of spherical lobes, each of which is composed of radially arranged, sac-like acini or follicles. The male and female portions of each acinus are separated by ovarian follicle cells and testicular accessory cells. A thick basal lamina serves as a barrier between adjacent acini. The surface of each ovotestis lobe is covered by several layers of myoepithelial cells resting on a connective tissue layer. Developing oocytes are intimately associated with follicle cells except in the last stages of vitellogenesis. Follicle cells are characterized by the presence of extensive arrays of rough endoplasmic reticulum (RER) and Golgi complexes and may play a role in vitellogenesis. An ultrastructural analysis of vitellogenesis suggests that oocytes utilize both auto- and heterosynthetic mechanisms of yolk formation. Autosynthetsis is suggested by the activity of the Golgi complex and RER, while heterosynthesis is indicated by high levels of endocytotic activity by the oocyte. Follicle cell development and high endocytotic activity in the oocytes may be a reproductive adaptation to accelerate yolk synthesis, resulting in more rapid egg production.     

Carbohydrate and protein histochemistry during oogenesis inHalobatrachus didactylus (Schneider, 1801) from the Bay of Cadiz (Spain)

Summary Histological and histochemical characteristics were studied inHalobatrachus didactylus (Schneider, 1801) during oogenesis. Three phases could be differentiated: previtellogenesis (oogonia and basophilic oocytes), vitellogenesis (yolk synthesis) and maturation-spawning. Glycogen, glycoproteins and proteins rich in certain amino acids were present in the previtellogenic as well as in the vitellogenic cytoplasm oocytes. No acid mucosubstances were detected. Three types of yolk (vesicles, vacuoles and granules) contained different types of organic reserves; granules were essentially proteic whereas globules were lipidic. Carbohydrates and proteins were present in vesicles.     

An ultrastructural study of oogenesis in a marine triclad

The ultrastructural features of oocyte differentiation were studied in the marine triclad Cercyra hastata. Oocytes at several stages of maturation, each surrounded by follicle cell projections, are present within each of the two ovaries. A pre-vitellogenic and a vitellogenic stage have been detected in the oogenesis of C. hastata. The pre-vitellogenic stage is mainly characterized by an increase in the nuclear and nucleolar volume and activity, and the appearance and development of cortical granule precursors which are elaborated by the Golgi complex. In early phases of the vitellogenic stage, intense delamination and blebbing of the nuclear envelope occurs which probably contributes to an increase in number of cytoplasmic membranes and to transfer of nuclear material to the cytoplasm. The rough endoplasmic reticulum is extensively developed and often assumes a ‘whorl’ array. Several areas of yolk precursor formation appear in the whorls. Numerous 2–5 μm protein yolk globules are subsequently formed which appear surrounded by a double membrane (cisternae of the smooth endoplasmic reticulum) and become randomly distributed throughout the cytoplasm of mature oocytes. The peripheral ooplasm is occupied by a monolayer of electron-dense cortical granules. Finally, the evolutionary significance of the autosynthetic mechanism of yolk production is discussed.     

Ultrastructural observations of previtellogenic ovarian follicles of dove

Dove ovarian follicle is a complex structure composed of oocyte surrounded by a somatic compartment consisting of theca externa, theca interna and granulosa. The structure of ovarian follicle (1 and 2 mm) of dove was studied by electron microscopy. The granulosa was pseudostratified in the 1-mm-diameter follicles and stratified with two or three irregular rows of cells in the 2-mm-diameter follicles. In the larger follicle indentations between oocyte and granulosa cells become more numerous and the microvilli of granulosa cell elongated to form a zona radiata with similarly elongated oocyte microvilli. Lining bodies were present at the tips of granulosa microvilli and in the cortical region of the oocyte. In the oocyte cortex were observed coated pits, coated vesicles, dense tubules, multivesicular bodies and primordial yolk spheres. Primordial yolk spheres may contain lining bodies and were observed fused with dense tubules and multivesicular bodies or associated with smooth cisternae.     

Resorption of unemitted gametes in Lithognathus mormyrus (Sparidae,Teleostei): a possible synergic action of somatic and immune cells

The resorption of unemitted gametes during the post-spawning period of the male and female reproductive cycles in Lithognathus mormyrus was studied by histochemical, histological and cytological methods. The resorption of residual spermatozoa involved the phagocytotic activity of Sertoli cells bounding the seminiferous cysts of spermatozoa, and those associated with spermatogonia lining the lobular lumen. Spermatozoa remaining in the sperm duct were phagocytozed by the lining epithelial cells. Eosinophilic granulocytes and macrophages were identified in the vicinity of residual spermatozoa. The remnants of oocytes underwent an atretic phenomenon in which follicle cells were firstly involved, inducing a progressive fragmentation of the oocyte cytoplasm. Subsequently, eosinophilic granulocytes invaded oocyte degenerative areas and clung to the remaining vitelline inclusions ensuring their biotransformation into waste products (brown bodies). The analogy of the resorption processes of both male and female unemitted gametes during the post-spawning period of natural reproductive cycle, involving first the enveloping somatic cells and then immune cells, is emphasized.