Ultrastructure of gametogenesis in the ovotestis of an estuarine pulmonate slug,Onchidium tigrinum (Stoliczka, 1869)

The pulmonate slug Onchidium tigrinum (Stoliczka, 1869) is an estuarine protandrous gastropod. Transmission electron microscopy of both the gonadal and somatic cell populations of the ovotestis of the slug is documented. The acini of smaller slugs are comprised of developing spermatogenic cells and three to four small ill-developed oocytes. Details of the microscopic structures of Sertoli cells, interacinar cells and acinar boundary are described in-depth, revealing their secretory function. Sertoli cells are more numerous in the ovotestes of smaller slugs than in those of larger slugs. Tunnelling nanotubes of 200–400?nm in diameter are described for the first time in the Sertoli cells of molluscan ovotestis. These nanotubes may help to supply various cellular materials into distantly developing spermatogenic cells. The acini of larger slugs possess 2–3 mature oocytes along with a few spermatogenic cells. Sertoli cells, interacinar cells and spermatogonial cells are fewer in number in the acini of the ovotestis of larger individuals establishing the predominance of oogenesis in this phase of life. The number of oocytes per acinus is analysed in relation to the habitat of the pulmonates.     

Influence of the mesonephros on the development of fetal mouse ovaries following transplantation into adult male and female mice

We previously reported that fetal mouse ovaries frequently develop testicular structure following transplantation into adult male mice. The mechanism involved in gonadal sex reversal of ovarian grafts is not known. In the present study, we examined the influence of the adjacent mesonephros on development of the ovarian grafts. The results show that (1) when fetal ovaries were transplanted with the attached mesonephros, the frequency of ovotestis development was higher in male hosts than in female hosts, (2) the fetal ovaries that had been separated from mesonephros developed testicular structures more frequently than those with the mesonephros, and the incidence of ovotestis development was comparable in male and female hosts, (3) removal of the cranial or caudal half of the mesonephros resulted in a similar frequency of ovotestis development, and (4) when fetal ovaries were separated and reattached to the mesonephros, they developed testicular structures at a frequency similar to that of ovaries left attached to the mesonephros, and the sex of mesonephroi reattached to ovarian grafts did not influence the incidence of ovotestis development. These findings suggest that fetal ovaries can develop testicular structures after transplantation regardless of the sex of host, and that the adjacent mesonephros protects ovarian grafts from masculinizing stimuli more efficiently in female host than male hosts.     

Gonadal morphogenesis and sex differentiation in intraovarian embryos of the viviparous fish Zoarces viviparus (Teleostei, Perciformes, Zoarcidae): a histological and ultrastructural study

It is essential to know the timing and process of normal gonadal differentiation and development in the specific species being investigated in order to evaluate the effect of exposure to endocrine-disrupting chemicals on these processes. In the present study gonadal sex differentiation and development were investigated in embryos of a viviparous species of marine fish, the eelpout, Zoarces viviparus, during their intraovarian development (early September to January) using light and electron microscopy. In both sexes of the embryos at the time of hatching (September 20) the initially undifferentiated paired bilobed gonad contains primordial germ cells. In the female embryos, ovarian differentiation, initiated 14 days posthatch (dph), is characterized by the initial formation of the endoovarian cavity of the single ovary as well as by the presence of some early meiotic oocytes in a chromatin-nucleolus stage. By 30 dph, the endoovarian cavity has formed. By 44 dph and onward, the ovary and the oocytes grow in size and at 134 dph, just prior to birth, the majority of the oocytes are at the perinucleolar stage of primary growth and definitive follicles have formed. In the presumptive bilobed testis of the male embryos, the germ cells (spermatogonia), in contrast to the germ cells of the ovary, remain quiescent and do not enter meiosis during intraovarian development. However, other structural (somatic) changes, such as the initial formation of the sperm duct (30 dph), the presence of blood vessels in the stromal areas of the testis (30 dph), and the appearance of developing testicular lobules (102 dph), indicate testicular differentiation. Ultrastructually, the features of the primordial germ cells, oogonia, and spermatogonia are similar, including nuage, mitochondria, endoplasmic reticulum, and Golgi complexes.     

Immuno-localization of ferritin polypeptides in oocytes and somatic tissue of the freshwater snails Lymnaea stagnalis L. and Planorbarius corneus L.

Summary Antibodies were raised in rabbits against the 19000 Mr and 24000 Mr polypeptides of snail ferritin from Lymnaea stagnalis L. Anti-24000 Mr polypeptide antibodies were purified by an affinity-purification step and were made monospecific for their antigen by preabsorption with the 19000 Mr antigen. These purified antibodies were then used for in situ detection of their respective antigens by the indirect immunofluorescence method. The 19000 Mr polypeptide was found widely distributed in tissues of both pulmonate snails investigated (Lymnaea stagnalis L. and Planorbarius corneus L.) with the most intense antigen-directed fluorescence in certain connective tissue cells, secretory cells of the midgut gland and Sertoli cells and epithelia of the gonadal acini. In contrast, the 24000 Mr polypeptide could be detected only in yolk platelets of vitellogenic oocytes. The results indicate that yolk and somatic cell ferritins differ in immunoreactivity and structure and, accordingly may differ in function.This investigation was supported by the Deutsche Forschungsgemeinschaft. I greatly appreciate the advice given to me by Drs. U. Mays and V. Riedel, Münster.     

Germinal and non-germinal lines in the ovotestis of Helix aspersa: a survey

Summary The study of gonadal organogenesis and differentiation by means of light and electron microscopy suggested the following in Helix aspersa: (1) the distal parts of the acini have components of mesodermal origin, whereas the neck and efferent duct comprise ectodermal elements; (2) a segregation of a germinal line occurs early, during the embryonic life; (3) in juvenile and adult animals, male and female cells arise from a germinal ring located at the base of the acinar neck. Apart from developing oocytes, the epithelium lining the distal region of the acini consists of somatic cells (Sertoli and follicle cells).     

Spatial and temporal expression of c-Kit in the development of the murine submandibular gland

The c-Kit pathway is important in the development of many mammalian cells and organs and is indispensable for the development of hematopoiesis, melanocytes, and primordial germ cells. Loss-of-function mutations in c-Kit lead to perinatal death in mouse embryos. Previously, c-Kit has been used as one of salivary epithelial stem or progenitor cell markers in mouse, its specific temporo-spatial expression pattern and function in developing murine submandibular gland (SMG) is still unclear. Here we used quantitative real-time PCR, in situ hybridization, and immunohistochemistry analysis to detect c-Kit expression during the development of the murine SMG. We found that c-Kit was expressed in the epithelia of developing SMGs from embryonic day 11.5 (E11.5; initial bud stage) to postnatal day 90 (P90; when the SMG is completely mature). c-Kit expression in the end bud epithelium increased during prenatal development and then gradually decreased after birth until its expression was undetectable in mature acini at P30. Moreover, c-Kit was expressed in the SMG primordial cord at the initial bud, pseudoglandular, canacular, and terminal end bud stages. c-Kit was also expressed in the presumptive ductal cells adjacent to the developing acini. By the late terminal end bud stage on P14, c-Kit expression could not be detected in ductal cells. However, c-Kit expression was detected in ductal cells at P30, and its expression had increased dramatically at P90. Taken together, these findings describe the spatial and temporal expression pattern of c-Kit in the developing murine SMG and suggest that c-Kit may play roles in epithelial histo-morphogenesis and in ductal progenitor cell homeostasis in the SMG.     

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.     

Transfer of blood containing primordial germ cells between chicken eggs development of embryonic reproductive tract

The present study was carried out to investigate development of recipient chicken embryonic reproductive tracts which are transferred chicken primordial germ cells (PGCs). It is thought that differentiation of PGCs is affected by the gonadal somatic cells. When female PGCs are transferred to male embryos, it is possible that they differentiate to W-spermatogonia. However, the relationship development between PGCs and gonads has not been investigated. At stage 12–15 of incubation of fertilized eggs, donor PGCs, which were taken from the blood vessels of donor embryos, were injected into the blood vessels of recipient embryos. The gonads were removed from embryos that died after 16 days of incubation and from newly hatched chickens and organs were examined for morphological and histological features. The survival rate of the treated embryos was 13.6% for homo-sexual transfer of PGCs (male PGCs to male embryo or female PGCs to female embryo) and 28.9% for hetero-sexual transfer PGCs (male PGCs to female embryo or female PGCs to male embryo) when determined at 15 days of incubation. The gonads of embryos arising from homo-sexual transfer appeared to develop normally. In contrast, embryos derived from hetero-sexual transfer of PGCs had abnormal gonads as assessed by histological observation. These results suggest that hetero-sexual transfer of PGCs may influence gonadal development early-stage embryos.     

Development of pancreatic acini in embryos of the grass snake Natrix natrix (Lepidosauria,Serpentes)

This study report about the differentiation of pancreatic acinar tissue in grass snake, Natrix natrix, embryos using light microscopy, transmission electron microscopy, and immuno-gold labeling. Differentiation of acinar cells in the embryonic pancreas of the grass snake is similar to that of other amniotes. Pancreatic acini occurred for the first time at Stage VIII, which is the midpoint of embryonic development. Two pattern of acinar cell differentiation were observed. The first involved formation of zymogen granules followed by cell migration from ducts. In the second, one zymogen granule was formed at the end of acinar cell differentiation. During embryonic development in the pancreatic acini of N. natrix, five types of zymogen granules were established, which correlated with the degree of their maturation and condensation. Within differentiating acini of the studied species, three types of cells were present: acinar, centroacinar, and endocrine cells. The origin of acinar cells as well as centroacinar cells in the pancreas of the studied species was the pancreatic ducts, which is similar as in other vertebrates. In the differentiating pancreatic acini of N. natrix, intermediate cells were not present. It may be related to the lack of transdifferentiation activity of acinar cells in the studied species. Amylase activity of exocrine pancreas was detected only at the end of embryonic development, which may be related to animal feeding after hatching from external sources that are rich in carbohydrates and presence of digestive enzymes in the egg yolk. Mitotic division of acinar cells was the main mechanism of expansion of acinar tissue during pancreas differentiation in the grass snake embryos.     

Visualization of 'water secretion' by confocal microscopy in rat salivary glands: possible distinction of para- and transcellular pathway

Visualization of water transport in cells, tissues and organs is an important, yet still difficult, task in morphological science. By using confocal microscopy and the fluid-phase fluorescent tracer technique, we visualized water secretion and estimated the routes of water transport across the acinar epithelia in rat parotid and submandibular glands. Confocal microscopy of whole glands perfused arterially with Lucifer yellow revealed a bright fluorescence at the basolateral space of acini. Luminal space was devoid of fluorescence, but revealed it after isoproterenol pretreatment, ductal infusion of fluorescent dextrans into the lumen, or tissue dissociation by collagenase. Under these conditions, stimulation of fluid secretion with carbachol caused a rapid decline of the luminal fluorescence intensity, indicating that the secreted water washed out the fluorescent probes in the acinar lumen. In the stimulated dissociated acini, the luminal fluorescence disappeared by 15 sec, but reappeared at 30-45 sec to maintain a low plateau level. By assuming that the tight junction was 'paralyzed' by the collagenase digestion and that the paracellular fluid transport could not influence the dilution of Lucifer yellow, we estimated that the initial water secretion by CCh occurs via the transcellular pathway, while later than 30-45 sec the additional water permeates through the paracellular pathway.     

Sex reversal and aromatase in the European pond turtle: treatment with letrozole after the thermosensitive period for sex determination

In the European pond turtle (Emys orbicularis), gonadal sex differentiation is temperature-dependent. The temperature sensitive period (TSP) of gonadogenesis lies between stages 16 and 22 of embryonic development. Previous studies have shown that embryos incubated at 30 degrees C, a temperature yielding 100% phenotypic females, can be sex reversed by treatments with an aromatase inhibitor administered during TSP or even somewhat after TSP (as of stage 22+). The goal of the present study was to determine whether the ovary still retains male potential at later stages of embryonic development and whether the induced male characters persist after hatching. For this purpose, eggs of E. orbicularis were treated with letrozole, a nonsteroidal aromatase inhibitor, at or as of stages 23, 24 or 25, then gonadal aromatase activity in each individual and the related gonadal structure were studied at hatching (stage 26) and for one year after hatching. Two kinds of treatments were carried out: 1) repeated applications of 10 microg of letrozole in ethanolic solution onto the eggshell; and 2) a single injection of 10 microg of letrozole in olive oil. Similar results were obtained with either application or injection of the aromatase inhibitor. In treatments as of or at stage 23, individuals with gonadal aromatase activity lower than 20 fmoles/hour/gonad had ovotestes, i.e., 22% of the treated individuals. At hatching, the inner part of these ovotestes contained testicular cords and also mixed lacunae presenting various degrees of transdifferentiation of the epithelium into a Sertolian epithelium. The cortex was maintained, although some germ cells degenerated within it. These processes continued after hatching. However, at 12 months, gonads were still ovotestes displaying some follicles with a growing oocyte in the remaining parts of the cortex. In treatments as of or at stages 24 or 25, only a few individuals were masculinized. One had ovotestes; in others, the cortex was absent in some parts and when it was present oocytes were degenerating. These results show that in the European pond turtle, differentiation of ovotestes from ovaries can be induced by treatment with an aromatase inhibitor starting at late stages of embryonic development (between the end of TSP and hatching), although such differentiation is less frequent as embryonic development proceeds. Sex reversal persists for at least one year after hatching. J. Exp. Zool. 290:490-497, 2001.     

Haploinsufficiency of Bcl-x leads to male-specific defects in fetal germ cells: differential regulation of germ cell apoptosis between the sexes

In germ cells, the function of which is to form the next generation, apoptotic cell death occurs during development, as in the case of somatic cells. In this study, we show that Bcl-x knockout heterozygous (Bcl-x(+/-)) mice exhibit severe defects in male germ cells during development. A substantial increase in apoptosis of male germ cells occurs at around embryonic day 13.5 (E13.5) in Bcl-x(+/-) embryos, leading to hypoplasia of postnatal testes and reduced fertility. On the other hand, female germ cells at the same stages do not show discernible differences between wild-type and Bcl-x(+/-) embryos. This phenotype of Bcl-x haploinsufficiency shows that regulation of apoptosis becomes different between the sexes at around the onset of sex differentiation. Through this study, we found that, in wild-type embryos, (1) apoptosis is much more frequent (approximately 10 times) in the male than in female germ cells, and (2) expression of Bcl-xL, but not that of Bax, is higher in female than in male germ cells, at around E13.5. Male fetal germ cells, cultured with gonadal somatic cells in vitro, showed higher frequencies of apoptosis than those cultured without gonadal somatic cells. On the other hand, in the absence of gonadal somatic cells, both male and female fetal germ cells in vitro showed similar frequencies of apoptosis to female fetal germ cells in vivo. Therefore, male germ cell apoptosis, of which the default pathway is similar to that of the female, is likely to be influenced by male gonadal environments.     

A study of gonadal organogenesis,and the factors influencing regeneration following surgical castration in Deroceras reticulatum (Pulmonata:Limacidae)

Summary At hatching, the hermaphrodite duct of Deroceras reticulatum consists of a single cell type designated the Gonadal Stem Cell (GSC). Proliferation of the GSC leads to the formation of numerous ductules each of which forms one of the acini of the gonad. The germinal and supporting cells are derived entirely from the GSC. The germ cells differentiate first, followed by the Sertoli and follicle cells. At the early sperm stage of gonadal development the hermaphrodite duct differentiates to function as a seminal vesicle. Once the GSC are committed to this change they lose their regenerative ability. The only remaining GSC are the cells of the acinar epithelium, and these retain their germinal potential until the death of the animal.Regeneration will occur from the hermaphrodite duct provided it is in the immature state, i.e., composed of GSC, and is exposed to the hormonal conditions of a young animal. Nervous connections and the presence of an artery are not necessary for this regeneration. The presence of a functional gonad does not inhibit regeneration.     

In vitro adhesion of mouse fetal germ cells to extracellular matrix components

Mouse primordial germ cells (PGCs) isolated from the dorsal mesentery and gonadal ridges of 10.5–12.5 days post coitum (dpc) embryos showed a progressively increasing adhesiveness to laminin and fibronectin coated substrates, whereas type I collagen and various glycosaminoglycans (hyaluronic acid, heparin and chondroitinsulphates) were poor adhesive substrates. At later stages germ cells appeared to lose their adhesiveness to fibronectin and laminin substrates; the ability to adhere to laminin decreased very rapidly in male and slowly in female germ cells. Oocytes and prospermatogonia from 15.5 dpc fetal gonads showed poor adhesiveness to all substrates tested. PGC adhesion to laminin and fibronectin substrates did not require calcium but was markedly trypsin sensitive. Antibodies against the fibronectin receptor of CHO fibroblasts and short peptides containing the Arg-Gly-Asp sequence greatly reduced PGC adhesion to fibronectin. Following adhesion to laminin or fibronectin, most PGCs did not exhibit a morphology typical of motile cells, but remained spherical. A significant proportion (about 30%) of oocytes from 13.5–14.5 dpc embryos appeared, however, able to spread and elongate following attachment to laminin. The results support the hypothesis that mouse PGCs may utilize laminin and/or fibronectin as adhesive substrates during migration and gonad colonization, but indicate that additional factors are probably required to promote PGC motility. In addition, our data provide indirect evidence that binding sites for specific components of extracellular matrix are present in PGCs, and that their expression may be developmentally regulated.     

南方鲶性腺分化的组织学观察

用芳香化酶抑制剂(Fadrozole)、雌激素受体拮抗剂(Tamoxifen)对人工孵化的南方鲶(Silurus meridionalis)幼鱼进行雄性化诱导处理(口服),获得雄鱼。对孵化后第5—130d的南方鲶幼鱼性腺进行组织学观察,结果表明,在实验条件下,南方鲶性腺分化发生在孵化后7d左右,雌雄性分化过程差异明显。雌鱼卵巢腔在孵化后12d左右形成,生殖细胞在孵化后35d左右快速增殖,成熟分裂最早发生在孵化后55d左右;雄鱼生殖细胞在孵化后130d左右快速增殖,成熟分裂最早发生在孵化后130d左右。雌性性腺分化早于雄性。     

Evidence That the Mechanism of Prenatal Germ Cell Death in the Mouse Is Apoptosis

Using fluorescence-activated cell sorting combined with fluorescence microscopy the mechanism of embryonic germ cell death in the mouse has been shown to be apoptosis. Primordial germ cells (PGCs) from embryos at specific developmental stages have been analyzed, and cells with apoptotic morphology have been isolated by cell sorting. In the female, apoptotic oogonia at Day 13 and apoptotic oocytes at Days 15 and 17 were found. In the male, apoptotic cells were seen on Day 13 through Day 17. Apoptotic germ cells were not detected at Day 12 (combined male and female PGCs). Examination of sorted cells by fluorescence microscopy and by light microscopic analysis after alkaline phosphatase staining confirmed that the cells are apoptotic germ cells. Electron microscopy further confirmed that cells showing the morphological characteristics of apoptosis are present.