Ultrastructure of the basal lamina of bovine ovarian follicles and its relationship to the membrana granulosa

Different morphological phenotypes of follicular basal lamina and of membrana granulosa have been observed. Ten preantral follicles (< 0. 1 mm), and 17 healthy and six atretic antral follicles (0.5-12 mm in diameter) were processed for light and electron microscopy to investigate the relationship the between follicular basal lamina and membrana granulosa. Within each antral follicle, the shape of the basal cells of the membrana granulosa was uniform, and either rounded or columnar. There were equal proportions of follicles 相似文献   

A novel basal lamina matrix of the stratified epithelium of the ovarian follicle.

Basal laminas are important sheets of specialized extracellular matrix that underlie and surround groups of cells, such as epithelia or endothelia, enabling the cells to orientate their basal/apical polarity and creating a microenvironment for them. Basal laminas can also individually encapsulate whole cells, such as muscle cells, thereby forming a microenvironment but not polarizing the enclosed cells. Other mesenchymal or stromal cells exist with no basal lamina. In the course of studying the bovine follicular basal lamina which underlies the multilayered epithelium of the ovarian follicle, we identified a developmentally regulated novel extracellular matrix (which we call focimatrix for focal intra-epithelial matrix). Focimatrix is composed of basal lamina-like material deposited as plaques or aggregates between the multilayers of the epithelial granulosa cells. The focimatrix does not encapsulate individual or groups of cells and therefore does not form a microenvironment for them. Focimatrix contains collagen type IV subunits alpha1 and alpha2 (but not alpha3-alpha6), and laminin chains alpha1, beta2 and gamma1 (but not alpha2 or beta1), and nidogen-1 and perlecan (but not versican). The amount of focimatrix increases with increasing follicular size, and its appearance precedes the expression by granulosa cells of the enzymes for steroid hormone synthesis, cholesterol side-chain cleavage cytochrome P450 (SCC) and 3beta-hydroxysteroid dehydrogenase (3beta-HSD), in the days preceding ovulation. The expression in granulosa cells of two components examined, nidogen-1 and perlecan, also increases substantially when follicles enlarge to a sufficient size capable of ovulating. Following ovulation the follicular basal lamina is degraded, and presumably focimatrix is too since it is not detected in corpora lutea that develop from the ovulating follicles. During this development the granulosa cells undergo an epithelial-mesenchymal transition (EMT) into luteal cells following ovulation, and substantially increase their expression of steroidogenic enzymes in the process. During EMT epithelial cells lose polarity. Since focimatrix exists on more than one side of the granulosa cells, we propose that it disrupts the polarity induced by the follicular basal lamina in the lead up to ovulation. Hence focimatrix maybe a key part of the follicular/luteal EMT.     

The fine structure of the cumulus oophorus during follicular development in sheep

Summary The cumulus and membrana granulosa of non-atretic ovarian follicles from primordial up to a stage shortly before ovulation were studied by electron microscopy.The follicular cells of primordial follicles were undifferentiated and rested on a thick basal lamina. In secondary follicles the endoplasmic reticulum had proliferated forming an anastomosing network. In early antral and antral follicles (0.5–2.0 mm dia.) the ER was composed of short cisternae, the mitochondria had elongated and gap junctions were first observed. In late antral follicles (3.0–5.9 mm dia.) gap junctions were frequent. In the cumulus the glycogen was associated with electron lucent areas whereas in the granulosa it was invariably associated with membranes. In large antral follicles large membrane bound bodies were present in the basal cells of the cumulus. At early oestrus a distinctive mitochondrial morphology was noted in the granulosa but not elsewhere in the follicles. At mid oestrus numerous annular nexuses were present in the granulosa but not in the cumulus. At late oestrus numerous lipid droplets were formed in both cumulus and granulosa, the boundary with theca interna became indistinct and the basal lamina became incomplete.Deceased     

Remodeling of extracellular matrix at ovulation of the bovine ovarian follicle

Using immunohistochemistry and RNA analyses we examined the fate of components of a newly identified matrix that develops between granulosa cells (focimatrix, abbreviated from focal intraepithelial matrix) and of the follicular basal lamina in ovulating bovine ovarian follicles. Pre- and postovulatory follicles were generated by treatment with estradiol (Day 1), progesterone (Days 1-10), and prostaglandin analogue (Day 9) with either no further treatment (Group 1, n = 6) and or with 25 mg porcine LH (Day 11, Group 2, n = 8 or Day 10, Group 3, n = 8) and ovariectomy on Day 12 (12-14 hr post LH in Group 2, 38-40.5 hr in Group 3). In the time frame examined no loss of follicular basal lamina laminin chains beta2 and gamma1 or nidogen 1 was observed. In the follicular basal lamina collagen type IV alpha1 and perlecan were present prior to ovulation; after ovulation collagen type IV alpha1 was discontinuously distributed and perlecan was absent. Versican in the theca interna adjacent to the follicular basal lamina in preovulatory follicles was not observed post ovulation, however, the granulosa cells then showed strong cytoplasmic staining for versican. Expression of versican isoforms V0, V1, and V3 was detected at all stages. Focimatrix was observed in preovulatory follicles. It contained collagen type IV alpha1, laminins beta2 and gamma1, nidogen 1 and perlecan and underwent changes in composition similar to that of the follicular basal lamina. In conclusion focimatrix and the follicular basal lamina are degraded at ovulation. Individual components are lost at different times.     

A New Model of Development of the Mammalian Ovary and Follicles

Ovarian follicular granulosa cells surround and nurture oocytes, and produce sex steroid hormones. It is believed that during development the ovarian surface epithelial cells penetrate into the ovary and develop into granulosa cells when associating with oogonia to form follicles. Using bovine fetal ovaries (n = 80) we identified a novel cell type, termed GREL for Gonadal Ridge Epithelial-Like. Using 26 markers for GREL and other cells and extracellular matrix we conducted immunohistochemistry and electron microscopy and chronologically tracked all somatic cell types during development. Before 70 days of gestation the gonadal ridge/ovarian primordium is formed by proliferation of GREL cells at the surface epithelium of the mesonephros. Primordial germ cells (PGCs) migrate into the ovarian primordium. After 70 days, stroma from the underlying mesonephros begins to penetrate the primordium, partitioning the developing ovary into irregularly-shaped ovigerous cords composed of GREL cells and PGCs/oogonia. Importantly we identified that the cords are always separated from the stroma by a basal lamina. Around 130 days of gestation the stroma expands laterally below the outermost layers of GREL cells forming a sub-epithelial basal lamina and establishing an epithelial-stromal interface. It is at this stage that a mature surface epithelium develops from the GREL cells on the surface of the ovary primordium. Expansion of the stroma continues to partition the ovigerous cords into smaller groups of cells eventually forming follicles containing an oogonium/oocyte surrounded by GREL cells, which become granulosa cells, all enclosed by a basal lamina. Thus in contrast to the prevailing theory, the ovarian surface epithelial cells do not penetrate into the ovary to form the granulosa cells of follicles, instead ovarian surface epithelial cells and granulosa cells have a common precursor, the GREL cell.     

Cholesterol side-chain cleavage cytochrome P450 and 3beta-hydroxysteroid dehydrogenase expression and the concentrations of steroid hormones in the follicular fluids of different phenotypes of healthy and atretic bovine ovarian follicles

Bovine ovarian antral follicles exhibit either one or the other of two patterns of granulosa cell death in atresia. Death can commence either from the antrum and progress toward the basal lamina (antral atresia) or the converse (basal atresia). In basal atresia, the remaining live antrally situated cells appeared to continue maturing. Beyond that, little is known about these distinct patterns of atresia. Healthy (nonatretic) follicles also exhibit either one or the other of two patterns of granulosa cell shape, follicular basal lamina ultrastructure or location of younger cells within the membrana granulosa. To examine these different phenotypes, the expression of the steroidogenic enzymes cholesterol side-chain cleavage cytochrome P450 (SCC) and 3beta-hydroxysteroid dehydrogenase (3beta-HSD) in granulosa cells and concentrations of steroid hormones in follicular fluid were measured in individual histologically classified bovine antral follicles. Healthy follicles first expressed SCC and 3beta-HSD in granulosa cells only when the follicles reached an approximate threshold of 10 mm in diameter. The pattern of expression in antral atretic follicles was the same as healthy follicles. Basal atretic follicles were all <5 mm. In these, the surviving antral granulosa cells expressed SCC and 3beta-HSD. In examining follicles of 3-5 mm, basal atretic follicles were found to have substantially elevated progesterone (P < 0.001) and decreased androstenedione and testosterone compared to healthy and antral atretic follicles. Estradiol was highest in the large healthy follicles, lower in the small healthy follicles, lower still in the antral atretic follicles, and lowest in the basal atretic follicles. Our findings have two major implications. First, the traditional method of identifying atretic follicles by measurement of steroid hormone concentrations may be less valid with small bovine follicles. Second, features of the two forms of follicular atresia are so different as to imply different mechanisms of initiation and regulation.     

The physiology of the ovary: Maturation of ovarian granulosa cells and a novel role for antioxidants in the corpus luteum

During folliculogenesis the granulosa cells divide whilst in contact with each other, and so exhibit some of the characteristics of stem cells. In vitro we have shown that bovine granulosa cells from 3–7 mm follicles, like stem cells, divide without the need for a substratum, and produce colonies of cells. Growth factors, bFGF and IGF's, stimulate their division. These cells secrete and assemble a basal lamina, suggesting that the follicular basal lamina is produced by the granulosa cells. They have the morphological characteristics of follicular granulosa cells. Thus this system is ideal for studying the functions of immature granulosa cells because the cells do not spontaneously differentiate or luteinize into luteal cells, as occurs in culture on a substratum. On differentiation into luteal cells in vivo the cells express the steroidogenic enzymes for progesterone production and accumulate β-carotene. During culture of bovine luteal cells we observed that a proportion of the steroidogenic enzyme cholesterol side-chain cleavage cytochrome P450 enzyme became chemically cross-linked to its electron donor, adrenodoxin. P450 enzymes produce oxygen free radicals and oxygen free radicals can cause cross-linking between proteins in close proximity. Cell protect against this damage by the use of antioxidant vitamins. Repleting the cultured luteal cells with β-carotene reduced the amount of cross-linking. We conclude that the high levels of β-carotene in corpora lutea are to protect against damage due to oxygen free radicals generated in the course of progesterone synthesis.     

Effect of basal lamina on progesterone production by chicken granulosa cells in vitro — influence of follicular development

Experiments were conducted in vitro to study the regulation of progesterone production in chicken granulosa cells by homologous basal lamina isolated from preovulatory follicles of chicken ovary. The majority of components of the basal lamina (90–95% by weight) were solubilized with guanidine-HCl (and designated fraction 1); the remaining components were solubilized with β-mercaptoethanol containing guanidine-HCl (and designated fraction 2). The ability of fraction 1 to regulate progesterone production in granulosa cells obtained from the largest (F₁, mature), third largest (F₃, growing), fifth to seventh largest (F_5–7, growing) follicles and a pool of small yellow follicles (SYF, immature) of chicken ovary was assessed. Granulosa cells isolated from SYF follicles were in the least differentiated (undifferentiated) and those obtained from F₁ follicles were in the most differentiated state. The ability of fraction 1 to regulate progesterone production by chicken granulosa cells was influenced both by the state of cell differentiation and the form of the matrix material (whether solid or liquid). When fraction 1 was added as liquid to the incubation mixture, it promoted progesterone production by granulosa cells at all stages of differentiation; however, it caused a greater relative increase in the amount of progesterone produced by undifferentiated (SYF) and differentiating (F₃) granulosa cells than by differentiated (F₁) ones. In the presence of the liquid-form of fraction 1, luteinizing hormone (LH) stimulated progesterone production in differentiated (F₁) and differentiating (F_5–7) granulosa cells. Similarly, follicle-stimulating hormone (FSH) stimulated progesterone production by differentiating (F₃) and undifferentiated (SYF) granulosa cells in the presence of the liquid-form of fraction 1 protein. In culture wells that had been pre-coated with fraction 1 (solid-form), progesterone production by less differentiated (SYF, F_5–7) granulosa cells was enhanced, whereas progesterone production by differentiated (F₁) cells was reduced. The solid-form of fraction 1 augmented LH-stimulated progesterone production by less differentiated (F_5–7) granulosa cells however, it attenuated LH-induced progesterone production in differentiated (F₁) cells. FSH-promoted progesterone production in granulosa cells from immature follicles (SYF) was augmented by solid-form of fraction 1 whereas the effect of FSH on cells obtained from older follicle (F₃) was suppressed by solid-form of fraction 1. In experiments in which gonadotropin action was attenuated by solid-form of fraction 1, the amount of progesterone produced in the presence of maximally inhibiting concentrations of fraction 1 protein was greater than control values (no fraction 1, no gonadotropin). These results show that basal lamina of the ovarian follicle can regulate progesterone production by granulosa cells. The data demonstrate that the interactions between the components of basal lamina and LH or FSH on granulosa cell function were dependent on the stage of follicular development and were influenced by the form of the matrix material. It is concluded that the basal lamina of the chicken ovarian follicle is biologically active and regulates granulosa cell function.     

Ruthenium red staining and tannic acid fixation of dental basement membrane

Summary Ruthenium red staining and tannic acid fixation were used to analyse the fine structure of embryonic mouse dental basement membrane in intact first mandibular molars or in EDTA-isolated dental papillae. Preameloblasts are separated from extracellular matrix proper by a basal lamina that contains regularly arranged proteoglycan granules of about 10 nm in diameter. This distribution pattern is particularly evident in the inner and outer lamina rara of the basal lamina associated with EDTA-isolated dental papillae. The plasmalemma of preameloblasts demonstrates electron dense plaques on the inner leaflet. Ruthenium red positive granules (50 nm in diameter) coat non-striated and striated fibrils of the matrix. Hyaluronidase treatment digested the ruthenium red positive granules. Tannic acid fixation allowed the demonstration of filaments within the lamina rara interna, connecting the lamina densa with plasmalemma of preameloblasts. These observations are discussed in the context of the terminal differentiation of odontoblasts.These studies were supported by INSERM, grant n 537785 and DGRST     

Identification and immunolocalization of decorin, versican, perlecan, nidogen, and chondroitin sulfate proteoglycans in bovine small-antral ovarian follicles

Proteoglycans (PGs) consist of a core protein and attached glycosaminoglycans (GAGs) and have diverse roles in cell and tissue biology. In follicles PGs have been detected only in follicular fluid and in cultured granulosa cells, and the composition of their GAGs has been determined. To identify PGs in whole ovarian follicles, not just in follicular fluid and granulosa cells, small (1-3-mm) bovine follicles were harvested. A proportion of these was incubated with (35)SO(4) for 24 h to incorporate radiolabel into the GAGs. The freshly harvested and cultured follicles were sequentially extracted with 6 M urea buffer, the same buffer with 0.1% Triton X-100 and then with 0.1 M NaOH. Proteoglycans were subjected to ion-exchange and size-exclusion chromatography. The GAGs were analyzed by chemical and enzymic digestion, and on the basis of their composition, we chose a list of known PGs to measure by ELISA analyses. Versican, perlecan, decorin, but not aggrecan or biglycan, were identified. These, excluding decorin for technical reasons, as well as a basal lamina glycoprotein, nidogen/entactin, were immunolocalized. Versican was localized to the thecal layers, including externa and the interna particularly in an area adjacent to the follicular basal lamina. Perlecan and nidogen were localized to the follicular basal lamina of antral follicles, both healthy and atretic, but not to that of preantral follicles. Both were localized to subendothelial basal laminas, but the former was not readily detected in arteriole smooth muscle layers. This study has confirmed the presence of versican and perlecan, but not the latter as a component of follicular fluid, and identified decorin and nidogen in ovarian antral follicles.     

Basal lamina of avian ovarian follicle: influence on morphology of granulosa cells in-vitro

Experiments were conducted to determine the influence of basal lamina on the morphology of ovarian granulosa cells in vitro. Pure and intact basal lamina was isolated from the large preovulatory follicles of the chicken ovary and designated basal lamina of avian ovarian follicle (BLAOF). Examination of the isolated basal lamina with electron microscope revealed an ultrastructure that is similar to that of basal lamina in the intact ovarian follicle. Pieces of the intact basal lamina were attached to the bottom of 32 mm culture dishes (BLAOF-coated dishes) in which differentiated granulosa cells isolated from the largest preovulatory follicle or undifferentiated granulosa cells isolated from immature small yellow chicken ovarian follicles were cultured; uncoated dishes served as controls. Granulosa cells incubated on intact basal lamina assumed spherical shape, whereas granulosa cells incubated directly on plastic in control dishes became highly flattened. Interestingly, granulosa cells that attached to plastic close to BLAOF (in BLAOF-containing dishes) became rounded. The storage of BLAOF-coated culture dishes at 4°C for 2 years had no apparent effect on its ability of the matrix material to induce changes in granulosa cell shape. Some components of the basal lamina could be solubilized with guanidine–HCl alone (fraction 1; 90–95% of total protein in BLAOF) with the remaining components solubilized with β-mercaptoethanol containing guanidine–HCl (fraction 2; 5–10% of total protein in BLAOF). Differentiated and undifferentiated chicken granulosa cells became rounded when incubated in fraction 1-pre-coated wells; whereas those incubated directly on plastic in control wells were flattened. Similarly, when fraction 1 of solubilized basal lamina was added as liquid to incubation mixture, it caused both differentiated and undifferentiated granulosa cells to assume spherical shapes. The storage of fraction 1-coated culture dishes at 4°C for 12 or more months had no apparent effect on its ability to influence granulosa cell shape. Fraction 1-induced changes in granulosa cell shape were similar to those observed for complete and intact basal lamina (BLAOF). These findings demonstrate that intact homologous basal lamina (BLAOF) or its solubilized (fluidized) form can induce normal (in vivo) morphology in granulosa cells. It is suggested that BLAOF or its solubilized form can be used to culture cells in experiments designed to examine the influence of the natural basal lamina microenvironment on cellular behavior and function.     

Ultrastructure and composition of Call-Exner bodies in bovine follicles

Call-Exner bodies are present in ovarian follicles of a range of species including human and rabbit, and in a range of human ovarian tumors. We have also found structures resembling Call-Exner bodies in bovine preantral and small antral follicles. Hematoxylin and eosin staining of single sections of bovine ovaries has shown that 30% of preantral follicles with more than one layer of granulosa cells and 45% of small (less than 650 microns) antral follicles have at least one Call-Exner body composed of a spherical eosinophilic region surrounded by a rosette of granulosa cells. Alcian blue stains the spherical eosinophilic region of the Call-Exner bodies. Electron microscopy has demonstrated that some Call-Exner bodies contain large aggregates of convoluted basal lamina, whereas others also contain regions of unassembled basal-lamina-like material. Individual chains of the basal lamina components type IV collagen (alpha 1 to alpha 5) and laminin (alpha 1, beta 2 and delta 1) have been immunolocalized to Call-Exner bodies in sections of fresh-frozen ovaries. Bovine Call-Exner bodies are presumably analogous to Call-Exner bodies in other species but are predominantly found in preantral and small antral follicles, rather than large antral follicles. With follicular development, the basal laminae of Call-Exner bodies change in their apparent ratio of type IV collagen to laminin, similar to changes observed in the follicular basal lamina, suggesting that these structures have a common cellular origin.     

Focal cell contacts detected by ruthenium red, triton X100 and saponin in the granulosa cells of mouse ovary

Granulosa cells in growing follicles of mouse ovary, observed after treatment with ruthenium red (RR) as described by Luft (1971a, b), appeared to be covered by a continuous well-defined layer. On the contrary, treating granulosa cells with 1% Triton X100 (Vaccaro and Brody, 1981), followed by RR staining, resulted in the complete extraction of the plasma membrane coat (Triton does not affect the basement membrane and extracellular matrix proteoglycans). The use of 0.02% saponin together, with the RR stain, or 0.1% Triton X100 followed by RR staining, allows good visualization of follicular basement membrane and extracellular matrix proteoglycans without destroying cell morphology. Using this technique, we observed the extraction of the plasma membrane coat, but focal RR-stained condensations that were unaffected by saponin or 0.1% Triton X100 treatment were observed between plasma membranes of granulosa cells located around the periphery of large Graafian follicles. In some cases, RR condensations were located at the apex of plasmalemmal evaginations, in proximity to adjacent granulosa cells. Focal condensations of RR stain were never observed in secondary follicles. Present evidence suggests that focal cell contacts are mediated by transmembrane intercalated glycoproteins or proteoglycans and consequently play a role in cell adhesion. Their presence among granulosa cells of only very large Graafian follicles may be related to the maturation process of granulosa cells.     

Extracellular matrix in ovarian follicular development and disease

The ovarian follicle contains several different cell types and separate compartments and undergoes substantial development during its growth and maturation. Extracellular matrix (ECM) could be expected to play a major role in these processes. Most research on ECM in follicles has focused on the follicular basal lamina and its changing composition during folliculogenesis and on the specialised matrix formed at ovulation by the cumulus cells surrounding the oocyte and the zona pellucida. We review these aspects. Few naturally occurring gene mutations have identified unique roles for ECM molecules in follicular function. Presumably, any mutations leading to reduced fertility are eliminated quickly by natural selection and, when mutations are not eliminated, considerable redundancy occurs to ensure successful reproduction. In mice, in which the genome can be easily manipulated, the modification of matrix components associated with cumulus and oocytes has often resulted in partial infertility, suggesting redundancy. We provide an update of basal lamina components focusing on newer discoveries. In addition, we review matrix associated with the occyte and cumulus cells (excluding the zona pellucida) and other components of ECM. Where possible, we examine evidence for the role of the ECM in follicular development and diseases.Research in the authors' laboratories has been supported by the National Health and Medical Research Council of Australia, The University of Adelaide, Wellcome Foundation, and the Clive and Vera Ramaciotti Foundation.     

Role of Thy-1+ and Ia+ cells in ovarian function

Cryostat sections of anovulatory ovaries from persistent estrous rats following a single postnatal dose of testosterone and from persistent diestrous rats following long-term postnatal estradiol treatment were investigated. The indirect immunoperoxidase technique was used to localize ovarian Thy-1 and Ia glycoproteins, as well as several other cell surface markers, and the results were compared with those obtained in normal ovaries of cycling females. Folliculogenesis in persistent estrous rats proceeded up to cystic antral follicles and was associated with the occurrence of Thy-1+ stromal cells under follicular basal lamina. In contrast to normal ovaries, Thy-1+ material did not invade the basal layers of granulosa cells. There was also no association of Ia+ cells with follicular basal lamina, but Ia+ cells were usually found associated with some thecal vessels. In persistent diestrous rats folliculogenesis was significantly retarded in both advanced antrum formation and thecal development. Thy-1+ cells were usually present in theca. No Thy-1+ material was found among basal layers of granulosa cells and the depletion of thecal Ia+ cells was almost complete. We suggest that normal follicular development may be dependent on the correct effects of Thy-1+ and Ia+ cells in addition to appropriate gonadotropin and steroid stimulation. On the other hand, anovulatory syndromes following postnatal androgen or estrogen treatment might be induced by temporary direct ovarian effects disturbing the establishment of the normal relationship between follicular structures and the immune system.     

A correlation between ovarian follicular maturity and anchorage-independent growth of bovine granulosa cells

Previous studies have shown that a subset of bovine ovarian granulosa cells can proliferate to form clones of functional cells in suspension in a semisolid agar matrix, without the requirement for attachment to the substratum. These clonogenic anchorage-independent granulosa cells are responsive to epidermal growth factor and exhibit properties of a primitive progenitor cell population. We have used this assay system to analyze the proliferation of granulosa cells during ovarian follicular maturation. As the follicle increases in size, there is a progressive decline in the ability of granulosa cells to clone in agar, and the proliferative potential of these cells as measured by colony size also decreases. The ratio of large colonies with high proliferative potential (greater than 250 micron in diameter) to small colonies with limited capacity for growth falls from 1.92 in follicles less than 7 mm in diameter, to 0.32 in follicles larger than 10 mm in diameter. This occurs as the follicular content of granulosa cells with more limited capacity for clonal growth in agar undergoes expansion. Analysis of colony-forming cells in follicles harvested at early and late estrus suggests that these cells are regulated by complex intraovarian factors rather than circulating gonadotropin levels. Our data indicate that the granulosa cell lineage is an age-structured continuum of maturing and differentiating cells with a progressively restricted proliferative capacity.     

Cytoskeleton reorganization mediates alpha6beta1 integrin-associated actions of laminin on proliferation and survival,but not on steroidogenesis of ovine granulosa cells

Background  

Laminin (LN) is one of the most abundant extracellular matrix components of the basal lamina and granulosa cell layers of ovarian follicles. Culture of ovine granulosa cells (GC) on LN substratum induces cell spreading, enhances cell survival and proliferation, and promotes luteinization. Previous investigations have shown that these effects are mostly mediated by the alpha6beta1 integrin, but its signalization pathways have not been investigated. This study aimed to assess the importance of the cytoskeleton in the alpha6beta1 integrin-mediated actions of laminin on survival, proliferation and steroidogenesis of ovine GC.     

Ultrastructural studies on developing follicles of the spotted ray Torpedo marmorata.

Light and ultrastructural investigations on sub-adult and adult sexually mature females, demonstrates that in Torpedo marmorata folliculogenesis starts in the early embryo and that the two ovaries in the adult contain developing follicles of various sizes and morphology. Initially, the follicle is constituted by a small oocyte, surrounded by a single layer of squamous follicle cells. The organization is completed by a basal lamina and, more externally, by a theca, that at this stage is composed by a network of collagen fibers. As the oocyte growth goes on, during previtellogenesis and vitellogenesis, the organization of the basal lamina and of the oocyte nucleus does not change significantly. The basal lamina, in fact, remains acellular and constituted by fibrils intermingled in an amorphous matrix; the nucleus always shows an extended network of chromatin due to the lampbrush chromosomes, and one or two large nucleoli. By contrast, the granulosa (or follicular epithelium), the ooplasm, and the theca cells significantly change. The granulosa shows the most relevant modifications becoming multi-layered and polymorphic for the progressive appearance of intermediate and pyriform-like cells, located respectively next to the vitelline envelope, or spanning the whole granulosa. The appearance of intermediate cells follows that of intercellular bridges between small follicle cells and the oocyte so that one can postulate that, as in other vertebrates, small cells differentiate into intermediate, and then pyriform-like cells, once they have fused their plasma membrane with that of the oocyte. Regarding the ooplasm, one can observe as in previtellogenic follicles, it is characterized by the presence of intermediate vacuoles containing glycogen, while in vitellogenic follicles by an increasing number of yolk globules. The theca also undergoes significant changes: initially, it is constituted by a network of collagen fibers, but later, an outermost theca esterna containing cuboidal cells and an interna, with flattened cells, can be recognized. The role of the different constituents of the ovarian follicle in the oocyte growth is discussed.     

Local regulation of granulosa cell maturation

Fluid from small antral follicles inhibits several functions of porcine granulosa cells from 3-10-mm follicles in vitro, whereas fluid from large follicles stimulates cells from small follicles. Local factors may be needed in vivo to enable granulosa cells to fully respond to gonadotrophins. Only those follicles containing local stimulators may develop while those containing inhibitors may become arrested in development or become atretic. We have compared the actions of GnRH analogs and chondroitin sulfate (CS) on porcine granulosa cell steroidogenesis with actions of follicular fluids. GnRH agonist mimicked follicular fluid inhibition of progesterone secretion but GnRH antagonist did not antagonize follicular fluid's inhibitory actions. GnRH antagonist mimicked follicular fluid enhancement of basal and LH-stimulated progesterone secretion, but did not mimic follicular fluid enhancement of FSH action or stimulation of estrogen secretion. GnRH agonist blocked the enhancement of LH-stimulated progesterone secretion by both GnRH antagonist and stimulatory follicular fluid. CS inhibited basal and LH-stimulated progesterone secretion but did not inhibit pregnenolone utilization, aromatase activity or estrogen secretion. GnRH-like molecules and CS may be partially responsible for follicular fluid actions on granulosa cells. The actions of other molecules are needed to explain the total effects of follicular fluids on granulosa cells.     

Evidence for exclusive adrenergic innervation of feather muscles (mm. pennati) in the chicken

Summary Feather follicles in the avian skin are interconnected by well-defined bundles of smooth muscle cells, which are responsible for the erection and depression of feathers and thus play an important role in thermoregulation. The depressing and erecting muscle bundles were found to receive a very dense supply of unmyelinated nerve fibres that displayed ultrastructural and histochemical characteristics of noradrenergic axons (formaldehyde- and glyoxylic acid-induced catecholamine fluorescence; uptake to 5-hydroxydopamine). No nerve fibres were encountered showing histochemical acetylcholinesterase activity. There was no indication of the presence of peptidergic or purinergic nerve endings.The neuromuscular space usually ranged from 40–60 nm in width and contained a basal lamina. Occasionally, this space was reduced to approximately 20 nm. At such close neuromuscular contacts a basal lamina was lacking, and focal densities beneath the pre- and postsynaptic plasma membrane were observed. Since no gap junctions between muscle cells were detected, the dense supply with noradrenergic nerve fibres indicates a high amount of directly innervated smooth muscle cells.An additional finding of the present study was the observation that high local concentrations of 5-hydroxydopamine led to degeneration of noradrenergic nerve endings.Supported by a grant from the Deutsche Forschungsgemeinschaft (Dr. 91)