Histochemical Study of the Rat Uterine Glycoconjugate Alteration following Treatment with Exogenous Gonadotropic Hormones during the Implantation Period

One of the female causes of infertility is anovulation which is treatable with gonadotropin hormones. These hormones affect the molecular organization of the uterus such as glycoconjugates that are the first site of contact between the blastocyst and the uterus. The objective of this project was to study the alteration of glycoconjugates on the uterine apical, Golgi zone, and basement membrane of epithelial cells and the uterine gland after hyperstimulation with pregnant mare serum gonadotropin (PMSG) (4, 8, 16, 24, and 40 IU), during the implantation period. Injection of PMSG (in experimental groups) and injection of distilled water (in the control group) were followed by HCG administration (10 IU), mating, isolation of positive vaginal plug rats, and killing at 5.5 days of pregnancy. Histochemistry was done on the pregnant uterine horns with the use of WGA, DBA, PNA, ConA, SBA, and UEA lectins. The intensity of the immunohistochemical staining was scored, and quantitative data were generated. 4 IU did not show any significant differences with the control, 8 IU had less effect on the alteration of the Golgi zone, and apical and basement membrane glycoconjugates and 40 IU had the least effects on the alteration of uterine gland glycoconjugates. Also, 24 IU had the most effect on the alteration of uterine glycoconjugates. Understanding of the effects of gonadotropin hormones at the uterine level in implantation time helps to optimize hormonal manipulation for improving the outcome of assisted reproductive procedures. It seems that the optimal dose for superovulation and less alteration in uterine glycoconjugates of rats at implantation time were induced by the administration of 8 IU PMSG.

Some ascites monoclonal antibody preparations contain contaminants that bind to selected Golgi zones or mast cells.

A small proportion of mouse ascites fluid induced by hybridomas producing monoclonal antibodies or myelomas secreting immunoglobulin yielded staining that was confined to the Golgi zone of certain epithelial cell types in rats and gerbils but not in mice. In addition, a commercial IgG fraction from mouse plasma similarly labeled the Golgi area, unlike IgG from mouse serum from another source. Culture supernatant from one hybridoma line contrasted with ascites fluid produced by the same hybridoma in failing to stain the Golgi region. The capacity of a fluid to react with the Golgi cisternae bore no relationship to the class of immunoglobulin secreted by the hybridoma or myeloma. Absorption of an ascites fluid with blood group A1 human erythrocytes eliminated its affinity for Golgi cisternae. Adsorption with blood group A2 or B or two type O cells used for screening for blood group antibodies had no effect on Golgi zone labeling by this ascites fluid. The positive cells included most serous secretory cells in rats, serous cells of sublingual and tracheal glands, and some endometrial and oviduct-lining cells in gerbils, and columnar lining cells of small intestine and cecum and all or part of the lining cells in some prostate lobes in both genera. Some of the tested ascites fluids stained mast cells. The agent accounting for mast cell labeling differed, however, from that reacting with Golgi cisternae in its distribution among the mouse ascites fluids examined, lack of relationship to the ABO blood group system, occurrence additionally in normal rat serum, and capacity to stain cells in mice as well as rats and gerbils.

Rapid and transient reorganization of the cytoskeleton in GH3B6 cells during short-term exposure to thyroliberin

The cytoskeletal organization of the rat pituitary tumor cell line GH3B6 was analysed using immunofluorescence, in basal conditions and after stimulation by thyroliberin (TRH). Under basal conditions, a dense and entangled cytoplasmic microtubule network, a perinuclear cage of cytokeratin fibers, and a diffuse distribution of F-actin were revealed. Short-term stimulation of these cells by TRH induces a first early phase of PRL release (0–2 min), concomitant with a rarefaction of cytoplasmic PRL-containing granules, followed by a second plateau phase (5–30 min), concomitant with modifications of the Golgi zone. We show that TRH induced early and transient modifications in the cytoskeletal distribution during these short periods of stimulation. First, after 2 min of stimulation, small fluorescent tubulin blebs appeared under the plasma membrane. Then, after 5 min they disappeared, and a thin actin network, accentuated by thicker fibers, organized transiently in the cytoplasm. After 30 min, the microtubules and cytokeratin networks had extended throughout the cytoplasm and the actin distribution was diffuse again. So, in this study, we have shown the existence of a parallelism between the redistribution of intracellular PRL compartments and the reorganization of cytoskeletal elements, during exposure to TRH. We could not clearly correlate these modifications with transduction mechanisms involved in TRH action.

Ultrastructural demonstration of tubular inclusions coinciding with von Willebrand factor in pig megakaryocytes

The appearance of von Willebrand factor (vWF) in bone marrow megakaryocytes was studied by standard electron microscopy (EM) and immuno-EM using an original purification technique. Eighty percent pure megakaryocytes were isolated from porcine rib bone marrow using Percoll gradients followed by counterflow centrifugation. Activation was prevented by prostacyclin and prefixation with low concentrations of glutaraldehyde. In early megakaryoblasts, standard EM revealed the presence of tubular structures in the small vesicles located in the Golgi area, in the small immature alpha-granules and in the rare mature alpha-granules. Immunolabeling for vWF was simultaneously observed in small vesicles and small alpha-granules, mainly in the Golgi zone. In mature megakaryocytes, standard EM showed that tubular structures were numerous, regularly spaced, and aligned in parallel. Immunolabeling for vWF was intense, restricted to the alpha-granules, and distributed in a similar manner to porcine platelets. Gold particles were located eccentrically at one pole of the alpha-granule, labeling only its periphery or outlining one side of an elongated granule. Tubule profiles could be seen underlying the immunolabeling and were usually located at one side of the granule. In conclusion, this study demonstrates the presence of tubular structures in megakaryocyte alpha- granules, their association with vWF, and the appearance of both in the Golgi-associated vesicles.

Ultrastructural demonstration of tubular inclusions coinciding with von Willebrand factor in pig megakaryocytes

The appearance of von Willebrand factor (vWF) in bone marrow megakaryocytes was studied by standard electron microscopy (EM) and immuno-EM using an original purification technique. Eighty percent pure megakaryocytes were isolated from porcine rib bone marrow using Percoll gradients followed by counterflow centrifugation. Activation was prevented by prostacyclin and prefixation with low concentrations of glutaraldehyde. In early megakaryoblasts, standard EM revealed the presence of tubular structures in the small vesicles located in the Golgi area, in the small immature alpha-granules and in the rare mature alpha-granules. Immunolabeling for vWF was simultaneously observed in small vesicles and small alpha-granules, mainly in the Golgi zone. In mature megakaryocytes, standard EM showed that tubular structures were numerous, regularly spaced, and aligned in parallel. Immunolabeling for vWF was intense, restricted to the alpha-granules, and distributed in a similar manner to porcine platelets. Gold particles were located eccentrically at one pole of the alpha-granule, labeling only its periphery or outlining one side of an elongated granule. Tubule profiles could be seen underlying the immunolabeling and were usually located at one side of the granule. In conclusion, this study demonstrates the presence of tubular structures in megakaryocyte alpha- granules, their association with vWF, and the appearance of both in the Golgi-associated vesicles.

Effect of monensin on secretory pathway in GH3 prolactin cells. A cytochemical study.

The effects of the carboxylic ionophore monensin have been studied on a rat prolactin cell line (GH3 cells) under basal conditions or after acute stimulation by thyrotropin-releasing hormone (TRH). It was found that 1) monensin induces a rapid dilatation of Golgi elements in these endocrine cells; 2) secretory product, prolactin, is localized by electron microscope immunocytochemistry attached to the inner face of the membrane of these dilated vacuoles; 3) monensin induces preferentially a dilatation of the cis face of the Golgi zone, since the "GERL" complex identified by acid phosphate cytochemistry is disorganized or fragmented rather than vacuolized; and 4) monensin decreases strongly the basal release of prolactin in the culture medium but does not prevent the stimulating effect of TRH on this release. This suggests that monensin blocks preferentially the pathway of release of secretory product under basal conditions in GH3 cells but that another pathway less sensitive to monensin is involved under acute stimulation by TRH.

The rough endoplasmic reticulum and the Golgi apparatus visualized using specific antibodies in normal and tumoral prolactin cells in culture.

Antibodies directed against membrane components of dog pancreas rough endoplasmic reticulum (A-RER) and rat liver Golgi apparatus (A-Golgi) (Louvard, D., H. Reggio, and G. Warren, 1982, J. Cell Biol. 92:92-107) have been applied to cultured rat prolactin (PRL) cells, either normal cells in primary cultures, or clonal GH3 cells. In normal PRL cells, the A-RER stained the membranes of the perinuclear cisternae as well as those of many parallel RER cisternae. The A-Golgi stained part of the Golgi membranes. In the stacks it stained the medial saccules and, with a decreasing intensity, the saccules of the trans side, as well as, in some cells, a linear cisterna in the center of the Golgi zone. It also stained the membrane of many small vesicles as well as that of lysosomelike structures in all cells. In contrast, it never stained the secretory granule membrane, except at the level of very few segregating granules on the trans face of the Golgi zone. In GH3 cells the A-RER stained the membrane of the perinuclear cisternae, as well as that of short discontinuous flat cisternae. The A-Golgi stained the same components of the Golgi zone as in normal PRL cells. In some cells of both types the A-Golgi also stained discontinuous patches on the plasma membrane and small vesicles fusing with the plasma membrane. Immunostaining of Golgi membranes revealed modifications of membrane flow in relation to either acute stimulation of PRL release by thyroliberin or inhibition of basal secretion by monensin.

Immunocytochemical localization of procollagen and fibronectin in human fibroblasts: effects of the monovalent ionophore, monensin.

The monovalent ionophore monensin inhibits the secretion of both procollagen and fibronectin from human fibroblasts in culture. The distribution of these proteins in control and inhibited (5 x 10(-7) M monensin) cells has been studied by immunofluorescence microscopy. In control cells, both antigens are present throughout the cytoplasm and in specific deposits in a region adjacent to the nucleus, which we identify as a Golgi zone by electron microscopy. Treatment of cells with monensin causes intracellular accumulation of procollagen and fibronectin, initially in the juxta-nuclear region and also subsequently in peripheral regions. Electron microscope studies reveal that in such cells the juxta-nuclear Golgi zone becomes filled with a new population of smooth-membraned vacuoles and that normal Golgi complexes are not found. Immunocytochemically detected procollagen and fibronectin are localized in the region of these vacuoles, whereas more peripheral deposits correspond to the dilated cisternae of rough endoplasmic reticulum, which are also caused by monensin. Procollagen and fibronectin are often codistributed in these peripheral deposits. Accumulation of exportable proteins in Golgi-related vacuoles is consistent with previous analyses of the monensin effect. The subsequent development of dilated rough endoplasmic reticulum also containing accumulated proteins may indicate that there is an additional blockade at the exit from the endoplasmic reticulum, or that the synthesized proteins exceed the capacity of the Golgi compartment and that their accumulation extends into the endoplasmic reticulum.

[Ciliogenesis in the mucous cells of the quail oviduct. I. Ultrastructural study in the laying quail]

The luminal epithelium of the oviduct (magnum) of laying quails is composed of ciliated cells and mucous cells. Ciliogenesis was observed in some of the mucous cells. Both centrioles of the diplosome migrate to the top of the cell, and one of them induces the formation of a rudimentary cilium. In some of the other cells, that are filled with mucous granules, the formation of basal bodies by an acentriolar pathway was observed. In these cells, numerous, dense fibrous masses are associated with the forming face of the Golgi apparatus. In the Golgi zone, generative complexes composed of a deuterosome and some forming procentrioles were found. Cilia develop from completed basal bodies. During ciliogenesis, the Golgi apparatus is disorganized, and generally the production of mucous granules is arrested. The nucleus is also modified: it becomes larger and the chromatin is dispersed. It is assumed that mucous cells are able to be transformed into ciliated cells in the oviduct of laying quails.

Alkaline phosphatase isozyme patterns and histochemistry in adult and differentiating skate spiral valve

This project was undertaken to study the histochemical alkaline phosphatase (AP; EC 3.1.3.1) localizations in the spiral valve of adult and developing skates (Raja binoculata). Secondly, it was decided to determine if spiral valve AP isozymes, separated by disc electrophoresis, are altered during spiral valve differentiation.The histochemical localization of AP in the adult spiral valve is similar to that observed in the intestines of other vertebrates. Enzyme activity was found in brush borders and the Golgi zone of the epithelial cells, as well as in the lamina propria. The developing spiral valves showed an accumulation of enzyme in some areas and a depletion at other sites with advancing differentiation.Spiral valve AP banding patterns do change during morphological differentiation of the organ. It appears that one electrophoretic zone, present alone in early stages, loses activity upon completion of spiral valve morphological differentiation with the formation of the villi. Each isozyme becomes demonstrable at different developmental stages.