Scanning electron microscopic changes in granulosa cells during follicular atresia in Caprine ovary

During this study, topographic changes in healthy and atretic granulosa cells have been investigated during follicular atresia in goat ovary. Under scanning electron microscopy atresia was marked by asymmetrical shrinkage and vacuolization of cytoplasm. The specific topographical alterations observed in atretic cells were loss of micro extensions, disruption of cell-cell interaction, and smooth-textured membrane with a number of uneven depressions and ruffles. Some portions of the cell membrane were marked by extensive shrinkage due to condensation of cytosol. Irregular membrane at occasions was studded with blunt microextensions. The findings of present investigation will help in understanding the cellular changes in granulosa cells during follicular atresia and will find applications in screening of follicles for in vitro culture, in vitro fertilization and Embryo transfer technology.     

Preovulatory rise of NGF in ovine follicular fluid: possible involvement in the control of oocyte maturation

Since nerve growth factor (NGF) is produced in vitro by granulosa cells after gonadotropin stimulation, the present research has been designed to investigate whether this neurotropin is involved in the events triggered by the gonadotropin surge that lead the follicle to ovulate a mature oocyte. To this aim, NGF levels in follicular fluid, collected before or 20 hours after the gonadotropin surge, was measured by ELISA. To evaluate whether NGF may have a non-neurotropic effect on follicle cells, the presence of NGF receptors was investigated by immunohistochemistry and further evaluated by analysing the tyrosine-phosphorylation pattern after NGF stimulation in vitro. The effect of NGF on the degree of cumulus expansion, cumulus-oocyte metabolic coupling, and meiotic maturation was finally studied by using the culture of follicle-enclosed oocyte. The results demonstrate that GnRH causes a dramatic rise of NGF in large follicles. Immunohistochemistry revealed a discrete positivity for trkA receptors localised in cumulus cells. Tyrosine phosphorylation pattern confirms that somatic cells are capable to transduce NGF signal. By contrast, all the oocytes examined were negative for trkA and did not change the phosphorylation pattern after NGF. In vitro NGF (100 ng/ml) induced a marked cumulus expansion and a progressive cumulus-oocyte uncoupling similar to that produced by gonadotropins. The addition of NGF also caused the resumption of meiosis in more than 70% of the oocytes analysed with an effect that is only slightly less pronounced than that of gonadotropins (80%). The increase in NGF secretion following gonadotropin surge suggests that this neurotropin may be involved in the control of oocyte maturation.     

Structural changes and cell properties of human ovarian surface epithelium in ovarian pathophysiology

The surface epithelial cells of the ovary, which are modified peritoneal cells, form a single, focally pseudostratified layer. The Müllerian ducts differentiate after invagination of the coelomic mesothelium over the gonadal ridges during the 6th week of embryonic life. On the basis of the embryologically putative Müllerian potential of this epithelium, endometriosis can be explained by coelomic metaplasia from the peritoneum, including ovarian surface epithelium. Some pelvic endometriosis specimens have shown that epithelial cells on the ovary or pelvis are serially changed to endometriotic gland cells. Immunohistochemistry as well as scanning electron microscopy also reinforce the light-microscopical findings. A three-dimensional culture system demonstrated that human ovarian surface epithelial cells exhibited a glandular-stromal structure when they were cocultured with endometrial stromal cells in an estrogen-rich environment. Ovarian carcinomas in the epithelial-stromal category are thought to arise from the surface epithelium and its inclusions. The ovarian surface epithelium is physiologically involved in follicular rupture, oocyte release, and the subsequent repair of follicle wall during reproductive age. Simultaneously, ovulation may cause a loss of integrity of the surface epithelium, followed by accumulation of multiple mutations. The cortical invagination, surface stromal proliferation, and Müllerian differentiation of these cells are likely not to be an early step in the cancer development. However, the inclusion cysts are closely related with carcinogenesis because they are significantly more common in ovaries contralateral to those containing epithelial cancers than in control ovaries. As an in vitro study, ovarian carcinoma cell lines were established from simian virus 40 large T antigen-transformed human surface epithelial cells of the ovary. Further investigations of these cell lines may lead to insights into the preneoplastic and early stages of carcinomas. To clarify the pathogenesis of endometriosis and epithelial ovarian cancer, specifically designed studies of ovarian surface epithelium are required.     

Oocyte-follicle cell interactions during ovarian follicle development, as seen by high resolution scanning and transmission electron microscopy in humans

The aim of this article is to summarize and update, through an integrated analysis by transmission electron microscopy (TEM) and high resolution scanning electron microscopy (SEM) after osmium-dimethyl sulfoxide-osmium (ODO) maceration, the studies of our research group on the morphodynamics of oocyte-follicle cell associations during follicle development in humans. In resting oocytes, follicular cells project few and short cytoplasmic processes in the perioocytic space. They often form bulbous terminals very close to the oolemma where zonulae adherentes, maculae adherentes, and gap junctions are present. The oolemma mostly appears smooth with short and scanty microvilli. In early growing follicles, follicular cell projections appear as (a) long and tortuous microvilli or (b) large and short extensions. The oolemma shows numerous short microvilli. By TEM, long and thin follicular "intraooplasmic processes" have been seen to penetrate deeply into some oolemma invaginations. In macerated samples, they are observed by SEM to come very close to the nucleus and contact different oocyte organelles. These processes are more likely involved in early oocyte growth. In late growing follicles, oocyte-somatic cell interactions-now established through the interposition of the zona pellucida (ZP)-preserve the general features of early growth stage, with the exceptions of "intraooplasmic processes," which are no more present. In mature follicles subjected to a long ODO maceration, corona cells appear to contact the oocyte through an apical plume of numerous very long "curly hair-like microvilli." Corona cell microvilli, quite likely provide a sort of cytoplasmic skeleton for the ZP and they are possibly involved in (a) release of nutrients or removal catabolites to/from oocyte and vice versa and (b) transfer of substances to build up ZP. In conclusion, among oocyte and somatic cells a structural and functional association is revealed. This association, certainly highly dynamic in vivo, plays a key role in regulating the healthy folliculogenesis to assure a correct and timed oocyte maturation and ovulation.     

Microglia-precursor cell interactions in health and in pathology

Until recently, microglia were mainly known as the resident phagocytes of the brain, i.e. the ‘immunological warriors’ of the brain. However, extensive knowledge is being accumulated about the functions of microglia beyond immunity. Nowadays, it is well accepted that microglial cells are highly dynamic and responsive, and that they intervene in a dual manner in many developmental processes that shape the central nervous system, including neurogenesis, gliogenesis, spatial patterning, synaptic formation and elimination, and neural circuit establishment and maturation. The differentiation and the pool of precursor cells were also shown to be under microglia regulation via bidirectional communication. In this concise review, I discuss our recent work in microglia-Pax6⁺ cell interactions in one of the circumventricular organs, the pineal gland. An analogy with the rest of the central nervous system is also presented. In addition, I briefly examine mechanisms of interaction between microglia and non-microglial cells in both health and disease. New avenues are also introduced, which may lead us to better comprehend the impact of microglia in physiological and pathological conditions.     

Role of the central and peripheral nervous system in the ovarian function

This review attempts to give a comprehensive overview of ovarian innervation, considering the whole nervous system and its different levels that may modify the ovarian function. The connection between the ovary and the central nervous system through the autonomic pathways, including the peripheral ganglia, is highlighted. The evidence obtained over the last years highlights the role of the superior ovarian nerve (SON) in the ovarian phenomena. Besides, the effect on the ovary of conventional neurotransmitters and others such as indolamines and peptides, which have been found in this organ, are discussed. Various reproductive diseases have been studied almost exclusively from the endocrine point of view. It is evident that a better knowledge about the role of the neural factors involved in the ovarian physiology may facilitate the understanding of some of these. A review of the concepts and an update of some experimental designs is made that permits clarifying several aspects of the relationship between the neural system and the ovary. At present, there is no doubt that the innervation of the ovary is involved in several physiological aspects of this gland function. However, the relationship of some levels of the nervous system and the ovary offer a wide avenue for future research.     

The effects of human menstrual blood stem cells‐derived granulosa cells on ovarian follicle formation in a rat model of premature ovarian failure

Many studies have reported that human endometrial mesenchymal stem cells (HuMenSCs) are capable of repairing damaged tissues. The aim of the present study was to investigate the effects of HuMenSCs transplantation as a treatment modality in premature ovarian failure (POF) associated with chemotherapy‐induced ovarian damage. HuMenSCs were isolated from menstrual blood samples of five women. After the in vitro culture of HuMenSCs, purity of the cells was assessed by cytometry using CD44, CD90, CD34, and CD45 FITC conjugate antibody. Twenty‐four female Wistar rats were randomly divided into four groups: negative control, positive control, sham, and treatment groups. The rat models of POF used in our study were established by injecting busulfan intraperitoneally into the rats during the first estrus cycle. HuMenSCs were transplanted by injection via the tail vein into the POF‐induced rats. Four weeks after POF induction, ovaries were collected and the levels of Amh, Fst, and Fshr expression in the granulosa cell (GC) layer, as well as plasma estradiol (E2) and progesterone (P4) levels were evaluated. Moreover, migration and localization of DiI‐labeled HuMenSCs were detected, and the labeled cells were found to be localized in GCs layer of immature follicles. In addition to DiI‐labelled HuMenSCs tracking, increased levels of expression of Amh and Fshr and Fst, and the high plasma levels of E2 and P4 confirmed that HuMenSC transplantation had a significant effect on follicle formation and ovulation in the treatment group compared with the negative control (POF) group.     

Role of the biomolecular interactions in the structure and tribological properties of synovial fluid

The analysis of healthy rat synovial fluid (SF) by electron microscopy proved the existence of lipid multilamellar vesicular discontinuities of several hundred nm in diameter. To understand the discontinuities’ role in biolubrication, their intermolecular and tribological properties were studied using AFM and biotribological techniques. Hyaluronic acid and lubricin exhibited high affinity and high friction coefficients for lipid bilayers while biomimetic SF vesicles including glycoproteic components conferred low friction coefficients. These results suggest that SF’s volume is composed by a network of vesicles containing glycoproteic gel separated by lipid multilayers, lubricin’s role being devoted to anchor lipid layers on the cartilage.     

Role of pyroptotic cell death in the pathogenesis of NASH

Nonalcoholic fatty liver disease (NAFLD) represents a huge threat to public health of the whole world.Around 25% of NAFLD patients will progress to nonalcoholic steatohepatitis (NASH), which has been predicted to bethe main reason for liver transplantation in the United States in 2020. Extensive effort has been devoted to investigatingthe underlying molecular mechanisms of NASH pathogenesis and developing new promising treatments. Recentstudies have demonstrated that pyroptosis, an inflammatory programmed cell death mediated by inflammasome andgasdermin-D (GSDMD), is involved in the development and progression of NASH. This review aims to summarize therecent findings regarding the role of pyroptosis and related molecules in the pathogenesis of NASH.     

Ultrastructural characterization of apoptotic granulosa cells in caprine ovary

The unique phenomenon of cell proliferation and apoptosis is encountered in the ovarian follicles undergoing early stages of atresia. The aim of this study was to verify the morphological variations in these two physiologically distinct processes operating in antral follicles of caprine ovaries using histological and ultrastructural techniques. Histologically the degenerating granulosa cells were characterized by condensed cytoplasm, and nucleus fragmentation in hazy cytosol. The pyknotic nuclei of degenerating cells stained darkly with haematoxylin and giemsa while the cytoplasm was eosinophilic. Under electron microscopy, apoptosis was marked by asymmetrical shrinkage, vacuolization of cytoplasm, swollen and vacuolated mitochondria, increased irregularity and/or fragmentation of nucleus, chromatin condensation and finally, production of membrane enclosed nuclear fragments containing intracellular material, the apoptotic bodies. The parallel use of these two methods on caprine ovaries has enabled us to analyse the decline in the frequency of granulosa cells during follicular atresia due to apoptosis.     

Dual-color fluorescence lifetime correlation spectroscopy to quantify protein-protein interactions in live cell

Dual-color fluorescence correlation spectroscopy is an interesting method to quantify protein interaction in living cells. But, when performing these experiments, one must compensate for a known spectral bleed through artifact that corrupts cross-correlation data. In this article, problems with crosstalk were overcome with an approach based on fluorescence lifetime correlation spectroscopy (FLCS). We show that FLCS applied to dual-color EGFP and mCherry cross-correlation allows the determination of protein-protein interactions in living cells without the need of spectral bleed through calibration. The methodology was validated by using EGFP-mCherry tandem in comparison with coexpressed EGFP and mCherry in live cell. The dual-color FLCS experimental procedure where the different laser intensities do not have to be controlled during experiment is really very helpful to study quantitatively protein interactions in live sample.     

Interference microscopic studies on wood plastic and cell wall—liquid interactions in beech

A method is described to measure the content of methylmethacrylate-polymer (poly-MMA) in wood-plastic composites and the influence of MMA on water and ethanol accessibility of beech wood fibre walls using interference microscopy. Although the S₂-layer of the fibre wall is capable of containing about 12% volume of plastic, the effect of treatment on ultimate water absorption of the cell wall is negligible. The ultimate absorption of ethanol is, however, completely reduced to zero. Increased dimensional stability of wood-poly-MMA composites is probably due to hindrance by the plastic in the lumina of the wood elements. The interference microscopic method is also shown to be suitable for determination of the fibre saturation point of cell wall areas.     

Role of cell cycle molecules in the pathophysiology of glomerular epithelial cells

The postmitotic characteristics of podocytes are the basis for the structural assembly and central function (filtration) of the glomeruli. Persistent cell cycle quiescence is required for stability in these cells. In cells of the podocyte lineage, transdifferentiation from the metanephric mesenchyme to mature podocytes is closely associated with tight regulation of the expression of cell cycle molecules. This cell cycle control in podocytes acts as a safeguard for the glomeruli; however, deregulation of this system might result in structural deterioration. This article focuses on the expression of cell cycle molecules in podocyte differentiation and pathology.     

Probing molecular interactions and mechanical properties of microbial cell surfaces by atomic force microscopy

Knowledge of the surface properties of microbial cells is a key to gain a detailed understanding of their functions in the natural environment and to efficiently exploit them in biotechnological processes. In this paper, we present force-distance curves recorded, by atomic force microscopy (AFM) in aqueous solutions, on various microbial samples: reconstituted S-layers, whole fungal spores and several bacterial strains. The approach and retraction curves exhibited important differences--depending on the type of microorganism, on the physiological state (dormancy versus germination) and on the environmental conditions (ionic strength)--which were shown to reflect differences in long-range surface forces, adhesion forces and mechanical properties. These data illustrate the great potential of AFM force measurements to elucidate the physical properties of microbial cells and to understand, at the molecular level, biointerfacial phenomena such as cell adhesion and cell aggregation.     

肿瘤干细胞标志物CD133和CD117在卵巢癌中的表达及意义

目的 :探讨肿瘤干细胞标志物CD133与CD117在卵巢癌中的表达及临床意义。方法:采用免疫组化SP法检测50例上皮性卵巢癌组织及20例正常卵巢组织中CDl33与CD117的表达情况。结果:(1)上皮性卵巢癌组织中CDl33及CD117的表达较正常卵巢组织明显增高,组间差异有统计学意义(P<0.05)。(2)低分化卵巢癌组CD133及CD117阳性表达率明显高于高、中分化组(P<0.05)。(3)Spearman相关性分析显示CD133表达与CD117的表达有相关性(r=0.364,P=0.04)。结论:高表达的CDl33及CD117可能与上皮性卵巢癌的发生、发展有一定关系。     

Regulatory cell interactions between retinal ganglion cells and radial glia during axonal and dendritic outgrowth

Neuronal differentiation and the formation of cell polarity are crucial events during the development of the nervous system. Cell polarity is a prerequisite for directed information flux within neuronal networks. In this article, we focus on neuro-glial cell interactions that influence the establishment of neural cell polarity and the directed outgrowth of axons versus dendrites. The cellular model discussed in detail is the retinal ganglion cell (RGC) of the chick retina, which is investigated by a comprehensive set of in vitro assays. The experiments demonstrate that retinal microenvironment determines axon vs. dendrite formation of RGCs. The instructive differences in different retinal microenvironments are substantially influenced by radial glia. Different glial domains support or inhibit axon vs. dendrite outgrowth. The data support the notion that neuro-glial interactions are crucial for directed neurite outgrowth.     

Role of insulin-like growth factor binding protein-3 in breast cancer cell growth

The mitogenic effects of insulin-like growth factors (IGFs) are regulated by a family of insulin-like growth factor binding proteins (IGFBPs). One member of this family, IGFBP-3, mediates the growth-inhibitory and apoptosis-inducing effects of a number of growth factors and hormones such as transforming growth factor-beta, retinoic acid, and 1,25-dihydroxyvitamin D3. IGFBP-3 may act in an IGF-dependent manner by attenuating the interaction of pericellular IGFs with the type-I IGF receptor. It may also act in an IGF-independent manner by initiating intracellular signaling from a cell surface receptor, or by direct nuclear action, or both. The possibility of a membrane-bound receptor is strengthened by recent studies which have identified members of the transforming growth factor-beta receptor family as having a role, either directly or indirectly, in signaling from the cell surface by IGFBP-3. A number of growth factors and hormones stimulate the expression and secretion of cellular IGFBP-3, which then signals from the cell surface to bring about some of the effects attributed to the primary agents. Within the cell, the apoptosis-inducing tumor suppressor, p53, can also induce IGFBP-3 expression and secretion. Since IGFBP-3 upregulates the cell cycle inhibitor, p21(Waf1), and increases the ratio of proapoptotic to antiapoptotic members of the Bcl family, it appears to exert the same effects on major downstream targets of cell signaling as p53 does. The nuclear localization of IGFBP-3 has been described in a number of cell types. IGFBP-3 may act to import IGFs or other nuclear localization signal-deficient signaling molecules into the nucleus. It may also act directly in the nucleus by enhancing the activity of retinoid X receptor-alpha and thereby promote apoptosis. All of the above phenomena will be discussed with particular emphasis on the growth of breast cancer cells.     

Role of ultrastructural studies in the analysis of cell lineage in the mammalian pre-implantation embryo.

Ultrastructural studies have contributed significantly to our understanding of cell lineage differentiation in the mammalian pre-implantation embryo. Such studies have documented, and continue to document, morphological, biochemical, and physiological characteristics of the cell lineages established during the pre-implantation period in eutherian embryos, principally that of the mouse. This review evaluates these contributions and identifies areas of study in which ultrastructural analysis is most likely to have an important role in the future.     

Ultrastructural correlates of meiotic maturation in mammalian oocytes

Immature mammalian oocytes reside in ovarian follicles with junctionally coupled granulosa cells. When released from a currently undefined meiotic arresting influence, these oocytes resume meiosis to progress from late diplotene (germinal vesicle stage) through the first meiotic division to metaphase II. Oocytes remain at metaphase II until fertilization activates them to complete meiosis. This review summarizes ultrastructural events that occur during meiotic maturation in mammals. Developmental correlates that promise a clearer understanding of regulatory mechanisms operating to control maturation are emphasized. By use of TEM of thin sections, freeze-fracture analysis, and replicated oocyte cortical patches, we demonstrate stage-specific changes in the oocyte nucleus, reorganization of cytoplasmic organelles, correlations between oocyte maturational commitment and the junctional integrity of associated granulosa cells, and definition of the components comprising the oocyte cortical cytoplasm.