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Examines the pregnancy-induced changes in substance P and neurokinin 1 receptor (NK1-R) expression in the rat uterus. Decrease in the immunoreactivity of substance P in the uterus; Accounts on the behavior of the peptidergic system; Factors influencing the synthesis of of NK1-R in protein on peritoneal macrophages; Transformation of myometrium into a functional syncytium.

Examines the microvessels in calf and cow ovaries to identify the presence of factor VIII-related antigen endothelial cells. Role of the microvascular bed in the initiation and maintenance of growth from primordial to primary follicles; Localization of the primordial and primary follicles; Cause of the poverty of the blood supply.

Abstract Obese melanocortin-4-receptor-deficient (MC4R-/-) male mice are reported to have erectile dysfunction, while homozygous MC4R-/- female mice are apparently fertile. A recently established obese mouse strain, carrying an inactivating mutation in the MC4R gene, revealed difficulties in breeding for the homozygous female mice. This prompted us to determine the presence of follicles and corpora lutea (CL) in ovaries of MC4R-/- mice aged 3–6 months in comparison to wild type (MC4R+/+) littermates. Serial sections of formaldehyde-fixed ovaries of mice with vaginal signs of estrus and metestrus were assessed for the number of healthy and regressing follicles and CL. The number of CL, as an estimate for the ovulation rate, decreased to zero during aging in MC4R-/- mice. The number of small- (diameter 100–200 micrometer) and large-sized follicles namely antral follicles (diameter >200 micrometer) were slightly increased in MC4R-/- compared to MC4R+/+ mice. Greater differences were found in very large to cystic follicles, which were more numerous in MC4R-/- mice. The number of regressing antral follicles was higher in the MC4R-/- group compared to the MC4R+/+ group. This was associated with a wide range in the number of collapsed zonae pellucidae as the last remnants of regressed follicles. A conspicuous hypertrophy of the interstitial cells was noted in 6-month-old MC4R-/- mice. In conclusion, cystic follicles and the reduction in CL number point to a decreased ovulation rate in obese MC4R-/- mice.

Abstract The cyclic nature of the capillary bed in the corpus luteum offers a unique experimental model to examine the life cycle of endothelial cells, involving discrete physiologically regulated steps of angiogenesis, blood vessel maturation and blood vessel regression. The granulosa cells and theca cells of the developing antral follicle and the steroidogenic cells of the corpus luteum produce and respond to angiogenic factors and vasoactive peptides. Following ovulation the neovascularization during the early stages of corpus luteum development has been compared to the rapid angiogenesis observed during tumor formation. On the other end of the spectrum, the microvascular endothelial cells are the first cells to undergo apoptosis at the onset of corpus luteum regression. Important insights on the morphology and function of luteal endothelial cells have been gained from a combination of in vitro and in vivo studies on endothelial cells. Endothelial cells communicate with cells comprising the functional unit of the corpus luteum, i.e., other vascular cells, steroidogenic cells, and immune cells. This review is designed to provide an overview of the types of endothelial cells present in the corpus luteum and their involvement in corpus luteum development and regression. Available evidence indicates that microvascular endothelial cells of the corpus luteum are not alike, and may differ during the process of angiogenesis and angioregression. The contributions of vasoactive peptides generated by the luteal endothelin-1 and the renin-angiotensin systems are discussed in context with the function of endothelial cells during corpus luteum formation and regression. The ability of two cytokines, tumor necrosis factor alpha and interferon gamma, are evaluated as paracrine mediators of endothelial cell function during angioregression. Finally, chemokines are discussed as a vital endothelial cell secretory products that contribute to the recruitment of eosinophils and macrophages. The review highlights areas for future investigation of ovarian microvascular endothelial cells. The potential clinical applications of research directed on corpus luteum endothelial cells are intriguing considering reproductive processes in which vascular dysfunctions may play a role such as ovarian failure, polycystic ovary syndrome (PCOS), and ovarian hyperstimulation syndrome (OHSS).

Abstract: Co-cultures of 3T3-L1 adipocytes with neurons from the rat dorsal root ganglia (DRG) showed enhanced neuritogenesis and synaptogenesis. Microarray analysis for upregulated genes in adipocyte/DRG co-cultures currently points to apolipoproteins D and E (ApoD, ApoE) as influential proteins. We therefore tested adipocyte-secreted cholesterol and the carrier proteins ApoD and ApoE3. Cholesterol, ApoD, and ApoE3 each increased neurite outgrowth and upregulated the expression of presynaptic synaptophysin and synaptotagmin, as well as the postsynaptic density protein 95. The neurotrophic effects of ApoD and ApoE3 were associated with an increased expression of the low-density lipoprotein receptor and apolipoprotein E receptor 2. Simultaneous treatment with receptor-associated protein, an apolipoprotein receptor antagonist, inhibited the neurotrophic function of both apolipoproteins. The application of ApoD, ApoE3, and cholesterol to DRG cell cultures corresponded with increased expression of the chemokine stromal cell-derived factor 1 and its receptor CXC chemokine receptor 4 (CXCR4). Surprisingly, the inhibition of CXCR4 by the antagonistic drug AMD3100 decreased the apolipoprotein/cholesterol dependent neurotrophic effects. We thus assume that apolipoprotein-induced neuritogenesis in DRG cells interferes with CXCR4 signaling, and that adipocyte-derived apolipoproteins might be helpful in nerve repair. [Copyright &y& Elsevier]

Objective: Multilamellar bodies associated with an organized endoplasmic reticulum (ER) arise in various somatic cell types, and a subtype called multivesicular bodies is described in oocytes. Both entities, so far undetermined in significance, may occur in oocytes of follicles under oxidative stress. In preovulatory follicles, oxidative stress appears to be caused by oxidized low-density lipoprotein (ox-LDL)., Method: Cultures of preantral mouse follicles were treated with 100 µg/ml ox-LDL or normal LDL (n-LDL) for 12-48 h or for 12 days during antral follicle growth followed by in vitro ovulation and harvest of cumulus oophorus complexes (COCs) with metaphase II (MII) oocytes on day 13. Preantral follicles, COCs, or MII oocytes were immunostained with anti-tubulin antibody or stained with actin-binding phalloidin for confocal microscopy. Ultrathin sections were prepared for electron microscopy., Results: Preantral follicles exposed to n-LDL or ox-LDL developed normally, and MII oocytes in COCs possessed normal spindles with well-aligned chromosomes. In contrast, treated cumulus cells underwent apoptosis. Only the ox-LDL-treated preantral follicle oocytes showed ER-derived multilamellar bodies (EMBs) of type I, consisting of rough ER membranes for the envelope. The MII oocytes of COCs showed type II EMBs consisting of smooth/vesicular ER and were more prominent after ox-LDL than after n-LDL exposure. Degenerating mitochondria were prominent in oocytes of the ox-LDL group and judged as a sign of oxidative stress., Conclusion: Oxidative stress presumably induces damage of proteins and organelles in the oocytes. The EMBs might sequester the damaged structures for oocyte survival. Thus, EMBs could represent a novel form of autophagy., (Copyright © 2012 S. Karger AG, Basel.)

Since previous in vitro experiments revealed that nicotine can impair endothelial intercellular communication via the downregulation of connexin43 (Cx43), we wanted to find out which nicotinic acetylcholine receptors are involved in the molecular mechanism of communication failure. Cultured human endothelial cells were exposed to 1 μM nicotine for 5 days. Intercellular communication was measured using dye transfer study with/without subtype-specific nicotinic acetylcholine receptor (nAChR) inhibitors. Reverse transcriptase (RT)-PCR was used to further investigate the regulation of nAChR subtypes. Electron microscopy together with MAP LC3-II western blot was used to investigate possible autophagy processes. In cultured human endothelial cells, nicotine decreased the Cx43 protein amount as shown by western blot and immunohistochemistry; however, together with an unaltered mRNA expression as shown by RT-PCR. The nicotine-induced Cx43 downregulation functionally impaired intercellular dye transfer, which could be prevented by mecamylamine, κ-bungarotoxin, lobeline, and dihydro-β-erythroidine but not α-bungarotoxin, indicating that the nAChR subtypes α4β2 and α3β2 but not α7 are involved in signal cascade. RT-PCR analysis revealed that nicotine exposure resulted in the upregulation of α3 and β4 and the downregulation of α4-nAChR, while α7- and β2-nAChR-mRNA expressions remained unaltered. Furthermore, nicotine increased total protein ubiquinylation and proteasome activity as was shown by immunohistochemistry and peptide degradation analysis. Evidence of enhanced autophagic processes was assured by the occurrence of autophagic vacuoles in transmission electron microscopy and enhanced formation of MAP LC3-II in western blot. Reduced intercellular endothelial communication together with programmed cell death helps to explain the toxic effect of nicotine leading to endothelial dysfunction. The nAChR involved in the impairment of intercellular communication seem to be α4β2 and α3β2 but not α7.

Neuropeptide Y (NPY) is expressed in adipose tissue and is involved in adipocyte metabolism. Although NPY impacts on glucose utilization in vivo, the underlying cellular mechanism is yet to be fully elucidated. In this study we investigated the effect of NPY on the insulin-stimulated translocation of glucose transporter 4 (GLUT4) from intracellular stores to the cell surface in vitro. Using cellular fractionation and immunofluorescence we analyzed the cellular localization and content of GLUT4 in 3T3-L1 adipocytes. Additionally we investigated the effect of NPY on insulin action in adipocyte cultures by assessing the phosphorylation of Akt and [(3)H]-deoxyglucose uptake. Our data suggest that in 3T3-L1 adipocytes NPY inhibits insulin-stimulated glucose uptake in a GLUT4-dependent manner. The insulin induced translocation of GLUT4 was attenuated by the Y1 receptor agonist [Phe(7),Pro(34)] pNPY, demonstrating an essential role of the Y1 receptor in GLUT4 translocation. Additionally, we observed an NPY dose-dependent impairment of Akt phosphorylation. This study provides evidence that NPY impairs the insulin sensitivity of adipocytes and suggests that the Y1 receptor could be a potential therapeutic target for type 2 diabetes., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

The morphology of sciatic nerves from leptin-deficient ob/ob mice and leptin receptor-deficient db/db mice, both models for obesity, peripheral diabetic neuropathy, and the metabolic syndrome, has yet to be examined for changes in nerve fibers and in endoneural microvessels. Sciatic nerves from three groups of 4-month-old mice (WT C57BL6, ob/ob, and db/db) were investigated. In ultrathin sections, the thickness of myelin sheaths was significantly reduced in small, medium-sized, and large axons of db/db mice compared with WT mice. In ob/ob mice, only large fibers showed a decrease in myelin sheath thickness. The number of nonmyelinated nerve fibers was lower in ob/ob mice than in the db/db group. A thickened basal lamina of Schwann cells occurred in the ob/ob group only. In contrast, the basement membrane of endoneural microvessels was thickened in both obese groups. For this reason, laminin expression in Western blot analysis was lower in the db/db group than in the ob/ob one. Endoneural microvessels, which had been injected with fluorescein isothiocyanate, depicted signs of vasodilatation in the ob/ob and vasoconstriction in db/db mice. Endoneural vessels displayed two receptors of oxLDL. LOX-1 was strongly expressed in db/db mice, whereas TLR4 was at its maximum in the ob/ob group. We conclude that changes in nerve fibers and in endoneural microvessels are present in sciatic nerve of both mouse models of type 2 diabetes. Upregulation of oxLDL-dependent receptors in endoneural microvessels might be connected to different degrees of oxidative stress in severe diabetic db/db mice and in the mild diabetic ob/ob group., (Copyright © 2011 Wiley Periodicals, Inc.)

KIT is a type III receptor protein tyrosine kinase, and KITL its cognate ligand. KIT can mediate its effects via several intracellular signalling pathways, or by formation of a cell-cell anchor with its ligand. Through these mechanisms, KIT controls fundamental cellular processes, including migration, proliferation, differentiation and survival. These cellular processes are modulated by soluble KIT, a cleavage product of KIT, generated at the cell membrane. A cell-retained KIT cleavage fragment also arises from this cleavage event. This cleavage fragment must be distinguished from truncated KIT (trKIT), which originates through cryptic promoter usage. The expression of trKIT is highly restricted to postmeiotic germ cells in the testis. In contrast, KIT, together with its cleavage products, is present in somatic cells and germ cells in the gonads of both sexes. A functional KITL/KIT system is mandatory for normal population of the gonads by germ cells. Signalling via the KITL/KIT system promotes the growth, maturation, and survival of germ cells within the gonads, and prevents meiotic entry and progression. In addition to its importance in germ cell biology, the KITL/KIT system is crucial for gonadal stromal differentiation. During foetal life, KIT is expressed by testicular stromal precursor cells, which develop into Leydig cells. In the ovary, stromal cell KIT expression accompanies theca layer development around advanced follicles. After ovulation, KIT-immunopositive cells translocate from the theca layer to the luteal ganulosa where they contribute to a delicate cellular network that extends between the fully luteinised large luteal cells. In the outer regions of the developing corpus luteum, a highly conspicuous subpopulation of KIT/CD14-double-immunopositive cells can be observed. KIT/CD14-double-immunopositive cells are also seen in the haematopoietic-like colonies of long-term granulosa cultures established from late antral follicles. These cultures demonstrate expression of pluripotency marker genes such as octamer binding transcription factor-3/4 and sex determining region Y-box 2. The KIT/CD14-double-immunopositive cells can be purified and enriched by KIT-immunopositive magnetic cell sorting. Subsequent exposure of the KIT-expressing cells to the hanging drop culture method, combined with haematopoietic differentiation medium, provides the signals necessary for their differentiation into endothelial and steroidogenic cells. This suggests that monocyte-derived multipotent cells are involved in ovarian tissue remodelling. In summary, multicelluar KITL/KIT signalling organizes the stroma in the ovary and testis; monocyte-derived multipotent cells may be involved., (Copyright © 2011 Elsevier GmbH. All rights reserved.)

Progress in understanding the background of structural luteolysis depends on insights into the physiological function of innate immunity (INIM), in particular the presence of dendritic cells (DCs) in the corpus luteum (CL). For this reason, the cultures of five endothelial cell-like phenotypes derived from the bovine CL and their long-lasting analysis (morphology, function, and origin) become important. Types 1 and 2 represent microvascular endothelial cells with cytokeratin (CK) expression, assumed to be danger-sensing cells. Types 3 and 4 express features of common endothelial cells. Type 5 indicates a steroidogenic cell type, which could be derived from steroidogenic CK(+) cells in the CL of development after loss of CK expression. Type 5 is a promising candidate to become a mature DC. It might act with the microvascular CK(+) cell/type 1 like a luteovascular unit, which connects INIM with adaptive/cell-mediated immunity (ADIM) in structural luteolysis., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

The ovulatory process is characterized by tissue wounding and, after oocyte expulsion, by healing being connected to the formation of a corpus luteum (CL). The ovulatory event thus compares with a sterile inflammation. The concept is forwarded that the ovulatory process depends on innate immunity (INIM) function. The ultimate trigger for INIM signaling are danger signals/alarmins from granulosa cells damaged by oxidative stress and reactive oxygen species (ROS), respectively. Alarmins like oxidized low density lipoprotein (oxLDL) are recognized by cytokeratin-positive (CK(+)) granulosa cells with the expression of toll-like receptor 4 (TLR4). The subsequent inside-out signaling from the antrum towards the thecal cell layer comprises inflammation and tissue disintegration, which might be dominated by the myeloid differentiation factor 88 (Myd88) gateway. Additive or co-regulatory function are expected from the complement cascade for vessel permeability and leukocyte immigration and the wingless (WnT)-signaling for cell adhesion of CK(+) granulosa cells. The outside-in signaling relates to the repair phase, which is primarily controlled by the TIR-domain-containing adaptor protein producing IFN type I (TRIF) gateway of TLR signaling. The KIT/CD117 tyrosine kinase receptor and the tachykinin-tachykinin receptor system could be involved. The appealing concept of INIM function in the ovary is novel and inaugurates a novel research field., (Copyright © 2010 Elsevier Ireland Ltd. All rights reserved.)

The existence of a cross-talk between nerves and fatty tissue is increasingly recognized. Using co-cultures of dorsal root ganglion (DRG)-derived cells and 3T3-L1 adipocytes, we have previously shown that the presence of fat cells enhances neurite outgrowth and number of synapses. Vice versa, neural cells induced expression of neurotrophic adipokines apolipoprotein D and E (ApoD, ApoE) and angiopoietin-1 (Ang-1) by adipocytes. Here, we tested whether pituitary adenylate cyclase-activating peptide (PACAP), which is released by sensory fibres and causes Ca(2+) influx into fat cells, is involved in ApoD induction. Using 3T3-L1 cell cultures, we found that PACAP at a dose of 1 nM up-regulated the expression of ApoD protein and mRNA approx. 2.5 fold. This effect was driven by ERK1/2 acting upon PAC1/VPAC2 receptors. In turn, PACAP-treated 3T3-L1 adipocytes in co-cultures with DRG cells enhanced neurite ramification of neurofilament 200 (NF200)-positive neurons (measured using fluorescence microscopy) and neurofilament 68 protein levels (measured using Western blot analysis). This effect could be blocked using the PAC1/VPAC2 antagonist PACAP(6-38). Scanning cytometry revealed PACAP/ApoD induced low density lipoprotein receptors (LDLR) and ApoE receptor 2 (apoER2) in NF200-positive cells. Thus, a bidirectional loop seems to exist regulating the innervation of fatty tissues: PACAP released from sensory fibres might stimulate fat cells to synthesize neurotrophic adipokines, which, in turn, support peripheral innervation., (Copyright © 2010 Elsevier Ireland Ltd and the Japan Neuroscience Society. All rights reserved.)

This monograph introduces innate immunity as a second force in the ovary in addition to the endocrine system. Innate immunity appears to orchestrate follicular atresia, follicle rupture, and follicle transformation into a corpus luteum (CL) and CL regression through sterile inflammation and tissue repair. The concept is new. It centers on cytokeratin-positive (CK+) cells being recognized as a potential non lymphoid dendritic cell (DC) type. Part I describes morphological aspects of immune privilege starting with hamster ovary implants into the chicken chorioallantoic membrane with a non reactive mesenchyme. Follicular atresia and follicle rupture correspond to mild and moderate tissue damage in ovaries of small rodents and rabbits. Superovulations cause severe tissue damage through intra-ovarian oocyte release with follicle wall remnants in oedema, rupture of vessel walls and thrombosis. The complement system and neuropeptides might play regulatory roles. Part IIa analyzes intact ovaries (cows, human) for the appearance of CK+ cells. In the fetal ovary, sex cords give rise to CK+ cells in primordial follicles. In the adult ovary, CK+ cells are absent in preantral follicles and reappear in mature and regressing follicles. In the CL of early development, steroidogenic CK+ cells build a peripheral zone in the previous granulosa cell layer, and are uniformly distributed in the following stages. A microvascular CK+ cell type is seldom found. Part IIb characterizes the morphology and function of CK+ cells in vitro. Isolated from human preovulatory follicles, the epithelioid CK+ granulosa cell subtype regulates TLR4 and CD14 at 36 h of treatment with oxidized lipoprotein (oxLDL, 150 microg/ml); non-apoptotic cell death and the increase of reactive oxygen species occur. In contrast, the CK-negative (CK) granulosa cell type regulates the lectin-like oxLDL receptor 1 (LOX-1) and survival autophagy under oxLDL stimulation. Isolated from bovine CL, the epitheliold CK+ cell type 1 is disclosed as a microvascular cell type with a single nonmotile cilium. The microvascular CK+ type strongly upregulates intercellular contacts under treatment with interferon-gamma (IFN-gamma). In the CK- cell type 5 of granulosa cell-like appearance, IFN-gamma treatment supports cell proliferation, N-cadherin upregulation, and the dramatic increase in major histocompatibility complex II peptides (MHC II) by 80-fold compared to basal levels. Type 5 could have been converted from the steroidogenic CK+ cell type. We summarize and conclude: CK+ granulosa cells express functionally active TLR4, which sense danger signals, such as oxidative stress in preovulatory follicles, and trigger inflammatory and immunoregulatory pathways. The final outcome regulates follicle rupture and transformation into CL. Luteolysis could start by danger-sensing through the microvascular CK+ type 1 cells and the DC-like type 5 cells, both sensitive to IFN-gamma. The future will witness a novel strategy in the therapy of ovarian disorders such as anovulations, luteal phase insufficiency and autoimmune failures.

Oxidized low-density lipoprotein (oxLDL) induces apoptosis or autophagy in dependence on the cell type. We here investigated the effect of oxLDL on the B104 neuroblastoma and RN22 schwannoma cells being popular in neuroscience research. Cells were cultivated with and without oxLDL. To generate oxLDL, we added 50 μg/ml nLDL and 50 μM CuSO(4) into the culture medium. After a 24-h-long treatment, oxLDL was detectable in media from both cell culture types and its concentration was approximately 16 μg/ml. In the oxLDL-treated B104 neuroblastoma cell cultures 75% cells died after the 24-h exposure. The intact cells showed impaired mitochondria at the ultrastructural level. Western blot analysis revealed the increased expression of AIF 57 kDa (AIF(57)) protein, as a sign of caspase-independent cell death. In RN22 schwannoma cell cultures, oxLDL did not have any effect on cleaved caspase-3 and AIF(57) protein levels indicating absence of cell death. Treated RN22 schwannoma cells underwent survival autophagy by forming conspicuous autophagosomes and by processing LC3-I into LC3-II protein. Collectively, oxLDL induces AIF-dependent cell death in B104 neuroblastoma cells whereas in RN22 schwannoma cells enhanced signs of survival autophagy are noted., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Background: In patients with acute mesenteric ischemia by occlusive thrombo-embolism, the superior mesenteric artery (SMA) is more affected than the inferior mesenteric artery (IMA)., Methods: This study investigated postmortem mesenteric arteries from aged subjects (n=21). Four atherosclerotic stages were defined by signs of degeneration and inflammation in sections stained with Elastica-van-Gieson and immunohistology, respectively., Results: In females and males, Stages 3 and 4 were found in 62% of the SMA and 24% of the IMA. Lumenal areas based on diameter measurements remained essentially unchanged between Stages 1 and 4. Compared to a Stage 1 reference, remodeling was associated with thinning of the media below the plaque base and with pronounced thickening below the shoulder in the IMA. In Stages 3 and 4, the adventitia of the IMA had more vasa vasorum and a higher number of CD45-positive leukocytes than the adventitia of the SMA. During atherosclerotic progression, a stable fraction of leukocytes represented mast cells (6%) and CD117-positive cells as potential progenitor cells (1%)., Conclusions: Outgrowth remodeling occurred in both the SMA and the IMA. Less severe atherosclerosis in the IMA than in the SMA was associated with stronger signs of inflammation., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Gross dissection for demonstrating anatomy of the human pelvis has traditionally involved one of two approaches, each with advantages and disadvantages. Classic hemisection in the median plane through the pelvic ring transects the visceral organs but maintains two symmetric pelvic halves. An alternative paramedial transection compromises one side of the bony pelvis but leaves the internal organs intact. The authors propose a modified technique that combines advantages of both classical dissections. This novel approach involves dividing the pubic symphysis and sacrum in the median plane after shifting all internal organs to one side. The hemipelvis without internal organs is immediately available for further dissection of the lower limb. The hemipelvis with intact internal organs is ideal for showing the complex spatial relationships of the pelvic organs and vessels relative to the intact pelvic floor.

Objective: To extend our recent observations on lectin-like oxidized low-density lipoprotein receptor (LOX-1) expression in human granulosa cell cultures with freshly harvested granulosa cells., Design: Clinical research., Setting: Institute of Anatomy and Clinic for Reproductive Medicine., Patient(s): Women undergoing IVF therapy were classified by total FSH dose, age, and body mass index., Main Outcome Measure(s): Purified granulosa cells were studied by Western blot and morphology for the presence of LOX-1, microtubule-associated light-chain protein 3 (LC3) and autophagosomes, which are both autophagic markers, cleaved caspase-3 for apoptosis, and apoptosis-inducing factor (AIF) for caspase-independent apoptosis., Intervention(s): None., Results: Active LOX-1 was found in all samples, being at its maximum in the younger obese group with a total FSH dose <2,000 IU. The LC3 II/LC3 I ratio, indicative of reparative autophagy, was at its maximum in younger normal-weight patients and increased under total FSH dose >2,000 IU. Autophagosomes in ultrathin sections were indicative of reparative autophagy. Cleaved caspase-3 was absent in all groups. The apoptotic AIF form was up-regulated in older patients. Unpurified granulosa cells consisted of approximately 20% dead cells in the younger normal-weight group compared with up to 50% in the older obese group., Conclusion(s): The regulation of LOX-1 and of cell death in granulosa cells depends on oxidative stress. It becomes excessive during aging and obesity, because the power of reparative autophagy fades and antioxidant efficiency declines., (Copyright 2010 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

Hematopoietic-like colonies develop in post-confluent granulosa cell cultures derived from bovine antral follicles. Previously, we had shown that these colonies gave rise to macrophages. In the present study, we validated the presence of somatic KIT-positive (KIT(+)) progenitor cells in colony-containing granulosa cell cultures. The cultures expressed the progenitor cell markers Sox-2, Oct 3/4, KIT, and alkaline phosphatase in western blot analysis. The successful double immunofluorescence localization of KIT and CD14, CD45, CD133, or VEGF-R2 revealed a specific subpopulation of progenitor cells. Flow cytometry showed that cells doubly positive for KIT and CD14 or CD45 comprised less than 10% of the population. The KIT(+) cells were purified by magnetic selection and differentiated with the hanging drop technique using haematopoietic differentiation medium. Pure cultures of either granulosa cells or endothelial cells were obtained. The spindle-shaped and epithelioid phenotypes indicated endothelial cell heterogeneity of microvascular source. We conclude that progenitor cells are obtained from the follicle harvest, which differentiate into endothelial cells. The cells are relevant for findings to angiogenesis and luteinization of the corpus luteum.

The mesonephros is often regarded as a simplified version of the terminal renal organ, the metanephros. Both renal organs result from an epithelio-mesenchymal interaction between the Wolffian duct and the nephrogenic ridge. It appears that the epithelio-mesenchymal interaction makes use of similar signal cascades for both renal organs and that key events required for the development of the metanephros occur at earlier stages. In murine metanephroi, the stem cell factor (SCF)/-KIT-signal transduction pathway has recently been shown to regulate ureteric bud branching and epithelial cell differentiation. We immunohistochemically defined the time-sequence of KIT and SCF presence in both renal organs using bovine embryos/foetuses with crown rump length (CRL) of 1.7-24 cm. In the mesonephroi, epithelial cells with strong KIT staining were scattered in distal tubules, and SCF was expressed in the epithelial wall of corpuscles and proximal tubules. KIT positivity occurred in the metanephroi of embryos prior to SCF; KIT was predominantly localised at the ureteric bud tips in the nephrogenic zone. In foetuses of 13 cm and more CRL, the SCF/KIT profile of developmentally advanced nephrons mirrored the situation in the mesonephros. Epithelial cells with strong KIT staining were scattered in the cortical areas of distal tubules, while SCF was expressed in the epithelial wall of corpuscles and proximal tubules. Our morphological findings agree with a potential role of KIT at the ureteric bud tips and demonstrate a similar expression of KIT and SCF along the areas of developmentally advanced mesonephric and metanephric nephrons.

Study Design: The ligaments of the human sacroiliac joint (SIJ) were investigated morphometrically., Objective: A macroscopical study was performed to measure the anterior sacroiliac ligament (ASL), the interosseous sacroiliac ligament (ISL), and the posterior sacroiliac ligament (PSL), applying different methods of ligament visualization., Summary of Background Data: Little is known about the SIJ ligaments, especially about the ISL. Pelvic computer simulations neglect these ligaments due to the lack of information. Computer simulations of the SIJ ligaments may help to improve the clinical outcome of SIJ operations., Methods: Seven-Tesla MR images, CT images, and corresponding thin slice plastinates of the SIJ of 1 male and 1 female specimen were obtained. Serial sections of the SIJ of 32 frozen specimens (13 males, 19 females) were generated to gather measurements of the SIJ ligaments., Results: By means of the MR images and the plastinates, a virtual reconstruction of the SIJ ligaments was accomplished. Parallelepipeds were attributed to the cranial, middle, and caudal parts of all SIJ ligaments. This allowed precise measurements and statistical comparison including positional relationships. The ISL volumes and origin surfaces were the largest. Statistically, the ASL and PSL parameters were larger in males, while the ISL parameters were larger in females. The height of the cranial ASL part showed large negative correlations in spite of positive correlations of the other heights., Conclusion: The combined use of high-resolution MRI and thin slice plastination allows precise reconstructions of the SIJ ligaments. With these techniques, the ligaments can be visualized in situ and described morphometrically if based on substantive data. The SIJ ligaments are gender-dependent. This has to be taken into account for pelvic computer simulations.

DRG cells have been found to undergo apoptosis and necrosis after oxidized low-density lipoprotein (oxLDL) stimulation in vitro. However, the mechanism of oxLDL-induced DRG cell death is unclear. For this reason, we studied the expression of two potential oxLDL receptors: lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) and toll-like receptor-4 (TLR4) in dorsal root ganglion (DRG) cell cultures from postnatal rats. Cells were cultivated with and without oxLDL. In oxLDL-treated DRG cell cultures, the increase of cleaved caspase-3 protein was observed as a sign of enhanced apoptosis. Untreated and oxLDL-treated DRG cell cultures expressed LOX-1 and TLR4 at similar levels. The LOX-1 expression remained unchanged after receptor blockade. However, the inhibition of LOX-1 caused a significant increase of cleaved caspase-3 and a decrease of TLR4 levels. The TLR4-inhibited DRG cell cultures lacked changes in LOX-1 expression for all experimental groups. The inhibition of TLR4 caused activation of jun N-terminal kinase (JNK) and a significant decrease of cleaved caspase-3 but did not change the TLR4 level. We conclude that LOX-1 and TLR4 are expressed in cultivated rat DRG cells and that the oxLDL-induced cell death in DRG cell cultures does not depend on the LOX-1 but on the TLR4., (2009 Wiley-Liss, Inc.)

The intrafollicular levels of oxidized low-density lipoprotein (oxLDL) and of enzyme antioxidants might contribute to reproductive disorders in obese and infertile women. Relevant data are missing. Eighty-four patients were grouped according to obese versus non-obese status and whether they had polycystic ovary syndrome (PCOS). The concentrations of oxLDL and the activities of superoxide dismutase (SOD), catalase, glutathione peroxidase (GPx) and glutathione reductase (GR) in the serum and follicular fluid were measured. Obese women with and without PCOS had significantly greater amounts of oxLDL in the follicular fluid as compared with non-obese women. The level of oxLDL in the follicular fluid was 1000 times lower than in serum. Obese women with and without PCOS had significantly higher catalase activity in the follicular fluid as compared with non-obese women. No differences were found for the SOD activity in the follicular fluid. The GPx and GR activities were up-regulated in obese patients without and with PCOS, yet not in respect to each serum and follicular fluid sample. We conclude that elevated levels of oxLDL in the follicular fluid of obese women are associated with higher catalase activity; both parameters are independent of PCOS. The levels of oxLDL and catalase activity appear to indicate different degrees of oxidative stress.

The origin of fetal Leydig cells (FLC) and whether they share a common lineage with adult Leydig cells (ALC) is still under debate, and a marker to reliably track and isolate fetal Leydig precursor cells remains to be identified. We analyzed KIT positive (KIT+) cells in gonads from bovine fetuses with crown-rump-length (CRL) 2.5-85 cm by immunohistochemistry, and found that KIT expression was gender-specific. In female gonads, expression was mainly associated with epithelial cell cords, which extended from the surface epithelium towards the KIT-negative inner stroma. In male gonads of fetuses, after CRL 2.9 cm, KIT expression was strikingly strong in interstitial cells (IC). Only a few KIT+ cells were detected in the epithelial cell cords and in the stromal layer under the surface epithelium after CRL 3.5 cm. In the male fetuses, KIT expression in IC was a continuous and characteristic feature until full term. At all developmental stages KIT+ areas alternated with anti-Müllerian hormone-positive areas. Platelet-derived growth factor receptor alpha production was initiated after the expression of KIT at CRL 4.5 cm. Detection of cytochrome P450 side chain cleavage enzyme and steroidogenic acute regulatory protein in KIT+ IC identified them as FLC. KIT+ cells, isolated from testes by magnetic-activated cell sorting, retained their steroidogenic capacity in vitro. Together, these findings show that KIT+ IC of fetal testis correspond to FLC, which can be successfully cultivated for advanced studies.

Deferoxamine (DFO) was granted orphan drug status for the treatment of traumatic spinal cord injury but its neuroprotective mechanism is not well understood. We therefore investigated the mode of action of DFO in serum-starved and/or iron-stressed cultures of rat dorsal root ganglion (DRG) cells. We probed for redox signaling by determining hemeoxygenase-1 activity and by measuring expression of intracellular iron metabolism-related proteins under pro-oxidative conditions. We also employed DNA microarrays to better understand the genomic response of DRG cultures to treatment with DFO thereby enabling the generation of hypotheses. Essentially, DFO treatment resulted in outgrowth of neurofilament 200-positive neurites and induction of synapse formation as determined by immunoblotting, transmission electron microscopy and immunofluorescence confocal microscopy. Furthermore, DFO treatment of DRG cell cultures activated neuroprotective and antioxidative programs such as matrix metallopeptidase 2 and apolipoprotein D to promote neurite regeneration. Indeed, DFO reduced markedly reactive oxygen species formation, increased the expression of hemeoxygenase-1 and improved iron management through regulation of transferrin receptor and ferritin. We propose DFO treatment of DRG cell cultures to completely abolish the oxidative effect of ferrous iron (Fe(2+)). Taken collectively, DFO reduced oxidative stress and induced synthesis of neuroprotective and antioxidative molecules to foster nerve repair and functional recovery. Our findings help to better understand the therapeutic benefit of DFO in the treatment of spinal cord injury.