Leptin-deficient (ob/ob) mouse ovaries show fatty degeneration, enhanced apoptosis and decreased expression of steroidogenic acute regulatory enzyme.

OBJECTIVE: Leptin-deficient (ob/ob) mice are obese and infertile. Dysfunctions of the ovaries are preferentially related to leptin-deficiency. DESIGN: Morphological and molecular biological obesity-dependent changes in ob/ob ovaries. SUBJECTS: Ovaries were obtained from three-month-old mice either homozygote (ob/ob) and heterozygote (ob/+) or wild-type (C57BL6, WT) for the investigation by light and electron microscopy, as well as for western blot analysis of lectin-like oxidised low density lipoprotein receptor (LOX-1), Toll-like receptor 4 (TLR4), CD36, cleaved caspase-3, microtubule-associated protein light chain 3 (LC3), and the steroidogenic acute regulatory protein (StAR). RESULTS: Compared with control ovaries with corpora lutea, ob/ob ovaries lacked corpora lutea, follicular atresia was at a higher rate; lipid droplets accumulated in follicle cells and in the oocyte with damaged mitochondria; the basement membrane of follicles was thickened. LOX-1 and CD36 expressions were comparable for all three groups. Ob/ob ovaries showed significantly higher levels of TLR4 and cleaved caspase-3 than the ones from the control groups. The high LC3-II/I ratio in the WT and ob/+ ovaries was related to the presence of corpora lutea. The StAR protein was lower in the ob/ob ovaries signifying reduced steroidogenesis. CONCLUSIONS: Excessive lipid storage causes disorders of ovarian function in ob/ob mice. The local lipid overload leads to advanced follicular atresia with apoptosis and defect steroidogenesis. We suggest that the changes in lipid metabolism lead to increased oxidative stress and thereby, they are an important reason of anovulation and infertility.

Granulosa cell subtypes vary in response to oxidized low-density lipoprotein as regards specific lipoprotein receptors and antioxidant enzyme activity.

CONTEXT: The oxidized low-density lipoprotein (oxLDL) and its lectin-like oxLDL receptor-1 (LOX-1) are found in the follicular fluid and in granulosa cells. Lipoprotein receptors and antioxidant enzymes could differ in granulosa cell subtypes. OBJECTIVE: Our aim was to reveal cell-specific responses under oxLDL treatment. DESIGN AND SETTING: We conducted basic research at the Institute of Anatomy and the Clinic of Reproductive Medicine. PATIENTS: Women undergoing in vitro fertilization therapy participated in the study. MAIN OUTCOME MEASURES: Cultures of cytokeratin-positive/negative (CK(+)/CK(-)) granulosa cells and of cumulus cells were treated with 150 microg/ml oxLDL or native LDL under serum-free conditions for up to 36 h. Dead cells were determined by uptake of propidium iodide. LOX-1, toll-like receptor 4, and cluster of differentiation 36 (CD36) were examined in lysates by Western blots. The enzyme activities were determined in lysates and in supernatants. RESULTS: Under oxLDL treatment, predominantly CK(+) cells underwent nonapoptotic cell death. Receptors showed a cell-specific pattern of up-regulation: toll-like receptor 4 in CK(+) cells, LOX-1 in CK(-) cells, and CD36 in cumulus cells. An antioxidant ranking occurred: superoxide dismutase activity in CK(+) cells, total glutathione in CK(-) cells, and catalase activity in cumulus cells. The supernatants of oxLDL-treated CK(+) cell cultures contained more catalase activity than in controls, whereas a moderate increase was noted for glutathione peroxidase (GPx) in supernatants of CK(-) and cumulus cells. CONCLUSIONS: Catalase/GPx activity in the supernatants may be due to cell death or to secretion. Oxidative stress could be sensed by CK(+) cells and indicated by changes in catalase/GPx activity in the follicular fluid during ovarian disorders.

Description of the iliolumbar ligament for computer-assisted reconstruction.

STUDY DESIGN: The iliolumbar ligament (IL) was examined using morphometric and virtual methods. OBJECTIVES: A macroscopic study was performed to measure the anterior (AIL) and the posterior part of the IL (PIL). SUMMARY OF BACKGROUND DATA: Though being a widely accepted cause of low back pain and lumbosacral instability, the IL is neglected in computer-based biomechanical studies due to the lack of morphometric information. METHODS: Frozen sections prepared from 29 human subjects were measured and 7-tesla MR images made to distinguish the AIL and PIL. Cuboids were designated as geometric figures to both parts of the ligament, allowing computer-based calculations of length, surface, volume and angle of positional relationships. RESULTS: Based on 7-tesla MR imaging, virtual reconstruction was conducted for one male pelvis, including the IL. While left- and right-side parameters varied at a statistically significant level, no gender-dependencies could be determined. Lengths of 30 and 25 mm were measured for the AIL and PIL, as well as heights of 17-19 mm, respectively, and a thickness of 4mm. CONCLUSIONS: Correlations between the side-dependent parameters and the AIL and the PIL of the same side indicate close functional relationships. Additional dependencies suggest that the IL is capable of compensating age-related as well as bone-attributed alterations in lumbosacral morphology. The IL data and the visualised ligament structures contribute to determination of the influence of the IL in spinal and sacroiliac stability by means of computer-assisted biomechanics.

Cryopreserved porcine tendons preserve cell viability after thawing.

Controlled cryopreservation is an important method for storage of tissue grafts in skin banking, reproductive medicine and other domains. Although the availability of cryopreserved flexor tendons would be highly beneficial in reconstructive surgery, especially in complex reconstructions for which grafting material is limited, only a few studies have dealt with transplanted tendons. We achieved successful cryopreservation of porcine flexor tendons in 2 cryoprotective media: dimethyl sulfoxide and glycerol. Their viability was shown using a quantitative colorimetric MTT (3-[4,5-dimethyl-2-thiazolyl]-2,5-diphenyl-2H-tetrazolium bromide) assay. For comparison of native and cryopreserved tendons (n = 7 samples each), the adopted viability index was the ratio of MTT-dependent optical density and tendon weight. The viability index of native samples did not change significantly after cryopreservation and thawing. The proliferative capacity of tendon fibroblasts after thawing was shown in primary cell cultures. The described cryopreservation protocol and MTT assay may provide a basis for future autografting of human tendons.

The sacrotuberous and the sacrospinous ligament--a virtual reconstruction.

Little is known about the morphometric properties of the sacrotuberous ligament (ST) and the sacrospinous ligament (SS). The influence of ligaments on pelvic stability and the extent of reconstruction in case of instability are controversially discussed. The ST and the SS of 55 human subjects fixed in alcohol solution and of four fresh cadavers were measured. Both ligaments were defined as geometric figures. The ST was a contorted bifrustum, while the SS was a contorted frustum, both with elliptic planes. In all cases investigated, the ST and the SS fibres were twisted. For men, the ST and the SS had a mean length of 64 and 38 mm. For women, lengths of 70 and 46 mm were measured in the ST and the SS. The ST length, height and cross-sectional area showed gender-specific differences at statistically significant level. The ST and the SS volumes correlated closely, regardless of gender or side. Measurements of fresh ligaments of four unfixed cadavers showed similar results. The data obtained were then used to generate computer-based three-dimensional models of both ligaments, using the Catia software. Conclusively, the virtually generated ST and SS are suitable models to be included in pelvic fracture simulation, using the finite element method.

Apolipoproteins D and E3 exert neurotrophic and synaptogenic effects in dorsal root ganglion cell cultures.

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.

Pregnancy-induced changes in substance P and neurokinin 1 receptor (NK1-R) expression in the rat uterus.

Adrenergic nerve fibres of the mammalian uterus degenerate during pregnancy. The behaviour of peptidergic fibres, such as substance P-positive fibres and of its preferred neurokinin 1 receptor (NK1-R), is poorly studied in the pregnant rat uterus. The present study analysed the changes in substance P immunoreactivity and in the expression of NK1-R protein in the uterus of non-pregnant, pregnant (days 7, 14 and 21) and postpartum rats (days 1, 8 and 22) by immunohistology, dot blot analysis and western blot analysis. In non-pregnant rats, substance P-positive fibres were localized to the myometrium; these fibres progressively disappeared during gestation and were almost absent at term (day 21). At day 22 post partum, substance P-positive fibres had recovered to numbers comparable with those in the non-pregnant uterus. Dot blot analysis revealed a significant decrease in the immunoreactivity of substance P in the uterus at mid-pregnancy (day 14) and especially at term. Expression of the NK1-R protein showed a progressive increase throughout pregnancy reaching a peak on day 1 post partum; downregulation of NK1-R protein occurred on day 8 post partum. The low and high expressions of NK1-R protein were coincident with a large number of eosinophils and almost no eosinophils in the uterus at oestrus and at term, respectively. It was concluded that substance P immunoreactivity is inversely correlated with NK1-R protein expression in the pregnant and postpartum uterus. The marked upregulation of NK1-R protein at term and after birth indicates that the NK1-R may be involved in the complex regulation of labour and postpartum physiology. However, it is likely that the NK1-protein is not involved in the recruitment of eosinophils into the uterus at oestrus.

Development and regression of non-capillary vessels in the bovine corpus luteum.

The corpus luteum life cycle is accompanied by capillary growth, maturation and degeneration. Arterial blood vessels are thought to undergo hyperplasia and hypertrophy during the stage of regression, as is the case with non-capillary vessels. In this study, we used morphological studies to show that the development of non-capillary vessels occurs at other corpus luteum stages. Non-capillary vessels were present at the developmental stage of the corpus luteum, and increased markedly in number in the subsequent stages. After double-staining for ASM-1 actin and Ki-67 nuclear antigen, the proliferation of smooth muscle cells (SMCs) was only detected during stages of development and secretion. When the capillaries had disappeared at the regression stage, the arterial blood-vessel walls thickened noticeably. This was attributed to the development of fibroelastosis as shown by staining for collagenous and elastic fibres. In conclusion, the bovine corpus luteum represents a physiological model for studying arteriolization at all stages of development and secretion. At the regression stage, arterioregression sets in.

Classification of a collection of malformed human hearts: practical experience in the use of sequential segmental analysis.

The inauguration of Sequential Segmental Analysis (SSA) a few decades ago provides basic conditions to establish a universally applicable classification system for cardiac malformations. To gain practical experience, we used this method to classify a collection of 292 congenitally malformed human hearts. Our aims were to determine advantages and problems of the SSA and to make recommendations for a better practical usability and documentation. SSA is an appropriate instrument for the description of complex cardiac malformations because it is a logical step-by-step approach based on the heart morphology. The method optimizes the diagnosis of different malformed hearts. However, there are some disadvantages of SSA concerning terminology, localization of simple septal defects and exact topographic description of heart structures. For these reasons, we recommend a graphic description of the malformed heart using symbols based on the SSA. The most important prerequisite for an interdisciplinary acceptable classification system of cardiac malformations is to include morphological as well as functional aspects of the hearts.

Correlation between expression of selectins and migration of eosinophils into the bovine ovary during the periovulatory period.

Leukocytes enter specific ovarian areas at a precisely defined moment, influencing cyclically changing structures such as follicles and corpora lutea. As yet, no studies have been published on the trafficking mechanisms involving the interaction between adhesion molecules on endothelial cells (ECs) and those on leukocytes. First, antibodies against human adhesion molecules were examined by flow cytometry with the aim of identifying the same bovine antigen. Western blot analysis and immunoprecipitation revealed that the molecules had the same molecular weight as their human counterparts. Afterwards, we investigated the distribution of these antigens in various ovarian stages using immunohistology. Among the molecules, P-selectin (CD62P) and L-selectin (CD62L) showed stage-dependent expression, and were thus examined further. In the preovulatory follicle, microvascular ECs were negative for CD62P. Few of the leukocytes expressed CD62L. In a freshly ruptured follicle, CD62P expression was found in the dilated vessels of the former thecal layer. Simultaneously, a large proportion of the rapidly increased numbers of leukocytes, mainly eosinophils, located around the microvessels of the outer thecal layer expressed CD62L. In the early corpus luteum development stage, CD62L showed peak expression with 70%-80% positive cells compared to leukocytes. In the secretory stage, the septal venules showed a consistent, but now weak, staining for CD62P. Few leukocytes expressed CD62L. During regression, the total number of leukocytes, now representing macrophages, increased significantly, but the proportion of CD62L-positive cells remained constant. In summary, we found a strong correlation of CD62P expression on activated ECs and the appearance of CD62L-positive leukocytes in the early corpus luteum development stage, suggesting the participation of both selectins in the migration of eosinophils under physiological conditions.

Evidence of leptin expression in normal and polycystic human ovaries.

Leptin, the 'obese' protein, is found in cultured granulosa cells derived from human pre-ovulatory follicles. However, the occurrence of leptin has not been studied in intact ovaries, either normal or polycystic, until now. Paraffin sections from 25 human ovaries of different cycle stages and 25 wedge resections of polycystic ovaries were investigated by means of immunochemistry. Additionally, three ovaries were available for reverse transcription-polymerase chain reaction analysis. Leptin-positive cells were located in the granulosa cells of pre-antral follicles, and distinctly in the thecal layer of intact and regressing antral follicles. In the corpus luteum (CL) in the developmental stage, the former epithelioid leptin-positive thecal cells became fibroblast-like in the septum. In the CL of the secretory stage, single leptin-positive cells were detected between luteal cells. In polycystic ovaries, leptin-positive cells were noted both in the hypertrophied thecal layer and in the luteinized granulosa layer. Our findings on leptin expression at the protein level were confirmed by a positive mRNA signal for leptin in granulosa cells and in the CL. Additionally, mRNA of the full-length leptin receptor OB-R and of the short isoforms B219.1-B219.3 was identified in granulosa cells and the CL, as well as in the cortex and medulla. We conclude that leptin is produced in the ovary and may act in autocrine and paracrine ways.

Limitation of microwave treatment for double immunolabelling with antibodies of the same species and isotype.

Microwave treatment (MW) involves completely blocking contaminating staining in the double-labelling technique, using primary monoclonal antibodies from the same species and the same isotype as well as the same secondary antibody (ab). However, we noticed some limitations when locating proliferating cell types in cryostat and paraffin sections using the advantages presented by MW. Control experiments have shown that MW does not diminish contaminating staining when cytoplasmic (desmin, ASM-1) or nuclear (Ki-67) antigens have been labelled with antibodies in the first round of immunolabelling. In contrast to the cell surface antigen, CD18, where the primary ab had to be crosslinked by a secondary ab to obtain contaminating staining, this was observed for the detection of cytoplasmic or nuclear antigens only labelled with a primary ab. In conclusion, for double immunolabelling with abs from the same species and the same isotype, MW is not able to completely abolish contaminating staining.

Pulmonary inflammation induced by a recombinant Brugia malayi gamma-glutamyl transpeptidase homolog: involvement of humoral autoimmune responses.

BACKGROUND: A major allergen from the lymphatic filarial parasite Brugia malayi implicated in the pathogenesis of tropical pulmonary eosinophilia (TPE) has recently been cloned and identified as the homolog of the membrane-bound mammalian enzyme gamma-glutamyl transpeptidase (gamma-GT). Patients with acute TPE show autoreactive antibodies against endogenous gamma-GT from the pulmonary epithelium. MATERIALS AND METHODS: Recombinant B. malayi gamma-GT, alone or adsorbed to aluminium hydroxide (AL), was used in a BALB/c mouse model to analyze its antigenic/allergenic potential, its potential to induce pulmonary inflammation, and its capacity to induce autoreacting antibodies. RESULTS: Mice immunized with B. malayi gamma-GT showed significant levels of gamma-GT-specific IgG1, IgG2a, IgG3, IgA, IgE antibodies, and mild blood eosinophilia, even in the absence of adjuvant. Intranasal challenge with B. malayi gamma-GT induced peribronchial and perivascular inflammation characterized by a mixed infiltrate of lymphocytes, neutrophils, eosinophils, and macrophages. Both IL-4 and IFN-gamma were detected in the peripheral blood and in the bronchoalveolar lavage fluid of immunized and intranasally challenged mice. Histological analysis of murine lungs using affinity-purified antibodies from mice immunized with the parasite's gamma-GT revealed the presence of autoimmune antibodies against pulmonary epithelium. Western blot analysis identified the 55 kDa heavy chain subunit of the murine gamma-GT as the target of autoreactive/crossreacting antibodies. CONCLUSION: Our data from the in vivo mouse model demonstrate the potent allergenicity/antigenicity of B. malayi gamma-GT, and its capacity to induce pulmonary inflammation upon intranasal challenge. This leads to breakdown of tolerance against endogenous murine gamma-GT. Thus, humoral autoimmunity against the airways epithelium may contribute to the pathogenesis of TPE.

Increase in nerve fibers and loss of mast cells in polycystic and postmenopausal ovaries.

OBJECTIVE: To quantify nerve fibers and mast cells in human ovaries at different functional stages. DESIGN: Retrospective study. SETTING: Research laboratory of the university. SPECIMEN(S): 8 human ovaries in the follicular (cyclic) phase, 7 polycystic ovaries, and postmenopausal ovaries with (n=5) or without (n=7) hyperthecosis. MAIN OUTCOME MEASURE(S): Single- and double immunohistology for the S100 antigen in glial cells of autonomic nerve fibers, for chymase and tryptase in mast cells, and for the common leukocyte antigen on leukocytes. Histometric evaluation was also performed. INTERVENTION(S): None. RESULT(S): Polycystic ovaries contained significantly more S100-positive nerve fibers in the corticomedullary region than did cyclic ovaries (mean +/- SD per 2-mm(2) area, 476 +/- 136 and 224 +/- 133; P<.01). Postmenopausal ovaries with or without hyperthecosis had the highest density of nerve fibers. In cyclic and polycystic ovaries, more tryptase-positive mast cells than chymase-positive mast cells were found in the interstitial cortex and the medulla. In cyclic ovaries, areas with a moderate density of nerve fibers contained many mast cells. Hence, with increasing nerve fiber density in polycystic ovaries, the number of mast cells decreased strikingly compared with cyclic ovaries (p<.001). Almost no mast cells were seen in postmenopausal ovaries with and without hyperthecosis. The number of leukocyte antigen-positive leukocytes was similar in all groups. CONCLUSION(S): The high density of nerve fibers in polycystic and postmenopausal ovaries, together with a conspicuous decrease in mast cells, indicates altered neuroimmune communication.

Increase in calcitonin gene related peptide (CGRP) and decrease in mast cells in dihydroepiandrosterone (DHEA)-induced polycystic rat ovaries.

UNLABELLED: The polycystic ovary is reported to correspond with a high density in intraovarian nerve fibers and their sympathetic hyperresponsiveness. Peptidergic nerves may also be involved in this process. An interaction between nerve fibers and mast cells is assumed because of nerve growth-factor production by mast cells. Here we investigated CGRP-positive nerve fibers and mast cells in polycystic ovaries induced in immature rats with dihydroepiandrosterone (DHEA). The DHEA treated ovaries contained less corpora lutea than controls (mean +/- SEM: 4.3 +/- 0.6 versus 11.3 +/- 0.9, P > 0.001) and less intact antral follicles (4.7 +/- 0.7 versus 8.1 +/- 1.1; P < 0.05) according to the histometric approach. By immunolabelling more CGRP-positive nerve fibers were found in the DHEA treated ovaries than in controls (mean +/- SEM per one section: 23.2 +/- 5.8 fibers versus 10.3 +/- 0.9 and 171 +/- 44.7 varicosities versus 84 +/- 9.5). This was confirmed by dot blot analysis, showing a significant higher CGRP signal intensity per microgram homogenized ovaries of the DHEA treated group compared to the untreated (P < 0.05). Toluidine-blue-stained mast cells populated the medulla in both groups, yet had strikingly decreased in the DHEA treated ovaries (23.5 +/- 3.9 versus 89 +/- 5.6, P < 0.005). CONCLUSION: The increase in CGRP-positive nerve fibers and the decrease of toluidine-blue-stained mast cells points to an altered neuroimmune function in DHEA-induced polycystic rat ovaries.

The expression of substance P and its neurokinin-1 receptor mRNA in the bovine corpus luteum of early developmental stage.

Related to all leukocytes, 90% of eosinophils are recruited into the bovine corpus luteum of early developmental stage. We here describe a simultaneous appearance of substance P (SP)-positive fibre-like structures and the neurokinin-1 (NK-1) receptor mRNA for SP. Substance P was depicted by using indirect immunohistology and immunofluorescence localization. The dot blot analysis confirmed the presence of SP at the protein level. Using nested reverse transcribed-polymerase chain reaction (RT-PCR) analysis, a 358 bp long partial bovine receptor mRNA for SP (NK-1) was sequenced in the spinal cord. The mRNA for SP and for the NK-1 receptor were then detected in the corpus luteum of early developmental stage with RT-PCR and nested RT-PCR. We conclude: The production of SP and the expression of NK-1 receptor mRNA may be involved in the selective recruitment of eosinophils into the bovine corpus luteum of early developmental stage.

Leucocyte proliferation in the bovine corpus luteum.

Leucocytes vary in type and number during the lifespan of a corpus luteum. The aim of this study was to determine whether there is an increase in the number of lymphocytes and macrophages as a result of local proliferation. Bovine corpora lutea were classified into stages of development, secretion and regression. A new double immunolabelling method was established for nuclear Ki-67 antigen (a marker for cell proliferation) and for leucocyte surface antigens (detection of CD2-, CD3-, CD4-, CD8-positive lymphocytes and CD14-positive monocytes). Differential cell counting was performed. Between the stages of development and regression there was an increase in the number of T-lymphocytes and macrophages. The percentage of proliferating leucocytes in relation to the total number of proliferating cells was approximately 20% at the stage of advanced secretion and 70% at late regression. The increase in the number of proliferating leucocytes at late regression was due to CD14-positive macrophages. These macrophages migrated from small blood vessels into the septa of corpora lutea at the early stage of regression. Macrophages showed local proliferation in the late stage of regression when capillaries were no longer present. It is concluded that the physiological involution of the corpus luteum is an inflammatory-like condition, which includes local proliferation of monocytes.

Increase in final stages of follicular atresia and premature decay of corpora lutea in Insl3-deficient mice.

The insulin-like factor 3 (Insl3), a member of the insulin-like hormone family, is exclusively synthesized in gonads. Our recent analysis of Insl3-deficient mice revealed the regulating role of the Insl3 factor on the gubernaculum development during the transabdominal descent of the testis. Here we define the role of the Insl3 factor by histometric analysis of wild-type and Insl3(-/-) ovaries. Ovaries from 40-day-old- and 6-month-old Insl3(-/-) mice as well as from wild-type littermates were serially sectioned. Sections were stained with periodic acid Schiff reaction (PAS) for counting the number of zonae pellucidae which indicated the final stages of follicular atresia. Corpora lutea were also determined. Some sections were processed using either a modified TUNEL method for in situ detection of apoptosis or a lectin labelling technique with Griffonia simplicifolia I agglutinin (GS I) for endothelial cell occurrence. The number of zonae pellucidae was higher in Insl3-deficient ovaries of both ages than in ovaries of wild-type sisters (P < 0.05 for 40-day-old ovaries; P < 0.01 for 6-month-old ovaries). Additionally, the wild-type mice of both ages possessed threefold more corpora lutea than their Insl3 littermates (P < 0.01 for 40-day-old; P < 0.001 for 6-month-old). In general, wild-type corpora lutea looked healthy, showed GS I-positive endothelial cells and no apoptotic cells. Corpora lutea from mutants were rich in regressing GS I luteal cells, and apoptotic cells appeared. We conclude: Follicular atresia and luteolysis are accelerated in ovaries of Insl3-deficient mice probably because of increased apoptosis. The Insl3 function thus appears to rescue endocrine cells from the apoptotic pathway.

Eosinophils in the human corpus luteum: the role of RANTES and eotaxin in eosinophil attraction into periovulatory structures.

We evaluated the presence and number of eosinophils at varying stages in the human corpus luteum from 27 ovaries of women at reproductive age. Eosinophils preferentially accumulated in dilated microvessels of the thecal layer transforming into septa of the corpus luteum. The granulosa layer under luteinization, the thecal layer, and haemorrhages in the former antrum each contained low, moderate and high numbers of extravasated eosinophils respectively. Eosinophils decreased rapidly during the stages of secretion and regression. Semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA) systems were used to investigate the expression and regulation of the eosinophil-attracting chemokines RANTES (regulated on activation, normal T cell expressed and secreted) and eotaxin in granulosa cells obtained from follicular aspirates from women undergoing IVF. Contaminating leukocytes were determined by CD18 mRNA quantification. Granulosa cells expressed RANTES (n = 3; 43 +/- 14 pg/ml, mean +/- SEM). 4ss-phorbol-12-myristate-13-acetate (PMA; 211 +/- 53) and tumour necrosis factor alpha (TNFalpha) (238 +/- 59), but not interleukin (IL)-1 up-regulated RANTES at significant levels. In general, higher basal and stimulated RANTES mRNA and protein were found in cultures with higher CD18 mRNA levels than in those with lower levels. We found only traces of eotaxin mRNA and no eotaxin secretion, even in stimulated granulosa cell cultures, independently of leukocyte levels. Taken together, this is the first study demonstrating the selective presence of eosinophils in human periovulatory structures. RANTES, but not eotaxin, may play an active process in the accumulation of these cells.

Microvascular endothelial cells differ in their basal and tumour necrosis factor-alpha-regulated expression of adhesion molecules and cytokines.

We recently located a rare cytokeratin-positive (CK+) type of microvascular endothelial cell (MVEC) in the corpus luteum and aorta. Bovine corpus luteum MVEC are known to be involved in the cyclic accumulation of eosinophils and macrophages. Since leukocyte migration is specifically mediated by adhesion molecules and the release of cytokines, we compared the expression of these factors in basal and TNF-alpha-stimulated CK+ MVEC and in common cytokeratin-negative (CK-) MVEC in order to obtain an initial insight into the functional capacities of CK+ MVEC. CK- MVEC revealed significantly higher basal RANTES mRNA expression than CK+ MVEC, and TNF- alpha up-regulated RANTES mRNA in both types of MVEC. Only resting and stimulated CK- MVEC expressed granulocyte-macrophage colony-stimulating factor mRNA. Both MVEC types expressed monocyte colony-stimulating factor mRNA, but remained negative for eotaxin and interleukin (IL)-5 mRNA even after stimulation. Resting CK+ MVEC were positive for CD29, CD31, CD49a and CD49e, but expressed most of these antigens at a significantly lower density than did CK- MVEC. In contrast to CK- MVEC, CK+ MVEC failed to express CD49b or MHC class II. The activation of CK+ MVEC with TNF-alpha induced the expression of CD62P, but not of CD49b or MHC class II. In summary, phenotypically variable MVEC derived from the microvascular bed of one organ differ in their TNF-alpha-regulated expression of cytokine mRNA and adhesion molecules. Morphological heterogeneity is related to a particular specialisation of functional MVEC.