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.

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.

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.

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.

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.

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.

The transient disappearance of cytokeratin in human fetal and adult ovaries.

Cells from the inner and outer granulosa cell layers of the ovarian follicles differ in function, probably because of their different origins from the surface epithelium and from the rete. This suggestion has not so far been thoroughly investigated in the human ovary. We examined fetal ovaries from the early, middle and late gestational periods, ovaries from fertile women, and preovulatory follicular cells obtained from patients under in vitro fertilization therapy (IVF). Indirect immunohistology and immunocytology were used to detect the presence of cytokeratin (CK)-positive epithelial cells. In fetal ovaries from the early gestational period, prominent rete tubules (sometimes with oocytes) appeared to be fused with the sex cords and primordial follicles. Both showed CK-positively, detected with the pan-CK antibody Lu-5. Cytokeratin 19 was clearly expressed in the fusion area. In the fetal and adult ovaries, CK-positive follicular or granulosa cells were noted in the primordial and primary follicles as well as the preovulatory follicles. Cytokeratin was not detected in the granulosa cells of growing follicles, CK-positive and -negative luteal cells were identified in the developing corpus luteum. We conclude for the human ovary: (1) the heterogeneous morphology of granulosa cells may be explained by their twofold origin from the surface epithelium and the rete, (2) the rete tubules appear to be involved in folliculogenesis, (3) the transient absence of CK expression in growing follicles compared to resting and mature follicles or to the developing corpus luteum indicates a particular role of CK-positive cells at the periovulatory period.

Cloning of bovine RANTES mRNA and its expression and regulation in ovaries in the periovulatory period.

RANTES may be one of the chemoattractants involved in stimulating eosinophils and macrophages to migrate selectively into bovine dominant follicles and into developing corpora lutea. We sequenced a 736 bp fragment of the bovine RANTES mRNA encoding the complete protein and defined the ovarian source of RANTES mRNA. As demonstrated by competitive RT-PCR, follicle-derived macrophages showed a 100-1000 times higher RANTES mRNA level compared to unpurified granulosa cells or follicle-derived fibroblasts. By means of in situ hybridization, RANTES mRNA positive macrophages were located in the former thecal layer of the developing corpora lutea.

Evidence for the maintenance of macrophage-like cells in long-term bovine granulosa cell cultures.

Ovarian macrophages may be involved in the degradation of the basal membrane after rupture of a preovulatory follicle, or in angiogenesis during follicle and corpus luteum development, or may support steroidogenesis. The present study describes easy access to macrophage-like cells in the ovary and characterizes them. Cells from bovine antral follicles with 1-4 ml of their naturally occurring fluid were plated on day zero. On day 10, the granulosa cell culture showed macrophage-like cells. They developed as non-adherent cells and also adhered to the monolayer as single cells or as hemopoietic-like clones. The macrophage-like cells contained strong activity of non-specific esterase, acid phosphatase or 3beta-hydroxy-steroid dehydrogenase (3beta-HSD). Only the steroid enzyme was absent on day 19. As detected by immunolocalization, macrophage-like cells of various sizes showed a response for the CD18 surface molecule of leukocytes, for the CD45 molecule related to hemopoietic progenitor cells, and for the CD14 molecule selectively expressed on cells of the monocyte-macrophage lineage. The macrophage-like cells exhibited a granular-like actin structure as revealed by fluorochrome-labeled phalloidin. The ultrastructure of small, intermediate or large macrophage-like cells was similar to that of monocytes or of macrophages. In situ immunolabeling of intact or regressing follicles revealed single cells positive for CD14, CD18 or CD45. In order to establish that in vitro proliferation of the macrophage-like cells had occurred, their number was assessed between days 1 to 19 of cultivation. The cultures derived from 23 follicles were classified into groups A, B, or C according to the number of non-adherent cells (<5000, <12 500, or >/=12 500 per 16-mm well) on day 12 of cultivation. The total number of macrophage-like cells (non-adherent and adherent) increased approximately fourfold in all groups between days 1 to 12. The total number decreased to day 5 levels between days 15 to 19. Thus, the macrophage-like cells probably represent a subpopulation of the macrophage family. They proliferate in co-culture with granulosa cells.

Evidence for the development of macrophage-like cells in long-term culture of bovine aortic endothelial cells.

Macrophages are known to be derived from monocytes which proliferate in the bone marrow. The proliferation of monocytes may occur in other places as well. In the present study, we describe the morphological behaviour of macrophage-like cells in endothelial cell cultures obtained from bovine aorta. These cells resembled hemopoietic clones containing progenitor-like cells. Immature and mature macrophage-like cells were rich in acid phosphatase activity, and expressed the CD18 molecule using immunolocalisation. Mature cells contained intracellular lipid droplets. "Actin" globules were apparent only in the peripheral cell areas without lamellipodia or filipodia. At the ultrastructural level, the mature cells were crowded with granules which could be lysosomes, phagolysosomes, or endocytotic vesicles. Multinuclear giant cells which behaved in a different way to the macrophage-like cells were observed. The development and maintenance of macrophage-like cells appears to be dependent on the coculture with endothelial cells. It may signify that endothelial cells are involved in the proliferation of monocytes outside the bone marrow.

Cytokeratin expression in the developing vagina of the postnatal gerbil (Meriones unguiculatus).

During postnatal development the vaginal epithelium of the Mongolian gerbil is transformed from two to three layers into a stratified, first mucified subsequently keratinized squamous epithelium. Changes in the expression of cytokeratins were studied and the immunohistochemical results compared with the ultrastructural findings at the corresponding stage. The first 10 postnatal days (days pn) were characterized by a moderate, positive immunoreaction for pancytokeratin in all vaginal cell layers. A faint reaction was caused by mAB CK 18.01 against CK 1, 5, 6 and 8. The appearance of mucous granules in the luminal cells after 15 pn seemed to coincide with an increase in cytokeratins. The immunoresponse for pancytokeratin in these cells was very intense compared with the reaction in the basal cell layers. Mucocytes during development and at proestrus were the only cells which reacted faintly positive with mAB against CK 18 alone. The keratinizing epithelium, which differentiates after day 40 pn, reacted strongly positive for pancytokeratin in the keratinizing layers, desquamating, fully keratinized cells, however, showed a negative reaction. The data indicate that mucocytes are not transformed squamous keratinized cells, but represent a cell category with its individual differentiation potential. Vimentin was not expressed. Neither the epithelium of the sinus vagina nor of the Müllerian vagina displayed any response.

Cytokeratin expression in bovine corpora lutea.

Cytokeratin (CK)-positive cells were obtained from bovine corpora lutea. When cultured, these cells behave like CK-positive endothelial cells obtained from bovine large blood vessels. The origin of CK-positive cells has now been studied in 45 bovine corpora lutea of different estrous cycle stages. Additionally, 7 corpora lutea of pregnant cows were examined. The tissues were grouped into early stage (days 2 to 4), secretory stage (days 5 to 17) and late stage (days 18 to 21) according to gross morphology, wet weight and total progesterone content. One portion of a corpus luteum was used for immunohistochemistry, and another for Western blot analysis. Twenty-six of the 45 corpora lutea showed CK expression, as confirmed by immunostaining and Western blotting. Cytokeratin expression was found in all corpora lutea from the early stage, in 14 of 26 corpora lutea from the secretory stage, and 3 of 10 from the late stage. Early stage corpora lutea displayed "zonation" such that a high number of CK-positive luteal cells occurred in the region of the previous granulosa layer and a very low number in the previous thecal layer. Secretory CK-positive corpora lutea showed uniformly distributed, predominantly large luteal cells. In secretory corpora lutea of group A, CK-positive cells and a distinct microvascular tree were seen, the latter visualized by factor VIII-related antigen immunolabelling of endothelial cells. Group B showed none or very few CK-positive cells. Corpora lutea of pregnant cows behaved like corpora lutea of group B. Roughly 1% of CK-positive cells closely associated with the capillary wall were sometimes reminiscent of endothelial cell sprouts.

Long-term co-culture of bovine granulosa cells with microvascular endothelial cells: effect on cell growth and cell death.

The intraovarian axis between granulosa cells and thecal cells is regulated by locally produced autocrine and paracrine factors. Until now, microvascular endothelial cells (MVEC) have not been included in such studies. Bovine granulosa cells from medium-sized antral follicles were plated at low density into the lower compartment of 24-well-culture plates on day 0. MVEC derived from bovine corpus luteum were seeded on appropriate inserts and placed as the upper compartment on day 1. Control granulosa cell cultures and MVEC co-cultures were maintained in serum-containing medium. On day 21, control cultures displayed an epithelioid monolayer and the coculture displayed a multilayer. Histochemical staining for 3 beta-HSD activity and for the lipid droplet stain with the fluorescent dye Nile Red were strong, suggesting augmented steroidogenesis in the multilayer. Yet the progesterone levels of supernants corrected for 10,000 cells were similar in monolayers and in multilayers. Co-cultures contained approximately three times more granulosa cells than control cultures as evaluated with a Coulter counter. Additionally, the occurrence of dead cells was quantified with the fluorescent DNA stain, ethidium homodimer, in 11-day-old control cultures and MVEC co-cultures which were deprived of serum, MVEC, or both for an additional 40 h. Serum and MVEC suppressed the occurrence of granulosa cell death. It is concluded that MVEC produce survival factors for the growth and maintenance of granulosa cells.

Isolation of granulosa-like cells from the bovine secretory corpus luteum and their characterization in long-term culture.

BACKGROUND: The isolation of cells termed type 5 from the bovine corpus luteum was recently reported. Since these cells were reminiscent of immature granulosa cells, their morphological and functional relationship requires further investigation in view of the novel concept of corpus luteum growth. It suggests that putative stem cells of unknown origin supply the pool of small luteal cells. METHODS: Bovine corpora lutea were mechanically dispersed, cell suspensions separated over a Percoll density gradient, and type 5 cells purified by colony transfer. Granulosa cells were harvested from small-sized antral follicles. Observations were carried out at the light and electron microscopical level. 3 beta-Hydroxy-steroid-dehydrogenase was localized histochemically in addition to intracellular lipid droplets stained with nile red. Immunolocalization was used to study Factor VIII antigen presence, the architecture of the cytoskeleton, as well as the occurrence of neuronal cell adhesion molecules, and of neuronal cadherin-like molecules. The uptake of acetylated low density lipoprotein was examined. As for progesterone concentration, cells were seeded at low density on day zero. Cell numbers and progesterone levels of supernatants were determined on day 10 in culture. RESULTS AND CONCLUSIONS: Type 5 cells behaved morphologically like immature granulosa cells, yet the total cell number and the progesterone concentration differed for type 5 cells compared to granulosa cells. The addition of LH had no influence on the progesterone concentration as seen for either type 5 cells or for granulosa cells. It is concluded that type 5 cells, which were originally mistaken for microvascular endothelial cells, display similarities with immature granulosa cells. Type 5 cells may play a role in renewal of luteal cells.

Isolation of blood granulocytes from golden hamsters for the study of chemotaxis.

Since the number of granulocytes is low in the circulating pool of adult golden hamsters, intraperitoneal injection of oyster glycogen was used to mobilize white blood cells from the non-circulating pool. The percentage of blood granulocytes increased from 7 to 49%. Mobilized granulocytes, purified by a discontinuous Percoll gradient, showed adherence to polycarbonate filters only when coated by collagen. Random migration was high using the modified Boyden chamber technique. While endotoxin-activated serum caused chemotactic response, it was absent when the tripeptide N-formyl-methionyl-leucyl-phenylalanine was applied at concentrations of 10(-7) or 10(-8) M. The true chemotactic response corresponded to the proportion of migrated cells versus the total cell number adherent to both sides of the filter. It is concluded that the golden hamster is a good model for the study of granulocyte sequestration.

Albumin localization in the testis of adult golden hamsters by use of immunohistochemistry.

There is an increasing interest in different biochemical roles of albumin (Alb) in respect to testicular function. For this reason, Alb was localized by the peroxidase-antiperoxidase technique in the testis of adult golden hamsters. A strong Alb immunoreactivity occurred in the interstitial space and on the surface of Leydig cells. A few Sertoli cells showed a strong Alb response under the appearance of so-called leakages traversing Sertoli-Sertoli cell junctions. Spermatogonia reacted strongly, while spermatids and sperms showed rather a mild, or moderate response. Spermatocytes remained unstained. When similar maturation stages of seminiferous tubules were compared, the number of reacting cells varied strikingly. Our results support the assumption that Alb or a protein with an Alb-like immunoreactivity exerts diverse functions of the testis.

The chick chorioallantoic membrane as test system for biocompatible materials.

Biologic and non-biologic materials clinically used as hemostyptica, as vascular prostheses, or as temporary skin substitutes were implanted to the chick chorioallantoic membrane (CAM) during days 9-14 of incubation. The histological study revealed an intact CAM after application of cellulose gauze (Tabotamp). The fibrin tissue adhesive (Tissucol), the collagen sponge (Tachotop), or the gelatin sponge (Gelfoam) induced different amounts of connective tissue. Some fibroblasts were about to grow into the fibrin adhesive. Fibroblasts and capillaries filled the interstices of the collagen sponge poorly, and in the case of the gelatin sponge markedly. Neutrophils and giant cells of the foreign body type occurred more often in the gelatin sponge than in the collagen sponge. With regard to the non-biologic materials, expanded polytetrafluorethylene induced squamous metaplasia of the chorionic epithelium. Dacron caused ulceration with the onset of connective tissue ingrowth, based on a reaction of giant cells of the foreign body type. Polyurethane foam (SYSpur-derm) showed bleeding into the implant due to spikes of the material. Eosinophilic granulocytes were absent in all materials studied. The results are related to different clinical findings. The correlation allows consideration of the CAM as an in vivo model in screening materials for their biocompatibility and their connective tissue reaction.

Effects of locally applied enzyme inhibitors of the arachidonic acid cascade on follicle growth and intra-ovarian oocyte release in hyperstimulated rabbits.

This study was conducted to examine follicle growth and intra-ovarian oocyte release in rabbit ovaries influenced by a local application of prostaglandin formation inhibitors. While five rabbits remained as unstimulated controls, twenty-seven mature rabbits were hyperstimulated by pure follicle stimulating hormone (FSH) followed by human chorionic gonadotropin (hCG) at time 0. At the same time, 1 mg lonazolac (lonazolac group), indomethacin (indomethacin group) or saline (so-called gonadotrophic stimulated controls) was applied into the vagina of each group. Both ovaries of each animal were evaluated morphometrically at 15, 20, 28 and 48 hr after hCG. The absolute numbers of smaller-sized preantral follicles were always higher in the gonadotrophic stimulated controls than in the unstimulated controls, the lonazolac, or the indomethacin group. The numbers of larger-sized preantral and antral follicles showed no differences. Unlike the gonadotrophic stimulated controls and the lonazolac group, the indomethacin group displayed an increase in its mature structures (large-sized antral and preovulatory follicles, follicle and luteinized cysts) from 20 to 28 hr after hCG. Call-Exner bodies underwent pseudomucification in preovulatory follicles. The absolute numbers of Call-Exner bodies were augmented by indomethacin. Maturation division of oocytes began to decrease in all three treated groups between 15 and 20 hr post hCG, but the height of intra-ovarian oocyte release (IOR) appeared at 28 hr. This coincided with a striking thrombus formation. It is assumed that: 1) lonazolac and indomethacin inhibit early development of proliferating follicles; 2) lonazolac and indomethacin have no effects on the maturation division of the oocyte and of IOR; 3) IOR extends over a longer period after ovulation induction in contrast to the maturation division of the oocyte; and 4) thrombus formation is related to IOR.