Matrilysin activity in the rat uterus during the oestrous cycle and implantation

The objective of this study was to follow changes in the activity of the small matrix metalloproteinase matrilysin (MMP-7) in the rat uterus during the oestrous cycle and embryo implantation. Matrilysin was extracted from rat uteri, partially purified and separated into active and latent forms. The two forms of the enzyme were quantified at all stages of the oestrous cycle and after oestradiol and progesterone treatment. The activity was also measured during the first 7 days of pregnancy. Both latent and active forms of MMP-7 reached a peak during the pro-oestrous stage of the cycle; the concentrations were three times higher than at dioestrus and metoestrus. In rats treated with 0.1 mg oestradiol at metoestrus, both latent and active forms of the enzyme increased by more than two-fold after 24 h. In rats treated at pro-oestrus with 0.4 mg progesterone, there was a 70% increase in latent MMP-7, but no change in the active form. The highest concentrations of MMP-7 were observed on the first day of pregnancy. Between days 3 and 7 of pregnancy, the concentrations were relatively constant and comparable to the low concentrations at dioestrus. Enzyme activities were not different at implantation sites compared with remote sites.     

Experimental mucopolysaccharidosis: preservation and ultrastructural visualization of intralysosomal glycosaminoglycans by use of the cationic dyes cuprolinic blue and toluidine blue

The cationic dyes Cuprolinic Blue (CB) and Toluidine Blue (TB) were used to preserve the intralysosomal storage material accumulating in tilorone-induced mucopolysaccharidosis. As shown in previous studies, the stored glycosaminoglycans (GAGs) are leached during the conventional fixation procedure, with the result that the lysosomes appear empty. In the present study, the liver, spleen, and cornea-conjunctiva of tilorone-treated rats were examined. The application of CB in the presence of 0.1 M or 0.3 M MgCl2 simultaneously with, or subsequently to the primary fixative yielded electron-dense precipitates within the storage lysosomes. When TB (0.1%) was added to the primary fixative, the storage lysosomes contained filamentous structures arranged in reticular patterns. With increasing TB concentrations (up to 1%) the lysosomes increasingly often showed apparently amorphous storage material which was continuous with the reticular filamentous structures. Similar ultrastructural patterns were obtained with GAG-TB complexes prepared in vitro. The intralysosomal storage material preserved by TB is interpreted as GAG-TB precipitates. In conclusion, the use of CB provides a method which allows direct cytochemical demonstration of the subcellular sites of GAG-storage. The use of TB represents an easy method to obtain electron micrographs pathognomonic of the mucopolysaccharidosis induced by tilorone and congeners. Either method may be helpful to detect this adverse drug effect at the subcellular level.     

Micro-organisms and the appearance of leucocytes in the vaginal wall of cyclic female rats at metoestrus.

The present study was undertaken to examine whether leucocytic infiltration of the vagina of the rat at metoestrus is dependent on its contamination by micro-organisms. Observations were made on vaginal tissue that had been transplanted under the kidney capsule in cyclic rats, taking care to avoid infection during the transplantation procedure. In such grafts, changes occurred that were associated with ovulation and formation of the CL, but leucocytosis was never obtained at metoestrus. Cyclic changes were observed in the cell patterns of the vaginal smears of germ-free rats, and could be correled exactly with the ovarian cycle. No leucocytes were present at metoestrus. Many micro-orgainisms were present in the vagina at pro-oestrus and oestrus in normal cyclic females, but not at metoestrus and dioestrus. It is concluded that the occurrence of leucocytosis in the vagina at metoestrus in normal cyclic female rats depends on the presence of micro-oranisms.     

Experimental mucopolysaccharidosis: Preservation and ultrastructural visualization of intralysosomal glycosaminoglycans by use of the cationic dyes cuprolinic blue and toluidine blue

Summary The cationic dyes Cuprolinic Blue (CB) and Toluidine Blue (TB) were used to preserve the intralysosomal storage material accumulating in tilorone-induced mucopolysaccharidosis. As shown in previous studies, the stored glycosaminoglycans (GAGs) are leached during the conventional fixation procedure, with the result that the lysosomes appear empty. In the present study, the liver, spleen, and cornea-conjunctiva of tilorone-treated rats were examined. The application of CB in the presence of 0.1 M or 0.3 M MgCl₂ simultaneously with, or subsequently to the primary fixative yielded electron-dense precipitates within the storage lysosomes. When TB (0.1%) was added to the primary fixative, the storage lysosomes contained filamentous structures arranged in reticular patterns. With increasing TB concentrations (up to 1%) the lysosomes increasingly often showed apparently amorphous storage material which was continuous with the reticular filamentous structures. Similar ultrastructural patterns were obtained with GAG-TB complexes prepared in vitro. The intralysosomal storage material preserved by TB is interpreted as GAG-TB precipitates. In conclusion, the use of CB provides a method which allows direct cytochemical demonstration of the subcellular sites of GAG-storage. The use of TB represents an easy method to obtain electron micrographs pathognomonic of the mucopolysaccharidosis induced by tilorone and congeners. Either method may be helpful to detect this adverse drug effect at the subcellular level.     

The mature mid-cycle follicle in the mare.

Oestrogen and progesterone concentrations in blood and follicular fluid and blood levels of LH were determined in 426 mares at different stages of the oestrous cycle. Mature follicles occur at all stages of the cycle; they ovulate readily in early metoestrus, occasionally in late metoestrus and very rarely in dioestrus. Maturation of a mid-cycle follicle is associated with intermediate levels of LH, which are less than those found during oestrus. This lower level of LH together with a high level of progesterone are probably responsible for the failure of ovulation and regression of most of the mid-cycle mature follicles found in the mare.     

Expression and localisation of thioredoxin in mouse reproductive tissues during the oestrous cycle

Thioredoxin expression within the reproductive tissues of the female mouse was analysed during the oestrous cycle stages of dioestrus, oestrus and metoestrus by Western blot analyses and immunocytochemistry. From Western blot analyses the expression of thioredoxin was found to be increased in oestrus compared to dioestrus and metoestrus. Localisation of thioredoxin within the reproductive organs of the mouse during the oestrous cycle has shown that the expression of thioredoxin is specific for distinct areas within the reproductive organs. These areas are the stratified squamous epithelium of the vagina, the simple columnar epithelium and the uterine glands of the uterus, the ciliated columnar epithelium of the oviduct, the corpus lutea, the interstitial cells and the secondary follicles of the ovary. The discrete cellular localisation and oestrous dependence of thioredoxin expression are suggestive of specific roles in various reproductive processes.     

Fixation and staining of granules in mucosal mast cells and intraepithelial lymphocytes in the rat jejunum, with special reference to the relationship between the acid glycosaminoglycans in the two cell types

Summary Various fixation and staining procedures have been examined in order to obtain optimal numbers and acceptable morphology of the mucosal mast cells and granular intraepithelial cells in the rat jejunum. For subsequent staining with Alcian Blue, the best fixation of the jejunum was obtained with a methanol-formaldehyde-acetic acid mixture. Specific staining of the granules of these cells has been obtained using Alcian Blue at pH 5.8, at which hydrogen ion concentration more cells stain than in the usual very acid conditions. Specificity is achieved by the use of magnesium chloride concentrations above the critical electrolyte concentrations for staining of protein and nucleic acid by Alcian Blue, and by the use of Safranin O as a competitive counterstain.The critical electrolyte concentration technique has also been applied to a comparative study of the glycosaminoglycan in the two cell types. Evidence is presented that the glycosaminoglycan in the granular intraepithelial cell has either a lower degree of sulphation or a lower molecular weight or both than the material in mucosal mast cells. This finding may support the possibility that the granular intraepithelial lymphocyte is a precursor of the mucosal mast cell.     

Changes in neurohypophysial cholecystokinin content during oestrous cycle in the rat

Cholecystokinin content in the neurointermediate lobe of the rat pituitary was measured by radioimmunoassay during the different stages of the oestrous cycle. Higher levels were observed in pro-oestrus and oestrus than in metoestrus and dioestrus rats.

This difference is similar to the variation observed in the same circumstance concerning oxytocin in the neurohypophysis and neurosecretory activity in magnocellular neurons. These results are discussed in relation to the coexistence of oxytocin and cholecystokinin in neurons of the hypothalamoneurohypophysial system.     

小鼠发情周期卵泡发育动态及其对超数排卵的影响

该文探讨了小鼠发情周期中阴门状态、阴道脱落细胞类型变化规律、卵泡发育规律及其相互关系,并比较了发情周期不同阶段的超排效果。结果表明,采用阴门状态观察法和阴道脱落细胞涂片法,能有效判断小鼠发情周期阶段。卵巢组织切片观察结果表明,在发情周期不同阶段,小鼠的卵泡发育和黄体的生成与消退存在明显的规律性变化;小鼠发情周期中,其阴门状态、阴道脱落细胞种类及卵泡发育动态之间存在相关关系;发情周期不同阶段开始超排的小鼠,其配种见栓率和回收胚胎平均数均存在明显差异,发情前期显著优于发情后期与间情期(P<0.05),并高于发情期,但差异不显著(P>0.05),即阴门状态观察法与阴道脱落细胞涂片法均可用于小鼠发情周期阶段的判断,发情前期为最适宜的小鼠超排时期。     

The acinar heterotopy of phosphoenolpyruvate carboxykinase activity in rat liver under the influence of the oestrous cycle

The acinar localization of phosphoenolpyruvate carboxykinase was investigated in livers from untreated female rats during the 4-day oestrous cycle. The results were correlated with plasma levels of insulin, glucagon and oestrogens. Total activity was highest in dioestrus and lowest in metoestrus. The highest activities were present in the periportal zone and decreased continuously towards the perivenous zone in all four phases of the cycle. This general pattern was modified under the influence of the cycle. The periportal-perivenous gradient was subject to cycle dependent changes, being steepest in proestrus. In addition to oestrogens the concentrations of plasma insulin also varied during the female cycle. Insulin was highest in oestrus and lowest in proestrus. Glucagon showed only minor variations. Oestrogen concentrations increased continuously from low values in oestrus to high values in proestrus. These changes were directly related to the changes in the slope of the activity gradient.     

Changes of progesterone content of rat uterine flushings in relation to serum concentrations of progesterone during the oestrous cycle.

Uterine fluid was collected from four-day cyclic rats at each stage of the oestrous cycle and assayed for progesterone and protein content. Progesterone was determined by radioimmunoassay either after ethanol (or 2.5% NaOH) denaturation of proteins from uterine flushings ('total' progesterone) or without protein denaturation ('ether-extractable' progesterone). The amount of 'ether-extractable' progesterone in the lumen was constant from metoestrus to pro-oestrus (340 pg per uterus) but lower in oestrus (200 pg per uterus). However, 'total' progesterone content of uterine fluid was subject to cyclic variations and was highest in dioestrus (890 pg per uterus) and lowest in oestrus (350 pg per uterus), in contrast to serum progesterone which is lowest in dioestrus and highest in oestrus. Protein content of uterine flushings peaked to 780 micrograms per uterus in pro-oestrus then fell to about 140 micrograms per uterus until the end of the oestrous cycle. Changes in protein content of the lumen were followed by qualitative variations since the mean amount of 'bound' progesterone ('total' progesterone minus 'ether-extractable' progesterone) released per milligram of denatured lumen protein rose from 1.8 pmol in pro-oestrus to 18.2 pmol in dioestrus. The changes of luminal 'bound' progesterone during the oestrous cycle suggest that progesterone binding to luminal proteins could be an important modulator of progesterone action in rat uterus. Moreover, the variations in progesterone content of the lumen, irrespective of serum progesterone concentrations, are consistent with the hypothesis that progesterone synthesis occurs in the uterus.     

Ovum transport in cyclic rats rendered hypo-oestrogenic by treatment with 4-hydroxy-4-androstene-3,17-dione

The effects of decreasing oestrogen secretion upon the rate of oocyte transport achieved by the administration of 4-hydroxy-4-androstene-3,17-dione were investigated in cyclic rats. Control animals examined during late pro-oestrus showed that the majority of oocytes had entered the uterus. In contrast, when inhibitor was administered from the afternoon of metoestrus or from late dioestrus through prooestrus, oviducal retention of oocytes was observed. When treatment was delayed until the morning of pro-oestrus, only uterine retention of oocytes was observed. The inhibitor decreased oestradiol concentrations in ovarian vein, while systemic testosterone values were increased. Treatment with exogenous oestradiol counteracted the effect of the inhibitor on ovum transport. The elevation of systemic testosterone concentrations by means of subdermal implants of testosterone failed to alter the normal pattern of ovum transport. These results demonstrate that normal oestrogen secretion during late metoestrus and dioestrus is required for the movement of oocytes from the oviduct to the uterus, whereas the preovulatory oestrogen surge is needed for the expulsion of ova from the uterus.     

Follicular growth and kinetics in the Indian gerbil (Tatera indica) ovary during oestrous cycle

The growth of the follicle and oocyte in the Indian gerbil (Tatera indica) was a continuous process. The relationship between follicle and oocyte or its nucleus was log linear, represented by the equation log Y =a +b logX.A linear relationship (Y =a +bX)existed between the oocyte and its nucleus. The number of stages I and II follicles varied significantly during the oestrous cycle. Maximum percentage of stage I follicles was observed during oestrus and metoestrus, while stage II follicles were abundant during dioestrus, metoestrus and pro-oestrus. These follicles were significantly more in number than other types of the follicles. The occurrence of comparatively larger follicles during pro-oestrus and the presence of newly formed corpora lutea at oestrus, indicated ovulation in the early oestrus.     

A rapid and simple staining method, using Toluidine Blue, for analysing mitotic figures in tissue sections

Summary Mitotic index is a clinically important parameter in cancer pathology. We developed a staining method using Toluidine Blue to detect efficiently and rapidly mitotic figures in sections of formalin-fixed paraffin-embedded human and rat tisues. Sections were stained at acid pH with a 0.01% Toluidine Blue solution after removal of RNA with hydrochloric acid or ribonuclease. The optimal pH of the TB staining solution was found to be 4.5 for rat tissues and 3.5 for human tissues. This procedure stained mitotic figures much more intensely than other (extra)cellular structures. A quantitative estimate of the total number of nuclei in the field where mitotic figures were counted, was obtained in an adjacent section hydrolysed in 5 N hydrochloric acid and stained by the Feulgen reaction with a Schiff-type reagent containing 0.01% Toluidine Blue. This method specifically stained interphase and mitotic nuclei and the field cellularity could be quantified by image cytometry. When these procedures were performed on two consecutive serial sections, a mitotic index could be determined accurately by relating the count of mitotic figures to the number of tumour cells.     

The effect of various fixation procedures on the digestability of sialomucins with neuraminidase

Synopsis The histochemical digestability with neuraminidase of sialomucin in mouse sublingual gland was studied in unfixed and formaldehyde vapour-fixed cryostat sections, and in sections prepared from paraffin-embedded material fixed in several alcohol- or formaldehyde-containing fixatives recommended for mucosubstances.The removal of sialic acid residues from sections treated with neuraminidase was followed histochemically with the following staining methods: Azure A pH 3.5, Alcian Blue pH 2.5, Low Iron Diamine and Alcian Blue pH 2.5 followed by periodic acid-Schiff. When Goland's methanolic cyanuric chloride was used as fixative, only a partial loss of tissue basophilia was evident after enzyme incubation, but in tissues fixed in other ways a complete loss of histochemically detectable sialic acid residues was observed.     

The effect of retinoic acid on proteoglycan biosynthesis in bovine articular cartilage cultures

The addition of retinoic acid to adult bovine articular cartilage cultures produces a concentration-dependent decrease in both proteoglycan synthesis and the proteoglycan content of the tissue. Total protein synthesis was not affected by the presence of retinoic acid, indicating that the inhibition of proteoglycan synthesis was not due to cytotoxicity. The proteoglycans synthesized in the presence of retinoic acid were similar in hydrodynamic size, ability to form aggregates with hyaluronate, and glycosaminoglycan composition to those of control cultures. However, the presence of larger glycosaminoglycan chains suggests that the core protein was substituted with fewer but longer glycosaminoglycan chains. In cultures maintained with retinoic acid, a decreased ratio of the large proteoglycan was synthesized relative to the small proteoglycan compared to that measured in control cultures. In cultures maintained with retinoic acid for 1 day and then switched to medium with 20% (v/v) fetal calf serum, the rate of proteoglycan synthesis and hexuronate contents increased within 5 days to levels near those of control cultures. Within 2 days of switching to medium with 20% (v/v) fetal calf serum, the relative proportions of the proteoglycan species were similar to those produced in cultures maintained in medium with 20% (v/v) fetal calf serum throughout. The rate of proteoglycan synthesis by bovine articular cartilage cultures exhibited an exponential decay following exposure to retinoic acid, with estimated half-lives of 11.5 and 5.3 h for tissue previously maintained in medium alone or containing 20% (v/v) fetal calf serum, respectively. The addition of 1 mM benzyl beta-D-xyloside only partially reversed the retinoic acid-mediated inhibition of proteoglycan synthesis. This indicates that the inhibition of proteoglycan synthesis by retinoic acid was due to both a decreased availability of xylosylated core protein and a decreased capacity of the chondrocytes to synthesize chondroitin sulfate chains.     

The synthesis of hyaluronic acid by ectoderm during early organogenesis in the chick embryo.

This study demonstrates that the dorsal ectoderm of the stage 14 chick embryo synthesizes hyaluronic acid. About 49 to 52% of the H3 glucosamine-labeled glycosaminoglycan that is synthesized by explanted ectoderm can be identified as hyaluronic acid on the basis of its susceptibility to Streptomyces hyaluronidase or isolation of chondroitinase ABC digestion products. In addition, autoradiographic evidence shows that the ectoderm, unlike adjacent tissues like epithelial somites or neural tube, incorporates glucosamine into hyaluronidase-sensitive material which becomes largely extracellular and localized in the subectodermal cell-free space. Ultrastructural evidence shows that there is a fine fibrillar matrix between the ectodermal cells and in the subectodermal spaces when tannic acid is included in the primary fixative. This material resembles authentic hyaluronate, similarly fixed, and is absent when tannic acid is omitted from the fixative or when embryos have been previously treated in ovo with Streptomyces hyaluronidase. The concomitant reduction in the intercellular and subectodermal cell-free spaces after in ovo treatment with Streptomyces hyaluronidase supports the hypothesis that the dorsal ectoderm plays a morphogenetic role by contributing hyaluronate to the forming extracellular spaces. It is proposed that ectodermally derived hyaluronate might influence the morphogenesis of subjacent tissues such as the dermatome and neural crest.     

The Synthesis of Hyaluronic Acid by Ectoderm During Early Organogenesis in the Chick Embryo

This study demonstrates that the dorsal ectoderm of the stage 14 chick embryo synthesizes hyaluronic acid. About 49 to 52% of the H³ glucosamine-labeled glycosaminoglycan that is synthesized by explanted ectoderm can be identified as hyaluronic acid on the basis of its susceptibility to Streptomyces hyaluronidase or isolation of chondroitinase ABC digestion products. In addition, autoradiographic evidence shows that the ectoderm, unlike adjacent tissues like epithelial somites or neural tube, incorporates glucosamine into hyaluronidase-sensitive material which becomes largely extracellular and localized in the subectodermal cell-free space. Ultrastructural evidence shows that there is a fine fibrillar matrix between the ectodermal cells and in the subectodermal spaces when tannic acid is included in the primary fixative. This material resembles authentic hyaluronate, similarly fixed, and is absent when tannic acid is omitted from the fixative or when embryos have been previously treated in ovo with Streptomyces hyaluronidase. The concomitant reduction in the intercellular and subectodermal cell-free spaces after in ovo treatment with Streptomyces hyaluronidase supports the hypothesis that the dorsal ectoderm plays a morphogenetic role by contributing hyaluronate to the forming extracellular spaces. It is proposed that ectodermally derived hyaluronate might influence the morphogenesis of subjacent tissues such as the dermatome and neural crest.     

Investigations of dye binding in the sequential staining of mucosaccharides by Alcian Yellow-Alcian Blue. II. Spot tests and experiments on dye-mucopolysaccharide (glycosaminoglycan) precipitates

Synopsis The amounts of dye bound to mucopolysaccharide in the histochemical sequential staining Alcian Yellow-Alcian Blue method were determined by studying dye-mucopolysaccharide (glycosaminoglycan) precipitates in test-tube and spot test experiments. In the second step (Alcian Blue) of the method, previously bound Alcian Yellow was released into the staining solution and simultaneously the precipitate took up Alcian Blue. The amounts of Alcian Yellow released and Alcian Blue taken up varied for different mucosaccharides, and depended on the staining time of the second step (Alcian Blue) of the sequence, as well as on the concentration of dye and salt in the Alcian Blue solution. It is thought that, among other things, the Alcian Blue in solution displaces some of the bound Alcian Yellow. Some observations could be explained by the aggregation of dye molecules. The results were in agreement with previous histochemical observations.     

Changes in rat ovaries of specific binding for LH, FSH and prolactin during the oestrous cycle and pregnancy.

In cyclic rats, the highest ovarian specific binding for LH was 6-0+/- 2-2% inpro-oestrus. During pregnancy, the specific binding of 125I-labelled bovine LH by rat ovaries increased gradually and reached a maximum of 24-1+/-4-9% between Days 14 and 18 of gestation; a slight decrease in binding was observed at Day 20 of pregnancy. Ovarian specific binding for FSH was also highest in pro-oestrus (8-9+/-2-1%), decreasing to about 50% in oestrus and metoestrus, but staying relatively constant during pregnancy. For prolactin, the specific binding in rat ovaries was highest (7-1+/-1-6%) in pro-oestrus, quite high in metoestrus and dioestrus and low in oestrus. Specific binding increased gradually only after Day 14 of pregnancy. Serum concentrations of rat LH, FSH and prolactin at different stages of the oestrous cycle and during pregnancy were determined by radioimmunoassays, and no obvious correlation was observed between levels of circulating hormones and the specific binding of these hormones in ovarian tissues. Affinity constants (Ka) for the hormones were very similar between ovaries from pro-oestrous rats and late-pregnant rats, being 0-31 X 10(9) M-1 for LH, 0-65 X 10(10)M-1 for FSH, and 1-14 X 10(10)M-1 for prolactin. Increases in specific binding for different hormones were due to increases of total binding sites in the ovary under different physiological states.