Quantitative studies on enzymes in structures in striated muscles by labeled inhibitor methods. II. Confirmation of radioautographic measurement by liquid-scintillation counting.

Fragments of mouse diaphragm and sternomastoid muscles were incubated in diisopropyl-fluorophosphate (DFP)-(3)H in conditions known to saturate all the available DFP-sensitive reaction sites. After being extensively washed, the enzyme acetylcholinesterase (AChase) was specifically reactivated by treatment with pyridine-2-aldoxime methiodide (2-PAM). The radioactive DP-groups released into solution by 2-PAM were measured by liquid scintillation counting, and related to the known number of motor endplates present. Considerable difficulty was encountered in reducing the excess, adsorbed radioactivity to acceptable levels: long washing routines, extraction with organic solvents, and removing excess muscle fiber by microdissection were necessary. Six experiments gave a mean value of 2.4 x 10(7)molecules AChase per sternomastoid endplate, in reasonable agreement with the previously reported measurements by radioautography.

Acetylcholinesterase in the fast extraocular muscle of the mouse by light and electron microscope autoradiography.

The distribution of acetylcholinesterase (ACHe) in the twitch fibers of the extraocular muscles of the mouse was examined by light and electron microscope autoradiography after labeling with radioactive diisopropyl fluorophosphate (DFP) with, and without, 2-pyridine aldoxime methiodide (2-PAM) reactivation. The values obtained were compared with those previously reported for the diaphragm and sternomastoid muscles. The extraocular muscles were studied because they differ from the other two muscles in that they are among the fastest of the mammalian muscles, yet their endplates have sparse junctional folds. They could thus provide information on the extent to which ACHe concentration is an invariant feature of endplate morphology and what, if any aspects may be related to their fast speed of response. We found, using light microscope autoradiography, that in the twitch fibers of the extraocular muscle, there is n average of 6.4 +/- 2.1 X 10(7) DFP-binding sites per endplate, of which 29% (1.8 X 10(7)) are reactivated by 2-PAM and are thus AChe. The morphology of the extraocular endplates allowed us to conclude, on statistical grounds, that the AChe site are probably localized not only along the surface area of the postjunctional membrane (PJM) but also along the surface of the presynaptic axonal membrane. Based on this localization, we calculate 7,800 DFP sites and 2,500 2-PAM-reactivated sites/micron 2 of surface area of pre-and postjunctional membrane. This stacking density of DFP-binding sites per surface area of membrane ( probably in the overlying sheets of basal lamina) is very similar to that in the diaphragm and sternomastoid muscles.

Quantitative studies on enzymes in structures in striated muscles by labeled inhibitor methods. I. The number of acetylcholinesterase molecules and of other DFP-reactive sites at motor endplates, measured by radioautography.

Di-isopropylfluorophosphate (DFP) labeled with phosphorus-32 was applied to fragments of the diaphragm and sternomastoid muscles of the mouse, in conditions in which it saturated all available sites at the motor endplates. After adequate washing and exchange with unlabeled DFP, single endplates were obtained by microdissection and their radioactivity was found by beta track radioautography. The number of sites phosphorylated by DFP-(32)P per endplate was relatively constant for each muscle: in the sternomastoid, about 9 x 10(7) sites per endplate, in the diaphragm, about 3 x 10(7). Reaction with DFP-(32)P was abolished by prior treatment with unlabeled DFP. Labeling was unaffected by prior fixation in formaldehyde, but was inversely proportional to the time of incubation in the Koelle staining medium, when this preceded labeling. The contribution of acetylcholinesterase (AChase) to this total number of DFP-reactive sites was determined by three methods. The first involved reactivation of the phosphorylated AChase by pyridine-2-aldoxime methiodide (2-PAM), in conditions in which the reactivation of other enzymes would be insignificant. The other two methods involved protection of the active centers of AChase from phosphorylation by labeled DFP by use of 284C51, an inhibitor highly specific for this enzyme, or by use of eserine. Each of these methods indicated that about 35% of the DFP-reactive sites at endplates of the sternomastoid and diaphragm are AChase. The mean number of AChase molecules was thus found to be 3.1 x 10(7) and 1.1 x 10(7)per endplate in sternomastoid and diaphragm, respectively. No significant reaction of labeled DFP with muscle and nerve was observed. Mast cells in the muscle had a concentration of DFP-reactive sites far higher than the endplates.

Autoradiography with tritiated methotrexate and the cellular distribution of folate reductase.

Bound methotrexate has been revealed by an autoradiographic procedure, presumed to introduce a method for cytochemical study of folate reductase. Preferential localization is seen in kidney proximal tubules, intestinal epithelium, and nuclei of parenchymal liver cells in mice. The extremely firm binding and prolonged retention of this drug should render it suitable as an inert label for the autoradiographic study of cell migrations and lifetimes.

The Zonal Architecture of the Mandibular Condyle Requires ADAMTS5.

Temporomandibular joint (TMJ) osteoarthritis (TMJOA) disrupts extracellular matrix (ECM) homeostasis, leading to cartilage degradation. Upregulated a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS)-5 leads to cleavage of its substrate aggrecan (Acan) and is considered a hallmark of TMJOA. However, most research on ADAMTS5-Acan turnover has focused on hyaline cartilage, not fibrocartilage, which comprises the TMJ. The mandibular condylar cartilage (MCC) of the TMJ is organized in zones, and chondrocytes are arranged in axial rows, yet the molecular mechanisms required to generate the MCC zonal architecture have not been elucidated. Here, we test the hypothesis that ADAMTS5 is required for development of the TMJ MCC. Adamts5+/+ and Adamts5-/- murine TMJs were harvested at postnatal day 7 (P7), P21, 2 mo, and 6 mo of age; histomorphometrics indicated increased ECM. Immunohistochemistry and Western blots demonstrated the expanded ECM correlated with increased Acan localization in Adamts5-/- compared to Adamts5+/+. Cell volume was also decreased in the MCC of Adamts5-/- due to both a reduction in cell size and less mature hypertrophic chondrocytes. Analysis of chondrogenic maturation markers by quantitative real-time polymerase chain reaction indicated Col2a1, Col10a1, and Sox9 were significantly reduced in Adamts5-/- MCC compared to that of Adamts5+/+. The older (6 mo) Adamts5-/- MCC exhibited changes in chondrogenic cell arrangements, including clustering and chondrogenic atrophy, that correlated with early stages of TMJOA using modified Mankin scoring. These data indicate a potentially novel and critical role of ADAMTS5 for maturation of hypertrophic chondrocytes and establishment of the zonal architecture that, when disrupted, may lead to early onset of TMJOA.

The number of acetylcholinesterase molecules in the rat megakaryocyte.

A megakaryocyte cell series from rat bone marrow has been examined by the isotopic di-isopropyl fluorophosphate (DFP) method for esterases. After complete reaction with 32P-DFP, the numbers of DFP-reacted molecules inindividual cells havebeen determined by beta trackauto-radiography. Previous work has shown the percentage of organophosphate-sensitive sites in these cells which can be taken as active centers of acetylcholinesterase (AChase). Combining these data, the absolute numbers of organophosphate-sensitive esterase molecules and AChase molecules per cell were determined. Histograms show a narrow spread of values within each of four size classes from megakaryoblast to fully mature megakaryocyte, but, with means increasing 4-fold through this series, approximately in proportion to cell volume. A rat megakaryoblast has 2 X 10(6) AChase molecules, and a megakaryocyte (of 48-micro diameter) has 7.6 X 10(6) molecules. The apparent turnover number of the enzyme for intracellular reaction with substrate is calculated and compared with turnover numbers available for other AChases.

How precise is histologic dating of endometrium using the standard dating criteria?

Sixty-three endometrial biopsies were dated histologically by using the standard criteria on two separate occasions by the same observer. Overall, it was found that exact agreement occurred in 15 (24%), but disagreement of more than 2 days occurred in 6 (10%). The proportion of exact agreement in the first half of the luteal phase (32%) was found to be significantly higher (P less than 0.05) than that in the second half of the luteal phase (9%). In a separate part of the study, 27 women had two endometrial biopsies, each performed in a separate cycle. The within-subject between-cycle variation of the results of endometrial dating (exact agreement: 4%, disagreement of more than 2 days: 41%) was found to be significantly different from intraobserver variation (P less than 0.01 for both). The amount of intraobserver variation suggests that the traditional dating criteria are not precise enough to quantify corpus luteum function in the second half of the luteal phase, whereas the amount of within-subject between-cycle variation implies that the result of endometrial dating in one cycle cannot be used reliably to predict that of another cycle.

A comparison between two methods of chronological dating of human endometrial biopsies during the luteal phase, and their correlation with histologic dating.

This prospective study was performed on 61 infertile women to examine the correlation between histologic dating using the same criteria by two independent observers and chronological dating by two different methods: (1) determination of luteinizing hormone (LH) peak by daily LH assay, (2) calculation based on the onset of the next menstrual period (NMP). The correlation between histologic dating and chronological dating was found to be significantly better if the LH peak was used to determine the chronological date than if the NMP was used (r = 0.70 and 0.37, respectively).

A new method of histologic dating of human endometrium in the luteal phase.

Morphometric analysis was performed on 70 endometrial biopsy specimens collected from a population of fertile women. Of the 17 morphometric measurements that were performed on each endometrial biopsy, only 5 were required to achieve a highly significant correlation with chronologic dating based on luteinizing hormone surge (R = 0.99). The result of histologic dating, based on morphometric analysis of endometrial biopsies collected from a separate, unbiased population, correlated strongly and significantly with chronologic dating (r = 0.98, P less than 0.0001). The correlation was better than that achieved when histologic dating was performed according to the conventional dating criteria (r = 0.88, P less than 0.001).

The effects of progesterone receptor blockade in the luteal phase of normal fertile women.

The effects of a single, variable dose (5 to 200 mg) of RU38486 (RU486) (Roussel-Uclaf, Paris, France) in the first half of the luteal phase of the menstrual cycle were studied in 30 normal fertile volunteers. Quantitative histologic techniques were employed, and the results were compared with normal ranges derived from a separate, normal, fertile population. It was found that RU486 inhibited glandular secretory activity, accelerated degenerative changes, induced various vascular changes, increased stromal but not glandular mitotic activity, and did not affect the predecidual reaction. The superiority of morphometric analysis over traditional dating criteria was demonstrated in this study of endometrial responses to an exogenously administered agent. In addition, it was found that temperature drop occurred in 56%, menstrual induction in 43%, luteolysis in 24%, mood change in 23%, and thirst sensation in 3% of women studied. Both menstrual induction and changes in hypothalamic function after the administration of RU486 occurred independently of luteolysis and so were likely to be the direct result of progesterone receptor blockade in these organs. Menstrual induction was significantly related to the dose given and the day on which RU486 was administered. Mood change was significantly related to the day on which RU486 was given. Most of the effects of RU486 occurred around 48 hours after its administration.

PIP: The effects of a single, variable dose (5-200 mg) of RU38486 (RU486; Roussel-Uclaf, Paris, France) in the 1st 1/2 of the luteal phase of the menstrual cycle were studied in 30 normal fertile volunteers. Quantitative histologic techniques were employed, and the results were compared with normal ranges derived from a separate, normal, fertile population. It was found that RU486 inhibited glandular secretory activity, accelerated degenerative changes, induced various vascular changes, increased stromal but not glandular mitotic activity, and did not affect the predecidual reaction. The superiority of morphometric analysis over traditional dating criteria was demonstrated in this study of endometrial responses to an exogenously administered agent. In addition, it was found that temperature drop occurred in 56%, menstrual induction in 43%, luteolysis in 24%, mood change in 23%, and thirst sensation in 3% of the women studied. Both menstrual induction and changes in hypothalamic function after the administration of RU486 occurred independently of luteolysis and thus were likely to be the direct result of progesterone receptor blockade in these organs. Menstrual induction was significantly related to the dose given and the day on which RU486 was administered. Mood change was significantly related to the day on which RU486 was given. Most of its effects occurred around 48 hours after administration.

Why does RU486 fail to prevent implantation despite success in inducing menstruation?

The administration of RU38486 (RU486) in the luteal phase may induce menstruation, but it may not be associated with shedding of the functional layer of the endometrium. This provides an explanation why, in some cases of successful menstrual induction by RU486, pregnancy continues undisturbed. The ability of RU486 to interrupt a very early pregnancy is more likely to be related to its ability to cause shedding of the endometrium than its ability to induce menstruation.

PIP: To see why RU-486, given in luteal phase, induces menstruation but does not always prevent pregnancy, daily progesterone assays and 3 endometrial biopsies were taken from 2 volunteers who received a single dose. Progesterone was assayed by direct radioimmunoassay from saliva samples. The 1st subject was a 36-year old multipara who took a single oral dose of 150 mg RU-486 on Day 4 after the LH surge. She menstruated apparently normally 2 days later, and again 10 days after RU-486, at the expected time of menstruation. Her endometrial biopsy resembled secretory phase, and her progesterone profile did not indicate luteolysis. The 2nd volunteer was a 38-year old multipara. She took 75 mg RU-486 on Day 6 after her LH surge, and had apparently normal menses 2 days later. The endometrial histology was suggestive of menstruation, and the progesterone profile had fallen to baseline, indicating luteolysis. Menstruation did not recur at the normal time. In this woman who had complete shedding of the endometrium, RU-486 had been taken later in the luteal phase, and in a smaller dosage.

Effect of culture in vitro and organ culture on the dry mass of preimplantation mouse embryos.

The dry mass of mouse embryos cultured in vitro in medium alone or in an organ culture system were measured by means of the Vickers M86 scanning microinterferometer. The data were compared with previous data on the dry mass of preimplantation embryos in vivo. The metabolism of embryos cultured in vitro differs from that of fresh embryos. In cultured embryos, dry mass decreases throughout the 2-cell stage whereas the dry mass is increasing at this stage in vivo. Embryos in an organ culture system regain a dry mass profile, similar to that observed in vivo at the late cleavage stage. These results support the view that conditions for embryo metabolism are suboptimal in vitro and that, although the oviduct may confer some advantage on developing embryos in vitro, it is unable fully to support the pattern of metabolism, as assessed by dry mass, observed in vivo.

An autoradiographic search for radioactive particles in the lungs of cigarette smokers.

Mucosal samples from the bifuracation of a major bronchus were examined from 23 patients undergoing diagnostic bronchoscopy. Samples were autoradiographed using a technique that avoids any contact between tissue and reagents until the end of autoradiographic exposure. The autoradiographs were scanned for a-particle tracks. No significant level of alpha -activity was detected, even in samples from heavy smokers who had continued to smoke within several hours of the bronchoscopy. The lower limit of detection of a-activity in this experiment was equivalent to about 55.5 becquerrels/kg (1,500 pCi/kg) Polonium-210. These results do not support the hypothesis presented by others that a radioactivity in particulate material of cigarette smoke contributes significantly to the association between cigarette-smoking and bronchial carcinoma.

An endometrial factor in unexplained infertility.

OBJECTIVE: To study a group of women with unexplained infertility to see whether they have a defect that is intrinsic to the endometrium. DESIGN: Evaluation of the functional response of the endometrium by examining endometrial biopsy specimens using immunohistochemical methods in a group of women with unexplained infertility and in a control group of women with normal fertility. PATIENTS: 27 Women with unexplained infertility (average age 33.2); median duration of infertility five years. A control group of 44 women with normal fertility (average age 33.8) who were requesting sterilisation or reversal of sterilisation. SETTING: Infertility clinic, Jessop Hospital for Women, Sheffield. INTERVENTION: Secretory phase endometrial biopsy specimens were taken, with informed consent, as an outpatient procedure. MAIN OUTCOME MEASURES: Immunohistochemistry with monoclonal antibody D9B1, was used to assess the production and secretion of an oligosaccharide epitope produced by endometrial gland cells between two and seven days after the luteinising hormone surge. A reflected light measuring system was used to assess the amount of epitope within the gland cells, and in the gland lumen. RESULTS: In the control group of women, mean reflected light measurements at the cell base and cell apex peaked at three and five days after the luteinising hormone surge respectively, and in the gland lumen the epitope accumulated rapidly from three days, reaching a peak at seven days. In the women with infertility the peaks of epitope at the cell base and cell apex were lower, broader, and delayed in onset, and the build up of epitope in the gland lumen was retarded. The synthesis and secretion of the epitope in the women with infertility was therefore significantly reduced and delayed, even in the presence of normal concentrations of circulating progesterone. CONCLUSIONS: The results suggest that a primary dysfunction of the endometrium might be associated with hitherto unexplained infertility.