Immunocytochemical identification of interstitial cells of Cajal in the murine fundus using a live-labelling technique.

Interstitial cells of Cajal (ICC) within the gastrointestinal (GI) tract play a critical role in the generation of electrical slow waves and as mediators of enteric motor neurotransmission. Kit immunohistochemistry has proven to be a reliable method to identify the location of these cells within the tunica muscularis and to provide information on how the distribution and density of these cells change in a variety of GI motility disorders. Because of the labile nature of Kit or its detection, ultrastructural immunocytochemistry using conventional chemical fixation methods has been difficult. We describe a novel in vivo technique to label ICC within GI tissues. Using antibodies directed against the extracellular domain of the Kit receptor, we have been able to live-label the stomach with Kit while the animal is under anaesthesia and the organ is still receiving normal blood supply. This approach provided optimum maintenance of ultrastructural features with significant binding of antibody to the Kit receptor. The loss of ICC in many human motility disorders suggests exciting new hypotheses for their aetiology. This method will prove useful to investigate the ultrastructural changes that occur in ICC networks in animal models of motility disorders that are associated with the loss of these cells.

Ultrastructural analysis of the transdifferentiation of smooth muscle to skeletal muscle in the murine esophagus.

The ultrastructure of the mouse esophagus at the level of the diaphragm was studied from embryo day 17 to adult. The transdifferentiation of smooth muscle into skeletal muscle was categorized into seven ultrastructural stages: during phase I normal smooth muscle myogenesis was observed. In phase II subpopulations of cells changed into aggregates of myoblast-like cells. At the center of these cell aggregates, phase III cells appeared that contained condensed myofilaments. Dense bodies and dense bands appeared enlarged by the accumulation of thin filaments. In phase IV the condensed myofilaments organized into sarcomere pretemplate structures. The dense bodies and dense bands formed rudimentary Z-lines. In phase V the sarcomere templates appeared as more defined structures and began to align. An elaborate perinuclear region appeared. During phase VI, skeletal muscle sarcomeres were apparent and myofilaments were arranged in a typical hexagonal array. Phase VII skeletal muscle fibers were unique with sarcomeric bifurcations and anastomoses between adjacent myofibrils. Non-contractile organelles were less organized in these cells than in skeletal muscles such as rectus and vastus lateralis muscles. During the transdifferentiation process, other cell types remained unchanged, except the number of interstitial cells of Cajal became reduced. Immunocytochemical studies with antibodies against smooth and skeletal muscle myosin were also performed during the process of transdifferentiation. An osmium tetroxide/potassium ferricyanide en bloc mordant enabled the use of ultrathin Unicryl sections for immunocytochemistry. Cells exhibited smooth muscle myosin-like immunoreactivity from the smooth muscle stage through the condensed myofilament stage. Cells were immunopositive for skeletal muscle myosin before the formation of sarcomere templates, during the condensed stage, and after development of mature skeletal muscle cells. We also observed a hybrid muscle cell with properties of both smooth and skeletal muscle cells.

Mock embryo transfer in early luteal phase, the cycle before in vitro fertilization and embryo transfer: a descriptive study.

OBJECTIVE: To investigate visually the uterine retention or sequestration of boluses of radiopaque dye, mimicking embryo transfer (ET). DESIGN: During the cycle before in vitro fertilization (IVF) and ET, patients underwent a mock ET of 40 microL of radiopaque dye into the uterine cavity. The patient was positioned supine for retroverted or axial and knee chest for anteverted uteri. The position of the dye at injection, during and after catheter removal, and during patient roll over and standing was monitored. SETTING: Treatment of infertility in a private practice. PATIENTS: Thirty-four IVF patients. INTERVENTIONS: During the cycle before IVF/ET, patients underwent a mock ET using a bolus of 40 microL of radiopaque dye. MAIN OUTCOME MEASURES: Planned before visual observation began. RESULTS: The dye remained primarily in the uterine cavity in only 68% (optimum ET position is knee chest for anteverted and supine for retroverted or axial uterus) and 48% (nonoptimum position is supine for anteverted uterus) at mock ET; in those groups, a 33% clinical pregnancy rate (PR) per retrieval resulted. Dye motility into the fallopian tube(s), cervix, and/or vagina is 38.2%, 8.8%, and 11.8%, respectively. CONCLUSIONS: If the mock ET had been the actual ET, 32% (optimum ET position) and 52% (nonoptimum ET position) of all patients would have lost their opportunity for pregnancy as a result of the ET procedure. Our 33% PR per retrieval among those patients who retained the dye in utero is more consistent with our expectations, given the advanced technologies of IVF/ET today.

In vitro fertilization (IVF) of mouse ova in HEPES-buffered culture media.

Some major drawbacks of a bicarbonate-buffered culture medium include the requirement of an elaborate incubator system able to maintain a 5% CO2 environment and the inability of the culture medium to maintain a physiological pH range (pH 7.3-7.4) in room air (0.03% CO2). This work resulted in the development of IVF culture media, BB (modified T6) and Hams-HEPES, which use HEPES-buffered systems not requiring the specialized CO2 environment to maintain a physiological pH range in room air. These media generate above-average cleavage rates in in vitro fertilized, superovulated B6CBAF1 mice ova. The effect of heparin and HEPES on cleavage was studied and neither had a significant effect at the concentrations used. Cleavage rates of nonfertilized ova (parthenogenic division) were 9 to 13%. There was no significant difference in parthenogenesis between any of the culture media and it appears to be a function of the strain of mice and the timing between human chorionic gonadotropin (hCG) injection and ovum collection. These results emphasize the need to account for parthenogenesis when determining cleavage rates of in vitro fertilized mouse ova. Also, the results suggest that because of individual mouse differences in cleavage rates, it is important to use an adequate number of mice per group to determine an accurate, average cleavage rate.

Lung lamellar body amphiphilic topography: a morphological evaluation using the continuum theory of liquid crystals: I. Closed surfaces: closed spheres, concentric tori, and Dupin cyclides.

The structure and symmetry of the bilayers of in vivo phospholipid lung lamellar bodies is shown to be analogous to thermotropic smectic-A liquid crystals and in vitro lyotropic multilamellar liquid crystalline liposomes. This structural similarity has led us to extend biophysical and geometrical principles that have long been used to predict the layer conformation in these in vitro systems to in vivo lung lamellar bodies. These configurations were demonstrated by electron micrographs of thin sections of rodent, monkey, and human lung lamellar bodies prepared by lipid-retaining embedment procedures. The bilayer configurations in all species were consistent with the two geometries predicted by minimal energy solutions of a continuum theory of liquid crystals subject to the boundary conditions imposed by the amphiphilic nature of lung surfactant lipid: concentric (either closed or partially closed) spheres and Dupin cyclides. These bilayer arrangements in lung lamellar bodies were virtually identical to the bilayer configuration of in vitro multilamellar liposomes. The agreement between the two predicted configurations and our observations shows that multilamellar liquid crystalline bilayer aggregate organization is universal in any aqueous environment.

Lung lamellar body amphiphilic topography: a morphological evaluation using the continuum theory of liquid crystals: II. Disclinations, edge dislocations, and irregular defects.

The bilayers in normal mammalian and human lung multilamellar bodies (LMB) are parallel, equally spaced, and continuous--a configuration that minimizes the large elastic strain energy associated with changing the equilibrium bilayer separation and the hydrophobic-hydrophilic repulsion energy between the hydrocarbon tails of phospholipid and the aqueous phase. This ideal behavior is disrupted at a limited population of large Burgers vector edge dislocations dissociated into +/- 1/2 disclination pairs. The configuration and interaction of the defects are explained by the continuum theory of liquid crystals and are shown to be identical to defects observed in in vitro surfactant liposomes and bilayers. We report the first observations with molecular resolution of the core structure of a liquid crystal dislocation. Defect cores are shown to be located between both headgroups and tailgroups in human LMB, suggesting that both types of core are similar in energy. This may be the result of partitioning of proteins or other nonlipid impurities in the LMB to the defect cores, which might also change the stability of the dislocations to favor their preservation. The edge dislocation defects interact in ways that minimize their overall strain energy. A population of edge dislocations may play an important role in the transport or localization of certain molecules through the lamellar body. Certain defects were observed in lung multilamellar bodies that have not been observed in in vitro systems; these are probably due to the complex, multicomponent nature of the LMB surfactant and the dynamic, in vivo environment of the LMB.

The development of a successful non-university-based ambulatory in vitro fertilization/embryo transfer program: phase I.

Most of the current in vitro fertilization and embryo transfer (IVF-ET) programs are university-based. The establishment of a successful ambulatory IVF program in association with a busy, two-man general obstetrics-gynecologic practice is described. Seventy-one infertile couples were screened between February 1 and October 15, 1983. Forty-three couples were judged eligible for IVF-ET. Forty-three women underwent a single attempt at ET. The first 13 of these women underwent controlled ovarian hyperstimulation (COH) with 150 mg clomiphene citrate and human chorionic gonadotropin (hCG), and the remaining 30 underwent COH with human menopausal gonadotropin (hMG) and hCG. One of the 13 patients who underwent COH with clomiphene citrate conceived but subsequently miscarried early in the first trimester, for an 8% pregnancy rate. There were 12 pregnancies among the 30 patients who received hMG and hCG, for a 40% pregnancy rate. Only three of these pregnancies miscarried in the early first trimester, and three of the nine viable pregnancies are twin gestations. The possible factors responsible for the high pregnancy rate with IVF-ET, using COH with hMG and hCG, are discussed, and the feasibility of its performance in a well-controlled, non-university program is demonstrated.

Localization on encapsulated Cryptococcus neoformans of serum components opsonic for phagocytosis by macrophages and neutrophils.

Previous studies have shown that the cryptococcal capsule inhibits phagocytosis of Cryptococcus neoformans by macrophages and neutrophils. In this study, the binding sites of potential serum opsonins in immune and nonimmune sera were determined by immunoelectron microscopy, and the results were compared with the results of phagocytosis of the yeasts by mouse peritoneal macrophages and human neutrophils. Immunoglobulin G (IgG) from normal human serum showed low-density binding at the capsular surface and at sites throughout the capsule. Complement component C3 from normal serum bound heavily at the capsular surface. IgG from rabbit capsular antiserum showed relatively dense deposition at the capsular surface and at sites throughout the capsule. Cells opsonized with heat-inactivated human serum were engulfed poorly by both macrophages and neutrophils, indicating that the low-density deposition of IgG produced by normal serum was not adequate for opsonization. Yeasts opsonized with normal human serum were engulfed in large numbers by neutrophils and to a lesser extent by macrophages, indicating that neutrophils in particular were able to effectively utilize the opsonically active C3 which normal human serum deposited at the capsular surface. Yeasts opsonized with rabbit anticapsular serum were engulfed by both macrophages and neutrophils, indicating that the high density of surface IgG produced by capsular antiserum is an effective opsonin for both cells. These results suggest that the complement-neutrophil system is a possible defense mechanism in the nonimmune host.

In vitro sperm capacitation and transcervical intrauterine insemination for the treatment of refractory infertility: phase I.

Fourteen couples with long-standing infertility, associated with cervical mucus insufficiency, male subfertility, or unexplained infertility, participated in a therapeutic trial. The female partners, who were all ovulatory, were given human menopausal gonadotropin from day 2 of the menstrual cycle (controlled ovarian hyperstimulation). When plasma estradiol concentrations reached 1000 to 2000 pg/ml, human chorionic gonadotropin was given. Approximately 32 hours and again 70 hours thereafter, a masturbation specimen of the husband's sperm was capacitated in vitro and inseminated transcervically into the uterine cavity. Five women (35%) conceived following a single cycle of treatment. Four of the pregnancies are currently progressing normally; one ended in a spontaneous miscarriage in the early first trimester. The potential role of in vitro sperm capacitation and transcervical intrauterine insemination in the treatment of refractory infertility unrelated to female organic pelvic disease is discussed.

Stereoscanning electron microscopy of argon laser excised laryngeal papilloma.

Laryngeal papillomas were removed from two patients with the argon laser and examined by transmission electron microscopy (TEM), scanning electron microscopy (SEM), and stereoscanning electron microscopy (SSEM). Two techniques, lipid-carbohydrate retention for TEM specimens and sonication for SEM, were applied in studying the papillomas. Lipid-carbohydrate retention results were in agreement with previous work in papilloma ultrastructure. The char interface was observed with the sonication technique. Peripheral to the laser impact site, four zones were observed: 1. coagulation necrosis, 2. contracted epithelial cells, 3. minimally damaged epithelial cells, and 4. morphologically undamaged epithelial cells. In Zone 2, cells were violently popped-off as heat was conducted through the epithelium. The potential for spread of papillomas is discussed and suction of the vapor plume recommended when removing laryngeal papillomas with the argon laser.

Argon laser use in papillomas of the larynx.

Papillomas of the larynx are discussed, including the use of lasers. Argon lasers' differences from CO2 lasers and techniques are outlined. Case reports are given in which laryngeal papillomas were removed from two patients with the argon laser and examined by transmission electron microscopy (TEM), scanning electron microscopy (SEM), and stereoscanning electron microscopy (SSEM). The depth and width of morphological change are noted. Peripheral to the laser impact site, four zones were observed: 1. coagulation necrosis, 2. contracted epithelial cells, 3. minimally damaged epithelial cells, and 4. morphologically undamaged epithelial cells. In Zone 2, cells were violently popped-off as heat was conducted through the epithelium. The potential for spread of papillomas was discussed and suction of the vapor plume recommended when removing laryngeal papillomas with the argon laser.

The lipid solubility of fixative, staining and embedding media, and the introduction of LX-112 and poly/bed-812 as dehydrants for epoxy resin embedment.

The manufacture of Shell, Epon-812 (E-812) resin has recently been discontinued. E-812 and two newly introduced E-812 substitutes, and Ladd-112 (LX-112) and the Polysciences, Poly/bed-812 (PB-812) resins, were studied biochemically and morphologically for their effectiveness as polar dehydrants. Their technical properties as general E-812 replacements were also explored. In the biochemical studies, acetone was more effective in retaining lung phospholipid components than ethanol, the resin dehydration was more effective than either acetone or ethanol. There was no appreciable difference in lipid solubility among the three resins. Acetone and uranyl magnesium acetate each had a loosening effect on previously fixed phospholipids. The PB-812 and E-812 resin dehydrated blocks of dense animal tissues, demonstrated serious technical difficulties during sectioning. The L-112 resin substitute, due to its low viscosity and improved infiltration, was found to be technically as effective a dehydrant as ethanol or acetone. None of the three resins was successful as dehydrating agents for the plant tissue. With organic solvent dehydration, both epoxy resin substitutes demonstrated excellent embedment properties with both animal and plant tissues.

The ultrastructural histochemistry and stereoscanning electron microscopy of the rodent and amphibian surfactant systems.

Ultrastructural histochemical precedures were employed to determine the carbohydrate components and their contributions to the rodent and amphibian surfactant systems. Zirconium stained the rodent (rat) cytoplasm surrounding the multilamellar bodies, the Golgi, and was associated with the membrane structures of the compact lamellae of alveolar multilamellar bodies. In the rodent and amphibian (Rana pipiens), ruthenium red stain was observed within all tubular myelin surfactant matricies. The "gutters," tubular myelin surfactant matrix, and intratubular myelin surfactant matrix materials all demonstrated a positive reaction product. The periodic acid-chromic acid-silver procedure revealed irregular channels extending from the multilamellar bodies to the surface of the rodent great alveolar pneumocyte. The extra-pulmonary and respiratory surfaces in both species were additionally studied by stereoscanning electron microscopy. The respiratory anatomy of the rodent was corroborated. The amphibian lung demonstrated three orders of septa, and in the expired state, tertiary septal pits. The amphibian primary septa were hollow, blind tubules containing respiratory surfaces.

The stereoscanning electron microscopy and ultrastructural histochemistry of the avian and reptilian surfactant systems: Indian dove, desert spiny and Taiwan golden skink lizards.

Lung tissues from the Indian dove, Scardafella inca, desert spiny lizard, Sceloporus magister, and the Taiwan golden skink lizard, Mabuya aurates, were studied by transmission electron microscopy utilizing ruthenium red as a carbohydrate stain and with the so-called lipid-carbohydrate retention procedures to elucidate the morphology of the surfactant systems. Stereoscopic scanning electron microscopic procedures were utilized for a comparative anatomical study of these three species, and the results were compared with the rat and frog in the companion article. The avian lung tissues demonstrated several perculiarities. The ciliated epithelial cells of the bronchus had cytoplasmic ciliated projections between the boundaries of mucus secreting cells. The discrete morphology of the main bronchus, secondary bronchi, parabronchi, and the air capillaries, and their three-dimensional morphologic perspective were elucidated. The skink illustrated an arrangement of primary, secondary, and tertiary septa, with elaborate tertiary septal pits, similar to the amphibian. All septa contained a solid connective tissue core. The desert lizard was similar to the skink except the tertiary septal pits were rudimentary. All three species had a modified great alveolar pneumocyte and a laminated surfactant which included a carbohydrate matrix material between layered phospholipid-based membranes. The ruthenium red additionally stained the homogeneous surface-lining material. A comparative analysis of the surfactant systems of these three species with each other, and with the rodent and amphibian in the companion article, were discussed in terms of phylogenetic origin.

The surfactant system of human fetal lung organotypic cultures: ultrastructural preservation by a lipid-carbohydrate retention method.

Human fetal lung organotypic cultures consisted of epithelial elements (congruent to 40- 100 micrometer in diameter) formed by the reaggregation of single cells from a monodisperse suspension of enzymatically dissociated fetal lung. These elements, termed alveolar-like structures, were composed primarily of type II alveolar epithelial cells whose apical surfaces bordered the central lumen of the alveolar-like structure. Pulmonary surfactant secreted by the type II cells was retained within the lumen and accumulated in close association with the epithelium. These characteristics made this culture system an advantageous model for the morphological study of human pulmonary surfactant in vitro. A lipid-carbohydrate retention procedure which reduced the extraction of tissue components and thus provided improved preservation of multilamellar bodies and tubular myelin surfactant was used in an ultrastructural study of organotypically cultured surfactant. Human surfactant was observed for the first time with most of its structural components intact. In vitro human surfactant was found to be similar to in vivo rodent and non-human primate surfactant, but with certain differences. Long surfactant tubules were not observed. There were more transformed multilamellar bodies present with more foci undergoing transformation. Each focus contained fewer layers of tubular myelin surfactant than occurs in rodent surfactant. No epiphase-hypophase areas were observed, only tubular myelin surfactant. In addition, a previously unreported intrasurfactant matrix material was observed.

The ultrastructure of multilamellar bodies and surfactant in the human lung.

Normal tissues from human lungs were dehydrated through Epon 812 resin to retain many of the lipids and carbohydrates in thin section. The three-dimensional structure of the multilamellar body was determined. The paired layer of phospholipid heads (PH) is 36 A thick; the layer of fatty-acid tails (FA) is 31 A, the same as reported previously for non-human primates and rodents. The human multilamellar body is apparently unique: the lamellae of the major focus divide into two or three lamellae; the matrix material of the core is without vesicular bodies and a projection core is present. When compared with those of the rat, human tissues contain a greater number of lamellar foci and fewer lamellae per focus. The presence of a peripheral layer of lamellae, an ever-present external limiting membrane, and the fusion of multilamellar bodies are also characteristic. Tubular myelin surfactant has the same appearance as in other mammals. Multilamellar bodies were observed in direct communication with Golgi vesicles. Their origin from multivesicular bodies and their maturation through secretion and exocytosis were demonstrated. Untransformed multilamellar bodies in the alveolar space demonstrated three periodicities (P): (1) compact regular lamellae, PH = 36 A, FA = 36 A, FA = 31 A, P = 66 A; (2) compact broad lamellae, PH = 72 A, FA = 22 A, P = 94 A; (3) loose lamellae, PH = 36 A, FA = 31 A with a variable interlamellar space.