A simple technique for nonsurgical embryo transfer in mice

A simple method was developed for nonsurgical transfer of mouse embryos which enabled transfer to both uterine horns. Embryos were picked up in a modified capillary tube and transferred through the cervix into each uterine horn of an unanesthetized mouse. A glass speculum was used to facilitate location of the cervix. The technique was found to be as successful (up to 60% of embryos transferred developed to term) as surgical methods yet was simpler and eliminated surgical trauma to the recipient mouse.     

CO2激光治疗宫颈及宫颈粘膜下肌瘤的临床观察

本文采用ＣＯ２激光和Ｎｄ：ＹＡＧ激光对１３例宫颈及宫颈粘膜下肌瘤进行了切除治疗,疗效满意。其中瘤体最大４ｃｍ,最小１ｃｍ均位于宫颈间质组织内和宫颈管内。术中除１例少许出血外,余例几乎无出血。术后无１例感染,伤口愈合好。经过临床观察,认为采用激光治疗,方法简便,不需缝合,不住院,宫颈无粘连狭窄,值得采用。     

A preliminary study on the usefulness of huIL-8 in cervical relaxation of the ewe for artificial insemination and for embryo transfer

The efficacy of using human interleukin 8 (huIL-8) as an agent for inducing cervical relaxation in estrous and diestrous sheep was assessed in a small pilot study. Multiparous, estrus-synchronized ewes were treated for either 2 or 5 consecutive days with vaginal suppositories with or without 5 micrograms cytokine. Cervical penetration with an insemination instrument was then assessed in vivo. After euthanasia, physical, histological and enzymological properties of the cervix were examined. Treatment of diestrous sheep with huIL-8 did not result in recruitment of neutrophils into the cervix. Treatment of estrous sheep with huIL-8 usually led to neutrophil recruitment to the cervix and to either full or partial penetration of the cervix. However, some animals receiving placebo treatment had neutrophil infiltration of both the vagina and cervix and, in one of these, partial penetration of the cervix was also achieved. Thus, treatment with IL-8 as the sole agent in the vaginal suppository was not sufficient to relax the cervix of the nonpregnant ewe in this study.     

Intrauterine insemination of sows with reduced sperm numbers: results of a commercially based field trial

Artificial insemination (AI) in pigs requires 2-3 billion spermatozoa to achieve consistently high fertility with current practice of inseminating into the posterior region of the cervix. We have investigated the potential advantages of inseminating through the cervix into the caudal region of the uterus using lower sperm numbers. Total sperm doses from 22 boars of 3, 2 or 1 billion spermatozoa were packaged in 80 ml volumes in X-Cell extender in gene-flat-pack (Cochette) bags. A novel inseminating device, the Deepgoldenpig, was employed which permits the ready introduction of a narrow catheter through the cervix into the uterus without traumatic injury to the mucosa. This was compared with the standard Goldenpig device that allows semen to be deposited in the posterior folds of the cervix. Sows of two different genotypes and of parities ranging from 2 to 11 were used. They were selected solely on the basis of a weaning to estrus interval of 4-6 days. Two inseminations, with a 24 h interval between them, were carried out on each sow. Pregnancy was determined at 35 days by ultrasound scan, and farrowing and litter size recorded. Pregnancy and farrowing data were very similar. The standard inseminating device produced farrowing rates of 91.1, 91.8 and 65.8% for insemination with 3, 2 and 1 billion spermatozoa, whereas the deep insemination device gave rates of 90.5, 90.5 and 86.9%. Only the 1 billion dose with the standard device was significantly different from the high dose control (P < 0.001). Similarly, the mean litter sizes with the standard device were 12.5, 12.6 and 10.3 and with the deep insemination device 12.3, 12.3 and 12.1. Only the 1 billion dose with the standard device was significantly lower (P < 0.001). None of the covariates differed significantly and there were no significant interactions with treatment. We conclude that transcervical insemination in the sow is simple, effective and safe, and allows the sperm dose to be reduced to 1 billion spermatozoa.     

A novel in vitro model system to grow films of oral bacteria for the study of human tooth root surface caries

AIMS: To develop a simple and flexible novel in vitro model system to grow films of oral bacteria that could be used to study aspects of dental caries. METHODS AND RESULTS: Standardized suspensions of bacteria were inoculated into Ultrafree-CL (Millipore) ultrafiltration units at various densities. These were incubated for varying time intervals with a range of carbon sources. The bacterial films reproducibly achieved between 107 and 108 cfu cm-2, irrespective of the number inoculated and with no significant changes for 14 d. However, Streptococcus mutans grew through membranes with pores of diameter greater than 0.1 microm after 6 d. Culture of films in sucrose or water for 6 d led to a decreased number of colony-forming units, but returning them to broth reversed this. CONCLUSION: Reproducible films of oral bacteria can be cultured in Ultrafree-CL units. SIGNIFICANCE AND IMPACT OF THE STUDY: This study has shown that Ultrafree-CL units can be used as a simple model system to grow biofilms that could be used for dental caries research.     

Characterization of a cell line (SW756) derived from a human squamous carcinoma of the uterine cervix

Summary An established cell line, SW756, derived from a primary squamous carcinoma of the uterine cervix is described by its morphology, ultrastructure, karyotype, genetic signature analysis, HLA typing, and tumorigenesis in the nude mouse. Cultured cells obtained from the SW756 derived nude mouse tumor also were studied for chromosome and isozyme markers. The original tumor was poorly differentiated carcinoma with minimal keratinization and is compared with that occurring in the nude mouse after the cultured cells were inoculated. The nude mouse tumor showed similar histological features, but better differentiation than the original tumor. Karyotype analysis of SW756 demonstrated a hyperdiploid stem line number and several marker chromosomes (MI-M6). No HeLa marker chromosomes were identified. The isozyme pattern for SW756 reported by others has been confirmed. The unique chromosome and isozyme features have been identified repeatedly in the cultured cells and, most importantly, in the post nude mouse culture. We recommend SW756 as a defined human tumorigenic cell line derived from a primary squamous carcinoma of the uterine cervix. This investigation was supported in part by Public Health Research Grant CA-06294 from the National Cancer Institute, Department of Health and Human Services.     

A device for nonsurgical transfer of bovine embryos and its effect on uterine contamination

A simple, inexpensive, concentric cannula device is described for the transfer of embryos through the cervix in cattle. Its effectiveness in minimizing the introduction of contamination into the uterus was tested by bacteriological surveillance of the various components after use, and of the uterus of 12 cows slaughtered 2 – 3 days after sham transfer. The results of the bacteriological examination of the outer cannula, middle cannula and inner catheter were positive in 100%, 66% and 50% respectively. Bacteria were always found in the samples obtained from the vulva and vagina, but in only 55% of those taken from the external os of the cervix. Pregnancy resulted in $\text{5}\text{10}$ cows that were inseminated and subsequently subjected to a sham transfer to the horn contralateral to the corpus luteum.     

Transport of Radiopaque Fluid into the Uterus After Vaginal Deposition in the Oestrous Bitch

A limited number of studies have been published concerning intrauterine infusions in the bitch, presumably because it is difficult to pass a catheter into the canine cervix. Cobb (1959) designed an apparatus for hysterosalpingography with which he reported fairly easy catheterization of the cervical canal in the anaesthetized bitch. Recent advances made in the field of deepfreezing of dog semen have emphasized the need for a simple method for intrauterine infusion in the unanaesthetized bitch. The first successful insemination with frozen dog semen was reported in 1969 by Seager. The semen was frozen in pellets and deposited vaginally. Over a six-year period 61 (39.1 %) out of 156 bitches inseminated with this method became pregnant (Seager et al. 1975). Andersen (1972), however, when inseminating dog semen frozen in French straws, reported no success after vaginal deposition of the thawed semen. Based on experience with insemination in Blue foxes Andersen (1975) developed a special catheter which he could introduce through the cervix to deposit the semen into the corpus uteri without anaesthetizing the bitches. With this method 19 (73.1 %) out of 26 bitches became pregnant (Andersen 1977, personal communication). This method of passing the catheter through the cervix requires training and the method is impractical in nervous or obese bitches in which palpation of the abdomen is difficult or impossible. In order to fully use the advantages offered to the dog breeders by deep-freezing of dog semen, it is necessary to develop a simple method of inseminating the bitch that can be employed by practising veterinarians without previous special training. The present investigation was undertaken as an introduction to further studies of the problems related to the use of frozen dog semen.     

Polyelectrolyte Flocculation as an Aid to Recovery of Enteroviruses from Oysters

A simple and rapid method for recovering enteroviruses from oysters is described. A polycation sewage flocculant promoted cohesion of oyster solids and thereby aided separation of these from the viruses. Recovery of 80 to 100% of experimentally inoculated virus was achieved, and the suspension or extract obtained could be inoculated directly into tissue cultures or concentrated first for greater sensitivity.     

Administration of 15-methyl-prostaglandin F2α as a pre-operative means of cervical dilatation

Pre-operative dilatation of the cervix prior to vacuum aspiration was accomplished in 67 volunteers by extra-amniotic or intra-muscular administration of 15(S)-15-methyl-PGF_2α (15-me-PGF_2α). Ninety-four per cent of the patients were in the 11th–13th week of gestation and 61% were nulliparae. A single extra-amniotic instillation (mean of 400 μg) or 3 intramuscular injections (300–800 μg per injection) of the compound induced a satisfactory outcome in terms of either abortion or sufficient dilatation of the cervix in 81% of the patients. In the remaining cases, the cervix was found at operation to be open for 7–9 mm which simplified the process of additional instrumental dilatation. In general the outcome of the trial turned the operation into an easy and safe procedure. Vacuum aspiration was performed in all cases after a mean time lag of 16 hours following the onset of the treatment. Extra-amniotic administration was associated with a low incidence of gastro-intestinal side-effects, but there was a transient and moderate degree of uterine pain reaction. The intramuscular route was technically more simple and caused less uterine pain but the high incidence of vomiting and diarrhoea constituted a clinical disadvantage. In late first trimester abortions, particularly cases where the uterus appears larger than expected, it is believed that dilatation of the cervix by PG prior to vacuum aspiration is a sound clinical indication. The method offers definite advantages that compensate for the price of some minor side-effects.     

CD4+ T Cells Are Necessary and Sufficient To Confer Protection against Chlamydia trachomatis Infection in the Murine Upper Genital Tract

Chlamydia trachomatis infection is the most common bacterial sexually transmitted disease in the United States. Chlamydia infections that ascend to the upper genital tract can persist, trigger inflammation, and result in serious sequelae such as infertility. However, mouse models in which the vaginal vault is inoculated with C. trachomatis do not recapitulate the course of human disease. These intravaginal infections of the mouse do not ascend efficiently to the upper genital tract, do not cause persistent infection, do not induce significant inflammation, and do not induce significant CD4(+) T cell infiltration. In this article, we describe a noninvasive transcervical infection model in which we bypass the cervix and directly inoculate C. trachomatis into the uterus. We show that direct C. trachomatis infection of the murine upper genital tract stimulates a robust Chlamydia-specific CD4(+) T cell response that is both necessary and sufficient to clear infection and provide protection against reinfection.     

Pathogenicity of Tritrichomonas mobilensis: subcutaneous inoculation in mice

The standard "subcutaneous mouse assay" was used to investigate the inherent pathogenicity of Tritrichomonas mobilensis, an intestinal parasite of squirrel monkeys. C57B1/6 mice given subcutaneous bilateral inocula of T. mobilensis died by day 4 postinoculation with lesions too small to be measured. Control mice similarly inoculated with pathogenic and nonpathogenic strains of Trichomonas vaginalis survived the challenge and produced lesions on day 6 with mean volumes in agreement with previous reports. CD1 mice similarly inoculated with standard and double doses of trichomonads (T. mobilensis) again produced small lesions. CD1 mice inoculated at double dosage were moribund or dead on days 5 and 6, respectively, postinoculation. Necropsies were performed on dead and sacrificed mice. Tissues were obtained from internal organs for histology and culture. Unexpectedly, trichomonads were cultured from liver and lung of C57B1/6 mice at the standard level of inoculation and liver, lung, and spleen of CD1 mice at the higher level of inoculation. Although trichomonads are normally considered surface-dwelling noninvasive organisms, the penetration of trichomonads to deep tissues is not without precedent. Tritrichomonas foetus and Trichomonas gallinae are known to invade tissues of their respective hosts. Trichomonas vaginalis has been demonstrated in subepithelial areas of both the prostate gland and cervix of humans. The ability of several species of trichomonads to invade tissues and/or migrate to other sites in their hosts suggests a need for revision of the concept of trichomonads as strictly lumen or surface-dwelling parasites.     

Isolation of epithelial stem cells from dermis by a three-dimensional culture system

Skin is a representative self-renewing tissue containing stem cells. Although many attempts have been made to define and isolate skin-derived stem cells, establishment of a simple and reliable isolation procedure remains a goal to be achieved. Here, we report the isolation of cells having stem cell properties from mouse embryonic skin using a simple selection method based on an assumption that stem cells may grow in an anchorage-independent manner. We inoculated single cell suspensions prepared from mouse embryonic dermis into a temperature-sensitive gel and propagated the resulting colonies in a monolayer culture. The cells named dermis-derived epithelial progenitor-1 (DEEP) showed epithelial morphology and grew rapidly to a more than 200 population doubling level over a period of 250 days. When the cells were kept confluent, they spontaneously formed spheroids and continuously grew even in spheroids. Immunostaining revealed that all of the clones were positive for the expression of cytokeratin-8, -18, -19, and E-cadherin and negative for the expression of cytokeratin-1, -5, -6, -14, -20, vimentin, nestin, a ckit. Furthermore, they expressed epithelial stem cell markers such as p63, integrin beta1, and S100A6. On exposure to TGFbeta in culture, some of DEEP-1 cells expressed alpha-smooth muscle actin. When the cells were transplanted into various organs of adult SCID mice, a part of the inoculated cell population acquired neural, hepatic, and renal cell properties. These results indicate that the cells we isolated were of epithelial stem cell origin and that our new approach is useful for isolation of multipotent stem cells from skin tissues.     

A fast method for monitoring the colonization rate of lactobacilli in a meat model system

A random amplified polymorphic DNA (RAPD) assay coupled to a fast and reproducible cell lysis method from Lactobacillus colonies were developed to type lactobacilli of different strains and species, with the aim of precisely enumerating each of the different Lactobacillus strains inoculated in a nutrient-rich environment, such as sausage meat batter. Colonization assays were carried out in an aseptic meat fermentation system for up to 14 d and the inoculated strains were challenged with mixtures of wild lactobacilli. The proportion of inoculated strains remaining at different times was compared with the total number of lactobacilli grown on MRS agar by RAPD. The colonization rate of the different strains tested was very different. The RAPD-fast lysis method developed is simple and, with a low cost per assay, could also be applied to other food fermentations.     

Measurement of growth of bifidobacteria on inulofructosaccharides

A simple method for continuous measurement of growth of bifidobacteria is described. An inoculated broth in a small tube was evacuated at 100mmHg through a two-way stopcock and incubated at 37°C. Growth was periodically measured by inserting the tube into a spectrophotometer. Doubling times of three bifidobacterium strains in broth containing various inulofructosaccharide preparations were compared. Inulofructosacchandes from the Jerusalem artichoke tubers supported good growth of these bacteria.     

Death of sperm in the cervix of ewes bearing intrauterine plastic spirals or threads

A plastic spiral intrauterine device (IUD) in the ewe inhibits sperm transport through the cervix. In Exp. 1, plastic spirals were inserted surgically into the lumen of one or both uterine horns. In Exp. 2, Dacron threads were placed in the lumen of each horn. At a subsequent estrus, ewes were mated and necropsied 2 hours later. Sperm were washed from the uterine body and from the anterior, middle and posterior one-third of the cervix and examined. Plastic spirals significantly reduced the percentage of sperm recovered from the uterine body and each segment of the cervix that were motile and live and had normal acrosomes. In the anterior one-third of the cervix, e.g., 49% of the sperm were motile and 66% were live in control ewes but only 17% were motile and 23% were live in IUD ewes. Intrauterine threads also reduced the percentages of sperm that were motile and live in the anterior cervix. Failure of sperm transport in IUD-bearing ewes may be caused by conditions which result in loss of sperm motility in the cervix, thereby inhibiting the establishment of a reservoir of live sperm for transport to the oviducts.     

A model for cystic endometrial hyperplasia/pyometra complex in the bitch

The objective of this study was to develop a reliable model for the study of the cystic endometrial hyperplasia and pyometra complex (CEH/P) in the bitch. Greyhound bitches (n = 15) were ovariectomised and allocated into three groups (Group 1, n = 5; Group 2, n = 5; Group 3, n = 10, including 5 used from Group 1). Simulated proestrus, estrus and diestrus were induced by treatment with estradiol benzoate and megestrol acetate. The duration of cervical opening during estrus was determined by the intra-vaginal infusion of radio-opaque medium and subsequent radiography of the uterus (Group 1). One milliliter of a culture of Escherichia coli (with five uro-pathogenic virulence factors as identified by PCR: pap, sfa, hlyA, cnf1 and fim) was inoculated intra-vaginally daily throughout the simulated estrus (Group 2). One milliliter of the culture (n = 6) or sterile Luria-Bertani broth (n = 4) was introduced directly into the uterus on simulated diestrus Days 8 or 12 (Group 3). Necropsies were performed 12 and 7-14 days after the inoculation (Groups 2 and 3). The cervix remained open throughout the duration of simulated estrus (5-6 days) in four out of five bitches, and for a shorter duration (3 days of a 6-day estrus period) in one bitch (Group 1). CEH/P was induced by inoculation of bacteria into the uterus (10/10 bitches) but not into the vagina (0/5 bitches), (P = 0.003). A model for the study of CEH/P has been validated.     

Cervical dilatation in late first trimester termination by prostaglandin,hylase and isogel

Pre-operative dilatation of the cervix was attempted in 223 cases prior to vacuum aspiration in patients seeking late first trimester termination beyond ten weeks. 15 Me PGF_2a was used in the form of vaginal suppositories, intramuscular and intracervical injections. Dilatation of cervix of 10 mm or more was achieved within 4 hours in 86% cases with intra-cervical injections. Gastro-intestinal disturbances caused by intra-muscular injections could be avoided by intra-cervical injections, as the amount of prostaglandin required was only 100 ugm, but the success rate was significantly lower. The success with multiple dose suppositories was 80%. There was no significant difference in the success with 1.5 mgm or 1.0 mgm dosage, but the side effects were significantly higher with 1.5 mgm suppositories.Intra-cervical Hylase did not dilate the cervix but successfully softened it within 5 minutes to make metallic dilatation simple. The hygroscopic Isogel tents achieved dilatation of 10 mm or more in 73% of the patients in 12 hours. The tents as well as intracervical prostaglandin injection had the disadvantage of requiring an additional theatre procedure prior to suction curettage.     

An efficient procedure for the isolation of PCR-competent DNA from Bacillus endospores germinated in soil

DNA extraction techniques for endospore-forming bacteria in soil are often labour-intensive and unreliable. Our objective in this work was to investigate whether good quality DNA could be obtained from spores germinated in soil. To this end, endospores from Bacillus subtilis, B. megaterium and B. thuringiensis were inoculated into soil microcosms and germination was induced by addition of LB medium supplemented with l-alanine, glucose, fructose and KCl. Heat resistance count was reduced to 80% for B. subtilis and more than 90% for B. thuringiensis and B. megaterium after a few minutes. Isolation of DNA from soil with a procedure which did not work on spores was shown to be as efficient for in situ-germinated spores as for inoculated vegetative cells. Furthermore, we developed a simple procedure that allowed us to use the recovered DNA in PCR amplifications. The present methodology is simple and efficient; it avoids the use of special equipment and harsh spore rupturing methods and can be carried out with multiple samples.