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Biomedical subjects

R B Gwatkin

Publications and source records attributed to R B Gwatkin.

At least 19 recordsLinked to original sources
PubMed1996

Human endometrial matrix metalloproteinase-2, a putative menstrual proteinase. Hormonal regulation in cultured stromal cells and messenger RNA expression during the menstrual cycle.

Proteinases are likely effectors of endometrial menstrual breakdown. We have investigated proteinase production by human endometrial stromal cells subjected in vitro to progesterone (P) withdrawal, the physiologic stimulus for menstruation. Culture media of cells exposed to estradiol, P, or estradiol plus P had low levels of proteolytic activity similar to cultures maintained in the absence of steroids. P withdrawal, or addition of RU486 to P-treated cultures, stimulated proteinase secretion. The stromal cell proteinase was characterized by gelatin zymography, inhibitor profile, and organomercurial activation, as a metalloproteinase present mostly as a 66-kD proenzyme with lower levels of a 62-kD active form. The P withdrawal-induced metalloproteinase was identified as matrix metalloproteinase-2 (MMP-2) by Western blotting. The increase of MMP-2 induced by P withdrawal was associated with the metalloproteinase-dependent breakdown of stromal cultures, involving dissolution of extracellular matrix and dissociation of stromal cells. Northern analysis showed the differential expression of MMP-2 mRNA in late secretory phase endometrium. These findings are consistent with the involvement of stromal cell-derived MMP-2 in the proteolysis of extracellular matrix promoting cyclic endometrial breakdown and the onset of menstrual bleeding.

Blotting, Western↗
PubMed1994

Expression of insulin-like growth factors I and II in conceptuses from normal and diabetic mice.

Insulin-like growth factors (IGF-I and IGF-II) play an important regulatory role in fetal growth and development. Alterations in expression of these growth factors may result in developmental abnormalities, macrosomia, and intrauterine growth retardation, which occur with a higher incidence in diabetic pregnancies. In situ hybridization histochemistry was employed to investigate the distribution and abundance of IGF-I and IGF-II in peri-implantation and postimplantation conceptuses from normal and streptozotocin-treated diabetic mice. Animals were sacrificed on gestational days 5, 6, 7, 8, and 9. The entire uterine horn was prepared for hybridization with antisense and sense alpha 35S-dATP labeled oligonucleotide probes for IGF-I, IGF-II, and mouse beta-actin. IGF-I transcript was apparent only in myometrium at 6 days of gestation in normal and diabetic mice. IGF-II transcripts were restricted to trophoectoderm cells within the implantation chamber on day 5. Following implantation, IGF-II transcripts were found in trophoectodermal derivatives, primitive endoderm, mesoderm, heart, walls of the foregut, and mesenchyme in normal and diabetic postimplantation conceptuses. There were no apparent differences between normal and diabetic samples in the distribution and abundance of the IGF-II transcript from gestational days 7, 8, and 9. The embryos from the diabetic mother at day 6 were growth retarded and had a significant decrease in the expression of IGF-II. These results suggest that maternal hyperglycemia may retard development of the early implanting conceptus in a narrow window around day 6 through a mechanism involving decreased IGF-II expression.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1993

Transgenic strategies in reproductive endocrinology.

The present discussion surveys some of the recently published studies utilizing transgenic strategies to address questions in reproductive endocrinology. Beginning with a brief introduction of the transgenic method itself, the following areas are covered: 1. Sexual development and Müllerian-inhibiting substance; 2. Hypogonadal mice and hypothalamic GnRH; 3. The GnRH neuron: generation of immortalized rare cell types; 4. Glycoprotein hormones: immortalized cells, development and evolution; 5. Growth hormone and reproduction; and, 6. Gestation and the insulin-like growth factors. In each section, the discussion attempts to be integrative with respect to the significance of the results to physiological, cellular and molecular biology. We believe this approach is appropriate, as transgenic science itself is necessarily an integration of all of these levels of investigation and participation from those working at all levels is needed.

Animals↗
PubMed1990

Zona drilling enhances fertilization by mouse caput epididymal sperm.

Spermatozoa from the caput epididymis are known to be much less capable of fertilization when compared to sperm from more distal segments of the epididymis. The purpose of this study was to determine if two micromanipulative techniques, zona drilling (ZD) and a modification of partial zona dissection (PZD), could be used to enhance fertilization with caput epididymal sperm. A mouse in vitro fertilization model was used. Inseminating oocytes with 500-1,000 sperm/oocyte from the cauda epididymis as a control resulted in fertilization of 98 of 300 (32.6%) oocytes. Of those fertilized, 47 developed to the blastocyst stage (47.9%). Caput sperm fertilized 13 of 116 (11.2%) nonmanipulated oocytes. Only 1 of 13 developed into a blastocyst, while with oocyte ZD, caput sperm fertilized 24 of 144 (16.7%) oocytes, 50% of those fertilized developing to blastocyst (P = 0.0129). When modified PZD was performed on oocytes, only one of 23 was fertilized, with no blastocyst development. These results indicate that acid Tyrode ZD enhances both fertilization and early embryonal development when caput epididymal sperm are used for insemination. These mouse studies suggest that ZD or other micromanipulation techniques may prove clinically useful in men with proximal epididymal obstruction where only caput sperm are available.

Animals↗
PubMed1990

The value of semen analysis and sperm function assays in predicting pregnancy among infertile couples.

Over a 2-year period, 227 couples were evaluated by an extended assessment of the male partner's ejaculate. This extended assessment comprised sperm penetration of denuded hamster oocytes, ability of sperm to penetrate synthetic mucus, and the adenosine triphosphate (ATP) content of whole semen. In proportional hazards analysis adjusting for the contribution of clinical and other seminal variables, the sperm penetration assay (SPA) test was a predictor of pregnancy in the subgroup with normal conventional seminal variables (greater than 40 million motile sperm per ejaculate). When the SPA result was 20% or more the probability of pregnancy was 3.7 times higher; the performance of the SPA as a diagnostic test was better in the couples with treated tubal disease than in other diagnostic categories.

Acrylamides↗
PubMed1989

Failure of spermatozoa from T/t mice to fertilize in vitro is overcome by zona drilling.

Failure of epididymal spermatozoa from T/t mutant mice, but not from t/t individuals, to fertilize oocytes in vitro was partially overcome by opening a small aperture in the zona pellucida with acidified Tyrode's solution to permit direct access of the spermatozoon to the vitellus. This study provides a model system to evaluate requirements for successful zona drilling in the treatment of human infertility and further insights into the effects of the t complex on sperm fertility.

Animals↗
PubMed1989

Gamete processing in an environmental chamber: effect on in vitro fertilization outcome.

A study of mouse gamete processing for in vitro fertilization (IVF) under various conditions showed that it is necessary to control the atmosphere if the temperature is raised from 22 degrees C to 37 degrees C. The data suggest that maximum IVF success is attained by processing the gametes at 37 degrees C, under an atmosphere of 5% O2 and 5% CO2, and overlaying the medium with silicone oil.

Animals↗
PubMed1989

Hamster oocyte penetration tests with oocytes frozen in propanediol: comparison with non-frozen oocytes.

Hamster oocytes were frozen using a 1,2-propanediol-sucrose procedure, which resulted in over 90% survival. After thawing and zona removal the oocytes were compared with non-frozen oocytes in a zona-free hamster egg test employing spermatozoa from human semen donors and suspected infertility patients. Similar data were obtained, indicating that propanediol-sucrose frozen hamster eggs may be used in place of fresh eggs for convenience and to avoid scheduling problems.

Animals↗
PubMed1989

Failed fertilization in human in vitro fertilization analyzed with the deoxyribonucleic acid-specific fluorochrome Hoechst 33342.

The degree and normality of nuclear maturation were assessed with the fluorochrome Hoechst 33342 in two groups of inseminated human oocytes that had failed to undergo fertilization. Group 1 consisted of 67 oocytes from 27 patients, each of whom had at least two other oocytes that had been fertilized and had cleaved. Group 2 consisted of 65 oocytes from 14 patients, none of whose oocytes had been fertilized. In group 1, 52.3% of the oocytes were found to be immature (germinal vesicle stage or metaphase-telophase I), whereas in group 2 only 26% were found to be immature. Thus oocyte nuclear immaturity was the major cause of fertilization failure when companion oocytes were fertilized. When no oocytes of a patient were fertilized, most oocytes were found to be mature, so other factors, such as sperm dysfunction or zona binding abnormalities, must account for most of the fertilization failure in this group of patients.

Benzimidazoles↗
PubMed1989

Hormonal regulation of human endometrial stromal cells in culture: an in vitro model for decidualization.

Stromal cells derived from proliferative or secretory human endometria, cultured in the absence of steroid hormones, grew as monolayers that showed only occasional areas of immunoreactive fibronectin and did not produce detectable levels of prolactin (PRL) or laminin. Treatment with physiological doses of estradiol and progesterone induced PRL production and stimulated cell proliferation, resulting in multilayering with an increase of the saturation density. Electron microscopy showed the development of gap junctions, whereas immunofluorescence revealed a dense pericellular matrix containing fibronectin and laminin. These findings show that human endometrial stromal cells in culture respond to physiological doses of ovarian hormones with ultrastructural, proliferative, and biochemical changes that are characteristic of decidualization in vivo. This culture system thus provides an in vitro model for human decidualization.

Cell Division↗
PubMed1988

Fertilization of zona-drilled mouse oocytes treated with a monoclonal antibody to the zona glycoprotein, ZP3.

Opening a small aperture in the zona pellucida of mouse oocytes by using micromanipulation and a stream of acidified Tyrode's solution (zona drilling) improved the efficiency of in vitro fertilization at low sperm concentrations without adversely affecting development to the blastocyst stage. Zona drilling also permitted in vitro fertilization and development when sperm penetration through the zona was blocked by a monoclonal antibody to the protein core of the zona glycoprotein, ZP3. These results provide a direct demonstration that sperm entry occurs through the aperture and also suggest that zona drilling of human oocytes may offer a therapeutic approach when autoantibodies to the zona pellucida are suspected as a cause of infertility.

Animals↗
PubMed1988

Pre-loading of mouse oocytes with DNA-specific fluorochrome (Hoechst 33342) permits rapid detection of sperm-oocyte fusion.

Mouse oocytes exposed to 1 microgram Hoechst 33342 (H-33342)/ml and then fertilized in vitro developed normally into blastocysts and blastocyst outgrowths. After penetration of the zona, the fertilizing spermatozoon showed intense fluorescence upon fusion with the vitelline membrane. Due to fluorochrome leakage from the perivitelline space a faint fluorescence was detected in zona-bound spermatozoa. This fluorescence of zona-bound spermatozoa intensified with increased fluorochrome concentration (10 micrograms/ml), obscuring the fluorescence of the fertilizing spermatozoa. Spermatozoa added to zona-free mouse oocytes (pre-loaded with 1 or 10 micrograms H-33342/ml) fluoresced within 10 min of insemination, provided the zonae were removed mechanically. Removal by protease digestion induced leakage of fluorochrome, so that all spermatozoa in the vicinity of an oocyte pre-loaded with 10 micrograms H-33342/ml became labelled. This leakage was not visibly apparent when protease-treated oocytes were exposed to only 1 microgram H-33342/ml. The technique could not be applied to zona-free hamster oocytes under our conditions, since the fluorochrome leaked freely from the oocytes whether the zona was removed mechanically or enzymically.

Animals↗
PubMed1988

Characterization of isolated acrosomal matrices from hamster spermatozoa.

The acrosomal matrix of hamster spermatozoa was enriched and characterized. Acrosomal matrices were released from spermatozoa with shaking in a pH 5.2 buffer containing Triton X-100 and protease inhibitors, and enriched on a glass-bead column. Phase-contrast microscopy indicated that 70-80% of the acrosomal matrices were released from the spermatozoa and only minor contamination from sperm heads was detected. Transmission electron microscopy confirmed the low level of contamination in the preparation and revealed a bilaminar structure similar but not identical to that of guinea-pig acrosomal matrix. One- and two-dimensional SDS-polyacrylamide gel electrophoresis (SDS-PAGE) showed the acrosomal matrix to be a complex structure enriched for several polypeptides. Proteinase activity was demonstrated by gelatin-SDS-PAGE. The major activity corresponded to bands of relative molecular masses (Mr) of 56,000, 51,000 and 48,000 with two minor bands of Mr 30,000 and 28,000. The lectin Pisum sativum agglutinin (PSA) bound to the anterior head of spermatozoa and isolated acrosomal matrix as judged by fluorescence microscopy using FITC-PSA. Western blots of spermatozoa and acrosomal matrices followed by overlay with biotinylated PSA indicated that there are at least two PSA-binding glycoproteins of Mr 60,000 and 72,000.

Acrosin↗
PubMed1987

Separation of motile human spermatozoa by means of a glass bead column.

Fertility potential of semen depends upon the presence of viable and motile spermatozoa. A variety of techniques has been reported whereby motile spermatozoa are separated from semen for use in homologous insemination. Using a column of glass beads, we tested various sizes for spermatozoa-separating efficiency. This procedure is quick and simple and results in a significantly better recovery of motile and viable spermatozoa from poor-quality semen. Therefore it has the potential for use in intrauterine insemination for treating male factor infertility.

Cell Separation↗
PubMed1987

Improvement in semen quality using glass bead column.

Fertilizing ability of semen requires the presence of viable and motile spermatozoa whose surface-membrane integrity is maintained. Male infertility is often the result of these variables being suboptimal. Various in vitro methods have been reported that separate good-quality spermatozoa from such ejaculates. In this study we describe a simple procedure that uses a column of glass beads to separate spermatozoa from poor quality semen. The filtered spermatozoa have significantly better progressive motility and viability and also have enhanced fertilizing capacity. Such a method may have clinical use for semen preparation prior to intrauterine insemination and in vitro fertilization. It may also have a role in the assessment of semen to determine fertilization potential and hence provide prognostic information to infertile couples.

Cell Separation↗
PubMed1983

Response of monkeys to porcine zona pellucida as detected by a solid-phase radioimmunoassay.

The immunological response of cynomolgus monkeys (Macaca fascicularis) to immunization was evaluated utilizing collagenase-isolated pig zona pellucida. Six weeks after initial immunization a high serum titer of antibody was exhibited. Serum antibody titers demonstrated a noticeable decline 5 months after booster injections were discontinued. The assay method used is rapid and is capable of detecting antibody in serum dilutions of 1:78,000, as compared to 1:125,000 with the indirect fluorescence assay.

Aluminum Hydroxide↗