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Biomedical subjects

G R Welch

Publications and source records attributed to G R Welch.

At least 37 records · Page 2Linked to original sources
PubMed1998

The relationship between membrane status and fertility of boar spermatozoa after flow cytometric sorting in the presence or absence of seminal plasma.

The motility, viability (percent live), capacitation status and in vitro fertility of boar spermatozoa were examined, after staining with Hoechst 33,342 and flow cytometric sorting in the absence or presence of seminal plasma. Viability was higher in unstained controls and when seminal plasma was present in the medium used to collect spermatozoa from the cell sorter than when seminal plasma was absent or in the staining extender only, but motility was highest when seminal plasma was included in the extender only, compared with the controls and other treatments. The proportions of capacitated spermatozoa were increased by sorting, but were lower when seminal plasma was present, rather than absent, from the staining extender and the collection medium. Compared with unstained controls, extension and staining without sorting only increased the proportion of capacitated spermatozoa after washing in preparation for in vitro fertilization. The percentages of polyspermic, penetrated and cleaved oocytes were lower when inseminated with unsorted (stained) than control (unstained) spermatozoa, regardless of the presence or absence of seminal plasma. These parameters were higher for sorted than for control spermatozoa in the absence of seminal plasma, but in its presence penetration and cleavage were substantially lower. The proportions of capacitated spermatozoa were lower when seminal plasma was present in the collection medium only than in the staining extender or when it was absent altogether, but the former treatment substantially reduced the proportions of polyspermic, penetrated and cleaved oocytes, and the proportion of blastocysts. These findings indicate that sperm capacitation associated with flow cytometric sorting can be reduced by the inclusion of seminal plasma in the collection medium, but this treatment reduces the ability of spermatozoa to fertilize oocytes in vitro under these conditions.

Animals↗
PubMed1997

Production of piglets preselected for sex following in vitro fertilization with X and Y chromosome-bearing spermatozoa sorted by flow cytometry.

In vivo-matured porcine oocytes were fertilized in vitro with X and Y chromosome-bearing spermatozoa, and sorted for sex on the basis of DNA content by flow cytometry. Developmental competence of the sexed embryos was determined through established pregnancies after embryo transfer. Spermatozoa were stained with Hoechst 33342 and sorted using a flow cytometry cell sorter. Purity of sorting was 83% for Y spermatozoa and 92% for X spermatozoa. A total of 387 mature cumulus-oocyte-complexes (COC) was collected from 18 superovulated prepuberal gilts shortly before ovulation. In vitro fertilization with sorted spermatozoa was performed in 4 replicates. After 18 h of sperm- oocyte co-culture at 39 degrees C, the zygotes were placed into culture medium (NCSU-23) for another 24 h. The average cleavage rate was 56.2%. Ninety-two embryos produced from X-sorted sperm cells were transferred surgically into the uterus of 2 recipients. Two gilts farrowed and delivered 6 and 4 healthy female piglets, respectively. Additionally, 2 gilts were inseminated intratubally via surgical laparotomy with either X or Y sorted spermatozoa (2 x 10(5)) per oviduct. The 2 sows farrowed producing 15 piglets. Thirteen of the 15 piglets were of the predicted gender (85%).

Journal Article↗
PubMed1996

Slit-scan flow cytometry for consistent high resolution DNA analysis of X- and Y-chromosome bearing sperm.

This paper describes the application of slit-scan flow cytometry for accurate DNA analysis of X- and Y-chromosome bearing sperm. The introduction of the slit-scanning technique was initiated to improve the consistency in resolution of the X and Y population from donor to donor. An optimal resolution is essential for high purity sorting of X and Y sperm, as the difference in DNA content is small (3-4%) in most mammals. This difference is the discriminatory parameter for the flow cytometric sorting of the two populations. Our approach was to focus on the role of the sperm tail in the detection process. Slit-scan flow cytometric analysis allows the whole sperm to be spatially analyzed along the direction of flow. Sperm were stained with Dansyl Lysine, a UV excitable fluorescent membrane dye, which stained the head, midpiece, and principal piece. Analysis of these stained sperm showed that there was no difference between the relative number of sperm that travel headfirst or tailfirst through the detection zone of the flow cytometer. The influence of sperm with coiled tails on DNA analysis was also investigated. The proportion of sperm with coiled tails influences semen quality. The standard X-Y separation procedure uses Hoechst 33342, which stains all intact sperm, both living and dead. Propidium iodide was added to discriminate the dead sperm population. Slit-scan analysis showed that measurement of a sample containing a high proportion of living sperm with coiled tails results in an inferior DNA histogram and reduced X-Y resolution. Sperm with coiled tails can result in a lower detected fluorescence intensity, but the reason for this is unclear. Slit-scan flow cytometry allows exclusion of sperm with coiled tails from the analysis, resulting in a restoration of high resolution of X- and Y-chromosome bearing sperm populations.

Animals↗
PubMed1996

Porcine sperm viability, oocyte fertilization and embryo development after staining spermatozoa with SYBR-14.

The objective of these experiments was to determine the efficacy of the new membrane permeant nucleic acid stain, SYBR-14, for assessing boar sperm viability and to determine it's effect on fertilization and early embryonic development using the pig as a model. We examined the staining patterns of SYBR-14 and another vital stain, Hoechst 33342, both in combination with the dead cell stain, propidium iodide (PI), to quantify the proportion of living and dead spermatozoa in ejaculated and epididymal semen. Flow cytometry analyses of semen from 4 boars revealed significant differences among boars for the proportion of SYBR-14-stained spermatozoa in both epididymal and ejaculated samples, but not for Hoechst 33342 or PI stained spermatozoa. Gilts were inseminated with unstained spermatozoa or spermatozoa stained with 2 levels of SYBR-14 or 2 levels of the reference stain, Hoechst 33342. Embryos recovered at 42 to 48 h postinsemination were morphologically evaluated, and only 4 to 8-cell embryos were continued in culture. Overall, fluorescent staining of boar spermatozoa with SYBR-14 or Hoechst 33342 neither affected their ability to fertilize oocytes, nor the developmental competence of the resultant embryos.

Journal Article↗
PubMed1996

Comparison of detergent-solubilized membrane and soluble proteins from flow cytometrically sorted X- and Y-chromosome bearing porcine spermatozoa by high resolution 2-D electrophoresis.

The only known and measurable difference between X- and Y-chromosome bearing spermatozoa is the small difference in their DNA content. The X sperm in the human carry 2.8% more DNA than the Y sperm, while in domestic livestock this difference ranges from 3.0 to 4.2%. The only successful sperm separation method, flow cytometric sorting, is based on this difference in DNA content. Using this technique, X and Y sperm populations with purities greater than 90% can be obtained. The number of spermatozoa that can be sorted in a given time period, however, is too low for application of this technique in routine artificial insemination. Therefore, the search for a marker other than DNA to differentiate between X and Y sperm remains of interest in order to develop a method for large scale X and Y sperm separation. The aim of the present study was to investigate whether porcine X and Y sperm contain some difference in their plasma membrane proteins. The flow cytometric sorting of sperm enabled a direct comparison of the proteins of the X and Y sperm populations. High resolution two-dimensional (2-D) electrophoresis was used; however, adaptations were needed to enable its use for analysis of proteins of flow cytometrically sorted sperm, both in the sorting procedure, membrane protein solubilization, and in the 2-D electrophoresis. Up to 1000 protein spots per gel could be detected and quantified. Comparison of the 2-D protein patterns revealed differences in protein spots between sperm of two individual boars. However, no differences in protein spots between the X and Y sperm fractions were found. These results provide additional support for the view that X- and Y-chromosome bearing spermatozoa are phenotypically identical, and cast doubt on the likelihood that a surface marker can provide a base for X and Y sperm separation.

Animals↗
PubMed1996

The enzymatic basis of information processing in the living cell.

Analogies are drawn between the computational aspects of information processing and the enzymatic events of cellular metabolism. The origin thereof is linked to the logic-elements involved in the reaction-diffusion processes of the metabolic operation. It is shown how such function entails a 'molecular machine' quality when associated with organized non-equilibrium energy sources in the cell.

Cells↗
PubMed1996

Viability and membrane integrity of spermatozoa after dilution and flow cytometric sorting in the presence or absence of seminal plasma.

Boar, bull and ram spermatozoa were examined after staining with the DNA-permeant Hoechst 33342 fluorochrome and flow cytometric sorting in the presence or absence of seminal plasma. Spermatozoa were assessed for viability with flow cytometry using the live cell nucleic acid stain SYBR-14 and propidium iodide (PI), and for membrane integrity using fluorescein isothiocyanate-conjugated Pisum sativum (FITC-PSA) and PI; motility and acrosome integrity were estimated by microscopy. Flow cytometric sorting was compared with pipette dilution of boar and bull spermatozoa into: (1) medium [boar: Test buffer containing 2% yolk (TY) or Beltsville thawing solution (BTS); bull: TY or HEPES buffer containing 0.1% bovine serum albumin (HEPES-BSA)] with or without 10% (v/v) seminal plasma; or (2) an empty tube containing no medium. Sorted spermatozoa were either not centrifuged or centrifuged before assessment during a 4-h holding period. The viability, motility and membrane integrity of boar, bull and ram spermatozoa centrifuged after sorting were also examined when seminal plasma was present or absent from the staining extender and/or the TY collection medium. The results indicate that the viability and membrane integrity of spermatozoa in vitro would be improved if: (1) seminal plasma (10%) was routinely included in the BTS and HEPES-BSA staining extenders for boar spermatozoa and ram spermatozoa, respectively, when used in preparation for flow cytometric sorting; and (2) 10% and 50% seminal plasma were included in the TY collection medium for boar or bull spermatozoa and ram spermatozoa respectively.

Animals↗
PubMed1995

Atresia in follicles grown after ovulation in the pig: measurement of increased apoptosis in granulosa cells and reduced follicular fluid estradiol-17 beta.

The incidence of atresia in the first group of follicles grown after ovulation was investigated in the pig. At slaughter, 113 follicles 3-6 mm in diameter were dissected from the ovaries of four pregnant pigs per day on Days 5, 6, and 7 after the onset of estrus. Granulosa cells were isolated from each follicle. The percentage of granulosa cells containing sub-diploid amounts of DNA (%Ao cells), a measure of apoptosis, was determined for each follicle by DNA fluorescence flow cytometry of propidium iodide (PI) stained nuclei of ethanol-fixed cells. Granulosa cell DNA condition was used to classify follicles. Follicles with > or = 10% Ao cells (n = 33) were designated biochemically atretic (BA), and follicles with < 10% Ao cells (n = 80) were designated biochemically healthy (BH). Internucleosomal cleavage, also indicative of apoptosis, was determined by autoradiographic analysis of [32P]-3'-end-labeled DNA from granulosa cells. Densitometric analysis showed that optical density of [32P]-3'-end-labeled DNA fragments in the 0.18-20-kb size range was correlated with the %Ao cells (R > or = 0.90, N = 22, p < 0.001). The incidence of pigs with BA follicles was 2 of 4, 3 of 4, and 4 of 4 on Days 5, 6, and 7, respectively. The %BA follicles per pig (mean +/- SEM) increased (p < or = 0.01) between Days 5 and 7; values were 6.2 +/- 3.6, 28.1 +/- 13.5, and 50.0 +/- 7.1, respectively, on Days 5, 6, and 7.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

Metabolic channeling versus free diffusion: transition-time analysis.

Metabolic channeling is the term used to describe the restricted flow of substrates and products in multienzyme systems. It has been argued for some time that free diffusion is sufficiently rapid to obviate the need for channeling and, furthermore, that it is also fast enough to prevent competing side reactions from interfering with the metabolic flow. In this article we argue that a thorough consideration of the temporal behavior of metabolite pools suggests that channeling is important in many cases.

Biological Transport↗
PubMed1994

Successful gamete intrafallopian transfer (GIFT) in the porcine.

Gamete intrafallopian transfer (GIFT) was successfully established in the pig. In Experiment 1 (6 replicates) 234 oocytes (39 +/- 5.5 per recipient) plus spermatozoa (4000 to 8000 per oocyte) were transferred bilaterally into the oviducts of synchronized gilts, and embryos were recovered 48 h thereafter. The recovery rate was 50.4% and 50% of the recovered oocytes were fertilized. A total of 55 embryos was cultured in vitro in NCSU-medium for 48 h and 63.6% developed to morula or blastocyst stages. In Experiment 2 (5 replicates) 220 oocytes (44 +/- 4.9 per recipient) plus spermatozoa (4000 per oocyte) were transferred to 5 recipients which were allowed to go to term. Three gilts delivered 16 (n = 3, 5, 8) piglets. In Experiment 3 (5 replicates) 183 oocytes (36.6 +/- 1.2 per recipient) plus flow cytometry gender sorted spermatozoa (4000 per oocyte) were transferred to 5 recipients. The recovery rate was 47.8%, and 27.6% of the oocytes were fertilized. From all cleaved oocytes 45.8% developed to expanded blastocysts, with the number of blastomeres varying from 20 to 85 (38.3 +/- 22.5). These results indicate that the GIFT procedure can be used successfully in pigs, and can be a valuable tool for the study of gamete interaction as well as in the continued development of biotechnological procedures such as sex pre-determination.

Journal Article↗
PubMed1994

Flow cytometric determination of degraded deoxyribonucleic acid in granulosa cells to identify atretic follicles during preovulatory maturation in the pig.

Granulosa cells of individual follicles were analyzed by DNA fluorescence flow cytometry to determine how the percentage of cells with degraded DNA and the distribution of cells in the phases of the cell cycle (Go/G1, S1, G2/M) related to the incidence of morphological atresia and to changes in follicular steroid concentrations. Follicles were dissected from ovaries recovered at slaughter on Days 1, 3, 5, or 7 of altrenogest-synchronized preovulatory maturation. Twenty-one follicles with debris among their isolated granulosa cells were classified as morphologically atretic (MA); 92 follicles with debris-free granulosa cells were classified as morphologically healthy (MH). Granulosa cells were prepared for flow cytometry by fixation in 80% ethanol and staining with propidium iodide (PI) containing RNase. DNA fluorescence intensity was determined by use of the 488-nm line of an argon laser. A subpopulation of granulosa cells with degraded DNA (Ao cells), containing less fluorescence than the Go/G1 peak, was found in the DNA histogram of every follicle. The percentage of Ao cells ranged from 0.02 to 83.6% per follicle. The percentage of Ao cells was inversely related to the percentage of Go/G1 cells (r = -0.9611, p = 0.0001). The percentage of Ao cells (mean +/- SEM) was greater (p = 0.0001) in MA (45.9 +/- 6.3%) than in MH follicles (5.3 +/- 1.6%). Follicular estradiol-17 beta was less in MA than in MH follicles, but androstenedione or progesterone did not differ significantly.(ABSTRACT TRUNCATED AT 250 WORDS)

Androstenedione↗
PubMed1993

Gender preselection in humans? Flow cytometric separation of X and Y spermatozoa for the prevention of X-linked diseases.

Human X- and Y-chromosome-bearing spermatozoa were separated based on their DNA content, using modified flow cytometric cell sorting technology. The resulting separation purity of the X-bearing from Y-bearing spermatozoa was evaluated using in-situ hybridization with alpha satellite DNA probes for the X- and Y-chromosomes. In the putative X-enriched-sorted populations, an average of 82% of the spermatozoa showed a hybridization signal with the X probe. Similarly, in the Y-sorted population 75% gave a signal with the Y probe. Sorted X- and Y-bearing spermatozoa were found to maintain their viability for several hours after sorting. These results demonstrate that the human sperm sex ratio can be significantly shifted to favour the selection of female-producing (X) spermatozoa or male-producing (Y) spermatozoa when spermatozoa are flow cytometrically sorted on the basis of DNA content. We propose that flow cytometrically sorted human spermatozoa, used in conjunction with in-vitro fertilization or intra-oviductal insemination, could be used by families who are at risk for X-linked diseases to preferentially produce female offspring. Sorted spermatozoa could also be used to pre-select for male offspring if that were medically indicated.

Cell Separation↗
PubMed1992

Patterns of spatiotemporal organization in an "ambiquitous" enzyme model.

Many enzymes in pathways such as glycolysis associate reversibly with cellular substructures. The spatiotemporal behavior of a "limit-cycle" oscillation model is studied under the condition that the "ambiquitous" oscillophor, phosphofructokinase, is partitioned between "bulk-phase" and "bound" forms in a heterogeneous system. Computer simulation demonstrates the occurrence of sustained, wave-like spatiotemporal patterns of chemical concentration in the bulk medium. Kinetic dissimilarity among the localized populations of bound enzyme leads to a "polarity" effect in the wave phenomenon. It is suggested that a key physiological role of the limit-cycle regime is to engender a rapid, site-to-site, signal-transmission modality in large eukaryotic (e.g., mammalian) cells.

Adenine Nucleotides↗
PubMed1992

Transforming growth factor-beta in synovial fluids modulates Fc gamma RII (CD16) expression on mononuclear phagocytes.

Mononuclear phagocytes in the synovium of patients with arthritis, in contrast to blood monocytes, were found to express a third receptor for the constant region of Ig (Fc gamma RIII), in addition to Fc gamma RI and Fc gamma RII. Previously identified on mature mononuclear phagocytes or phagocytes exposed to transforming growth factor-beta (TGF-beta) in vitro, this study documents the presence of Fc gamma RIII (CD16) expressing cells at an inflammatory site. Furthermore, the presence of CD16 on the majority of the LeuM3 (CD14) positive synovial monocytic cells could be mimicked by exposing blood monocytes to synovial fluids from patients with rheumatoid arthritis (17 of 19) and synovial fluids from patients with osteoarthritis (4 of 4). In additional studies, the soluble factor in inflammatory synovial fluids responsible for regulating CD16 expression was found to be consistent with the presence of TGF-beta. Inhibition of the activity in synovial fluids with a neutralizing antibody to TGF-beta confirmed a role for this peptide in synovial phagocytic cell CD16 expression. Moreover, signal transduction through CD16 on synovial phagocytes resulted in augmented extracellular release of superoxide anion that may contribute to tissue damage and other inflammatory sequelae. Identification of TGF-beta and its association with upregulation of CD16 at sites of chronic inflammation may provide insight into the destructive lesions associated with inflammatory arthropathies.

Antigens, Differentiation↗
PubMed1991

IL-4 antagonizes induction of Fc gamma RIII (CD16) expression by transforming growth factor-beta on human monocytes.

The multifunctional cytokines, transforming growth factor (TGF)-beta 1 and beta 2, have the capability of inducing human peripheral blood monocytes to express the type III receptor for the Fc portion of IgG (Fc gamma RIII/CD16). In this study we show that the T cell-derived cytokine, IL-4, antagonizes the ability of TGF-beta to induce the expression of CD16. Furthermore, this ability to down-regulate expression of CD16 is completely abrogated after treatment with polyclonal anti-IL-4, suggesting that IL-4 is solely responsible for the observed inhibition. The mechanism for negating the effect of TGF-beta is not due to decreased expression of surface receptors for TGF-beta, but appears to occur at the mRNA level. Nuclear run-off assays indicate that regulation occurs predominantly through a posttranscriptional mechanism(s), although a transcriptional process cannot be ruled out. Normally, CD16 appears on only a small population of circulating monocytes, however, expression is apparent on the majority of mature tissue and inflammatory macrophages likely due to the release of TGF-beta in these sites. Inasmuch as this receptor binds immune complexes and opsonized particles, it is associated with enhanced immunophagocytosis. Suppression of CD16 expression and its ability to suppress a number of other monocyte functions suggests that IL-4 may play an important role in the resolution of inflammatory and tissue repair responses.

Antigens, CD↗