Biomedical subjects

D W Dresser

Publications and source records attributed to D W Dresser.

At least 19 recordsLinked to original sources

PubMed2001

An expressed GNRP-like gene shares a bi-directional promoter with SF3A2 (SAP62) immediately upstream of AMH.

A region of homology, containing the contiguous SF3A2 (formerly called SAP62) and AMH genes, exists between human chromosome 19 (HSA19p) and mouse chromosome 10 (MMU10). In a previous study it was shown that SF3A2/Sf3a2 is very highly conserved between the two species and that AMH/Amh is somewhat less conserved although both human and mouse genes encode a protein (AMH) playing the same critical role during early male sex differentiation. The close association between SF3a2/Sf3a2 and AMH/Amh was thought to maintain open chromatin in the AMH/Amh promoter region, thus facilitating the necessary precise timing of AMH/Amh expression following that of SRY/Sry at the onset of testis differentiation. Further investigation of DNA upstream of Amh has revealed that there is another gene, in close association (about 400 bp) with Sf3a2, which has significant similarities to the N-terminus of a known guanine nucleotide releasing protein (GNRP) and consequently is provisionally named GNRPx/Gnrpx. The Gnrpx-Sf3a2-Amh (GSA) locus of the mouse (MMU10) is conserved in the human (HSA19p). Mapping the Sf3a2 transcription start site eventually led us to locate and characterize its promoter. We found that Sf3a2 and Gnrpx share a bi-directional promoter, with the latter being transcribed in an antisense direction. It has now been shown by RT-PCR analysis that both Sf3a2 and Gnrpx are widely expressed and therefore are likely to be 'housekeeping' genes. GNRPx/Gnrpx messenger RNA codes for a C-terminally truncated protein (149/164 aa), which contains an as yet uncharacterized domain common to GNRPs (and related proteins) and which may therefore act as a specific antagonist of a complete GNRP protein (>1200 aa) involved in the regulation of the GTPase (G-protein/Ras) cycle.

Amino Acid Sequence↗

PubMed2001

In vivo analysis of the regulation of the anti-Müllerian hormone, as a marker of Sertoli cell differentiation during testicular development, reveals a multi-step process.

Anti-Müllerian hormone (AMH) is a member of the TGF-beta family which elicits its main action during male sex differentiation. This hormone is probably the most convenient marker of Sertoli cell differentiation and maturation throughout testicular development. Studying AMH gene regulation may thus be one way of identifying effectors of Sertoli cell differentiation. To this end we first tried to locate and then to characterise DNA elements responsible for in vivo transcriptional control of AMH expression. We obtained transgenic mice expressing a reporter gene (LacZ), under control of various putative AMH regulatory sequences. Analysis of transgenic animals revealed that activation of the AMH gene probably requires a two-step regulatory process. The first step corresponds to the initial activation of the AMH gene occurring at around 12.0 dpc. It requires the presence of regulatory DNA encompassed within a maximum of 370 bp upstream of the translation start site of the gene, delimited by the presence of an upstream housekeeping gene (SAP-62). Following this initial transient phase, a second phase seems to account for the persistence of AMH gene expression until the onset of puberty. As the 370 bp regulatory region is not sufficient on its own to allow the triggering of this second phase, it seems possible that additional control elements are required for normal AMH expression throughout testicular development. The complete array of regulatory elements remains to be located. Mol. Reprod. Dev. 59:256-264, 2001.

Animals↗

PubMed2001

Haptoglobin is present in human endometrium and shows elevated levels in the decidua during pregnancy.

Assuming that haptoglobin, by virtue of its immunomodulatory properties, could be a regulatory factor during reproduction, its presence in the human uterus was determined. Protein extracts from endometrial tissue samples of pregnant and non-pregnant women were analysed by the immunoblot technique and the intensities of specific bands were quantified. Bands corresponding to haptoglobin were identified in tissue samples obtained from both sources. Protein, purified by high-performance liquid chromatography and monitored by Western blot analysis for its haptoglobin identity, was used for amino-terminal sequencing. Sequencing of the 42 kDa protein identified it as the beta chain of haptoglobin. Immunohistochemistry was used to corroborate the findings and to visualize the distribution of haptoglobin in the tissue. The intensity of the 42 kDa band derived from decidua graviditatis was significantly higher than the intensity of bands derived from non-pregnant endometrium in the proliferative phase (P < 0.01) and in the secretory phase (P < 0.05). Immunohistochemical staining with anti-human haptoglobin antibody elicited strong signals in the decidua graviditatis and weaker signals in the normal endometrium, with the latter showing menstrual cycle-dependent variation. Moderate staining of stroma and a lack of staining of epithelium in the proliferative phase contrasted with the strong staining of stroma and moderate level of staining of epithelium observed in the secretory phase. Haptoglobin in the uterus may exert several functions such as the known binding of haemoglobin, but could also be involved in the multi-factorial mechanism protecting the fetus from a maternal allograft-like immune response.

Blotting, Western↗

PubMed1995

The genes for a spliceosome protein (SAP62) and the anti-Müllerian hormone (AMH) are contiguous.

During an investigation of the regulatory potential of a region 5' of the mouse anti-müllerian hormone (Amh) gene, we identified a region of homology with the known cDNA sequence of a human spliceosome gene (SAP62). In mouse, the Sap62 termination codon (TGA) is just 434 bp 5' of the Amh start of translation (ATG); in the human the equivalent distance is 789 bp. RNase protection analysis shows the majority of Sap62 transcripts use an uncommon polyadenylation signal (ATTAAA) lying in the intragenic region, 87 bp 3' of the TGA. This analysis also shows that Sap62 is transcribed in all tissues examined, whereas specific Amh transcription initiating 10 bp 5' of the ATG is limited to the developing testis of the fetus from 11.5 days post coitum and in the ovary from 3 days post partum. However, in all tissues a significant number of Sap62 transcripts fail to polyadenylate in the intragenic region and continue through the Amh locus. This implies that the Amh locus is in an open chromatin state in all tissues despite a requirement for precise regulation. Human SAP62 can now be mapped to HSA19p and mouse Sap62 to MMU10.

Amino Acid Sequence↗

PubMed1993

Analyses of DNA content of living spermatozoa using flow cytometry techniques.

A two-dimensional fluorescence analysis of spermatozoa stained with Hoechst 33342 was carried out using an Epics V flow cytometer. This analysis involved the measurement of fluorescence both in the conventional manner at 90 degrees and at a narrow forward angle (nominally 0 degree) to the direction of the interrogating laser beam. The arrangement provided an optical means of distinguishing the orientation of flattened particles passing through the interrogation system and enabled the verification and reinterpretation of one-dimensional (90 degree) fluorescence histogram data obtained previously. We now consider that the distinction between live and dead spermatozoa was over simplified and that the observed differences are due partly to an artefact of orientation. It has been confirmed that our earlier use of the low fluorescence peak, consisting of (bull, rabbit, sheep and pig) spermatozoa passing through the interrogation point obliquely to both detectors, as criterion for sorting X- from Y-bearing spermatozoa, is more practical than the use of a very much smaller subpopulation, based on limits imposed by selecting only spermatozoa accurately orientated with their narrow edges to one detector, while measurement of total fluorescence (DNA content) was made simultaneously from a broad face by the other detector. We report marked differences in the rate of staining and in attainment of a staining equilibrium between spermatozoa from different animals or from different ejaculates collected on the same or different days from the same animal. This variation introduces an element of subjectivity into the use of flow cytometry for sorting X- and Y-spermatozoa.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1991

Apparent decline in fertility in heifers after repeated oestrus synchronisation with cloprostenol.

Over several years groups of heifers which were repeatedly treated with the PGF2 alpha analogue, cloprostenol, to synchronise oestrus were artificially inseminated. When sperm treatment was optimised, a reduction in conception rate was observed which was related to the number of synchronisation treatments the animals had received. Although factors such as a seasonal reduction in fertility may have contributed to the effect, there appeared to be a decrease in the proportion of animals becoming pregnant after successive synchronisations. Possible explanations for this observation are suggested.

Animals↗

PubMed1991

The potentiating effect of pregnancy on humoral immune responses of mice.

Previous work has shown that in the mouse, pregnancy significantly increases the level of immunoglobulin secreting cells in the lymph nodes draining the uterus. In this report it is demonstrated that there is also a widely disseminated potentiating effect on levels of immune responsiveness. T-cell responses, manifest as bystander B-cell responses, T-dependent and T-independent B-cell responses to injected antigens and non-specific "responses" to injected mitogens, have all been shown to be potentiated. Ablation of the fetus with retention of a healthy placenta leads to a loss of the potentiating effect. Feeding pregnant mice on indomethacin results in a lowering of immunoglobulin-forming cell levels in the para-aortic lymph node but has no effect on the disseminated potentiation effect detected by the popliteal lymph node assay. It is concluded that the fetus or the placenta under direct control of the fetus, is the source of the "pregnancy factor" responsible for the disseminated potentiation effect, that this factor is unlikely to be a prostaglandin and that the B-cell is the likely target of the potentiating factor. The possible nature of the "factor" is discussed.

Abortion, Veterinary↗

PubMed1991

A potential weak allelic difference in male-specific antigen between two inbred strains of mice.

Differences were detected between male-specific antigen(s) from BALB/c and C57BL/10 mice. Reciprocal crosses between these strains were analysed by means of a popliteal lymph node assay enumerating cells secreting IgM and IgG in a T-dependent bystander B-cell response. The responses, while being indirect, weak and variable, suggested that there are differences in 'immunogenicity' coded by Y-linked (male-specific) genes. This conclusion was strengthened by the results of experiments carried out in Y-backcross mice, where spleen cells from male C57BL mice with a BALB/c-Y (B10.C-YC) stimulated low popliteal lymph node responses in female littermates in comparison with C57BL mice with their own Y-chromosome. In contradistinction, male spleen cells from a BALB/c with a C57B1/10-Y chromosome (C.B10-YB), injected into a hind footpad of a female littermate, induced a relatively higher popliteal lymph node response.

Alleles↗

PubMed1990

Fast plaque sizing and some applications of the technique in antibody feedback experiments.

A method has been devised to make rapid assessments of plaque size simultaneously with plaque enumeration. The method depends on an inexpensive and simple interfacing of a weighted counter to a microcomputer. Comparisons made between relative (weighted counter) and absolute (photographic) assessments of plaque size, show that the relationship is not linear. It has been shown that assay conditions must be rigidly controlled for valid size comparisons to be made. IgM and IgG PFC specific for sheep RBC, burro RBC and TNP-, in primary and secondary, active and adoptive responses, were used in these experiments. An application of the method in antibody feedback experiments is described.

Animals↗

PubMed1990

Feedback by early and late primary antisera on the primary and secondary adoptive immune responses of mice to burro erythrocytes.

Experiments on antibody feedback inhibition of the immune response have confirmed that control is more effective against a primary response than against a secondary response. The cells producing antibodies in primary and secondary responses are different both in terms of number of IgFC and amount of antibody produced by individual IgFC (plaque size). Late primary anti-burro RBC sera (greater than 200 days), despite low titers, are, on a volume for volume basis, feedback inhibitors at least as good as early (8-12 days) primary antisera on primary responses but are more effective in suppressing secondary responses (B memory cells). Late primary antisera, due to the process of affinity maturation, have a high affinity for antigen. The suppressive effect of early and late antisera is equally removable by absorption with burro erythrocytes: a result which it is thought, decreases the likelihood of feedback by anti-idiotype being involved in the observed suppression. It is suggested that feedback antibody acts (a) in competition with receptors, inter alia removing antigen into immunologically irrelevant pathways, (b) by a process involving the linking of antigen to Fc receptors, and (c) as a blocking antibody coating B cells (Bm) or APC which are already binding epitopes, thus preventing their cooperation with specific helper or other accessory cells.

Animals↗

PubMed1990

The influence of maternal humoral responsiveness on the specific immunocompetence of the progeny in mice.

The progeny of mice immunized during pregnancy with burro erythrocytes have an impaired ability to respond to that antigen. The suppression, which is shown to be of relatively short duration, lasting for between 8 and 12 weeks, is mediated by antibody transferred from mother to offspring via the milk, thus confirming the observations of previous workers. The experiments described in this paper show that passive immunization of the dams with "late" (greater than 200 days) low titre but high affinity antisera is as effective in feedback suppression as "early" (8-12 days) high titre but low affinity antisera. The high affinity antibodies in "late" antisera, like the low affinity antibodies of "early" antisera, are mainly transmitted via the milk and are apparently not, or are only minimally, transferred across the placenta.

Animals↗

PubMed1990

Establishment of functional T cells in SCID mice does not lead to termination of pregnancy.

Syngeneic (CB20) thymus and spleen T cells transferred to SCID mice, and shown to be present by flow cytometric analysis and functional by their ability to reject third-party skin grafts, do not lead to a detectable failure of syngeneic or allogeneic pregnancy. Unless the small number of B cells contaminating the T-cell innocula are capable of mounting a blocking antibody response, it is concluded that these results show that a humoral ('blocking antibody') response is not essential for successful pregnancy.

Animals↗

PubMed1989

Immunoglobulin-secreting cells in close proximity to the mid-gestation mouse embryo.

A reverse haemolytic plaque assay has been used to detect cells secreting IgM and IgG from areas in close proximity to mid-gestation mouse embryos. These cells were obtained from the fetal-placental unit and from the decidual mass of mice on the 9th or 10th day of syngeneic pregnancy. Cryohistology using affinity prepared goat antibodies specific for mouse Ig isotypes, coupled to horseradish peroxidase by a second antibody, showed that free Ig and cells containing Ig, are located closely but externally to the extra-embryonic membranes and also in areas close to the developing placenta in the decidua basalis. We suggest that some of the identified Ig-forming cells may be producing antibodies specific for antigens on the extra-embryonic membranes and may thus be acting as blocking (enhancing) antibodies.

Animals↗

PubMed1989

Offspring from inseminations with mammalian sperm stained with Hoechst 33342, either with or without flow cytometry.

The technique of vital staining with a fluorochrome and subsequent flow cytometry has been employed to investigate the DNA content of living sperm. There have been several reports of chromosomal damage caused by staining cells with a bis-benzimidazole dye. Hoechst 33342, either with or without subsequent flow cytometry. Therefore the use of this technique might be expected to affect adversely the fertilising capacity of inseminated sperm, or to result in the production of congenital deformities in the young. Insemination experiments to date have resulted in the production of more than 400 offspring in four species, including 5 successive generations of rabbits, where stained or sorted sperm were used. All progeny were observed to be normal by anatomical criteria. There was no evidence to suggest that sperm treated in this manner were unable to fertilise oocytes in vivo or that embryonic development was affected.

Animals↗

PubMed1988

Sexing of sperm by flow cytometry.

Economics dictate that livestock producers will be under increasing pressure to optimise output. A technique for sex pre-selection could help by reducing the number of females required to produce a given number of progeny of the desired sex; the technique would be particularly useful to the dairy industry. Live mammalian sperm, stained with a vital dye and analysed by flow cytometry, show a bimodal fluorescence distribution. Such bimodality may represent two overlapping subpopulations of X- and Y-chromosome bearing sperm. To test this hypothesis, sperm from the two subpopulations were separated using the sorting capacity of a flow cytometer and were used for the insemination of suitably prepared females. The sex of the resulting progeny was determined either by anatomical criteria or by identification of the sex chromosomes by karyotyping. Insufficient data are available so far to provide statistically significant evidence in support of the hypothesis, but a preliminary sequential analysis indicates a progressive tendency towards significance.

Animals↗

PubMed1988

Generation in vivo of non-T suppressor cells with the use of anti-allotype antibody.

Anti-Igh-1b antiserum induced allotype-specific suppression of adult mouse spleen cells in an adoptive transfer system. Suppression of Igh-1b anti-sheep red blood cell plaque-forming cells was measured as late as 4 wk after the injection of allotype heterozygous (Igha/b) spleen cells, antiserum, and sheep red blood cells. Suppression was maintained on retransfer of the allotype-suppressed spleen cells to further irradiated recipients in the absence of additional exogenous anti-allotype antibody. Mixing experiments were performed to test the putative inhibitory effects of allotype-suppressed spleen cells from the first adoptive transfer (stage I) on the antibody response of normal spleen cells in a second adoptive transfer (stage II). No suppression was observed by using unfractionated stage I spleen cells. In contrast, when these allotype-suppressed spleen cells were depleted of T cells, they strongly inhibited the antibody production of admixed normal spleen cells in stage II. This inhibitory activity of antibody-induced stage I spleen cells was directed primarily toward the target allotype, but some suppression of the Igh-1a plaque-forming cell response and total IgG production also occurred. Although removal of adherent cells did not affect the inhibitory activity of allotype-suppressed spleen cells from stage I, removal of Ig+ cells completely abrogated the inhibitory activity. These results suggest that antibody-induced regulatory B cells may play a role in maintaining long term allotype suppression.

Animals↗

PubMed1987

Limitations of a hemolytic plaque assay for IgG-anti-IgG rheumatoid factor-producing cells.

An attempt has been made to develop a hemolytic plaque assay capable of detecting homophile IgG rheumatoid factor (RF)-producing cells. Anti-immunoglobulin allotype-developing reagents were used to distinguish between target and effector IgG. The hemolytic assay has been used to demonstrate an apparently high level of homophile IgM and IgG RF-producing cells in the spleens and lymph nodes of mice stimulated by LPS. However, it appears that a large proportion of the plaques obtained in these assays are due to an artefact resulting from cross-linking of target and effector molecules by the developing reagents. In the case of IgM RF the artefact depends on the presence of a small contamination of the target IgG by IgM, allowing cross-linking of target and effector IgM by the anti-mu-specific developing reagent. With the IgG RF, cross-reactivity of the rabbit anti-Ighb allotype-developing serum for the 'wrong' (Igha) allotype, normally undetectable, becomes sufficient to be biologically relevant when the developing antibody is complexed by being bound to its target (Ighb) allotype. Nevertheless anti-allotype reagents may afford an accurate means of detecting homophile IgG RF producing cells using other assay systems.

Animals↗

PubMed1985

B-cell responses to male-specific antigen(s) in mice.

It has been found that B-cell responses to male-specific antigen(s) can be clearly demonstrated by reversed plaque assays. Female mice injected with syngeneic male spleen cells showed significant increases (greater than 100 X in some strains) in the number of immunoglobulin-secreting cells in lymph nodes draining the injection site. There was a variation in B-cell responsiveness between strains and this correlated only partially with previously reported T-cell responsiveness to the H-Y antigen. C57BL (H-2b) mice were among the most responsive, while CBA (H-2k), (CBA X C57BL)F1, and BALB/c (H-2d) were all much less responsive. These results apparently open up a new approach to the investigation of B-cell responses to male-specific antigen(s).

Animals↗