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Biomedical subjects

A A Kiessling

Publications and source records attributed to A A Kiessling.

At least 19 recordsLinked to original sources
PubMed2000

Interaction of gametes with exogenous genes: possible opportunities for incorporation into embryonic genome.

The mechanism of incorporation of foreign DNA into newly fertilized eggs is poorly understood. It is not known with certainty if S phase DNA replication is required or if integration could occur at other times of the cell cycle that involve DNA strand breaks, such as chromatin rearrangements. We have investigated DNA strand breaks in mouse eggs and zygotes with a sensitive terminal uridine nucleotide end labeling (TUNEL) assay. Greater than 90% of all polar bodies and metaphase II chromosomes in freshly ovulated mouse eggs are TUNEL-assay positive. Approximately one-third of zygotes assayed 6 hr after fertilization contain at least one TUNEL assay positive pro-nucleus and/or decondensing sperm head. These results indicate that early embryonic DNA contains multiple transient DNA breaks that could play a role in the incorporation of foreign DNA.

Animals↗
PubMed2000

Multiple drug resistance mutations in human immunodeficiency virus in semen but not blood of a man on antiretroviral therapy.

The concept that the male reproductive tract harbors isolated reservoirs of human immunodeficiency virus (HIV) infection has now been widely accepted. The significance of semen viral burden to sexual transmission of HIV is obvious; however, its contribution to disease progression is unknown. We report a case study that demonstrates the emergence of resistance-conferring mutations to antiviral therapy in infected seminal leukocytes from a man with asymptomatic prostatitis associated with leukospermia. This finding demonstrates the potential importance of male reproductive tract organs to the development of therapy resistance in HIV-infected men.

Anti-HIV Agents↗
PubMed1999

Prostate specific antigen is detectable in formalin-fixed semen.

OBJECTIVE: To conduct a pilot study to test whether or not prostate specific antigen (PSA) and/or PSA-positive cells can be detected and characterized in semen specimens archived in aldehyde fixative. MATERIALS AND METHODS: Specimens from 12 men were examined, six before elective vasectomy and six undergoing infertility evaluation. Fixed semen elements were assessed immunocytologically using monoclonal antibodies against PSA and leucocyte common antigen, CD 45, as a control. RESULTS: PSA was detectable in the semen from the fertile men as amorphous protein and contained within round vesicles (prostasomes). Semen from men undergoing infertility evaluation contained a greater variation in detectable forms and amount of PSA than the specimens from the fertile men, including PSA associated with some nonspermatozoal cells (NSCs). CONCLUSION: PSA is detectable by immunocytological analysis of semen specimens archived in aldehyde-based fixative. Three forms of PSA were detected; within round vesicles characteristic of prostasomes, associated with some NSCs, and as amorphous protein. The detected variations suggest that analysis of PSA-positive semen elements may provide important insights into prostate physiology.

Fixatives↗
PubMed1998

Sperm-mediated gene transfer in mice.

Sperm-mediated DNA transfer to offspring has the potential to markedly simplify the generation of transgenic animals, but the efficiency in mice has been controversial. To determine the basis of the variability of the procedure in mice, we undertook a large, collaborative study of sperm-mediated DNA transfer to mouse eggs in well-established laboratory conditions for in vitro fertilization and offspring development following embryo transfer. Sperm were incubated with plasmid DNA during the capacitation period and then added to freshly ovulated mouse oocytes for fertilization; cleaved embryos were then transferred to the oviducts of pseudopregnant recipients for gestation. From a total of 75 experiments, 13 produced 130 transgenic offspring, amounting to 7.4% of total fetuses. In five experiments, more than 85% of offspring were transgenic, but the factors leading to this high success rate were not discovered. Clustering of such a low frequency event could account for the disparate reports of transgenic success with sperm-mediated DNA transfer to mouse offspring. Discovering the factors important to success would not only allow this simplified approach to become an important tool in the generation of transgenic mice, but could also lead to important insights into natural protective mechanisms against sperm-mediated transfer of foreign DNA.

Animals↗
PubMed1998

Expression of human immunodeficiency virus long terminal repeat-coupled genes in early cleaving embryos.

Early studies suggested that exogenous retroviral genes are not expressed by mouse embryos infected at early cleavage stages. The general view has therefore been that, since human eggs are non-dividing cells, they could not be infected with HIV before fertilization, nor would HIV be expressed if infection occurred during early cleavage stages following fertilization. In contrast with this view, more recent work has shown that genes coupled to the long terminal repeat (LTR) promotor elements in Rous Sarcoma Virus are readily expressed in early cleaving mouse embryos; in addition, pHIV-LTR, a plasmid construct with HIV-LTR coupled to the reporter gene, lacZ (which encodes beta-galactosidase), is expressed when transfected into a human embryonic carcinoma cell line. To determine if HIV LTR coupled genes would be expressed in early cleaving embryos, one nucleus of mouse zygotes were microinjected with approximately 5000 copies of the pHIV-LTR plasmid. Following microinjection, zygotes were cultured for 48 h to the four cell stage and stained for expression of beta-galactosidase. A plasmid construct containing lacZ without HIV LTR was microinjected as control. A total of 25 of the 111 mouse zygotes microinjected with pHIV-LTR stained positively for beta-galactosidase activity. Blastomeres within individual embryos were not uniformly stained, suggesting a mosaic pattern of distribution of the microinjected plasmids among the cleaving blastomeres. None of the 22 mouse zygotes microinjected with control plasmid stained positively. A total of six polypronuclear human eggs were obtained as discarded by-products of a human IVF program. All four human eggs microinjected with approximately 6000 copies of HIV-LTR reporter gene construct and cultured for 48 h stained highly positive for beta-galactasidase. Staining intensity varied among blastomeres, suggesting a mosaic pattern similar to the mouse embryos. The two human eggs microinjected with control plasmid were negative for beta-galactosidase activity. These results suggest that HIV-LTR is active in both early cleaving mouse embryos and in early cleaving polypronuclear human eggs. These findings indicate that human eggs infected at the time of fertilization with HIV could express viral proteins during early embryonic development.

Animals↗
PubMed1998

Analysis of human immunodeficiency virus in semen: indications of a genetically distinct virus reservoir.

It is well established that HIV is found in semen, either as cell-free or cell associated virus, yet many questions remain about the source of the virus. A number of factors, including anatomic features of the male reproductive tract, the restricted access of the immune system to the germ cell compartment, and the results from sexually transmitted virus studies, suggest that the source of HIV in semen may be different from that in the peripheral blood. In this study, we examine the HIV in the infected cells of semen as indicators of the virus producing reservoir. The frequency of HIV positive leukocytes in semen is compared to that of concurrent blood samples from eight donors and these values are found to be highly variable and frequently discordant. The protease gene sequences of HIV strains isolated from semen cells and blood cells were determined and phylogenetic analyses were performed which indicate the virus populations in the two sources are genetically distinct. In one patient receiving anti-HIV protease inhibitor therapy, gene sequences indicative of protease inhibitor resistance were found in the blood, but not the semen cell compartment. These results suggest that HIV in the semen and blood compartments are distinct, and further, may respond differently to antiviral therapy.

Amino Acid Sequence↗
PubMed1998

Human immunodeficiency virus in semen arises from a genetically distinct virus reservoir.

The reservoir of human immunodeficiency virus (HIV) in semen is unknown. Several lines of evidence suggest that semen HIV may not arise from the same reservoir of infection as peripheral blood. If true, the viral burden in the two compartments could be qualitatively and quantitatively different, a scenario of potentially profound significance for the design of effective strategies of treatment, disease monitoring, and infection containment. We report here that the ratio of infected to uninfected leukocytes in ejaculated semen specimens is highly discordant with paired blood samples, demonstrating that they derive from distinct populations of infected cells. In addition, infectious HIV was isolated from semen cells, but not from blood cells, of an individual on triple antiretroviral therapy; the absence of major resistance-conferring mutations in the semen virus indicates that it was replicating in isolation from the antiviral agents. The compartmentalization of blood and semen infection was further supported by genetic analysis of several infectious HIV clones isolated from semen cells and peripheral blood cells of another donor not on antiretroviral therapy. Protease gene sequence analyses revealed significant divergence of the two viral populations. These findings confirm the distinct compartmentalization of HIV in the semen of this study cohort, and support the concept that semen HIV arises from an isolated reservoir of infection that may function independently in the pathobiology of HIV disease.

Amino Acid Sequence↗
PubMed1997

Preimplantation development of mouse and human embryos biopsied at cleavage stages using a modified displacement technique.

A modified embryo biopsy method was tested on four- and eight-cell stage mouse embryos and used on human embryos to obtain blastomeres for preimplantation genetic diagnosis. The biopsy method tested combines zona drilling and fluid displacement to force one or two cells through an opening in the zona pellucida of the cleavage-stage embryo. Rates of cell division and the percentage of mouse embryos forming blastocysts following biopsy at the eight-cell stage were not significantly different from those observed in unoperated control embryos. The percentage blastocyst formation was not significantly different in embryos biopsied at the four-cell stage and in control embryos, although cell division was significantly retarded following biopsy. 96% of the mouse blastomeres isolated at the eight-cell stage were recovered intact and 96% of those placed in culture underwent cell division. Survival and division of cells isolated at the four-cell stage were 92 and 84% respectively. Most of the cultured blastomeres cleaved several times and formed small trophoblast vesicles. Chromosomes were observed in 59% of blastomeres incubated in the presence of colcemid. In the initial use of this biopsy technique for human preimplantation genetic diagnosis, blastocyst formation was observed in 9 of 13 human embryos biopsied at the 7- to 10-cell stage. These findings support the use of this biopsy method as an alternative to aspiration techniques.

Animals↗
PubMed1995

The developmental potential of mouse oocytes matured in serum-free culture conditions.

Studies were undertaken to identify serum-free conditions for the maturation of mouse oocytes in vitro. Oocytes were recovered from the antral follicles of juvenile mice 48 h after injection with gonadotrophin and allowed to resume meiosis in modified Ham's F-10 (mHF-10) medium unsupplemented or supplemented with bovine serum albumin (BSA), fetal calf serum, human pre-ovulatory serum, human follicular fluid or EDTA. They were inseminated 14-16 h later, scored for polar body extrusion after 4-6 h with spermatozoa, and transferred to protein-free mHF-10 for further development. In-vivo matured ova were inseminated and cultured in parallel as controls. Fertilization and development were scored as two cells 24 h after insemination and blastocysts 4 days following insemination respectively. Surprisingly, 41% of oocytes cultured in unsupplemented mHF-10 completed meiosis I, and of those, 50% fertilized; serum supplementation did not improve maturation or fertilization rates. Although the addition of human follicular fluid to the mHF-10 improved meiosis (69%) and fertilization (68% of eggs with polar bodies) to levels comparable with the in-vivo control eggs (79 and 66% respectively), BSA supplementation was equally beneficial. Blastocyst development varied, but within each maturation/fertilization group, the development from in-vitro matured eggs was comparable with embryos from in-vivo matured eggs. In addition, two out of eight 4-cell embryos from oocytes cultured in mHF-10 with BSA and EDTA gave rise to apparently normal pups following transfer to pseudopregnant recipients.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1995

A preliminary study of the effect of growth hormone on mouse preimplantation embryo development in vitro.

The role of growth hormone (GH) in follicular development, ovulation and embryo development is currently under reconsideration. In this study, we have tried to investigate the effect of GH on preimplantation development of mouse embryos in vitro. Zygotes and two-cell mouse embryos were cultured without (control) or with GH. For zygotes, the addition of 0.2 micrograms/ml of GH resulted in 77 +/- 1% of blastocysts formed and 66 +/- 3% rate of hatching (control 64 +/- 4 and 31 +/- 3%, p < 0.05 and p < 0.01, respectively). For two-cell embryos, the addition of 0.2 micrograms/ml of GH resulted in 87 +/- 2% of blastocysts formed and 60 +/- 4% hatching rate (control 76 +/- 4 and 47 +/- 5%, p < 0.05 for both). This positive effect of GH addition implies that the latter can support mouse preimplantation development in vitro and it suggests, along with its local action on the ovary and its possible effects, via the insulin-like growth factor system, on the tubal and uterine epithelium, a continuous role of this hormone in reproductive physiology from follicular maturation to embryonic development and, possibly, implantation.

Animals↗
PubMed1995

Semen leukocytes: friends or foes?

OBJECTIVE: To test the hypothesis that male reproductive tract leukocytes function in the elimination of abnormal spermatozoa from ejaculated semen. DESIGN: Semen specimens with > or = 2 x 10(6) nonspermatozoal cells/mL were examined for leukocytes and for mature sperm with ideal morphology. SETTING: Andrology laboratory of a Center of Assisted Reproductive Technology. RESULTS: Semen specimens with elevated concentrations of leukocytes contained a significantly higher frequency of sperm with ideal morphology than semen specimens with elevated numbers of immature germ cells and low numbers of leukocytes. CONCLUSIONS: The direct correlation between leukocyte density and sperm with ideal morphology supports the concept that sperm surveillance is a normal function of male reproductive tract leukocytes. Understanding such germ cell-leukocyte interactions may provide valuable new insights into immunologic control mechanisms in male reproductive tract tissues.

Adult↗
PubMed1994

The effect of compounds altering the cAMP level on reversing the 2-cell block induced by hypoxanthine in mouse embryos in vitro.

The possibility of reversing the hypoxanthine induced 2-cell block in mouse embryos when cultured in conditions supplemented with compounds that increase (FSH, hMG, IBMX, hCG) or inhibit (GnRH-analogue) cAMP was assessed. When embryos were cultured in Ham's F-10 without hypoxanthine supplemented with each of the above compounds, no inhibition of blastocyst development was observed. Embryos were then cultured in Ham's F-10 with hypoxanthine supplemented again with each compound. For the addition of GnRH-analogue or FSH, the rate of blastocyst formation was comparable with that of the control medium with hypoxanthine alone. Instead, the addition of IBMX or hMG reversed the induced block. There was no reversible effect for the addition of 2 micrograms/ml hCG while the latter was observed with higher doses. The results from GnRH-analogue and IBMX addition show that, contrary to what was found for oocytes, stimulation of cAMP reverses the hypoxanthine-induced block in mouse embryos. FSH and hCG also had effects opposite to those observed for oocytes. It is unknown why hMG (FSH + LH) reverses the block. A lower cAMP degradation rate resulting in a higher cAMP level is a possible explanation. Our results provide further evidence that cleavage arrest by hypoxanthine has a different mechanism than the hypoxanthine-induced arrest of meiosis.

1-Methyl-3-isobutylxanthine↗
PubMed1994

The Ras/Raf signaling pathway is required for progression of mouse embryos through the two-cell stage.

We have used microinjection of antisense oligonucleotides, monoclonal antibody, and the dominant negative Ras N-17 mutant to interfere with Ras expression and function in mouse oocytes and early embryos. Microinjection of either ras antisense oligonucleotides or anti-Ras monoclonal antibody Y13-259 did not affect normal progression of oocytes through meiosis and arrest at metaphase II. However, microinjection of fertilized eggs with constructs expressing Ras N-17 inhibited subsequent development through the two-cell stage. The inhibitory effect of Ras N-17 was overcome by simultaneous injection of a plasmid expressing an active raf oncogene, indicating that it resulted from interference with the Ras/Raf signaling pathway. In contrast to the inhibition of two-cell embryo development resulting from microinjection of pronuclear stage eggs, microinjection of late two-cell embryos with Ras N-17 expression constructs did not affect subsequent cleavages and development to morulae and blastocysts. It thus appears that the Ras/Raf signaling pathway, presumably activated by autocrine growth factor stimulation, is specifically required at the two-cell stage, which is the time of transition between maternal and embryonic gene expression in mouse embryos.

Animals↗
PubMed1994

Expression and potential function of the c-mos proto-oncogene in human eggs.

OBJECTIVE: To investigate the expression and possible function of the c-mos proto-oncogene in human eggs. DESIGN: Eggs obtained as discarded material from assisted reproductive technology procedures were analyzed for c-mos messenger RNA by reverse transcriptase-polymerase chain reaction. As an approach to investigating c-mos function, we measured maturation-promoting factor (MPF) activity (histone H1 kinase) in eggs without and with inhibition of protein synthesis. Detection of RNA transcripts of c-raf was included as control. RESULTS: Transcripts of c-mos were detected in small fractions of individual eggs, indicating that c-mos is abundantly transcribed. Inhibition of protein synthesis resulted in loss of MPF, leading to chromatin decondensation and reformation of a nucleus. C-raf maternal messages were also detectable in individual human eggs. CONCLUSION: The c-mos proto-oncogene is an abundant maternal message in human eggs as in other species. The effects of inhibiting protein synthesis in human eggs are similar to those obtained in mouse and Xenopus eggs, either as a consequence of protein synthesis inhibition or specific ablation of c-mos RNA by injection of anti-sense oligonucleotides. The c-mos gene product is thus likely to play a critical role in human oocyte meiosis by regulating the activity of MPF.

Base Sequence↗