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Biomedical subjects

H D Moore

Publications and source records attributed to H D Moore.

At least 37 records · Page 2Linked to original sources

Parthenogenetic activation of marmoset (Callithrix jacchus) oocytes and the development of marmoset parthenogenones in vitro and in vivo.

Mammalian oocytes can be induced to resume meiosis without fertilization, and the resulting parthenogenetic embryos carry only maternal chromosomes. Human oocytes can be activated by many chemical and physical stimuli, but postimplantation studies of human parthenogenetic embryos are not ethically acceptable. The common marmoset monkey (Callithrix jacchus) is a good model for studying primate parthenogenetic development postimplantation, since follicular aspiration, embryo transfer, and early postimplantation development of biparental embryos have already been described. Marmoset oocytes were either subjected to two series of six electrical pulses (DC; 2 kV/cm and 70 microsec) or were incubated in 7% ethanol in PBS. Ninety-two percent (68 of 74) and 20% (8 of 40) of marmoset oocytes were activated by electrical stimulus or ethanol, respectively. Parthenogenetic (n = 3) or in vitro-fertilized (n = 2) embryos were transferred at the 4-cell stage to synchronized recipient female marmosets (n = 5). Progesterone, chorionic gonadotropin, and inhibin in the peripheral plasma of recipient animals were measured. After 33 days of gestation, recipient animals were perfused and the uteri were collected. The 2 females that had received biparental embryos and 2 of the 3 females that had received parthenogenetic embryos displayed biochemical and histological evidence of implantation. This is the first report that a primate embryo comprising only parthenogenetic cells is capable of implantation. This highlights the need to scrutinize levels of parthenogenesis associated with human assisted reproductive technologies. Marmoset parthenogenones also provide a unique model for elucidating the roles of parental genomes in primate development.

Animals↗

Sperm maturation in vitro: co-culture of spermatozoa and epididymal epithelium.

Sperm maturation involves an intimate interaction between spermatozoa and the epididymal epithelium. Aspects of this relationship can be examined by co-incubating epididymal spermatozoa with epididymal epithelium in vitro. Plaques of epididymal epithelium from a variety of species (for example rodents, dogs, humans) can be maintained in culture medium supplemented with growth factors and androgens. When co-incubated with these epithelial cultures, immature epididymal spermatozoa undergo maturation changes that lead to the acquisition of progressive motility, zona binding and, in some instances, fertilizing capacity in vitro. The use of such co-culture techniques for the understanding of sperm maturation in vitro and in vivo is reviewed with reference to recent experiments.

Animals↗

cDNA cloning and characterization of a rat spermatogenesis-associated protein RSP29.

RSP29, a protein secreted by rat round spermatids, stimulates the secretory function of Sertoli cells in the testis. By making use of the N-terminal sequence homology of RSP29 and a human protein hDP1 that we had previously isolated, we cloned the full length cDNA sequence that encodes RSP29. The entire amino acid sequence of RSP29 showed significant homology with that of hDP1, which was later identified as glyoxalase II. Southern analysis showed that the RSP29 protein sequence is highly conserved in eukaryotes and possibly in prokaryotes. The RSP29 mRNA is expressed in many tissues but has an extremely high abundance in testis. These data suggest that RSP29 may have an important function in most tissues of enkaryotic organisms. The high expression of RSP29 in testis and its stimulatory effects on Sertoli cells suggest that RSP29 could be especially important in the regulation of spermatogenesis.

Amino Acid Sequence↗

Immunocontraception in rodents: a review of the development of a sperm-based immunocontraceptive vaccine for the grey squirrel (Sciurus carolinensis).

The strategy for developing contraceptive vaccines for wild rodents will depend on the species. In rats and mice, high all-year birth rates, high levels of dispersal and promiscous mating systems suggest that, if immunocontraception was used alone, > 90% of the population would have to sterilized to achieve the desired control. In Britain, the grey squirrel (Sciurus carolinensis) may be a better candidate to investigate the feasibility of a contraceptive vaccine in rodents. This introduced species is a seasonal breeder with a much lower population turnover than rats or mice. As well as causing damage to woodland, it has ousted the native red squirrel (S. valgaris) from most of the UK. A human and selective method for the control of grey squirrels is therefore highly desirable. Numerous sperm-specific antigens have been identified on rodent spermatozoa. Monoclonal antibodies to particular components block sperm-egg interactions in laboratory animals and cross-react with grey squirrel spermatozoa. In vitro fertilization assays indicate that squirrel sperm-egg binding may be inhibited also. Currently, a cDNA library obtained from grey squirrel testis is being screened to identify genes encoding specific sperm antigens involved in fertilization. Methods of enhancing immunogenicity after oral immunization using microparticle carriers and immune-stimulating complexes are currently under investigation.

Administration, Oral↗

Prolonged survival of human spermatozoa when co-incubated with epididymal cell cultures.

Human epididymal tissue was recovered from 11 patients undergoing orchidectomy without anti-androgen treatment. Everted epithelial fragments from the caput and corpus epididymis of six patients were successfully cultured in a modified RPMI 1640 medium supplemented with HEPES and androgens for up to 110 days (mean 56 +/- 28) in 5% CO(2) in air at 37 degrees C. Epithelial cells from human oviduct and non-reproductive tract cells (breast epithelial cells, fibroblasts) were also cultured for comparison. The proportion of epididymal epithelial cells in primary cultures assessed by immunofluorescent localization using a cytokeratin monoclonal antibody was shown to be >70% for the first 6-8 weeks of culture. Light and electron microscopy indicated that epithelial cells maintained polarity and some normal morphology during the culture period. Washed epididymal or ejaculated spermatozoa prepared by a 'swim-up' procedure were co-incubated (i) directly with epididymal cells in culture wells, (ii) in 12 mm Millicell inserts within culture wells, thereby preventing contact of spermatozoa with culture cells; and (iii) in culture medium alone. A significant proportion of spermatozoa in direct contact with culture cells or in Millicell inserts were viable after 6 days of co-incubation (30-45%) and exhibited progressive motility, while all spermatozoa in medium alone were non-motile by 3 days. Using computer-assisted sperm analysis it was shown that the progressive motility of viable spermatozoa decreased gradually for the first 5 days in culture and then remained constant (approximately 30 microm/s, average path velocity). After 12 days of co-incubation, 15 +/- 4% of spermatozoa in direct contact with epithelial cells remained motile; in one experiment, a few spermatozoa (<1%) were motile at 17 days. Light and electron microscope observations indicated that prolonged sperm survival was associated with close apposition of spermatozoa (by equatorial segment) to the apical membrane of epithelial cells. Oviductal epithelial cells were also beneficial for sperm survival, but other cell types had no effect.

Adult↗

A novel anti-inflammatory drug, SDZ ASM 981, for the topical and oral treatment of skin diseases: in vivo pharmacology.

There is a need for safe and effective therapies for inflammatory skin diseases. Current topical and systemic treatment of psoriasis is effective but suffers from side-effects or is inconvenient. The therapeutic armamentarium for atopic dermatitis is very limited and far from satisfactory. In vivo preclinical data are presented for SDZ ASM 981, a novel ascomycin macrolactam derivative with high anti-inflammatory activity. Anti-inflammatory activity was observed in mouse, rat and pig models of allergic contact dermatitis. In the pig model, topical SDZ ASM 981 was as effective as the ultrapotent corticosteroid clobetasol-17-propionate, and when compared with a series of commercial topical corticosteroid preparations, 0.1% SDZ ASM 981 had equivalent efficacy to clobetasol-17-propionate (0.05%), the most potent product on the market. Unlike the corticosteroid, however, SDZ ASM 981 did not cause skin atrophy in pigs. SDZ ASM 981 potently inhibited allergic contact dermatitis in mice and rats when given systemically, and oral treatment was more effective than cyclosporin A in rats. Furthermore, SDZ ASM 981 has a low potential for affecting systemic immune responses, as demonstrated in rat models of localized graft vs. host reaction and allogeneic kidney transplantation. Preclinical results suggest that SDZ ASM 981 has the potential to be a well-tolerated and effective drug for topical as well as oral treatment of inflammatory skin diseases.

Administration, Cutaneous↗

Molecular mechanisms of gamete recognition and fusion at fertilization.

Advances in many areas of reproductive technology have been rapid and, in many respects, have outstripped our knowledge of the fundamental processes of human and animal sperm-egg interactions at fertilization. This is particularly true of human fertilization, where the availability of eggs for research purposes is severely restricted. As a consequence of this, most of the significant advances in our understanding of mammalian fertilization have resulted from studies on animals, particularly the mouse. This review summarizes our current knowledge of the molecular aspects of mammalian fertilization from the point of view of the fertilizing spermatozoon. Particular reference is made to those advances in our knowledge of human fertilization mechanisms. Further understanding of the molecular basis of human fertilization is of paramount importance for the development of new methods of contraception and also for the rational diagnosis and treatment of certain forms of infertility.

Acrosome↗

Endogenous D-type (HERV-K) related sequences are packaged into retroviral particles in the placenta and possess open reading frames for reverse transcriptase.

All primates studied to date produce retroviral-like particles in their placentae. We have purified these particles from two primate species, one Old World (human) and one New World (marmoset), and have identified the retroviral sequences which are packaged into these particles. Three families of sequences have been detected in these particles in human, all of which have the highest homology to B- and D-type retroviruses and to the human endogenous retrovirus HERV-K10. Previous studies have reported that the New World monkeys do not possess sequences with homology to HERV-K10. We have identified a new family of low-copy-number sequences which are present in New World monkeys and which possess 70% homology to the HERV-K family. Particles from both species possess reverse transcriptase activity and we have found that some of these retroviral particles package sequences which encode long open reading frames in pol, as revealed by expression cloning in Escherichia coli. These open reading frames could encode the reverse transcriptase enzyme activity found in the particles.

Animals↗

Gamete biology of the new world marsupial, the grey short-tailed opossum, Monodelphis domestica.

Mammalian gametes undergo a series of functional and developmental changes that culminate in fertilization. In order to appreciate the necessity for such complex processes as sperm maturation, capacitation and the intimate sperm-egg interactions leading to gamete fusion, it is important to understand how gametes may have evolved. In this respect, marsupials are particularly relevant since they exhibit features reminiscent of both non-mammalian vertebrates and eutherian mammals. The grey short-tailed opossum, Monodelphis domestica, is a New World marsupial from Brazil. It breeds well under laboratory conditions and is an excellent animal model to investigate marsupial gamete biology. As in other American marsupials, the spermatozoa of the opossum form pairs in the epididymis. Here, a number of studies carried out in this laboratory, related to sperm maturation, capacitation and fertilization in M. domestica, are reviewed and the gamete biology in this species is compared with what is known in other marsupials and eutherian mammals.

Acrosome↗

Biological activity of recombinant human ZP3 produced in vitro: potential for a sperm function test.

The human zona binding test is the most predictive test of sperm function yet the availability of human zona severely restricts its clinical use. The primary aim of this study was to use a commercially available in-vitro transcription and translation system to produce immobilized recombinant human ZP3 (rhuZP3) on agarose beads. The biological activity of this preparation was examined using sperm binding and the acrosome reaction. Significantly higher levels of sperm binding to rhuZP3 beads (n = 12, P < 0.05) compared with controls were observed and there was a significant induction (n = 12, P < 0.01) in the acrosome reaction after overnight incubation at 37 degrees C in 5% CO2 in air. In conclusion, the in-vitro transcription and translation system can produce sufficient quantities of purified immobilized biologically active rhuZP3. These preliminary experiments will enable further refinements to be made so that a solid-phase sperm function test based on rhuZP3 coated beads is likely to be developed in the near future.

Acrosome↗

Recombinant human zona pellucida glycoprotein 3 induces calcium influx and acrosome reaction in human spermatozoa.

Recombinant human ZP3 (rhuZP3) generated by Chinese hamster ovary cells transfected with a plasmid containing human ZP3 cDNA was used to study the acrosome reaction (AR) and intracellular calcium fluxes in capacitated human spermatozoa. Conditioned medium containing rhuZP3 significantly induced the AR (P < or = 0.005) in 59.4 +/- 4.7% of spermatozoa (control = 8.5 +/- 3.1%) and caused complete acrosomal loss in a further 17.2 +/- 3.8% of cells (control = 3.7 +/- 0.7%; mean +/- SEM, n = 5). Sperm motility was not affected and acrosomal exocytosis in response to rhuZP3 was also shown to be time-dependent. Basal concentrations of sperm intracellular calcium were measured (82 +/- 7 nM; mean +/- SEM, n = 9). A transient increase in intracellular calcium (typically up to 400-450 nM) occurred within 1 min of rhuZP3 addition and was followed by sustained lower values of calcium (200-400 nM). These responses were dependent on the amount of rhuZP3. This is the first report of zona protein-induced changes in intracellular calcium levels in human spermatozoa. The results support the premise that ZP3 is an agonist of the human sperm AR and that rhuZP3 generated in a eukaryotic cell is effective in this respect.

Acrosome↗

Direct assessment of cryopreservation of human spermatozoa using a cryomicroscope and computer-aided sperm analysis.

Use of a cryostage has enabled direct observation of human spermatozoa as they are cryopreserved and thawed. Crystallization and recrystallization events are readily observed. In combination with computer-aided semen analysis (CASA) equipment it was possible to determine the consequence of altering the cooling, freezing and thawing rates of a temperature-rate profile on sperm motility. Increasing the cooling rate to 50 degrees C/min resulted in significantly lower pre-freeze to post-thaw ratios for average path velocity (VAP, 13%), mean straight line velocity (VSL, 35%), mean linearity (LIN, 28%) and straightness (STR, 24%), while the ratio of the number of cells crossing the field of view (NCF) significantly increased (30%) compared to a standard freeze-thaw temperature rate profile. The NCF pre-freeze to post-thaw ratio was associated with the percentage of cell recovery after cryopreservation. Faster thaw rates resulted in better survival of the cells, perhaps due to the shorter time during which recrystallization occurred. The NCF ratios were significantly higher (33 and 30% for thaw rates of 50 and 100 degrees C/min respectively) than for the standard profile samples. Previous studies on cell survival have shown a link between the cooling and thaw rates. The cryostage should prove invaluable in future studies to identify the causes of cryodamage to spermatozoa. When used in combination with CASA, changes to sperm function during cryopreservation can be accurately measured.

Computers↗

In vitro maturation of mammalian spermatozoa.

During epididymal transit, mammalian spermatozoa undergo maturation and acquire full fertilizing capacity. The contribution of factors from the epididymal epithelium appears to be essential for this process. Although complete in vitro maturation of epididymal spermatozoa has not been achieved, stages of maturation can be induced under various conditions. The most successful results have been obtained by incubating epididymal spermatozoa with primary cultures of epididymal epithelium. These co-incubation methods promote sperm motility and the capacity of spermatozoa to bind to and fertilize oocytes, and extend the viability of spermatozoa in vitro. Specific androgen-dependent secretory proteins from epididymal principal cells that may be involved in this maturation process have been identified using pulse-labelling techniques.

Animals↗

Interaction of a tyrosine kinase from human sperm with the zona pellucida at fertilization.

A 95-kilodalton mouse sperm protein with characteristics of a protein tyrosine kinase has been identified as a receptor for ZP3, a glycoprotein in the egg's extracellular matrix. The structure of the human homolog was determined by screening an expression library from human testis; a testis-specific complementary DNA was isolated that encodes a protein similar to receptor tyrosine kinases and appears to be expressed only in testicular germ cells. Antibodies against a synthetic peptide from the intracellular domain recognized a 95-kilodalton human sperm protein that contains phosphotyrosine; human ZP3 stimulates the kinase activity of this sperm protein. Synthetic peptides corresponding to regions of the predicted extracellular domain inhibited sperm binding to human zona pellucida. Availability of the primary sequence of a receptor for ZP3 provides a rational starting point for sperm-targeted contraceptive development.

Amino Acid Sequence↗

Separation of round spermatids from the rat using an immunoselection panning technique.

A method was devised for the isolation of round spermatids from the rat using a positive immunoselection technique (panning). A testis suspension was prepared from adult rats by enzymatic digestion of seminiferous tubules with collagenase. Specific mouse monoclonal antibody (97.25) was indirectly attached to Petri dishes and used in a panning protocol to purify spermatids from the testis cell suspension. The quantity and purity of cells isolated were determined by cell counts and histochemical (periodic acid-Schiff stain) or by immunostaining with acrosome-specific antibodies. A mean yield of 1.38 +/- 0.15 x 10(7) cells per dish was obtained with a purity of more than 90%. The viability of the cells was confirmed by epifluorescent microscopy with propidium iodide/carboxyfluorescein acetate probes. Northern blot analysis of RNA extracted directly from the dish indicated good integrity of a spermatid-specific transcript of glyceraldehyde-3-phosphate dehydrogenase (GAPDH).

Animals↗

Sperm pairing in the opossum increases the efficiency of sperm movement in a viscous environment.

In order to understand why sperm pairing has evolved in most American marsupials, the movement parameters of spermatozoa from Monodelphis domestica were analyzed after incubation in capacitating medium for 15 min, 2 h, and 24 h to induce a proportion of sperm pairs to uncouple. Motility characteristics of paired and single spermatozoa were measured in media of differing composition and viscosity by means of computer-aided semen analysis. In minimum essential medium or in RPMI 1640 medium alone, the absolute mean straight-line and curvilinear velocity values of paired spermatozoa (342 +/- 34 and 361 +/- 19 microns/sec, respectively, at 37 degrees C) were significantly greater than those of single spermatozoa (247 +/- 14 and 319 +/- 16 microns/sec), while mean lateral head displacement for paired spermatozoa (5.6 +/- 2.1 microns) was significantly less than for single spermatozoa (11.4 +/- 2.6 microns). However, when medium was made more viscous with polyvinyl pyrrolidone (0.8-82 poise) and sperm motility was calculated as a percentage of maximum attained velocity (in medium alone), there was no significant difference in straight-line or curvilinear velocity for single or paired spermatozoa in medium of the lowest viscosity (0.8 poise). In contrast, paired spermatozoa in medium of higher viscosity (above 1.92 poise) maintained straight-line velocity (e.g., 54 +/- 3% of maximum straight-line velocity in medium of 2.28 poise) while single sperm moved in tight circles and exhibited poor straight-line velocity (5 +/- 1% of maximum velocity). The data show that paired spermatozoa exhibit a significant motility advantage over single spermatozoa in a viscous medium.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

A strategy for identifying candidate sperm antigens for immunocontraception: isolation of human testis cDNA clones using polyclonal antisera directed against hamster acrosomal membrane preparation.

Components of the mammalian sperm acrosome that have been conserved during evolution are probably essential for fertilization and are therefore potential antigens for the development of an immunocontraceptive vaccine. In order to identify such protein components, a series of specific polyclonal antisera were generated by immunizing rabbits with purified acrosomal membrane fractions from hamster epididymal spermatozoa. Antisera were finally selected using immunological and in-vitro fertilization assays, and used to then screen a human testis lambda gt11 cDNA library. As a result of this screening over 70 clones were identified, selected and purified. The cDNAs were amplified by polymerase chain reaction (PCR) and the inserts characterized by restriction enzyme digestion and oligonucleotide probing techniques. The functional activity beta-galactosidase fusion proteins expressed by these clones (HA5-2, HA6-2 and HB4-1) inhibited significantly fertilization and reduced spermatozoa binding compared to controls. To date, sequence data has been obtained from HB4-1 (1.75 kb). The first 1132 nucleotides displayed > 96% homology to human testis-specific lactate dehydrogenase (LDH-C4) gene, the product of which is a known candidate antigen for a contraceptive vaccine. This finding suggests that a strategy involving the screening across species for conserved moieties of the mammalian acrosome may be useful for identifying candidate antigens for immunocontraception.

Acrosome↗

Why do spermatozoa of American marsupials form pairs? A clue from the analysis of sperm-pairing in the epididymis of the grey short-tailed opossum, Monodelphis domestica.

In order to understand the evolutionary significance of sperm-pairing in American marsupials, an ultrastructural investigation was made of this process in the South American grey short-tailed opossum, Monodelphis domestica. One epididymis from each animal (5) was fixed for light and electron microscopy and divided into 18 segments. The contralateral tract was divided into similar segments and assessments made of the total number of spermatozoa and the proportion of sperm-pairs. The mean total sperm number was 4.20 +/- 0.62 x 10(6)/epididymis. Sperm-pairing commenced around segment 9 in the proximal corpus epididymidis and reached a maximum of 80% in the caudal sperm storage region of the duct. The sperm-pairing process was characterised by four stages. Spermatozoa exhibited parallel alignment as indicated by the positioning of identical cross-sections of sperm heads. This was followed by close apposition with acrosomal faces parallel rather than opposite. Rotation of the sperm heads around each other then apparently occurred as indicated by the morphological alignment of sections of paired sperm heads. Sperm-pairing was complete when the acrosomal faces were precisely aligned and joined. Misalignment and failure to pair was observed in about 20% of spermatozoa in the cauda epididymis. Such a complex sperm-pairing process may ensure that conjugated spermatozoa are precisely aligned so that flagella movement can be accurately coordinated for maximal progressive motility.

Acrosome↗