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T Moore

Genomic imprinting is the process that differentially modifies the parental alleles at certain genetic loci in the parental germlines. Such modifications of DNA and chromatin are somatically heritable and cause unequal expression of the parental alleles during subsequent development. In mammals, imprinted genes encode a relatively small number of functionally heterogeneous proteins. Nevertheless, imprinted genes exert important effects, primarily on fetal development, and their deregulation is implicated in a variety of pathologies including sporadic, inherited and induced growth disorders. Imprinted loci show several unusual structural and functional characteristics that may be related to mechanistic aspects of mono-allelic expression or to modes of evolution of imprinted genetic loci. Typically, imprinted genes are clustered in certain genomic regions and have relatively reduced intronic DNA content relative to non-imprinted genes. In addition, their regulatory regions frequently contain a combination of features including tandem repeats associated with differentially methylated CpG islands and overlapping transcription of coding or non-coding RNAs. The evolution of imprinting can be understood as the stable outcome of sexual selection acting differently on the parental alleles of genes that influence parental investment in offspring. Consistent with this explanation, imprinted genes are expressed predominantly during embryonic and postnatal development in mammals and in the developing endosperm of plants, and maternal or paternal expression at imprinted loci is associated with reduced or increased parental investment, respectively. Such selective forces have implications for understanding mechanistic aspects of genome reprogramming in the early mammalian embryo.

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T Moore and W Reik

Parental (genomic) imprinting is the process by which the differential expression of maternal and paternal alleles at certain genetic loci in mammalian embryos occurs. Such loci are implicated in the control of fetal, placental and neonatal growth, and, more generally, in diverse aspects of fetal nutrient acquisition and maternal-fetal interactions. Not surprisingly, the aberrant expression of imprinted genes is implicated in a range of embryonic and fetal abnormalities. We outline how an evolutionary theory, based on classic parent-offspring conflict theory, relates to certain fetal growth abnormalities. In particular, we suggest that growth abnormalities resulting from the manipulation of preimplantation mammalian embryos in vitro (for example large calf syndrome) may reflect the occurrence of genetic conflict over the fetal growth programme in the early preimplantation period.

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H. D. M. Moore and T. D. Hartman

Summary. The epididymides of adult male hamsters were surgically ligated at the junction of the distal corpus and proximal cauda regions. After 3 days, spermatozoa recovered from the distal corpus displayed greater progressive motility and head to head agglutination in capacitating medium than did those from intact controls, but had low fertilizing ability (3% fertilization rate) in vitro or in vivo. When these spermatozoa were incubated for 6 h with epithelial cells from the proximal cauda epididymidis, previously cultured for 3 days, they maintained motility and exhibited a significant increase in fertilizing ability (30% and 29% in vitro and in vivo respectively). The fertilizing ability of distal corpus spermatozoa incubated with 3-day-old cultures without androgens, or 8–12-day-old epithelial cells with fibroblast overgrowth, or without epithelial cells, remained low (5%). Increase in sperm fertilizing ability was associated with increased sperm binding to the zona pellucida in vitro.

These results demonstrate that, under suitable culture conditions, the final stages in the development of hamster sperm fertilizing ability can be achieved in vitro. Factors secreted by cultured epithelium from the proximal cauda epididymidis are implicated in this maturation process.

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H. D. M. Moore and T. D. Hartman

Summary. To determine the importance during fertilization of various plasma membrane components of the hamster spermatozoon, monoclonal antibodies were generated in the mouse against specific sperm surface antigens. BALB/C mice were immunized with washed hamster spermatozoa from the cauda epididymidis and immune splenocytes fused with myeloma cells (P3 × 63 Ag8). The sperm-specific immunoglobulins were detected in hybridoma cultures by a solid-phase assay (ELISA). Five monoclonal antibodies bound specifically to the surface of intact hamster spermatozoa, three immunoglobulins to restricted regions of the head and tail plasmalemma as detected by immunofluorescence. In two cases, the affinity of the membrane antigen was modified during passage through the epididymis. Monoclonal antibodies to the sperm head or to the head and tail inhibited fertilization in vitro by blocking sperm attachment to the zona pellucida and the oolemma.

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N. P. Moore, T. J. B. Gray and J. A. Timbrell

The testis synthesizes creatine from both arginine and glycine precursors, but when rat testicular tissue is separated into seminiferous tubules and interstitial cells, creatine synthesis occurs only in the tubular fraction. The purpose of the work presented here was to define the locus of creatine synthesis within the seminiferous tubules, by using cell separation and culture techniques to examine synthesis in the Sertoli cells and germ cells. The total creatine content, in the cellular compartment and incubation medium, of Sertoli–germ cell co-cultures and of Sertoli cell-enriched cultures, largely free of germ cells, increased by similar amounts over a 24 h incubation period. Sertoli cell-enriched cultures incorporated radioactivity from l-[guanidino-14C]arginine and [1-14C]glycine into both creatine and its biosynthetic precursor, guanidinoacetic acid. Isolated germ cells did not incorporate radioactivity from l-[guanidino-14C]arginine into either creatine or guanidinoacetic acid when incubated at a similar density and protein concentration under similar conditions. It is concluded that the synthesis of creatine osberved in isolated rat seminiferous tubules occurs within the Sertoli cells and not the germ cells.

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C. A. Smith, T. D. Hartman and H. D. M. Moore

Summary. A murine monoclonal antibody raised against hamster cauda epididymal spermatozoa was shown to recognize an M r 34 000 component of epididymal epithelium. Antigen was localized by immunocytochemistry on the surface and in the apical cytoplasm of principal cells in the proximal corpus epididymidis but not in the caput or initial segment regions. Spermatozoa from the corpus epididymidis expressed antigen on their post-acrosomal plasma membrane and annulus.

Epididymal principal cells from the proximal corpus region when cultured in vitro bound antibody on their apical surface for at least 5 days. Spermatozoa from the caput epididymidis co-cultured with epithelium expressed antigen after incubation for 8 and 24 h. These results suggest that a surface change to epididymal spermatozoa during maturation in vivo may also be elicited during in-vitro culture.

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N. P. Moore, T. J. B. Gray and J. A. Timbrell

The Sertoli cells have been identified as the primary locus for creatine synthesis within the seminiferous epithelium. The purpose of the studies reported here was to examine the effect of modulators of Sertoli cell function on creatine secretion by primary cultures of these cells. Sertoli cell-enriched cultures, maintained in a defined medium, secreted creatine into the incubation medium in a manner that was linear with time over at least 6 h, but which had reached a plateau within 24 h. Secretion was stimulated by physiological and toxicological modulators of Sertoli cell function. Incubation of Sertoli cell-enriched cultures in the presence of FSH (≥40 mU ml−1), dibutyryl cyclic AMP (≥0.1 mmol l−1), mono-(2-ethylhexyl) phthalate (≥ 1 μmol l−1) or cadmium (≥3 μmol l−1) increased the secretion of creatine into the incubation medium by at least 85% over 24 h. Creatine secretion by Sertoli cell-enriched cultures, incubated over 4 h in a balanced salt solution, was independent of exogenous l-glutamine. However, the stimulation of secretion induced by 1 mmol dibutyryl cyclic AMP l−1 was dependent on the presence of 4 mmol l-glutamine l−1 in the incubation medium, which suggests that an increase in creatine secretion occurs as a consequence of stimulated glutamine oxidation.

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Dianne Moore Smith, C. H. Conaway and W. T. Kerber

Summary. Oocytes obtained from antral follicles of adult and adolescent rhesus monkeys during the annual breeding season extruded polar bodies in vitro at significantly higher rates (50–60%) than oocytes from animals of similar age during the non-breeding season (20–30%) or from infant and prepubertal females at any time of the year (20–30%). The proportion of oocytes degenerating in culture was greatest in groups where maturation was highest.

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H. D. M. Moore, T. D. Hartman and C. A. Smith

Summary. Epithelium from the proximal corpus region of the epididymis of adult hamsters was cultured in modified RPMI 1640 medium supplemented with growth factors and androgens at 37°C in 5% CO2 in air. Prepared plaques of epithelium formed spheres of tissue with epithelial cells outermost. At the light and electron microscope level, these epithelial balls displayed morphology consistent with continued secretory and absorptive function. After 3–5 days, cultured cells either plated out over the bottom of plastic wells or formed vesicles which expanded as their interior became fluid filled.

Spermatozoa recovered from the caput epididymidis were co-cultured with epithelium. After 8 and 24 h, a proportion of spermatozoa (30%) exhibited slow but persistent flagellum beats with slow progressive motility. Spermatozoa in control incubations were immotile. This change in motility pattern would suggest that some sperm maturation processes had occurred in vitro.

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D. Whyman, D. L. Johnson, T. W. Knight and R. W. Moore

Summary. The timing of ovulations in 42 PMSG-treated ewes was determined by repeated endoscopy. The first ovulation occurred at a median time of 23·6 ± 0·5 (s.e.m) h after the onset of oestrus. The median interval between first and second ovulations was less than 1 h, and that between first and last ovulations was approximately 6 h.

In 59 untreated ewes, probit regression analysis was applied to the number of ovulations which were found by endoscopy to have occurred by 23, 25 and 27 h after the onset of oestrus. The median time of first ovulation was 25·5 ± 0·5 h after the onset of oestrus, this interval being similar in single- and twin-ovulating ewes. The median interval between twin ovulations was 1·2 ± 0·6 h. Ovulation occurred after the end of oestrus in at least 75% of ewes.