Search PubMed⌕ Search

Biomedical subjects

H D Moore

Publications and source records attributed to H D Moore.

At least 55 records · Page 3Linked to original sources
PubMed1993

Ultrastructural characteristics of in vivo and in vitro fertilization in the grey short-tailed opossum, Monodelphis domestica.

To establish the mode of fertilization in a marsupial, a morphological investigation was made of the gametes of the South American grey short-tailed opossum. Monodelphis domestica, at the time of fertilization in vivo and in vitro. Oestrus was induced in females by the introduction of an unfamiliar male. To obtain oocytes recently fertilized in vivo, females were killed 18-24 hours after the first mating and the region of the oviduct containing eggs excised and fixed. Unfertilized mature oocytes were recovered from ovarian follicles 15-18 hours after first mating and fertilized in vitro with cauda epididymal spermatozoa in a modified MEM medium supplemented with bovine serum albumin at 37 degrees C in 5% CO2 in air. Following sperm-egg binding and fertilization, oocytes were fixed and prepared for light and electron microscopy. Spermatozoa unpaired prior to fertilization in vivo and in vitro and single spermatozoa bound to the zona surface by their plasmalemma overlying the acrosome on the dorsal face of the sperm head. The acrosome reaction was only observed at the zona surface (suggesting that it may be induced by zona components) and involved a vesiculation of sperm plasma and acrosomal membranes over the main body of the acrosome but not over the narrow, marginal region which persisted after the acrosome reaction was complete. Sperm penetration of the zona pellucida caused a large breach in the zona and the dispersal of perivitelline material. The fusion of the spermatozoon with the oolemma occurred first over the marginal acrosomal region and was accompanied by a fertilization cone which protruded through the zona penetration hole. Activation of the egg was characterized by the release of material from vesicles in the peripheral cytoplasm and extrusion of the second polar body. The mode of fertilization in Monodelphis was compared with what is known in other marsupials (New World and Australian) and eutherian (placental) mammals. It was concluded that the general features of the acrosome reaction and sperm-egg fusion may be essentially similar in both groups and that an evolutionary schism did not occur following the development of the eutherian mode of fertilization.

Acrosome↗
PubMed1993

Human sperm-egg binding is inhibited by peptides corresponding to core region of an acrosomal serine protease inhibitor.

An antiserum, designated R4 and raised against denatured hamster acrosomes, was shown to localize specifically to the acrosomal region of hamster, rat, mouse, and human spermatozoa, and to inhibit both hamster and human sperm-oocyte binding in vitro. Following screening of a human testis lambda gt11 cDNA expression library with the antiserum R4, a series of cDNA clones were isolated. One (cDNA 134) was selected based on the ability of the beta-galactosidase fusion protein to inhibit human and hamster sperm-zona binding in vitro. The fusion protein was also shown to inhibit the penetration of zona-free hamster oocytes by human spermatozoa. Sequence analysis revealed that cDNA 134 coded for a portion of a serine protease inhibitor (serpin) closely related to plasma Protein C inhibitor. Sequencing of an additional cDNA clone (261) and Northern blot analysis confirmed that a Protein C inhibitor-like mRNA is synthesised in the human testis. Affinity-purified anti-134 antibody specifically localized to the acrosomal region of both hamster and human sperm. Synthetic peptides corresponding to the conserved core region responsible for the interaction of the serpin with its cognate protease also blocked human sperm-zona binding in vitro. The results suggest that this acrosomally located inhibitor plays an important role in the series of binding events that results in human fertilization.

Acrosome↗
PubMed1993

In vitro fertilization and embryo development in the marmoset monkey (Callithrix jacchus).

Oocytes aspirated from preovulatory (i.e. > or = 2 mm) follicles of marmoset monkeys were graded for maturity according to the degree of cumulus expansion, grade I being most mature and grade IV least mature. After preincubation for 2-5, 9-11 or 21-29 h, 82% of oocytes could be fertilized using epididymal spermatozoa and only 2.3% were polyspermic. Fertilization rate was lowest (60%) in grade IV oocytes and all oocytes preincubated for 2-5 h (53%). Fertilization rate increased to 92% in oocytes preincubated for 21-29 h. Embryos developed in vitro to a mean of eight cells. Embryo development was unaffected by oocyte maturity but correlated with preincubation time. Oocytes preincubated for 2-5 h developed into embryos with significantly fewer cells than those preincubated for 9-11 or 21-29 h (P < 0.001). Fifty-six per cent of embryos showed delayed cleavage and these had fewer cells than non-delayed embryos (P < 0.001). When oocytes were preincubated for 2-5 h, development of all resulting embryos was delayed. However, only 17 and 58% of embryos developing from oocytes preincubated for 9-11 and 21-29 h, respectively, were delayed and this was independent of oocyte maturity.

Animals↗
PubMed1993

In vitro fertilization and embryo culture in the grey short-tailed opossum, Monodelphis domestica.

In vitro fertilization and early embryo culture was undertaken in the South American marsupial, the grey short-tailed opossum, Monodelphis domestica. Adult females were induced into oestrus by a system of pairing with an unfamiliar male and mature oocytes were recovered from the ovary 15-18 h after mating and placed in pre-warmed modified MEM medium at 33 degrees C (basal body temperature) or 37 degrees C. Spermatozoa recovered from the cauda epididymides of adult males were preincubated in medium for 2 h during which time paired spermatozoa separated and initiated hyperactivated motility. Oocytes were transferred to 0.4 ml drops of spermatozoa containing 0.5-1.0 x 10(6) spermatozoa ml-1. Only single spermatozoa bound to the zona pellucida, and fertilization occurred within 1-2 h as indicated by a breach in the zona and confirmed by electron microscopy. At 37 degrees C, 95 of 152 (62.5%) oocytes were fertilized and 64 (67%) developed to two-cell stage or beyond. At 33 degrees C, 5 of 28 (18%) oocytes were fertilized. This is the first report of complete in vitro fertilization in a marsupial.

Animals↗
PubMed1993

A new method for cryopreservation of mouse spermatozoa.

A new method for the cryopreservation of mouse spermatozoa was developed using a modified egg-yolk TES-Tris diluent containing 0.1% sodium lauryl sulfate and 1.25% (v/v) glycerol (mouse sperm cryoprotectant, MSC). Epididymal spermatozoa collected from 10-week-old CBA males were frozen at a rate of 5 degrees C min-1 to 4 degrees C and 50 degrees C min-1 to -70 degrees C using a programmable cell freezer. A percentage of the spermatozoa (25%) regained motility after thawing. In vitro fertilization with frozen-thawed spermatozoa resulted in 50% of oocytes developing to the two-cell stage. These two-cell embryos were placed in the oviducts of pseudopregnant recipients (C57BL/CBA) and 16% developed to be viable fetuses, or in the oviducts of pregnant recipients (MF1) and 17% developed to live offspring.

Animals↗
PubMed1993

Gonadal sex differentiation in embryos and neonates of the marsupial, Monodelphis domestica: arrest of testis development in postterm embryos.

Growth and histological differentiation were studied in 8 litters of embryos and 4 litters of neonate grey short-tailed opossums, Monodelphis domestica. The embryonic litters included 2 that had passed their expected birth date, and whose weights exceeded the usual birthweights; we refer to these litters as 'postmature'. There was an abrupt increase in the growth rate of XY gonads after birth, but this was not seen in XX gonads. Although there was evidence of testicular differentiation in XY gonads on the day before the expected birth, testicular differentiation was found to be blocked in postmature litters. The growth of XX gonads in postmature embryos was not affected. In view of evidence that exogenous oestrogens feminise the gonads of genetic males in some species of marsupials including Monodelphis domestica, the question arises whether oestrogen is responsible for the failure of testes to continue their development in utero. We suggest that the ability of functional testes to develop in the presence of oestrogen may be a fundamental requirement distinguishing eutherian mammals from other vertebrates, including marsupials.

Animals↗
PubMed1992

The culture of human epididymal epithelium and in vitro maturation of epididymal spermatozoa.

OBJECTIVE: To promote human sperm maturation in vitro. DESIGN: Spermatozoa from the proximal epididymis were coincubated with epididymal epithelial fragments. SETTING: Hospital and research institute. PATIENTS, PARTICIPANTS: Tissue samples were obtained from men undergoing epididymovasostomy procedures or vasectomy. INTERVENTIONS: Fragments of epididymal epithelium formed everted epithelial spheres that in the presence of androgen maintained cell integrity. Coincubation for up to 48 hours of caput epididymal spermatozoa with 3-day-old epithelial cultures from the cauda epididymis was undertaken. MAIN OUTCOME MEASURES: Morphology of epididymal epithelium was assessed by light and electron microscopy. Pulse labeling of tissue in vitro with 35S-methionine was performed with analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis technique and fluorography. Spermatozoa were assessed for progressive motility and their capacity to bind to salt-stored human zona pellucidae. RESULTS: Epididymal fragments formed everted epithelial spheres that maintained cell integrity and functional morphology for 5 to 7 days. Specific proteins were synthesized in culture, in particular, proteins of 20, 22, 40, and 66 kd. Coincubation of caput epididymal spermatozoa with cultures from the cauda epididymis induced a significant increase in progressive sperm motility and sperm binding to salt-stored human zona pellucidae compared with control cultures of epithelium incubated in the absence of androgens or overgrown with fibroblasts. CONCLUSIONS: Aspects of human sperm maturation processes can be mimicked in vitro using coculture techniques with epididymal epithelium. This method may be valuable for improving the fertilizing capacity of human spermatozoa retrieved from the proximal region of the excurrent ducts.

Cells, Cultured↗
PubMed1991

Differential expression of the mouse U1a and U1b SnRNA genes is not dependent on sequence differences in the octamer motif.

The mouse U1b SnRNA gene is expressed in only a limited range of cell types, whereas the U1a SnRNA gene is expressed in all cells. These two genes differ in the sequence of the octamer motif, which plays a critical role in SnRNA gene regulation. We show that the U1b octamer binds the octamer-binding protein Oct-1 with higher affinity than does the U1a octamer in both U1b-expressing and -non-expressing cell lines and tissues. Moreover, the U1b octamer can direct a higher level of gene expression than the U1a octamer when linked to a heterologous promoter and introduced into a non-U1b-expressing cell line. Hence the tissue-specific expression of the U1b gene is not determined by the failure of its octamer motif to bind Oct-1 or the weak affinity of this binding.

Animals↗
PubMed1990

Acrosome reaction of stallion spermatozoa evaluated with monoclonal antibody and zona-free hamster eggs.

The acrosome of the stallion spermatozoon was visualized by indirect immunofluorescence with monoclonal antibody (18.6) which recognized an integral acrosomal membrane component. Localization was confirmed by electron microscopy using peroxidase labelled antibody. In fresh semen samples (n = 19), 73.9 +/- 9.1% of the spermatozoa from five fertile stallions displayed a uniform bright fluorescence over their acrosome region. In two semen samples from an infertile stallion only 28% and 35% of spermatozoa showed the same pattern of fluorescence. Spermatozoa from fertile stallions incubated for up to 12 hours in TALP medium maintained motility and exhibited a significant progressive loss of acrosomes as detected by immunofluorescence. Alternatively, a similar loss of acrosomes could be induced with calcium ionophore A23187 over a 90 minute incubation. Ultrastructural observations and incubation with zona-free hamster eggs indicated that only with ionophore treatment was immunofluorescent acrosome loss correlated with a physiological acrosome reaction, while prolonged sperm incubation led to degenerative membrane changes. It was concluded that, if carefully validated, immunofluorescent localization of the acrosome of stallion sperm with monoclonal antibody could be used to monitor the acrosome reaction. Furthermore, definitive acrosome visualization would be valuable in assessing semen quality.

Acrosome↗
PubMed1990

An ultrastructural study of early chorionic villus formation in the marmoset monkey (Callithrix jacchus).

The ultrastructural morphology of developing chorionic villi in the marmoset monkey (Callithrix jacchus) placenta was studied in pregnant monkeys at known time intervals after ovulation. In samples obtained at 45 days after ovulation the mesoderm, which consists of primitive foetal blood vessels, is seen to extend down into cytotrophoblast columns. Syncytiotrophoblast completely surrounds maternal blood vessels and both basal laminae and endothelial cells of maternal origin show signs of disorganisation and degradation. Syncytiotrophoblast is first observed to breach the maternal circulation in samples collected from animals at 60 days after ovulation; this results in discrete haemochorial villi randomly distributed throughout the placental bed. Samples obtained at 80 days after ovulation and term placental samples (145 days after ovulation) exhibit tertiary haemochorial villi throughout the placenta, similar to those seen randomly distributed at 60 days after ovulation.

Animals↗
PubMed1990

The use of in vitro fertilization to detect reductions in the fertility of male rats exposed to 1,3-dinitrobenzene.

1,3-Dinitrobenzene (DNB) is an intermediate chemical in the manufacture of dyes and explosives and its toxic effects include specific damage to the Sertoli cells of the testis. This investigation determined the effect a toxic insult to Sertoli cells had on the functional capacity of developing germ cells as assessed by in vitro fertilization. Male rats were given a single, oral dose of 5, 15, or 25 mg DNB/kg. At selected times after treatment, spermatozoa recovered from the cauda epididymidis were tested for fertilizing capacity using in vitro fertilization techniques and the testicular response to DNB was determined by histological examination. Treatment with 15 and 25 mg DNB/kg resulted in substantial exfoliation of germ cells between 0.5 and 3.5 weeks after exposure and again after 4.5 weeks; seminiferous tubules which were not depleted showed signs of disrupted spermatogenesis. Reduced sperm fertilizing capacity in vitro was observed from 1.5 to 5 weeks and between 7.5 and 8.5 weeks after treatment with 15 and 25 mg DNB/kg. There were slight, but significant, reductions in fertility at 3, 5.5, 7.5, and 8.5 weeks after dosing with 5 mg DNB/kg. These data suggested that DNB did not affect all Sertoli cells equally, but acted in a stage-specific manner. Stages III, IV, XII, and XIV were most vulnerable to the toxicant. Germ cells associated with an affected Sertoli cell were usually sloughed off, resulting in lowered fertility at the time when these cells should have reached maturity in the epididymis. The extent of the testicular lesions and the loss of fertility were dose dependent. This investigation confirmed the use of in vitro fertilization to detect the effects of testicular toxicants.

Animals↗
PubMed1990

The use of rat in vitro fertilization to detect reductions in the fertility of spermatozoa from males exposed to ethylene glycol monomethyl ether.

An in vitro fertilization (IVF) assay sensitive enough to detect changes in the fertilizing capacity of spermatozoa would be a useful tool with which to investigate the action of testicular toxicants. A known testicular toxicant, ethylene glycol monomethyl ether (EGME), was used to induce specific lesions in the germinal epithelium so that the ability of a rat IVF system to detect changes in fertility could be tested. Male rats were given single, oral doses of 50, 100, and 200 mg EGME/kg. Spermatozoa were recovered from the cauda epididymides of these males at intervals after treatment; their fertility was assessed using IVF, and the testes were processed for histologic examination. The fertility of the control males was consistently greater than 65%. Spermatozoa from males treated with EGME had reduced fertility at specific times after dosing. Thus, after 50 mg EGME/kg there was reduced fertility at 5 weeks; after 100 mg EGME/kg there was reduced fertility at 3.5, 4.5, 5, 6, and 6.5 weeks, and after 200 mg EGME/kg there was reduced fertility at 2 and 3 weeks, between 4.5 and 6 weeks, and at 7 weeks. This corresponded to damage to the elongated spermatids (2, 3, and 3.5 weeks), pachytene spermatocytes (4.5 to 6 weeks), and leptotene and preleptotene spermatocytes (7 weeks). This accords well with the data from serial breeding trials and reports of histologic damage after exposure to EGME. Therefore, using IVF it was possible to detect EGME-induced changes in fertilizing capacity which correlated closely with observations of testicular damage. It was also possible to demonstrate a clear dose response to EGME.

Animals↗
PubMed1990

Sperm motility, velocity and migration.

Four different methods for evaluating sperm motility were analysed for experimental error: subjective assessment of a wet film preparation, sperm velocity measured by time-lapse photography, sperm velocity measured by computer analysis and sperm migration across a nucleopore membrane. Subjective assessment of motility was found to be inaccurate, within single observer and between 2 observers. Both methods of measuring mean sperm velocity were accurate, particularly that using the computer analysis system; a high technical failure rate was found using time-lapse photography. Sperm migration across a nucleopore membrane was found to be highly inaccurate. Two groups were then analysed for the predictive value of these tests (excluding sperm migration): 104 proven fertile men and 53 infertile men. Although subjective motility was able to predict from which group the sample came at optimum cut-off with 78% accuracy, computer analysed sperm velocity could predict with overall 91% accuracy at optimum cut-off. Computer analysis of sperm velocity offers a rapid, objective and predictive assessment of sperm function.

Humans↗
PubMed1990

Gonadal sex differentiation in the neonatal marsupial, Monodelphis domestica.

A quantitative and histological study of the gonads of newborn grey short-tailed opossums, Monodelphis domestica, is described. The pups were karyotyped, and comparisons were made within litters segregating for XX and XY sex chromosomes. A total of four litters including 25 pups were available. On the day of birth, developing testes were significantly larger than the ovaries of litter mates, and testes could be histologically distinguished by the formation of sex cords and a tunica albuginea. The data suggest that in this marsupial species gonadal differentiation may be initiated in utero.

Animals↗
PubMed1989

The value of sperm swimming speed measurements in assessing the fertility of human frozen semen.

The purpose of this study was to evaluate relationships between measured sperm velocity and in-vivo fertility, using donor semen samples from an artificial insemination (AID) programme. Seventy-one frozen semen samples were examined; measurements of sperm velocity were made immediately after thawing, upon a motile 'swim-up' fraction, and finally after 3.5 h incubation at 37 degrees C in the freezing mixture. Zona-free hamster egg penetration assays were performed upon all samples. Two groups of samples were identified; seven donors (11 samples) had failed to produce any pregnancies through AID from a range of 3 to 14 cycles tested, whilst the remaining samples (from 25 donors) had achieved at least one pregnancy each. The mean sperm velocity (+/- SEM) for the latter 'fertile' group was significantly higher than the corresponding value for the 'infertile' group; (i) after thawing, 65.9 +/- 1.8 versus 50.4 +/- 3.2 microns/s (P less than 0.001) and (ii) after 3.5 h incubation, 42.1 +/- 2.1 versus 24.7 +/- 5.7 microns/s (P less than 0.002). Using the maintenance of sperm velocity during incubation as an indicator of survival, life-table analyses were used to calculate monthly conception rates on various sub-groups of the semen samples. Poor survival (greater than 40% decline in velocity over 3.5 h) was associated with a monthly pregnancy rate of only 11.58% (362 cycles), whilst better survival (less than 40% decline) was associated with the significantly higher (P = 0.024) pregnancy rate of 16.87% (480 cycles).

Evaluation Studies as Topic↗
PubMed1988

Expression of C-type viral particles at implantation in the marmoset monkey.

Type C RNA retroviral particles have been observed budding from the plasma membrane of syncytiotrophoblast during a study of embryo implantation in the common marmoset (Callithrix jacchus). These viral particles appear in profusion at the interface of syncytiotrophoblast with cytotrophoblast. They appear to be a non-infective, endogenous viral component of the host genome, although the precise role of the C-type virus in this context is unclear at present. Such viral particles have been reported previously from studies of human term placenta; however, this is the first report indicating that expression of viral particles occurs at the earliest stages of embryo implantation.

Animals↗