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K Hardy

Publications and source records attributed to K Hardy.

At least 37 records · Page 2Linked to original sources
PubMed2001

Stage-specific and differential expression of gap junctions in the mouse ovary: connexin-specific roles in follicular regulation.

Gap junction communication plays an essential role in follicle growth. Immunocytochemistry and confocal microscopy were used to examine the expression of gap junction connexins of the alpha and beta subfamilies in follicles from primordial to preovulatory stages in the ovaries of prepubertal and adult mice. Connexin-specific antibodies detected alpha(1), alpha(4), alpha(6), beta(1), beta(2) and beta(4) connexins within follicles. In adult ovaries connexin immunolabelling was stronger in larger (more mature) follicles than it was in smaller follicles, with comparatively reduced labelling detected in prepubertal ovaries. In healthy follicles, labelling for alpha subfamily connexins was detected between granulosa cells, whereas labelling for beta subfamily connexins was found in the theca. Labelling for beta subfamily connexins and alpha(4) connexin (preantral stage) was detected on the oocyte surface membrane. In atretic follicles, labelling for beta(4) connexin appeared between the granulosa cells. These results demonstrate that alpha and beta connexin subfamilies are segregated to separate cellular compartments in the mouse follicle. The results are discussed in the light of possible roles for differential gap junctional communication in the regulation of folliculogenesis, oocyte maturation and atresia.

Animals↗
PubMed2000

Benzo[c]quinolizin-3-ones: a novel class of potent and selective nonsteroidal inhibitors of human steroid 5alpha-reductase 1.

The synthesis and biological evaluation of a series of novel, selective inhibitors of isoenzyme 1 of human 5alpha-reductase (5alphaR) (EC 1.3.99.5) are reported. The inhibitors are 4aH- (19-29) or 1H-tetrahydrobenzo[c]quinolizin-3-ones (35-47) bearing at positions 1, 4, 5, and 6 a methyl group and at position 8 a hydrogen, methyl group, or chlorine atom. All these compounds were tested toward 5alphaR-1 and 5alphaR-2 expressed in CHO cells (CHO 1827 and CHO 1829, respectively) resulting in selective inhibitors of the type 1 isoenzyme, with inhibitory potencies (IC(50)) ranging from 7.6 to 9100 nM. The inhibitors of the 4aH-series, having a double bond at position 1,2, were generally less active than the corresponding inhibitors of the 1H-series having the double bond at position 4,4a on the A ring. The presence of a methyl group at position 4 (as in compounds 39-40 and 45-47), associated with a substituent at position 8, determined the highest inhibition potency (IC(50) from 7.6 to 20 nM). Compounds 39 and 40, having K(i) values of 5.8+/-1.8 and 2.7+/-0.6 nM, respectively, toward 5alphaR-1 expressed in CHO cells, were also tested toward native 5alphaR-1 in human scalp and 5alphaR-2 in human prostate homogenates, in comparison with finasteride and the known 5alphaR-1-selective inhibitor LY191704, and their mechanism of inhibition was determined. They both inhibited the enzyme through a reversible competitive mechanism and again were selective inhibitors of 5alphaR-1 with IC(50) values of 41 nM. These specific features make these inhibitors suitable candidates for further development as drugs in the treatment of DHT-dependent disorders such as acne and androgenic alopecia in men and hirsutism in women.

5-alpha Reductase Inhibitors↗
PubMed2000

Noninvasive assessment of glucose and pyruvate uptake by human embryos after intracytoplasmic sperm injection and during the formation of pronuclei.

OBJECTIVE: To improve in vitro culture conditions and human embryo selection before transfer after IVF with intracytoplasmic sperm injection (ICSI). DESIGN: A controlled, randomized, prospective study. SETTING: University hospital-based IVF-ET program. PATIENT(S): Couples undergoing ICSI. INTERVENTION(S): Culture of human embryos in the presence of 1 mM or 5.56 mM glucose and metabolic measurements with the use of noninvasive microfluorescence assays immediately after ICSI to the time of transfer. MAIN OUTCOME MEASURE(S): Embryo development, implantation rate, and glucose and pyruvate uptake. RESULT(S): Fertilization rates, early embryo development, and implantation rates were not significantly different between 1 mM and 5.56 mM glucose. Pyruvate uptake was significantly higher during the formation of the pronuclei, at 15 +/- 0.7 and 11.4 +/- 1.3 pmol/embryo/h for fertilized and unfertilized oocytes, respectively. Pyruvate uptake did not correlate with cleavage stage or embryo morphology. However, during the second day of incubation, pyruvate uptake was significantly higher for the untransferred embryos of pregnant women compared with nonpregnant women, at 17.9 +/- 1.5 and 10.8 +/- 1.0 pmol/embryo/h, respectively. CONCLUSION(S): The increased level of pyruvate uptake during fertilization reflects the increased demand for energy necessary for the formation of the pronuclei. However, the metabolic measurements could not improve the selection of embryos with the best implantation potential. Finally, the reduction of glucose concentration in the culture medium failed to improve embryo viability.

Embryo Implantation↗
PubMed2000

Anti-apoptotic action of insulin-like growth factor-I during human preimplantation embryo development.

Insulin-like growth factor I (IGF-I) has been shown to increase the proportion of embryos forming blastocysts and the number of inner cell mass cells in human and other mammalian preimplantation embryos. Here we examined whether the increased cell number resulted from increased cell division or decreased cell death. Normally fertilized, Day 2 human embryos of good morphology were cultured to Day 6 in glucose-free Earle's balanced salt solution supplemented with 1 mM glutamine, with (n = 42) and without (n = 45) 1.7 nM IGF-I. Apoptotic cells in Day 6 blastocysts were identified using terminal deoxynucleotidyl dUTP terminal transferase (TUNEL) labeling to detect DNA fragmentation and 4'-6-diamidino-2-phenylindole (DAPI) counterstain to evaluate nuclear morphology. The number of nuclei and extent of DNA and nuclear fragmentation was assessed using laser scanning confocal microscopy. IGF-I significantly increased the proportion of embryos developing to the blastocyst stage from 49% (control) to 74% (+IGF-I) (P < 0.05). IGF-I also significantly decreased the mean proportion of apoptotic nuclei from 16.3 +/- 2.9% (-IGF-I) to 8.7 +/- 1.4% (+IGF-I) (P < 0.05). The total number of cells remained similar between both groups (61.7 +/- 4.6 with IGF-I; 54.5 +/- 5.1 without IGF-I). The increased number of blastocysts combined with reduced cell death suggests that IGF-I is rescuing embryos in vitro which would otherwise arrest and acting as a survival factor during preimplantation human development.

Apoptosis↗
PubMed2000

In vitro maturation of oocytes.

Only about 400 of the one million oocytes present at birth will be ovulated, while the rest will die by atresia. The ability to rescue oocytes destined to die and mature them in vitro would provide invaluable information about folliculogenesis and oocyte maturation, and could provide oocytes for infertile women. In vitro maturation (IVM) is challenging in the human because folliculogenesis is a lengthy process encompassing many complex cellular changes in the oocyte and its surrounding follicle cells. A few live births have resulted from the maturation and fertilization of immature human oocytes aspirated from small antral follicles. Furthermore, it is possible to grow primordial follicles to pre-antral stages in slices of ovarian tissue, and support antrum formation in isolated pre-antral follicles. However, we are still a considerable way from growing and maturing pre-antral follicles to pre-ovulatory stages in vitro. The importance of the follicular environment for producing a healthy and developmentally competent oocyte is illustrated by the oocyte's susceptibility to errors during meiosis. This counsels considerable caution in the development of IVM for clinical application.

Animals↗
PubMed2000

New protocol for DNA extraction of stool.

Present methods for DNA isolation of stool have various limitations such as the amount of stool used, the requirement of lavage fluids or the use of fresh stool. In this paper, a new method is described for the isolation of human nucleic acids from stool, which is independent from the moment of collection. Fecal samples as dry as possible were collected from 75 patients; two grams of stool were mixed with a lysis buffer containing phenol. DNA yields of crude stool were variable and ranged from 9-1686 micrograms/g of feces. With dot blots in 9 of the 75 cases, the human DNA was identified and ranged from 0.06%-46%. In the remaining 66 cases, human genomic DNA was detected by nested PCR, using human K-ras gene amplification as an example. Amplification products were confirmed for human K-ras with the exonuclease-amplification coupled capture technique (EXACCT). In conclusion, the developed DNA isolation method can be used for the study of large numbers of stool samples, is independent of the age or method of stool collection and is suitable for large-scale screening studies.

Base Sequence↗
PubMed2000

[Thoracoscopic treatment of pulmonary hydatidosis: a case report].

Hydatidosis is a ubiquitous parasitic condition observed in a pulmonary localization in 30 to 40% of cases. The hydatid cyst develops slowly and is well tolerated by the host who presents no signs for a long period. Complications include compression, fissuration, rupture, anaphylactic shock or infection after a latency phase of variable duration. Treatment of pulmonary hydatidosis is classically surgical with enucleation of the cyst by cleavage between the adventice and the anhistic membrane via thorachotomy using the Ugon and Barret procedure. Needle aspiration is also possible via thorachotomy or thoracoscopy. Finally resection of the pulmonary parenchyma can be used to excise the hydatic cyst. We describe a thoracoscopic treatment using specific material, in a man with complications due to a voluminous pulmonary hydatid cyst.

Adult↗
PubMed1999

Excitotoxic cell death dependent on inhibitory receptor activation.

Although excitotoxic cell death is usually considered a Ca(2+)-dependent process, in certain neuronal systems there is strong evidence that excitotoxic cell death is independent of Ca2+ and is instead remarkably dependent on extracellular Cl-. We have shown (in isolated chick embryo retina) that at least some of the lethal Cl- entry is through GABA and glycine receptors. Here we show that when all the GABA and glycine receptors are blocked by using an appropriate cocktail of inhibitors, agonist-induced excitotoxic cell death can be completely prevented. To determine if ligand-gated Cl- channels contribute to excitotoxic cell death in other neurons, we examined KA-induced cell death in cultured rat cerebellar granule cells. GABA receptor blockade with either a competitive or noncompetitive antagonist provides complete neuroprotection. KA stimulates Cl- uptake by the granule cells, and this is blocked by the GABA antagonists. Granule cell cultures take up [3H]GABA and release it in response to KA treatment. A subpopulation of neurons in the cultures is shown to have GAD and high concentrations of GABA, and this presumably is the source of the GABA that leads to receptor activation and lethal Cl- entry. Finally, we show that retinal cell death due to 1 h of simulated ischemia (combined oxygen and glucose deprivation) is completely prevented by blocking the inhibitory receptors. These results indicate that, paradoxically, excitotoxic cell death is completely dependent on activation of inhibitory receptors, in at least some neuronal systems, and this pathological process may contribute to disease.

Action Potentials↗
PubMed1999

Regional distribution in the mouse of proteins homologous to artery-specific antigenic proteins (ASAPs).

We have reported the sequences of four novel proteins derived from extracts of human aortic tissue and a cDNA library from human aortic adventitia. These proteins are immunoreactive with serum immunoglobulins from patients with abdominal aortic aneurysms (AAA), and they have homologies of amino acid sequence with microfibrillar proteins of connective tissue. We are reporting separately that two of these proteins are artery-specific antigenic proteins (ASAPs) in man. The present work investigates the regional distribution of these two proteins (AAAP-40 and MatCAM-1) in mouse (E-beta-b). Antibodies were raised in rabbit against polypeptides encoding novel amino acid sequences, unique to these proteins (e.g., not reported in GenBank). Immunohistochemical studies with these two specific antibodies show conspicuous immunoreactivity of collagen-associated microfibrils in the aortic adventitia of the murine abdominal and thoracic aorta. Immunoreactive peptides were not present in brain, muscle, or kidney. These findings support the hypothesis that proteins occur in the mouse that are homologous to a unique family of aortic microfibrillar proteins in man.

Actin Cytoskeleton↗
PubMed1999

Human primordial, primary and secondary ovarian follicles in long-term culture: effect of partial isolation.

Ovarian cortical tissue, donated by 20 women aged 25-43 years during gynaecological laparoscopies or laparotomies, was first cultured for 7-9 days as tissue slices, 0.1-0.3 mm in thickness, in extracellular matrix, to initiate the growth of the primordial and primary follicles. It was then divided into two parts, one of which was cultured further as slices, and the other one used for enzymatic (collagenase at 1, 0.5 or 0.25 mg/ml; 17 patients) or mechanical (four patients) partial isolation of the follicles. The tissue slices and the partially isolated follicles were cultured for a further 1-3 weeks in the matrix. After approximately 2 weeks in culture, some oocytes began to extrude from the follicles, which were usually at the secondary stage. They were small, 20-80 micrometer in diameter, and had a thin or absent zona. Polar bodies and meiotic chromosomes could be seen in these naked oocytes. This premature extrusion probably resulted from sub-optimal culture conditions. It occurred sooner in follicles that had been partially isolated using collagenase. Histologically, larger numbers of oocytes were observed in non-isolated slice cultures than in the partially isolated cultures. Initiation of growth of the follicles occurred during the first 7-9 days in culture within slices. In non-isolated slices and following mechanical partial isolation there were significantly more secondary follicles after 11-18 days in culture than following isolation with collagenase. The proportion of atretic follicles increased during all cultures, and it was significantly higher after partial isolation. Because partial isolation did not improve the survival or development of the follicles the optimal method for human ovarian follicles could be to culture them non-isolated within small tissue slices.

Adult↗
PubMed1999

Effects of follicle-stimulating hormone and serum substitution on the in-vitro growth of human ovarian follicles.

In-vitro maturation (IVM) of human ovarian follicles and oocytes could benefit infertile women, and allow the development of in-vitro systems for the study of human follicular development. Little is known about the initiation of growth of primordial follicles and the regulation of early folliculogenesis. An ovarian tissue-slice culture system was used to examine the effects of media composition, follicle stimulating hormone (FSH) and serum substitution on the development of small human follicles in vitro. Human ovarian cortex biopsies were cut into small pieces and cultured for 5, 10 or 15 days. Control (non-cultured) and cultured tissue was fixed, serially sectioned, and stained. The follicles contained within the tissue pieces were counted, measured, and assessed for stage of development and viability. Comparison of the ability of alpha-minimum essential medium (alpha-MEM), Waymouth's, or Earle's balanced salt solution (EBSS) culture media (all with 10% human serum) to support follicle growth demonstrated significantly increased initiation and growth of follicles in alpha-MEM during the first 10 days of culture. The supplementation of alpha-MEM with 300 mIU/ml FSH significantly reduced levels of atresia and increased the mean diameter of healthy follicles. Follicles in tissue cultured for 10 days with human serum albumin and ITS (insulin/transferrin/selenium mix) were significantly larger, more developed and showed significantly less atresia than those cultured with serum alone. Primordial to small preantral follicles can be grown under serum-substituted conditions in tissue-slice culture, and are responsive to FSH, which is thought to be acting mainly as a survival factor at these early stages.

Adult↗
PubMed1999

Effects of taurine on human embryo development in vitro.

Glutamine and taurine are reported to be beneficial for mouse embryo development in vitro, and we have recently shown that glutamine improves human blastocyst formation in vitro. This randomized study compared the development of supernumerary human embryos in the presence of 1 mmol/l glutamine and/or 5 mmol/l taurine from the 2-4-cell stage to the blastocyst stage. Blastocyst development and cell numbers were similar in the presence of glutamine or taurine: 52.6% and 58.3% of the embryos reached the blastocyst stage, respectively. Pyruvate uptake was similar in the presence of glutamine or taurine throughout development, as was lactate production after the 8-cell stage. Before this stage, lactate production was 4-fold higher in the presence of taurine (P < 0.001). The proportion of embryos reaching the blastocyst stage was similar with glutamine alone or with glutamine and taurine (62.5% and 47.2% respectively), as were the blastocyst cell numbers (63.0 +/- 4.6 and 61.0 +/- 5.1 respectively). In conclusion, taurine supports development of 2-4-cell human embryos to the blastocyst stage, although it does not further augment the beneficial effects of glutamine.

Blastocyst↗
PubMed1999

Apoptosis in the human embryo.

Preimplantation human embryos are characterized by various degrees of cytoplasmic fragmentation, and a high incidence of developmental arrest before the blastocyst stage. This review examines the current morphological and biochemical evidence that apoptosis plays a role in early human development and embryonic loss. Embryos examined 24 h or more after arrest often show characteristic features of apoptosis including cytoplasmic, nuclear and DNA fragmentation. In contrast, embryos of good morphology that appear to be developing normally show no evidence of apoptosis before compaction. However, at the morula and blastocyst stages, scattered cells with fragmented nuclei and DNA characteristic of cells undergoing apoptosis are common features. Apoptosis may result from suboptimal culture conditions, or may be involved in the elimination of abnormal cells. However, the causes, role and regulation of apoptosis in the human preimplantation embryo remain to be determined.

Apoptosis↗