Live birth after sperm retrieval from a moribund man.

OBJECTIVE: To report a live birth with IVF-ET and intracytoplasmic sperm injection (ICSI) using sperm retrieved from a moribund man being maintained on life support systems. DESIGN: Case report. SETTING: Nonprofit private teaching hospitals. PATIENT(S): A 27-year-old man who was decorticate after an accident. INTERVENTION(S): Epididymal sperm retrieval and sperm cryopreservation, pastoral psychologic counseling, and subsequent IVF-ET with ICSI. MAIN OUTCOME MEASURE(S): Pregnancy and delivery. RESULT(S): Birth of a healthy child after IVF-ET with ICSI and single blastocyst transfer. CONCLUSION(S): First report in peer-reviewed medical literature of a live birth after sperm retrieval from a moribund man.

Small molecule peptidomimetics containing a novel phosphotyrosine bioisostere inhibit protein tyrosine phosphatase 1B and augment insulin action.

Protein tyrosine phosphatase 1B (PTP1B) attenuates insulin signaling by catalyzing dephosphorylation of insulin receptors (IR) and is an attractive target of potential new drugs for treating the insulin resistance that is central to type II diabetes. Several analogues of cholecystokinin(26)(-)(33) (CCK-8) were found to be surprisingly potent inhibitors of PTP1B, and a common N-terminal tripeptide, N-acetyl-Asp-Tyr(SO(3)H)-Nle-, was shown to be necessary and sufficient for inhibition. This tripeptide was modified to reduce size and peptide character, and to replace the metabolically unstable sulfotyrosyl group. This led to the discovery of a novel phosphotyrosine bioisostere, 2-carboxymethoxybenzoic acid, and to analogues that were >100-fold more potent than the CCK-8 analogues and >10-fold selective for PTP1B over two other PTP enzymes (LAR and SHP-2), a dual specificity phosphatase (cdc25b), and a serine/threonine phosphatase (calcineurin). These inhibitors disrupted the binding of PTP1B to activated IR in vitro and prevented the loss of tyrosine kinase (IRTK) activity that accompanied PTP1B-catalyzed dephosphorylation of IR. Introduction of these poorly cell permeant inhibitors into insulin-treated cells by microinjection (oocytes) or by esterification to more lipophilic proinhibitors (3T3-L1 adipocytes and L6 myocytes) resulted in increased potency, but not efficacy, of insulin. In some instances, PTP1B inhibitors were insulin-mimetic, suggesting that in unstimulated cells PTP1B may suppress basal IRTK activity. X-ray crystallography of PTP1B-inhibitor complexes revealed that binding of an inhibitor incorporating phenyl-O-malonic acid as a phosphotyrosine bioisostere occurred with the mobile WPD loop in the open conformation, while a closely related inhibitor with a 2-carboxymethoxybenzoic acid bioisostere bound with the WPD loop closed, perhaps accounting for its superior potency. These CCK-derived peptidomimetic inhibitors of PTP1B represent a novel template for further development of potent, selective inhibitors, and their cell activity further justifies the selection of PTP1B as a therapeutic target.

Effect of Percoll wash on sperm motion parameters and subsequent fertility in intrauterine insemination cycles.

PURPOSE: This study examined sperm motion parameters as measured by computerized automated semen analysis before and after a Percoll wash and determined if differences in any parameter were correlated with fertility subsequent to intrauterine insemination (IUI). RESULTS: Total motile sperm decreased following the washing procedure from 79.0 +/- 9.0 to 37.2 +/- 7.6 million sperm. Motility increased from a mean of 43.4% to 61.7% (P < 0.001). Other motility parameters also changed significantly (P < 0.001) as follows: curvilinear velocity (VCL), 43.4 to 61.7 microns/s; straight-line velocity (VSL), 21.3 to 26.7 microns/s; linearity 53.1 to 45.2%; lateral head displacement (ALH), 2.97 to 3.94 microns. Similar changes occurred following a swim-up preparation, although changes in mean motility, VCL, and ALH were significantly greater when compared to Percoll. The postwash changes were not accounted for merely by time lapse in preparation since reanalyzed untreated controls did not show the same changes in motion parameters. Prewash linearity in those specimens which resulted in pregnancies was greater than in those which did not (P = 0.28). No other significant differences in pre- or post-Percoll washed sperm motion parameters were found between pregnant vs nonfertile cycles. CONCLUSION: Following Percoll wash all CASA-generated motility parameters were significantly altered, but there was little association between these parameters and pregnancy achieved in IUI cycles.

Pioglitazone promotes insulin-induced activation of phosphoinositide 3-kinase in 3T3-L1 adipocytes by inhibiting a negative control mechanism.

Activation of phosphoinositide 3-kinase (PI 3-kinase) is an early event in insulin signal transduction that is blocked completely in adipocytes from insulin-resistant KKAy mice. Treatment of KKAy mice with pioglitazone, an anti-diabetic thiazolidinedione, partially restores insulin-dependent changes in PI 3-kinase. The mechanism of this effect of pioglitazone was investigated using murine 3T3-L1 cells as an experimental model. Insulin and insulin-like growth factor I (IGF-I) each elicited rapid (within 2 min) and large (2-5-fold) increases in PI 3-kinase activity that could be immunoprecipitated using anti-phosphotyrosine (pY) antibodies. Maximal insulin-induced activity of PI 3-kinase in pY-immunoprecipitates was similar in 3T3-L1 adipocytes and mouse adipocytes, but the kinetics of activation differed. Insulin- and IGF-I-induced changes in PI 3-kinase were each half-maximal at 3-5 nM of hormone and were not additive. Increases in both insulin-induced and IGF-I-induced pY-immunoprecipitable PI 3-kinase activity were observed when 3T3-L1 fibroblasts became confluent and when they adopted the adipocyte phenotype. Pioglitazone (10 microM), administered either acutely or chronically to either 3T3-L1 adipocytes or 3T3-L1 fibroblasts, did not greatly alter the kinetics, magnitude or sensitivity of changes in PI 3-kinase elicited by either insulin or IGF-I. In contrast, the attenuation by isoproterenol of insulin-induced changes in PI 3-kinase was prevented in cells pretreated with pioglitazone. This effect of pioglitazone did not involve inhibition of isoproterenol-elicited accumulation of cyclic AMP. Pioglitazone also prevented attenuation of insulin induced changes in PI 3-kinase by cell penetrating analogs of cyclic AMP. Pioglitazone, therefore, has no direct effect on insulin-stimulated PI 3-kinase activity, but interferes with a cyclic AMP-dependent mechanism that normally antagonizes this action of insulin. These data support the proposition that the facilitation of insulin action by pioglitazone involves, at least in part, an inhibition of a negative control mechanism.

Pioglitazone promotes insulin-induced activation of phosphoinositide 3-kinase in 3T3-L1 adipocytes by inhibiting a negative control mechanism.

Activation of phosphoinositide 3-kinase (PI 3-kinase) is an early event in insulin signal transduction that is blocked completely in adipocytes from insulin-resistant KKAy mice. Treatment of KKAy mice with pioglitazone, an anti-diabetic thiazolidinedione, partially restores insulin-dependent changes in PI 3-kinase. The mechanism of this effect of pioglitazone was investigated, using murine 3T3-L1 cells as an experimental model. Insulin and insulin-like growth factor I (IGF-I) each elicited rapid (within 2 min) and large (2- to 5-fold) increases in PI 3-kinase activity that could be immunoprecipitated using anti-phosphotyrosine (pY) antibodies. Maximal insulin-induced activity of PI 3-kinase in pY-immunoprecipitates was similar in 3T3-L1 adipocytes and mouse adipocytes, but the kinetics of activation differed. Insulin- and IGF-I-induced changes in PI 3-kinase were each half-maximal at 3-5 nM of hormone and were not additive. Increases in both insulin-induced and IGF-I-induced pY-immunoprecipitable PI 3-kinase activity were observed when 3T3-L1 fibroblasts became confluent and when they adopted the adipocyte phenotype. Pioglitazone (10 microM), administered either acutely or chronically to either 3T3-L1 adipocytes or 3T3-L1 fibroblasts, did not alter greatly the kinetics, magnitude or sensitivity of changes in PI 3-kinase elicited by either insulin or IGF-I. In contrast, the attenuation by isoproterenol of insulin-induced changes in PI 3-kinase was prevented in cells pretreated with pioglitazone. This effect of pioglitazone did not involve inhibition of isoproterenol-elicited accumulation of cyclic AMP. Pioglitazone also prevented attenuation of insulin induced changes in PI 3-kinase by cell penetrating analogs of cyclic AMP. Pioglitazone, therefore, has no direct effect on insulin-stimulated PI 3-kinase activity, but interferes with a cyclic AMP-dependent mechanism that normally antagonizes this action of insulin. These data support the proposition that the facilitation of insulin action by pioglitazone involves, at least in part, an inhibition of a negative control mechanism.

Hepatic insulin resistance in KKA(y) mice and its amelioration by pioglitazone do not involve alterations in phospholipase C activity.

It has been proposed that an abnormality in the regulation of cytosolic-free Ca2+ may be the cause of some forms of insulin resistance. In support of this proposition, it was reported that phospholipase C-catalyzed hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) by liver plasma membranes from obese patients with non-insulin-dependent diabetes mellitus (NIDDM) was abnormally augmented (Thakker et al., J. Biol. Chem. 264, 7169-7175). The objective of this investigation was to determine if a novel antidiabetic agent, pioglitazone, ameliorated hepatic insulin resistance in KKA(y) mice and to identify any alterations in PIP2-phospholipase C activity of liver plasma membranes that may accompany changes in insulin sensitivity. Treatment of KKA(y) mice for 4 days with pioglitazone (20 mg/kg per day) decreased blood glucose and insulin and improved a variety of indices of hepatic insulin resistance, but did not alter the rate of PIP2 hydrolysis by liver plasma membranes. Acute treatment of isolated liver plasma membranes with pioglitazone (1-100 microM) also failed to alter PIP2-phospholipase C activity. Furthermore, the specific activity, Ca(2+)-requirement, pH-dependence and sensitivity to guanosine 5'-thiotriphosphate of the PIP2-phospholipase C in KKA(y) liver membranes were indistinguishable from those of C57BL/6J (normal) mice. Among C57BL/6J and KKA(y) mice fed either a control or pioglitazone-supplemented diet, there was no correlation between PIP2-phospholipase C activity in isolated liver membranes and either glucose or insulin concentrations in the circulation. These data indicate that an alteration in PIP2-phospholipase C activity of liver plasma membranes is neither a cause nor an obligatory consequence of insulin resistance in KKAy mice or its amelioration by pioglitazone. Alterations of liver membrane phospholipase C activity in NIDDM, therefore, may reflect diabetic pathology other than the insulin resistance associated with this disease.

The expression of thromboxane A2 synthase and thromboxane A2 receptor gene in human uterus.

The expression of thromboxane (TX) A2 synthase and thromboxane A2 receptor gene in human uterus was investigated by immunoblotting, immunocytochemistry, Northern blot, in situ hybridization, and autoradiographic analyses. Human uterus contains a single immunoreactive protein of 55 kDa that corresponds to the molecular size of human TXA2 synthase. Human uterus also contains a single 2.8-kb TXA2 receptor mRNA transcript and a receptor protein that can bind TXA2 antagonist, 125I-PTA-OH. The immunoreactive TXA2 synthase, TXA2 receptor mRNA, and protein are present in endometrial glands, stromal cells, myometrial smooth muscle, and uterine blood vessels. The TXA2 synthase and TXA2 receptors in different uterine cells varied within as well as between various reproductive states. There were differences in the binding site numbers even between elongated and circular myometrial smooth muscle in all reproductive states except postmenopause. In summary, the data presented demonstrate for the first time that different human endometrial and myometrial cells and uterine blood vessels express TXA2 synthase as well as TXA2 receptor gene. The expression, as well as changes during various reproductive states, suggests that TXA2 could be an autocrine/paracrine regulator of human myometrial contractions, endometrial secretory functions, and intrauterine blood flow and could play a role in the initiation and/or progression of labor in women.

High affinity insulin binding in the human placenta insulin receptor requires alpha beta heterodimeric subunit interactions.

Insulin binding to human placenta membranes treated at pH 7.6 or 8.5 in the presence or absence of 2.0 mM DTT for 5 min, followed by the simultaneous removal of the DTT and pH adjustment to pH 7.6, displayed curvilinear (heterogeneous) insulin binding plots when analyzed by the method of Scatchard. However, Triton X-100 solubilization followed by Bio-Gel A-1.5m gel filtration chromatography of the placenta membranes previously treated with DTT at pH 8.5 generated a nearly straight line (homogeneous) Scatchard plot. 125I-insulin affinity crosslinking studies coupled with Bio-Gel A-1.5m gel filtration chromatography demonstrated that the alkaline pH and DTT treatment of placenta membranes followed by detergent solubilization generated an alpha beta heterodimeric insulin receptor complex from the alpha 2 beta 2 heterotetrameric disulfide-linked state. The ability of alkaline pH and DTT to produce a functional alpha beta heterodimeric insulin receptor complex was found to be time dependent with maximal formation and preservation of tracer insulin binding occurring at 5 min. These data demonstrate that (i) a combination of alkaline pH and DTT treatment of placenta membranes can result in the formation of a functional alpha beta heterodimeric insulin receptor complex. (ii) the alpha beta heterodimeric complex displays homogeneous insulin binding. (iii) the insulin receptor membrane environment maintains the alpha 2 beta 2 association state, which displays heterogeneous insulin binding, despite reduction of the critical domains that are responsible for the covalent interaction between the alpha beta heterodimers.

Heterogeneity in the human erythrocyte band 3 anion-transporter revealed by Triton X-114 phase partitioning.

Triton X-114 phase partitioning used in conjunction with countercurrent distribution was utilized to examine the phasing properties of the human erythrocyte Band 3 anion-transport protein. Phase partitioning and countercurrent distribution of Band 3 protein followed by electrophoresis and immunoblotting revealed that Band 3 protein possesses biphasic properties with approx. 65% of the Band 3 97,000-Mr species being localized in the detergent phase and 35% isolated in the aqueous phase. The bidirectional phasing of the anion-transporter does not appear to be a result of glycosylation or phosphorylation, since treatment of alkali-washed ghosts with glycosidases or phosphatase respectively did not significantly alter the phasing profiles. Chymotrypsin treatment of erythrocytes followed by the purification of the 60,000-Mr fragment, and exposure of this fragment to phase separation and countercurrent distribution also revealed biphasic partitioning with 70% of the species being isolated in the aqueous phase and 30% in the detergent phase. These data demonstrate that the human erythrocyte Band 3 anion-transport protein is heterogenous by Triton X-114 phase partitioning and that this heterogeneity is preserved in the 60,000-Mr chymotryptic fragment of Band 3 protein.

Insulin-dependent covalent reassociation of isolated alpha beta heterodimeric insulin receptors into an alpha 2 beta 2 heterotetrameric disulfide-linked complex.

The purified human placental alpha 2 beta 2 heterotetrameric insulin receptor complex was reduced and dissociated into functional alpha beta heterodimers by a combination of alkaline pH and dithiothreitol treatment. Insulin treatment of the isolated alpha beta heterodimeric complex was observed to induce the complete reassociation to an alpha 2 beta 2 heterotetrameric state when analyzed by nondenaturing Bio-Gel A-1.5m gel filtration chromatography. Nonreducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis of 125I-insulin affinity cross-linked and 32P-autophosphorylated alpha beta heterodimers demonstrated that the insulin-dependent reassociation to the alpha 2 beta 2 heterotetrameric state occurred both covalently and noncovalently under these conditions. Comparison by reducing and nonreducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that the insulin-dependent covalent reassociation to an alpha 2 beta 2 heterotetrameric complex was due to the formation of a disulfide linkage(s) between the alpha beta heterodimers. beta subunit autophosphorylation of the control alpha 2 beta 2 heterotetrameric insulin receptor preparation was maximally stimulated within 5 min of insulin preincubation and occurred exclusively in the Mr = 400,000 alpha 2 beta 2 complex. Similarly, maximal insulin-stimulated beta subunit autophosphorylation of the alpha beta heterodimeric preparation occurred within 5 min of insulin pretreatment in the Mr = 210,000 alpha beta complex. However, 4 h of insulin pretreatment of the alpha beta heterodimer preparation induced the formation (6-fold) of a covalent 32P-labeled alpha 2 beta 2 heterotetrameric complex. Maximal stimulation of substrate phosphorylation for the alpha 2 beta 2 heterotetrameric complex was also observed to occur within 5 min of insulin treatment, whereas maximal insulin-stimulated substrate phosphorylation of the alpha beta heterodimeric complex required greater than 4 h. These data demonstrate that (i) insulin treatment can induce the reassociation of the alpha beta heterodimeric complex into a covalent alpha 2 beta 2 heterotetrameric state, and (ii) insulin-dependent protein kinase activation of the alpha beta heterodimeric insulin receptor correlates with the covalent reassociation into a disulfide-linked alpha 2 beta 2 heterotetrameric complex.

Functional properties of an isolated alpha beta heterodimeric human placenta insulin-like growth factor 1 receptor complex.

Treatment of human placenta membranes at pH 8.5 in the presence of 2.0 mM dithiothreitol (DTT) for 5 min, followed by the simultaneous removal of the DTT and pH adjustment to pH 7.6, resulted in the formation of a functional alpha beta heterodimeric insulin-like growth factor 1 (IGF-1) receptor complex from the native alpha 2 beta 2 heterotetrameric disulfide-linked state. The membrane-bound alpha beta heterodimeric complex displayed similar curvilinear 125I-IGF-1 equilibrium binding compared to the alpha 2 beta 2 heterotetrameric complex. Triton X-100 solubilization of the alkaline pH and DTT-pretreated placenta membranes, followed by Bio-Gel A-1.5m gel filtration chromatography, was found to effectively separate the alpha 2 beta 2 heterotetrameric and alpha beta heterodimeric IGF-1 receptor species, 125I-IGF-1 binding to both the isolated alpha 2 beta 2 heterotetrameric and alpha beta heterodimeric complexes demonstrated a marked straightening of the Scatchard plots, compared to the placenta membrane-bound IGF-1 receptors, with a 2-fold increase in the high-affinity binding component. Similar to the membrane-bound IGF-1 receptor species, the 125I-IGF-1 binding properties between the alpha 2 beta 2 heterotetrameric and alpha beta heterodimeric complexes were not significantly different. IGF-1 stimulation of IGF-1 receptor autophosphorylation indicated that the ligand-dependent activation of alpha beta heterodimeric protein kinase activity occurred concomitant with the reassociation into a covalent alpha 2 beta 2 heterotetrameric state.(ABSTRACT TRUNCATED AT 250 WORDS)

Functional differences in insulin receptors in rat adipocyte and human placenta membranes.

Incubation of human placenta membranes with low concentrations (0.1-0.2 mM) of dithiothreitol (DTT) increased insulin binding approximately 1.4-fold, while 10 mM DTT completely inhibited insulin binding. In contrast, treatment of rat adipocyte membranes with 0.5-2.0 mM DTT increased tracer insulin binding 3- to 6-fold, while higher levels of DTT (10 mM) also fully inhibited insulin binding. Scatchard analysis of insulin binding revealed that DTT treatment of adipocyte membranes resulted in an increase in both the high and low affinity dissociation constants. Purification of adipocyte insulin receptors by wheat germ agglutinin-Sepharose chromatography, followed by insulin-agarose affinity chromatography, resulted in loss of DTT stimulation of insulin binding. Comparison of insulin receptors purified from rat adipocytes or human placenta membranes revealed no significant differences in the DTT sensitivities of insulin binding or protein kinase activities. These data suggest that the functional properties of the rat adipocyte insulin receptor are modified by its membrane environment compared to those of insulin receptors in placenta membranes or purified insulin receptors in detergent solution.

Alteration of intramolecular disulfides in insulin receptor/kinase by insulin and dithiothreitol: insulin potentiates the apparent dithiothreitol-dependent subunit reduction of insulin receptor.

Dithiothreitol (DTT) was observed to increase both beta-subunit autophosphorylation and exogenous substrate phosphorylation of the insulin receptor in the absence of insulin. The natural protein reducing agent thioredoxin was also observed to increase the insulin receptor beta-subunit autophosphorylation. The activation of the insulin receptor/kinase by both DTT and thioredoxin was found to be additive with that of insulin. Further, the increase in the insulin receptor beta-subunit autophosphorylation in the presence of DTT and insulin was demonstrated to be due to an increase in the initial rate of autophosphorylation without alteration in the extent of phosphorylation. Similarly, the increase in the exogenous substrate phosphorylation was due to an increase in the Vmax of phosphorylation without significant effect on the apparent Km of substrate binding. In the presence of relatively low concentrations of DTT, insulin was found to potentiate the apparent insulin receptor subunit reduction of the native alpha 2 beta 2 heterotetrameric complex into alpha beta heterodimers, when observed by silver staining of sodium dodecyl sulfate-polyacrylamide gels. N-[3H]Ethylmaleimide ([3H]NEM) labeling in the absence of DTT pretreatment demonstrated that only the beta subunit had accessible sulfhydryl group(s). However, treatment of insulin receptors with DTT increased the amount of [3H]NEM labeling in the beta subunit as well as exposing sites on the alpha subunit. Further, incubation of the insulin receptors with the combination of DTT and insulin also demonstrated the apparent insulin-potentiated subunit reduction without any increase in the total amount of [3H]NEM labeling.(ABSTRACT TRUNCATED AT 250 WORDS)