Epilepsia en el paciente oncológico: prevención primaria e importancia en la selección del paciente de alto riesgo / Epilepsy in cancer patients: primary prevention and the importance of high-risk patient screening

Introducción: La epilepsia en el paciente oncológico presenta una prevalencia del 13%, especialmente elevada en pacientes con tumores cerebrales, así como una mayor morbimortalidad respecto de la epilepsia no tumoral. Sus mecanismos fisiopatógenos son diferenciadores, e incluyen la distorsión de la arquitectura cortical y la alteración del microambiente molecular tumoral y peritumoral favorecedor de glutamato. A pesar de ello, existe evidencia científica escasa e inconsistente acerca de aspectos fundamentales, como la profilaxis primaria postoperatoria, el perfil farmacológico idóneo o el tiempo de retirada de fármacos anticrisis tras la libertad de éstas. Desarrollo: Características como el bajo grado tumoral, el número/tamaño de las lesiones corticales, la localización (frontal, cortical/subcortical o área elocuente), las crisis tempranas y las alteraciones moleculares, como mutación IDH1/2, son factores favorecedores para la aparición de crisis. Dentro del tratamiento, la cirugía aportará citorreducción y control de crisis por escisión del área epileptógena, con libertad de crisis incapacitantes del 75-90%. Aunque sigue siendo un tema controvertido, el uso postoperatorio de fármacos anticrisis está contraindicado por las principales sociedades científicas por la escasa evidencia y el amplio espectro de efectos secundarios. Sin embargo, se emplean frecuentemente en la práctica clínica diaria. Conclusiones: Todo ello nos obliga a establecer un grupo de pacientes de ‘alto riesgo’ de crisis postoperatorias, que precisará seleccionar el fármaco anticrisis idóneo en prevención primaria, con una vía de administración que facilite un rápido efecto de acción y una farmacocinética que evite el metabolismo hepático y la inducción de CYP450 para conseguir un menor número de interacciones con quimioterápicos, corticoides y radioterapia. A pesar de ello, se describen tasas de farmacorresistencia del 20-40% y recidiva del 25-29%.(AU)

Introduction: Epilepsy in cancer patients has a prevalence of 13%, and is especially high in patients with brain tumours, with a higher morbidity and mortality rate compared to non-tumour-related epilepsy. Its physiopathogenic mechanisms are distinct and include distortion of the cortical architecture and alteration of the glutamate-enhancing tumoural and peritumoural molecular microenvironment. Nevertheless, there is scarce and inconsistent scientific evidence on some fundamental aspects, such as primary post-operative prophylaxis, the ideal pharmacological profile or the withdrawal time of antiseizure drugs after their release. Development: Characteristics such as low tumour grade, number/size of cortical lesions, location (frontal, cortical/subcortical or eloquent area), early seizures and molecular alterations, such as IDH1/2 mutation, are factors that favour the occurrence of seizures. Within the treatment, surgery will provide cytoreduction and seizure control by excision of the epileptogenic area, with 75-90% freedom from disabling seizures. Although still a controversial issue, the post-operative use of antiseizure drugs is contraindicated by the main scientific societies due to the scarce evidence and the wide spectrum of side effects. However, they are frequently used in daily clinical practice. Conclusions: All this forces us to establish a group of patients at ‘high risk’ of postoperative seizures, who will need to select the ideal antiseizure drug for primary prevention, with a route of administration that facilitates a rapid action effect and pharmacokinetics that prevents hepatic metabolism and CYP450 induction to achieve a lower number of interactions with chemotherapy, corticosteroids and radiotherapy. Despite this, drug resistance rates of 20-40% and relapse rates of 25-29% have been reported.(AU)

[Epilepsy in cancer patients: primary prevention and the importance of high-risk patient screening]. / Epilepsia en el paciente oncológico: prevención primaria e importancia en la selección del paciente de alto riesgo.

INTRODUCTION: Epilepsy in cancer patients has a prevalence of 13%, and is especially high in patients with brain tumours, with a higher morbidity and mortality rate compared to non-tumour-related epilepsy. Its physiopathogenic mechanisms are distinct and include distortion of the cortical architecture and alteration of the glutamate-enhancing tumoural and peritumoural molecular microenvironment. Nevertheless, there is scarce and inconsistent scientific evidence on some fundamental aspects, such as primary post-operative prophylaxis, the ideal pharmacological profile or the withdrawal time of antiseizure drugs after their release. DEVELOPMENT: Characteristics such as low tumour grade, number/size of cortical lesions, location (frontal, cortical/subcortical or eloquent area), early seizures and molecular alterations, such as IDH1/2 mutation, are factors that favour the occurrence of seizures. Within the treatment, surgery will provide cytoreduction and seizure control by excision of the epileptogenic area, with 75-90% freedom from disabling seizures. Although still a controversial issue, the post-operative use of antiseizure drugs is contraindicated by the main scientific societies due to the scarce evidence and the wide spectrum of side effects. However, they are frequently used in daily clinical practice. CONCLUSIONS: All this forces us to establish a group of patients at 'high risk' of postoperative seizures, who will need to select the ideal antiseizure drug for primary prevention, with a route of administration that facilitates a rapid action effect and pharmacokinetics that prevents hepatic metabolism and CYP450 induction to achieve a lower number of interactions with chemotherapy, corticosteroids and radiotherapy. Despite this, drug resistance rates of 20-40% and relapse rates of 25-29% have been reported.

TITLE: Epilepsia en el paciente oncológico: prevención primaria e importancia en la selección del paciente de alto riesgo.Introducción. La epilepsia en el paciente oncológico presenta una prevalencia del 13%, especialmente elevada en pacientes con tumores cerebrales, así como una mayor morbimortalidad respecto de la epilepsia no tumoral. Sus mecanismos fisiopatógenos son diferenciadores, e incluyen la distorsión de la arquitectura cortical y la alteración del microambiente molecular tumoral y peritumoral favorecedor de glutamato. A pesar de ello, existe evidencia científica escasa e inconsistente acerca de aspectos fundamentales, como la profilaxis primaria postoperatoria, el perfil farmacológico idóneo o el tiempo de retirada de fármacos anticrisis tras la libertad de éstas. Desarrollo. Características como el bajo grado tumoral, el número/tamaño de las lesiones corticales, la localización (frontal, cortical/subcortical o área elocuente), las crisis tempranas y las alteraciones moleculares, como mutación IDH1/2, son factores favorecedores para la aparición de crisis. Dentro del tratamiento, la cirugía aportará citorreducción y control de crisis por escisión del área epileptógena, con libertad de crisis incapacitantes del 75-90%. Aunque sigue siendo un tema controvertido, el uso postoperatorio de fármacos anticrisis está contraindicado por las principales sociedades científicas por la escasa evidencia y el amplio espectro de efectos secundarios. Sin embargo, se emplean frecuentemente en la práctica clínica diaria. Conclusiones. Todo ello nos obliga a establecer un grupo de pacientes de 'alto riesgo' de crisis postoperatorias, que precisará seleccionar el fármaco anticrisis idóneo en prevención primaria, con una vía de administración que facilite un rápido efecto de acción y una farmacocinética que evite el metabolismo hepático y la inducción de CYP450 para conseguir un menor número de interacciones con quimioterápicos, corticoides y radioterapia. A pesar de ello, se describen tasas de farmacorresistencia del 20-40% y recidiva del 25-29%.

Kinematic adjustments in the basketball jump shot against an opponent.

The aim of this study was to analyse the adjustments in technique made by a basketball player when shooting against an opponent. The subjects used consisted of 10 professional basketball players of the Spanish First Division League. Three-dimensional motion analysis based on video recordings (50 Hz) was used to obtain the kinematic characteristics of basketball jump shots with and without an opponent. It was found that when performing against an opponent the release angle of the ball increased, the flight time was reduced and postural adjustments as determined by the angles at the knee and shoulder increased, all significantly. There were several other non-significant differences that helped to interpret the changes in technique imposed by the presence of an opponent. It was suggested that when shooting with an opponent, players attempted to release the ball more quickly and from a greater height. This strategy will lessen the chance of the opponent intercepting the ball. It was concluded that the differences noted in the technical execution of the skill had implications for practice. It was suggested that training would benefit from practice with an opponent for at least some of the time to condition players to the demands which they were more likely to meet in the game situation.

Involvement of the calcium-specific protease, calpain, in the fertilizing capacity of human spermatozoa.

We recently reported the novel finding that human spermatozoa contain the calcium (Ca2+)-dependent protease, calpain. In somatic cells this protease mediates several cellular activities regulated by Ca2+ including membrane fusibility during cell-to-cell interactions. In this paper we examined the participation of sperm calpain in sperm-oocyte penetration, a process that is dependent on Ca2+ and involves membrane fusion between the two cells. Oocyte penetration was assessed using ejaculated spermatozoa from fertile men and zona-free hamster oocytes. Penetration rate was impaired by the presence of the active-site calpain inhibitor, calpain inhibitor-I, in a dose-dependent manner. At 1 mM, penetration scores were reduced by 65% (p < 0.01; n=5). The effects did not involve the oocyte, nor did the inhibitor alter sperm motility. Similar inhibitory effects on sperm penetration capacity were observed with specific antibodies directed either against calpain-I or calpain-II, the two forms of calpains described in somatic cells. At 1:1000 antibody dilution, penetration was inhibited 50 and 60% with anti-calpain-I and anti-calpain-II antibodies, respectively (p < 0.01; n=6). Furthermore, a combination of these two antibodies reduced the penetration rates by 75% (p < 0.01; n=6). We conclude that calpain inhibitor and anti-calpain antibodies impair human sperm capacity to fuse and penetrate the oocyte. These findings suggest that sperm calpain is a novel component of the biochemical processes that regulate the fertilizing capacity of human spermatozoa.

Short-term prediction of chaotic time series by using RBF network with regression weights.

We propose a framework for constructing and training a radial basis function (RBF) neural network. The structure of the gaussian functions is modified using a pseudo-gaussian function (PG) in which two scaling parameters sigma are introduced, which eliminates the symmetry restriction and provides the neurons in the hidden layer with greater flexibility with respect to function approximation. We propose a modified PG-BF (pseudo-gaussian basis function) network in which the regression weights are used to replace the constant weights in the output layer. For this purpose, a sequential learning algorithm is presented to adapt the structure of the network, in which it is possible to create a new hidden unit and also to detect and remove inactive units. A salient feature of the network systems is that the method used for calculating the overall output is the weighted average of the output associated with each receptive field. The superior performance of the proposed PG-BF system over the standard RBF are illustrated using the problem of short-term prediction of chaotic time series.

Changes in expression of adenyl cyclase activity in human endometrium during hormone replacement therapy and ovarian stimulation.

We have investigated membrane fractions prepared from human endometrium for activity of the signalling adenyl cyclase (AC). We characterized the AC guanine nucleotide-binding proteins (G proteins) and examined the changes in AC activity during evaluation cycles of oestrogen and progesterone replacement therapy as well during ovarian stimulation cycles. AC activity was determined by the conversion of substrate ATP into cyclic AMP under basal conditions and in the presence of guanine nucleotide or forskolin. G proteins were determined by Western Blot using specific polyclonal antibodies against Gsalpha, Gi1,2alpha and Gi3alpha. Our results indicate that endometrial AC was highly responsive to activation by both guanine nucleotide and forskolin and its rate of cyclic AMP production was highly pronounced. Mean activity reached 920 pmol/l/min/mg membrane protein in the presence of forskolin, a value approximately 5-fold higher than those detected in corpus luteum. Hormonal induction of AC activities increased Gsalpha protein, which couples with and stimulates the catalytic component of AC. We conclude that human endometrium is rich in AC and that enzyme activity is induced by oestrogen and progesterone treatment. These data strongly support the concept that the transmembrane signalling AC system and its messenger cyclic AMP are major regulators of endometrial function in the human.

Calpain-calpastatin: a novel, complete calcium-dependent protease system in human spermatozoa.

Calpain, a calcium (Ca2+)-activated cysteine protease presents in several somatic mammalian cells, has been demonstrated to mediate specific Ca2+-dependent reactions including cell fusion. Because spermatozoa cells have an absolute Ca2+ requirement for penetration of oocytes, we have postulated that calpain would also be found in mammalian spermatozoa. Here we show that whole sperm homogenate and cell fractions prepared from ejaculated human spermatozoa contain calpain activity. Specific calpain inhibitors impaired this proteolytic activity. Unlike the enzyme described in somatic cells, sperm calpain was mostly particulate in nature and its activity was maximal at pH 9.0. Presence of sperm calpain was confirmed by immunoblot analysis using specific anti-calpain I and anti-calpain II antibodies. A 67 kDa calpain II protein and a 75 kDa calpain I protein were detected. Also spermatozoa contain the endogenous calpain inhibitor, calpastatin. We detected 158.8 +/- 24.5 (mean +/- SD) fmol calpastatin/mg sperm protein. Immunoblot analysis using specific antibodies showed a 68 kDa calpastatin protein located in the cytosolic fraction. This is the first demonstration that a complete calpain-calpastatin system exists in mammalian spermatozoa. Because calpain is a unique effector system for calcium-dependent processes, our data reveals a novel mechanism by which calcium exerts its regulatory functions in spermatozoa.

Serum levels of androstenedione, testosterone and dehydroepiandrosterone sulfate in patients with premature ovarian failure to age-matched menstruating controls.

Serum concentrations of androstenedione, testosterone and dehydroepiandrosterone sulfate (DHEAS) were measured in 29 patients with premature ovarian failure (POF) and an identical number of age-matched normal control subjects. The study was aimed at determining possible differences in androgen concentrations of ovarian and adrenal origin in POF patients and age-matched normal menstruating controls. Serum testosterone and DHEAS concentrations in the 2 populations were not significantly different. The serum androstenedione concentration in the POF patient group (3,077.50 +/- 1,122.33 pmol/l) was significantly lower than in age-matched normal control subjects (4,167.70 +/- 1,381.09 pmol/l, p < 0.005), possibly reflecting the loss of ovarian androstenedione secretion and/or a subtle defect in adrenal steroidogenic capacity.

Evidence that hormone receptors couple and activate a common signal transducer adenylyl cyclase in corpus luteum.

OBJECTIVES: To investigate whether membrane-bound hormone receptors in corpus luteum (CL) couple to one common adenylyl cyclase (AC) or whether each receptor is coupled to its own AC. DESIGN: Plasma membranes from rat CL were used to assess the coupling of hormone receptors to AC under conditions allowing full expression of the enzyme system. The response to hCG, prostaglandin E2 alpha (PGE2), and isoproterenol were analyzed. MAIN OUTCOME MEASURE: Adenylyl cyclase activity was monitored by the direct conversion of [infinity-32P]ATP into [32P]cyclic AMP. Results were expressed as pmol/min per mg membrane protein. RESULTS: Addition of hCG (200 mIU), PGE2 (100 microM), and isoproterenol(100 microM) in the presence of a saturating (100 microM) concentration of guanine nucleotide resulted in a marked stimulation of the enzyme compared with controls, reaching 552 +/- 28, 537 +/- 42, and 558 +/- 32 (mean +/- SEM), respectively. Addition of hormones in pairs or all three together did not result in an additive response of the individual effects. Thus, in the presence of the three hormones together, AC stimulation was 582 +/- 41 (n = 5), a value that was not significantly different from the stimulation observed with each hormone alone. CONCLUSION: These data indicate that luteal membranes contain a single AC system. Therefore, hormone receptors couple and activate a common signal transducer AC in luteal membranes.

Enzymatic amplification of specific deoxyribonucleic acid sequences from single cells: evaluation of a simplified and rapid method for use in preimplantation genetic diagnosis.

OBJECTIVE: To develop a simplified polymerase chain reaction (PCR) protocol on single cells for the purpose of preimplantation genetic diagnosis. Also to evaluate a new thermal cycler, RoboCycler 40 (Stratagene, La Jolla, CA), for reducing the time to complete PCR amplification. DESIGN: PCR amplification without DNA purification or reamplification of a 149 base pair (bp) segment of the human Y chromosome was used as a model. The assay was tested in human fetal cells, single lymphocytes and single human blastomeres. RESULTS: Amplification of the 149 bp segment using fetal cells was 100% correct. Results on single lymphocytes were concordant in all but one of the 15 male cases. However, 2 of the 25 female cases were identified as male suggesting the occurrence of DNA contamination. Analysis of 61 blastomeres were concordant in 57 cases (93%); results for male blastomeres showed 12% of false negatives. No false positives were detected for female cells. Amplification using the simplified PCR protocol in combination with the RoboCycler was completed in 2 hours. CONCLUSION: These data show that this PCR assay performed directly, without DNA extraction or purification and without re-amplification is a practical and effective approach for amplification of specific DNA sequences in single cells. Furthermore, the simplified PCR protocol significantly reduced the time to complete DNA amplification. The reduced time is expected to facilitate the management of a routine program for preimplantation genetic diagnosis.

Relationship between serum estradiol concentration and IGF-I, IGF-II and IGF-binding proteins in patients with premature ovarian failure on short-term estradiol therapy.

OBJECTIVE: Insulin-like growth factors (IGFs) exert stimulatory effects on follicular growth and development, and early embryogenesis. In view of this, we studied the effect of short-term estradiol treatment, as used in preparing the uterus for embryo implantation, on the serum concentrations of IGFs and their binding proteins (IGFBP) in patients with premature ovarian failure (POF). PATIENTS AND METHODS: Twenty-four patients with POF, enrolled in an assisted reproduction program, were treated with increasing doses of estradiol up to 8 mg daily for 6 weeks. Blood was sampled for measurement of serum estradiol, IGF-I, IGF-II, and IGFBP 1, 2 and 3 at various times during estradiol treatment. RESULTS: There was no significant correlation between serum estradiol concentrations and the serum concentrations of IGF-I and IGF-II. As expected, IGF-I and IGF-II concentrations in serum correlated positively with the serum concentration of IGFBP-3, the major IGF-binding protein in serum. CONCLUSION: The results of this study suggest that estradiol therapy as used to prepare the uterus for implantation has no significant effect on serum IGF-I and IGF-II concentrations, and therefore probably does not influence, via an IGF-mediated mechanism, the success of implantation and early embryonic development.

Status of hCG/LH receptor and G proteins in human endometrium during artificial cycles of hormone replacement therapy.

OBJECTIVES: We examined the existence of hCG/LH receptors and associated GTP-binding (G) proteins in membrane fractions of nonpregnant human endometrium and investigated whether their expression is affected, in vivo, by estrogen and progesterone replacement therapy. METHODS: A pool of normal endometrial biopsy specimens (n = 5) was initially used to characterize receptors and G proteins. Subsequently, biopsy specimens (n = 22) were obtained from 11 patients undergoing evaluation cycles of hormone replacement therapy (HRT). From each patient, two specimens were collected on successive cycle days: on day 0 (last day of estrogen) and on either day 3, 6, or 9 of progesterone supplementation. Both hCG/LH receptor and G proteins were determined in membrane (10,000 x g) fractions by immunoblot analysis using specific polyclonal antibodies against synthetic fragments of hCG/LH receptor and against G proteins. Membrane fractions from rat brain and rat corpus luteum were used as controls. Proteins were loaded on the gel under reducing conditions. RESULTS: The receptor antibody immunoreacted with a protein of approximately 68 kd in endometrial membranes. A similar protein was detected in rat corpus luteum. The G-protein antibodies detected Gs alpha, Gi3 alpha, Gi1 alpha/Gi2 alpha, and common beta subunits in endometrial membranes with a molecular weight of 48-42 kd, 41 kd, 40 kd, and 37 kd, respectively. Analysis of membranes obtained during HRT indicated that levels of hCG/LH receptors remained fairly constant throughout the cycle days (days 0, 3, 6, and 9). Similar results were observed for Gi1 alpha/Gi2 alpha and Gi3 alpha. In great contrast, Gs alpha was low at day 0 but increased with the administration of progesterone (days 3, 6, and 9). CONCLUSIONS: Human endometrium contains both membrane-bound hCG/LH receptors and associated G proteins. During HRT, progesterone supplementation to estrogen therapy enhances the expression of Gs alpha protein subunit, but not hCG/LH receptors.

Stimulation of ovarian adenylyl cyclase activity by gonadotrophins during the natural and gonadotrophin-induced cycles in the hamster.

In this study we utilized the hamster ovary as a model to investigate the effects of ovulation induction with gonadotrophin on the activation of the signal transducer effector system, adenylyl cyclase (AC). For this purpose, we prepared membrane particles from the ovary and analysed both gonadotrophin-sensitive AC and non-receptor-mediated activation during a cycle in which ovulation and luteinization were achieved by pregnant mare's serum gonadotrophin (PMSG)/human chorionic gonadotrophin (HCG) administration. Results were directly compared with AC activation in similarly prepared membranes obtained at different stages of the natural unstimulated cycle. AC activity was quantified by the direct conversion of ATP substrate into cyclic adenosine monophosphate (cAMP). Measurements of ovarian weights, serum oestradiol and progesterone concentrations provided a solid base from which to evaluate the functional status of the ovary at each time period during the natural and stimulated cycles. We found that ovarian membranes contain functional components of the AC system and demonstrated that AC is highly dependent on hormonal changes and the functional state of the ovary. Thus, during the natural cycle, ovarian AC showed relatively constant responsiveness to follicle-stimulating hormone (FSH) throughout the cycle, whereas responsiveness to luteinizing hormone (LH)/HCG reached its peak during the luteal phase. On the other hand, during the stimulated cycle, sensitivity to FSH and LH/HCG varied considerably, being absent during the peri-ovulatory period. AC responsiveness to gonadotrophins was only regained 48 h after ovulation. Also during the peri-ovulatory period of the gonadotrophin-induced cycle, stimulation of ovarian AC with non-hormonal activators declines.(ABSTRACT TRUNCATED AT 250 WORDS)

The predictive value of a single beta human chorionic gonadotropin in pregnancies achieved by assisted reproductive technology.

OBJECTIVE: To investigate whether a single serum beta-hCG in pregnancies achieved by assisted reproductive technologies (ART) can accurately predict pregnancy viability and, in viable pregnancies, multiple gestation. DESIGN: Four hundred sixty-one consecutive successful ART pregnancies were studied retrospectively. Seventy-one of the 461 patients were excluded because their beta-hCG was either drawn on the incorrect day or outside our facility. Three hundred ninety subjects had a serum beta-hCG drawn 14 days after ET or 16 days after gamete transfer. The beta-hCG samples were analyzed by immunoradiometric assay based on the Third International Reference Standard (IRS) (First International Reference Preparation (IRP)). Pregnancy status was followed, at minimum, through the first trimester. RESULTS: One hundred fifty (38%) of the 390 were found to be nonviable, resulting in spontaneous abortion (n = 38, 10%), ectopic pregnancy (n = 27, 6%), or biochemical pregnancies (n = 85, 22%). A statistically significant difference by the Scheffe F-test was found between the mean beta-hCG value of the nonviable (115 mIU/mL) (conversion factor to SI unit, 1.00) and viable (428 mIU/mL) pregnancies. The positive predictive value of a single beta-hCG > 100 mIU/mL in distinguishing viable from nonviable pregnancies was 0.83 (sensitivity 91%, specificity 71%). Of the 240 viable pregnancies, 74 (32%) were multiple gestations (57 twins, 14 triplets, and 3 quadruplets). The mean beta-hCG of the singleton pregnancies (266 mIU/mL) was significantly different from that of the multiple gestations (792 mIU/mL). The positive predictive value of a single serum beta-hCG < or = 400 mIU/mL in distinguishing singleton from multiple gestations was 0.92 (sensitivity 86%, specificity 82%). CONCLUSION: A single early serum beta-hCG may be used in ART pregnancies to predict which pregnancies will continue beyond the first trimester and to identify multiple gestations. Early reassuring tests may reduce anxiety.

Evidence for a novel adenylyl cyclase in human epididymal sperm.

Since cAMP is considered to play a major role in the acquisition of maturation and fertilizing capacity of mammalian sperm, we investigated the expression of cAMP-synthesizing adenylyl cyclase (AC) in sperm retrieved directly from the human epididymis. Particulate fractions were prepared from purified epididymal sperm samples and AC was monitored by the direct conversion of ATP into cAMP. We report that in great contrast to human ejaculated sperm and other mammalian sperm cells, the human epididymal sperm do not express a Mn(2+)-sensitive AC. However, a functional AC was readily detectable in these sperm cells in the presence of saturating concentrations of Ca2+ (50mM) and bicarbonate (HCO3-, 50mM), a combination that causes maximal activation in human ejaculated sperm. Using these conditions, human epididymal sperm AC showed similar capacity to generate cAMP compared to human ejaculated sperm AC. When assays were performed in the presence of Mg2+ and a saturating concentration of GMP-P(NH)P (50 microM), the hydrolysis-resistant GTP analog, and forskolin (100 microM), no activity was detected indicating that the epididymal sperm AC differs from that in somatic cells. These data demonstrate that human epididymal sperm contain an AC that is unique and different from the enzyme system described in somatic cells and other mammalian sperm cells, including human ejaculated sperm.

Melatonin and gonadotropin secretion after acute exercise in physically active males.

Serum concentrations of luteinizing hormone (LH), follicle stimulating hormone, testosterone (T) and melatonin were measured in seven physically active male volunteers after exercise on a treadmill using the Bruce protocol. Measurements were made on blood samples obtained before exercise, within 30 s after exercise, at 15 min after exercise, and subsequently at 30-min intervals after exercise for a total duration of 180 min. Serum LH concentration fell from a peak post-exercise level of 15.7 (4.7) IU.l-1 [mean (SD)] to a nadir of 10.3 (2.4) IU.l-1 (P < 0.004). Nadir values in individual volunteers were seen between 60 and 150 min after exercise. This fall in serum LH was paralleled by a similar fall in the concentration of serum T. Serum melatonin concentrations did not change significantly after exercise. It is concluded that melatonin, despite is reported anti-gonadotropic properties, does not play a role in the depression of serum LH after acute strenuous exercise in physically active males.

Penetration of zona-free hamster oocytes using human sperm aspirated from the epididymis of men with congenital absence of the vas deferens: comparison with human in vitro fertilization.

OBJECTIVES: To assess the ability of sperm aspirated from the epididymis of men with congenital absence of the vas deferens to penetrate zona-free hamster oocytes. To directly compare the performance of human epididymal sperm in the zona-free hamster oocyte sperm penetration assay (SPA) with the results of human in vitro fertilization (IVF). DESIGN: Sperm penetration assay was carried out with epididymal sperm retrieved microsurgically, and with ejaculated sperm obtained from fertile donors (internal controls). For direct comparison, SPA was performed with the same epididymal sperm sample used for IVF. PATIENTS, PARTICIPANTS: Men with congenital absence of the vas deferens undergoing sperm aspiration as part of their infertility treatment and control donors who provided ejaculated sperm. RESULTS: Epididymal sperm penetrated SPA with a score of 0% to 30%. The SPA scores for internal controls using ejaculated sperm was 30% to 71%. Linear regression analysis of the association between penetration scores in SPA and fertilization rate in IVF indicated a positive correlation that was highly significative. CONCLUSIONS: These findings using SPA confirm previous reports on the fertilizing potential of human epididymal sperm and its ability to produce normal pregnancies. The good correlation between SPA and human IVF using epididymal sperm suggest that SPA is an excellent bioassay to test laboratory experimental conditions for improving fertilizing capacity of human epididymal sperm.

Regulation of cyclic adenosine monophosphate synthesis in human ejaculated spermatozoa. I. Experimental conditions to quantitate membrane-bound adenylyl cyclase activity.

Although cyclic adenosine monophosphate (cAMP) is an important regulator of motility and metabolism in human spermatozoa, little is known on the cellular system responsible for its synthesis. Here, we investigated the experimental conditions directly to quantitate adenylyl cyclase (AC) activity synthesizing cAMP in human ejaculated spermatozoa and analysed the general properties of the enzyme. A 10,000 g membrane fraction was prepared from washed sperm cells homogenized by sonication. AC activity was monitored by the direct conversion of [alpha-32P]adenosine triphosphate (ATP) into [32P]cAMP. Using a nucleoside triphosphate regenerating system to ensure availability of ATP substrate, the human sperm AC showed a steady production of cAMP for at least 1 h. The assay was optimized for pH, buffer concentration, membrane protein and substrate concentration. Activity was dependent upon the presence of Mn2+ as a divalent cation and showed a pH optimum between 7.0 and 8.5. Optimal activity required 5 mM ATP, 1 mM ethylenediamine tetraacetic acid (EDTA) and 20-40 mM total MnCl2. Dependence on Mn2+ was not mandatory; Mg2+ at 5-40 mM also supported significant activity, but the activity was 4-6 times lower than that with Mn2+. Regardless of the presence of Mn2+ or Mg2+ as divalent cation in the assay, human sperm AC was insensitive to the regulatory ligands NaF, guanine nucleotide or forskolin. Insensitivity to these ligands supports the proposal that this enzyme system does not contain a stimulatory guanine nucleotide-binding regulatory protein and that its catalytic component is unique and different from that of somatic cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Regulation of cyclic adenosine monophosphate synthesis in human ejaculated spermatozoa. II. The role of calcium and bicarbonate ions on the activation of adenylyl cyclase.

In this study, we have applied our previous data describing the experimental conditions necessary for expression of cyclic adenosine monophosphate (cAMP)-synthesizing adenylyl cyclase in human ejaculated spermatozoa, to investigate the direct effects of calcium (Ca2+) and bicarbonate (HCO3-) upon activation of the enzyme in vitro. We report that the effects of Ca2+ and HCO3- were significantly dependent on the status of the enzyme activity. Thus, at a near saturating (10 mM) concentration of MnCl2 giving high enzyme activity, addition of less than 10 mM HCO3- did not affect adenylyl cyclase activity and higher concentrations inhibited the enzyme, with 50 mM HCO3- reducing the activity by 33%. Also, addition of less than 20 mM CaCl2 alone or in combination with 10 mM HCO3- did not significantly change the enzyme activity. In great contrast, enzyme activation was highly responsive to Ca2+ and HCO3- when MnCl2 was present at a concentration giving submaximal enzyme activity. Thus, at 2 mM MnCl2, adenylyl cyclase was markedly increased by CaCl2 concentrations between 10 and 100 mM. The activation was further enhanced by increasing concentrations of HCO3-, with 50 mM HCO3- giving the highest activity at 50-100 mM CaCl2. Activation by CaCl2 was also observed in the absence of added MnCl2, being significantly greater than basal activity at 10 mM CaCl2 and maximal at 100 mM CaCl2.(ABSTRACT TRUNCATED AT 250 WORDS)