Oocyte ERM and EWI Proteins Are Involved in Mouse Fertilization.

In mammalian fertilization, the link between the oocyte plasma membrane and underneath cytoskeleton has often been associated to key elements of successful gamete fusion, like microvilli shaping or CD9 function, but its effective role has poorly been studied. EWI-2 and EWI-F as cis partners of CD9, and ERM proteins (Ezrin, Radixin and Moesin) that both attach to the actin cytoskeleton and to the EWI are part of the molecules that make the link between the oocyte membrane and its cytoskeleton. This study aims to assay through siRNA inhibition, the involvement of these ERM and EWI molecules in mouse fertilization, their role in the microvilli morphology of the egg but also their possible contribution to the cortical tension, a parameter that reflects the mechanical behavior of the oocyte cortex. Whereas inhibiting separately the expression of each protein had no effect on fertilization, the combined inhibition of either EWI-2/EWI-F or the three ERM triggered a significant decrease of the fertilization index. This inhibition seems to correlate with an increase in the radius of curvature of the oocyte microvilli. It also causes a decrease of the oocyte cortical tension. These results show the importance of EWI-2 and EWI-F and ERM proteins in the smooth running of a fertilization event and support their involvement in the microvilli architecture of the oocyte and in its mechanical properties.

Overweight and obesity in Eastern Morocco: Prevalence and associated risk factors among high school students.

BACKGROUND: Overweight and obesity in children and adolescents have become a major public health problem affecting most countries worldwide. The purpose of the study was to assess the prevalence and risk factors of overweight and obesity among public high school students in Eastern Morocco. METHODS: A cross-sectional survey was conducted between February and May 2014 among a sample of 2271 students (1086 girls and 1185 boys). References from the International Obesity Task Force (IOTF) were used to determine the prevalence of overweight and obesity. RESULTS: The prevalence of overweight and obesity reached 12.2% (14.2% in girls vs 10.4% in boys, P<0.01) and 3.0% (3.1% in girls vs 2.8% in boys), respectively. Risk factors associated with overweight and obesity were urban residence (OR=1.76; [1.18-2.63]; P<0.01), father's income≥5000MAD (OR=1.32; [1.02-1.70]; P<0.05), father's overweight (including obesity) (OR=1.87; [1.38-2.54]; P<0.001) and female sex (OR=1.31; [1.02-1.68]; P<0.05). CONCLUSION: The prevalence of overweight/obesity has reached an alarming rate among high school students in the Eastern region of Morocco. The findings of the present study suggest an urgent need to set up a strategy to prevent and combat this epidemic.

JUNO, the receptor of sperm IZUMO1, is expressed by the human oocyte and is essential for human fertilisation.

STUDY QUESTION: Is JUNO protein present at the surface membrane of human oocytes and involved in the fertilisation process? SUMMARY ANSWER: JUNO protein is expressed on the plasma membrane of human oocytes and its inhibition by a monoclonal antibody completely blocks gamete fusion. WHAT IS KNOWN ALREADY: Fusion of gamete membranes is the culminating event of the fertilisation process, but its molecular mechanisms are poorly understood. Until now, three molecules have been shown to be essential: CD9 tetraspanin in the oocyte, Izumo1 protein on the sperm and Juno, its corresponding receptor on the oocyte. Oocyte CD9 and sperm IZUMO1 have been identified in human gametes and their interaction is also well-conserved among several mammalian species. The presence of JUNO on human oocytes, however, has not yet been reported, nor has its role in fertilisation been investigated. STUDY DESIGN, SIZE, DURATION: We selected an anti-human JUNO antibody in order to investigate the presence of JUNO on the oocyte membrane surface and studied its potential involvement in gamete membrane interaction during fertilisation. PARTICIPANTS/MATERIALS, SETTING, METHODS: Monoclonal antibodies against human JUNO (anti-hJUNO mAb) were produced by immunisation of mice with HEK cells transfected with the putative human JUNO sequence (HEK-hJUNO). These antibodies were used for immunostaining experiments and in vitro fertilisation assays with human gametes (GERMETHEQUE Biobank). MAIN RESULTS AND THE ROLE OF CHANCE: Three hybridoma supernatants, verified by immunostaining, revealed specifically HEK-hJUNO cells. The three purified monoclonal antibodies, FJ2E4 (IgG1), FJ8E8 (IgG1) and FJ4F5 (IgG2a), recognised the soluble recombinant human JUNO protein and, in a western blot of HEK-hJUNO extracts, a protein with an expected MW of 25 kDa. In addition, soluble recombinant human IZUMO protein inhibited the binding of anti-hJUNO mAbs to cells expressing hJUNO. Using these anti-hJUNO mAbs in immunostaining, we identified the presence of JUNO protein at the plasma membrane of human oocytes. Furthermore, we revealed a progressive expression of JUNO according to oocyte maturity. Finally, we showed that human zona-free oocytes, inseminated in the presence of anti-hJUNO mAb, were not fertilised by human sperm. These results suggest that, as seen in the mouse, JUNO is indeed involved in human gamete membrane fusion during fertilisation. LARGE-SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: In accordance with French bioethics laws, functional tests were performed using zona-free oocytes, which of course does not fully encompass all normal in vivo physiological conditions. However, these in vitro tests do provide direct information regarding sperm-oocyte membrane interactions. WIDER IMPLICATIONS OF THE FINDINGS: Mechanisms of gamete fusion appear to be homologous between mice and humans. However, some differences do exist and analysing the human mechanisms is essential. In fact, this is the first report describing the presence of JUNO on human oocytes and its involvement in human fertilisation. This discovery allows further examination of the understanding of molecular mechanisms that drive gamete fusion: a crucial challenge at a time when infertility affects 16% of reproductively active couples. STUDY FUNDING/COMPETING INTERESTS: This work was supported by the Agence Nationale pour la Recherche, Grant no. ANR-13-BVS5-0004, and by Association Institut du Cancer et d'Immunogénétique (ICIG). There are no competing interests.

Effects of Juglans regia Root Bark Extract on Platelet Aggregation, Bleeding Time, and Plasmatic Coagulation: In Vitro and Ex Vivo Experiments.

Platelets have an important role in thrombosis and haemostasis. Hyperactivity of the platelets has been associated with thromboembolic diseases and represents the main cause of complications of cardiovascular diseases. Crude aqueous extract (CAE) of Juglans regia root bark was evaluated for bleeding time, antiaggregant activity by using agonists, thrombin, ADP, collagen, or arachidonic acid (in vitro and ex vivo), and anticoagulant activity by measuring the clotting parameters: activated partial thromboplastin time, prothrombin time, thrombin time, and fibrinogen dosage (in vitro and ex vivo). The result of this study reported that the strongest antiaggregant effect of CAE in vitro was observed on the ADP-induced aggregation with inhibitions up to 90 %, while, in ex vivo experiments, the inhibition (more than 80 %) was observed with all agonists. Anticoagulant effect of CAE significantly prolonged the TT and decreased the fibrinogen level in vitro and ex vivo without interfering with APTT and PT. The bleeding time in mice and rats was significantly increased by CAE. The antiplatelet and anticoagulant effect observed in this study suggest that Juglans regia could have antithrombotic and/or thrombolytic activities and provide an alternative therapy against thrombotic complications related to cardiovascular diseases.

Growth arrest specific 1 (Gas1) and glial cell line-derived neurotrophic factor receptor α1 (Gfrα1), two mouse oocyte glycosylphosphatidylinositol-anchored proteins, are involved in fertilisation.

Recently, Juno, the oocyte receptor for Izumo1, a male immunoglobulin, was discovered. Juno is an essential glycosylphosphatidylinositol (GIP)-anchored protein. This result did not exclude the participation of other GIP-anchored proteins in this process. After bibliographic and database searches we selected five GIP-anchored proteins (Cpm, Ephrin-A4, Gas1, Gfra1 and Rgmb) as potential oocyte candidates participating in fertilisation. Western blot and immunofluorescence analyses showed that only three were present on the mouse ovulated oocyte membrane and, of these, only two were clearly involved in the fertilisation process, namely growth arrest specific 1 (Gas1) and glial cell line-derived neurotrophic factor receptor α1 (Gfrα1). This was demonstrated by evaluating oocyte fertilisability after treatment of oocytes with antibodies against the selected proteins, with their respective short interference RNA or both. Gfrα1 and Gas1 seem to be neither redundant nor synergistic. In conclusion, oocyte Gas1 and Gfrα1 are both clearly involved in fertilisation.

Effect of induced peritoneal endometriosis on oocyte and embryo quality in a mouse model.

PURPOSE: To assess the impact of peritoneal endometriosis on oocyte and embryo quality in a mouse model. METHODS: Peritoneal endometriosis was surgically induced in 33 B6CBA/F1 female mice (endometriosis group, N = 17) and sham-operated were used as control (sham group, N = 16). Mice were superovulated 4 weeks after surgery and mated or not, to collect E0.5-embryos or MII-oocytes. Evaluation of oocyte and zygote quality was done by immunofluorescence under spinning disk confocal microscopy. RESULTS: Endometriosis-like lesions were observed in all mice of endometriosis group. In both groups, a similar mean number of MII oocytes per mouse was observed in non-mated mice (30.2 vs 32.6), with a lower proportion of normal oocytes in the endometriosis group (61 vs 83 %, p < 0.0001). Abnormalities were incomplete extrusion or division of the first polar body and spindle abnormalities. The mean number of zygotes per mouse was lower in the endometriosis group (21 vs 35.5, p = 0.02) without difference in embryo quality. CONCLUSIONS: Our results support that induced peritoneal endometriosis in a mouse model is associated with a decrease in oocyte quality and embryo number. This experimental model allows further studies to understand mechanisms of endometriosis-associated infertility.

[Prostatitis and fertility: the biologist's point of view]. / Prostatite, fertilité : le point de vue du biologiste.

There are two leukocytospermia: the leukocytospermia below 10(6) cells/ml comes from the epididymis. It is physiological, improves sperm quality and ART outcomes and therefore must be respected. Leukocytospermia above 10(6) cells/ml are of prostatic origin and reflect a chronic prostatitis. The results of IVF and ICSI with these sperm are always surprisingly improved when compared to those obtained using semen without leukocytes at all. But this improvement is offset by a dramatic increase in the miscarriage rate. Should we treat this leukocytospermia or its cause? A clinical trial is conducted in Cochin hospital with the PHRC Sigma (Male Genital Track Inflammatory Syndrome) that will help us answer this question. It seems, a priori, that it is better to treat the cause and to respect the leukospermia.

[Sperm reservoir in mice: involvement of ADAMs]. / Réservoir spermatique chez la souris : implication des ADAMs.

Mammalian sperm migrate over long distances through the female genital tract before reaching the oviduct where fertilization occurs. This process is more complex than predicted by the movement of sperm. The oviduct is composed of three major segments: the uterotubal junction, the isthmus and the ampulla. These structures appear to play roles for the success of fertilization. Gene knockout approaches of several genes in mice suggest that the migration of spermatozoa in the oviduct is regulated to allow competent gametes encounter ensuring the success of fertilization with minimum risk of polyspermy. The sperm of male mice deleted for following genes: Calmegin, Calsperin, Angiotensin-Converting-Enzyme, Adam1a, Adam2 or Adam3 are morphologically normal and motile, but not able to pass through the uterotubal junction. The precise mechanism of how these molecules facilitate the passage of spermatozoa through the uterotubal junction is still unknown, but Adam3 seems to be the major factor in this process since it is implicated in these six lines of mutant mice.

Spermatogenetic inhibition in men taking a combination of oral medroxyprogesterone acetate and percutaneous testosterone as a male contraceptive method.

BACKGROUND: We previously demonstrated in a small pilot study that oral medroxyprogesterone acetate and percutaneous testosterone (OMP/PT) induce reversible spermatogenesis suppression. The aims of this study were to determine the rate of spermatogenetic inhibition and recovery and to obtain preliminary data on efficacy for a larger population under OMP/PT. METHODS: A total of 35 healthy men with normal spermiograms requesting male hormonal contraception were treated with OMP (20 mg/day) and PT (50-125 mg/day) for periods up to 18 months. Couples were included in a contraceptive efficacy phase after a value of ≤1 million/ml spermatozoa was reached between 1 and 3 months of treatment. RESULTS: Sperm counts decreased by 47% at 1 month, reaching 90% at 2 months and 98-100% between 4 and 8 months. At 3 months, 80% of men had ≤1 million/ml spermatozoa. Follicle-stimulating hormone and luteinizing hormone decreased to 35% of pretreatment levels after 1 month of treatment and to 75-80% at 2 and 6 months, respectively. Plasma testosterone and estradiol levels were in the eugonadal range at 3, 6, 9 and 12 months of treatment. Dihydrotestosterone concentrations were 2-4 times higher than pretreatment values. The rate of spermatogenetic recovery was rapid (73 ± 29.5 days). During the efficacy phase (211 months for 25 couples), one pregnancy attributable to poor compliance of the male partner was observed. CONCLUSIONS: OMP/PT efficiently inhibits spermatogenesis in 80% of men, maintains testosterone at physiological levels and avoids the need for parenteral administration, which is poorly accepted by French men. These results justify larger studies to define a more adequate dosage of OMP/PT and to confirm its efficacy and safety.

Hypertensive effects of oral administration of the aqueous extract of Solanum torvum fruits in L-NAME treated rats: evidence from in vivo and in vitro studies.

ETHNOPHARMACOLOGICAL RELEVANCE: Solanum torvum fruits are commonly used in Cameroonian traditional medicine for treatment of arterial hypertension. It has been previously shown that intravenous administration of aqueous extract from dried fruits (AEST) reduced blood pressure. AIM: The present work evaluates acute toxicity and effects of oral administration of AEST in chronic arterial hypertension induced by L-NAME. Effects of AEST were also evaluated on isolated aorta. MATERIALS AND METHODS: AEST (200 mg/kg/day, p.o.) was given solely or concomitantly with L-NAME (40 mg/kg/day, p.o.) for 30 consecutive days. Animal body weight, systolic blood pressure and heart rate were measured before stating the treatment and at the end of each week. Urinary volume and urinary sodium and potassium contents were quantified before and at days 1, 15 and 30 of the treatment. Aorta from treated animals was tested for their sensitivity to noradrenaline and carbachol. Aorta from normal untreated rats was used to evaluate the in vitro vascular effect of AEST. RESULTS: The results showed that AEST did induce neither mortality nor visible signs of toxicity. When given solely or in co-administration with L-NAME, AEST significantly reduced animal's body weight. It amplified the hypertensive and cardiac hypertrophy effect of L-NAME and did not affect these parameters in normotensive animals. AEST increased the sensitivity to noradrenaline in normotensive and significantly reduced it in hypertensive animals. AEST significantly increased urinary volume and sodium excretion in L-NAME treated animals while reducing the sodium excretion in normotensive. In vitro, AEST induced a potent partial endothelium-dependent contraction of aortic ring; contractions that were partially antagonized by prazosin and verapamil and were not relaxed by carbachol. CONCLUSION: These results suggest that oral chronic administration of AEST induced potentiation of arterial hypertension and cardiac hypertrophy in L-NAME treated rats. These effects may result from a reduction in sensitivity to vasorelaxant agents and increase in hypersensitivity to contractile factors. AEST possess potent in vitro vasocontractile activity that may result from activation of both alpha(1)-adrenergic pathway and calcium influx.

Short gamete co-incubation during in vitro fertilization decreases the fertilization rate and does not improve embryo quality: a prospective auto controlled study.

PURPOSE: Evaluate the effect of short gamete incubation on fertilization rate and embryo quality. METHODS: A prospective study has been performed. Two thousand five hundred and forty seven sibling oocytes from 240 couples undergoing IVF attempts were allocated to a short (1 h) or a standard (18 h) insemination procedure. Diploid fertilization rate (two pronuclei, 2PN), polyspermy (>2PN) and embryo quality were compared. RESULTS: The fertilization rate was statistically lower in the short insemination group compared to the standard insemination one (64.9% and 70.1%; P = 0.039), with a similar polyspermy rate observed between the two groups. A slight, but non significant, increase was observed concerning good embryo quality rate in the short insemination group when compared to the standard insemination, both at day 2 (60.1 vs. 58.1%; P = 0.06) and day 3 (53.2 vs. 48.5%; P = 0.22). CONCLUSION: This new study highlights that a 1 h gamete exposure decreases the fertilization rate and does not improve embryo quality compared with a standard 18 h insemination procedure.

Anti-hypertensive effects of the methanol/methylene chloride stem bark extract of Mammea africana in l-NAME-induced hypertensive rats.

AIM OF THE STUDY: The methanol/methylene chloride (CH(3)OH/CH(2)Cl(2)) extract from the stem bark of Mammea africana was showed to possess vasodilating effect in the presence and the absence of N(omega)-nitro-l-arginine methyl ester (l-NAME). The present study was designed to evaluate the effects of the methanol/methylene chloride from the stem bark of Mammea africana. MATERIALS AND METHODS: The extract (200 mg/(kg day)) was administered orally in rats treated concurrently with l-NAME (40 mg/(kg day)). l-Arginine (100 mg/(kg day)) and captopril (20 mg/(kg day))were used as positive controls. Bodyweight, systolic arterial blood pressure and heart rate were measured weekly throughout the experiment period (28 days). At the end of treatment, animals were killed and the cardiac mass index evaluated. The aorta was used to evaluate the endothelium-dependant relaxation to carbachol. The aorta contraction induced by noradrenalin was also examined and expressed as a percentage of that induced by KCl. RESULTS: The extract neither affected the body weight nor the heart rate. The extract as captopril completely prevented the development of arterial hypertension. Both the substances failed to restore the endothelium-dependent vascular relaxation and increased the vascular contraction to norepinephrine in relation to KCl contraction. They also significantly reduced the left ventricular hypertrophy induced by l-NAME. CONCLUSION: These findings are in agreement with the traditional use of Mammea africana in the treatment of arterial hypertension and indicate that it may have a beneficial effect in patients with NO deficiency but will be unable to improve their endothelium-dependent vasorelaxation.

Antihyperglycemic activity of Arbutus unedo, Ammoides pusilla and Thymelaea hirsuta.

The effect of the water extract (WE) of three medicinal plants used as antidiabetic medication in Eastern Morocco (Arbutus unedo: Au, Ammoides pusilla: Ap and Thymelaea hirsuta: Th) was tested in rats with the Oral Glucose Tolerance Test (OGTT) and Intravenous Glucose Tolerance Test (IVGTT). In the OGTT the rats received water, glibenclamide (2 mg/kg) or WE (500 mg/kg for Au and 250 mg/kg for Th and Ap) 30 min before glucose loading (glucose: 1 g/kg). The WE of Au, Ap and Th produced a significant decrease in glycemia after glucose loading. In the IVGTT the WE of Ap and Th produced a significant decrease in glycemia 60 min after i.v. glucose loading (0.5 g/kg). The addition of the WE of Au (500 mg/kg), Ap or Th (250 mg/kg) induced a significant inhibition of jejunal glucose absorption, (31.6%, 28.5% and 40.5% respectively). This effect could explain in part the significant antihyperglycemic effect observed in the OGTT model but it does not exclude other effects on glucose homeostasis, particularly for Ap and Th. Toxicity tests (high LD50 value) suggest no adverse effect of the use of these plants.

Cyclic FEE peptide increases human gamete fusion and potentiates its RGD-induced inhibition.

BACKGROUND: Alpha6beta1 integrin has been proposed to act as a sperm receptor on the mouse oocyte by interacting with spermatozoon fertilin beta. We investigated, in humans, whether oocyte integrins could act similarly in gamete fusion, using a cyclic peptide containing the putative disintegrin-binding domain of human fertilin beta [cyclic FEE (cFEE)] and RGD peptide. METHODS: Zona-free eggs were inseminated in the absence or presence of peptides. To maintain the membrane protein pattern, the zona pellucida was removed by microdissection. Immunofluorescence and confocal microscopy were used to detect integrin subunits on the oocyte. RESULTS: Unexpectedly, cFEE alone increased human gamete fusion by 94% instead of inhibiting fertilization. Furthermore, cFEE together with RGD potentiated the RGD-induced inhibition of fertilization in a dose-dependent manner. The data suggested the hypothesis of integrin cross-talk, further supported by the co-localization of alpha6beta1 and alphavbeta3 integrins, the putative receptors of cFEE and RGD peptides, respectively. CONCLUSIONS: RGD-sensitive and -insensitive integrins may be associated in a multimolecular complex working as a sperm receptor on the human oocyte membrane. Supplementation of human IVF culture medium with cFEE peptide might improve fertilization rates in ART.

Differential gene expression in pre-implantation embryos from mouse oocytes injected with round spermatids or spermatozoa.

BACKGROUND: The use of immature male germ cells to fertilize human oocytes raises several questions. Spermatozoa are normally quiescent, but many genes are transcribed post-meiotically in round spermatids. This creates a novel situation for the oocyte. We have therefore explored the effects on early embryonic development of introducing a fully transcriptionally active round spermatid into the oocyte. METHODS AND RESULTS: Following the micro-injection of spermatozoa or spermatids into mouse oocytes we have analysed the expression, at various times, of six genes in the resulting embryo. Spermatozoa and spermatids produced similar fertilization rates. Hprt was expressed in all embryos at all stages tested. Hsp70.1 was found normally during the 2-cell stage and repressed by the 4-cell stage in embryos from both spermatozoa and round spermatids. However, the amplitude of the signal was greatly reduced in 2-cell embryos from round spermatids. Smcy also showed a disturbed pattern of expression in embryos from round spermatids. Protamine 2, which is normally restricted to the spermatid stage, was expressed following fertilization with round spermatids, but was already repressed at the two pronuclei stage. Ube1Y, which is normally expressed post-meiotically and not during the post-implantatory development, was expressed up to the 2-cell stage in embryos from round spermatids only, and then repressed. Ube1X was also expressed up to the 2-cell stage, but in both embryo types. CONCLUSIONS: We therefore suspect that in embryos fertilized with round spermatids, regulatory mechanisms for inhibiting the inappropriate transcription of male post-meiotically expressed genes are activated following fertilization, permitting the zygotic genome activation to occur, though with some disturbances.

Acute diuretic, natriuretic and hypotensive effects of a continuous perfusion of aqueous extract of Urtica dioica in the rat.

This study was performed on anaesthetized male Wistar rats that received a continuous intravenous perfusion during 1.25 h of an aqueous extract of aerial parts of Urtica dioica L. (Urticaceae) at a low dose of 4 mg/kg/h or at a high dose of 24 mg/kg/h, or furosemide (control diuretic) at a dose of 2 mg/kg/h. As compared with a control period in each rat, the arterial blood pressure was reduced proportionally to the dose of the perfusion of the plant extract (15 and 38%, P<0.001, respectively). These effects were accompanied by a correlative increase of diuresis (11 and 84%, P<0. 001, respectively) and natriuresis (28 and 143%, P<0.001, respectively). In the rats perfused by furosemide, the arterial blood pressure was reduced by 28% (P<0.001). The diuresis and natriuresis were also increased proportionally in this case (85 and 155%, P<0.001, respectively). Nevertheless, the hypotensive action of U. dioica was reversible during the recovery periods in about 1 h with the lower dose of the plant extract and furosemide, while the effect of the higher dose was persistent, indicating a possible toxic effect. In conclusion, the results demonstrate an acute hypotensive action of U. dioica that indicates a direct effect on the cardiovascular system. Moreover, diuretic and natriuretic effects were also observed, suggesting an action on the renal function. Finally, the plant extract seems to have a toxic effect at the higher dose.

Flow cytometry isolation and reverse transcriptase-polymerase chain reaction characterization of human round spermatids in infertile patients.

Flow cytometry coupled to cell sorting is proposed as a method to isolate round spermatids from testicular biopsies in obstructive azoospermic patients. The cells were separated on the basis of their size and density only. We obtained homogenous populations of alive round spermatids free of lymphocytes and diploid germ cells. The detection of protamine 1 gene (PRM1) and PRM2 expression in the sorted cells proves that these cells are round spermatids. On the contrary, neither the expression of CD3-delta, which is specific to lymphoid cells, nor that of MAGE1, which has been demonstrated in diploid germ cells, could be observed in the round spermatid population even after using a nested polymerase chain reaction (PCR) assay. The flow cytometry procedure failed to isolate round spermatids from ejaculates in non-obstructive azoospermic patients. In > 39 ejaculates tested by reverse transcriptase-PCR, only nine revealed the presence of some round spermatids, as demonstrated by the expression of PRM1. However, these round spermatids did not express PRM2.

Flow cytometric method to isolate round spermatids from mouse testis.

The purpose of this study was to isolate pure populations of round spermatids from mouse testis by flow cytometry followed by cell sorting. Cell suspensions from mouse testis were enriched in germ cells by centrifugation on a discontinuous Percoll gradient, then analysed using a FACScalibur flow cytometer measuring the cell size and density. A large and well-delimited population of cells (R1) expected to contain round spermatids was observed on the dot plot diagram. Sorted R1 cells were very homogeneous in size (approximately 11 microns) and displayed the characteristic cytological aspect of round spermatids. Spermatid-specific gene expression was confirmed by reverse transcriptase-polymerase chain reaction (RT-PCR) analysis of R1 cells using primers for protamine 2 gene (PRM2) and SP-10. A positive signal for SP-10 was obtained with a single cell using nested primers. The 5.5 kb transcript of c-kit, which is not expressed in spermatids, was not detected by nested RT-PCR, excluding a contamination with spermatogonia. Our results clearly established that flow cytometry followed by cell sorting allows the isolation of a highly homogeneous population of round spermatids from the testis.

Phytotherapy of hypertension and diabetes in oriental Morocco.

In order to select the main medicinal plants used in folk medicine to treat arterial hypertension and/or diabetes, a survey was undertaken in different areas of oriental Morocco. The patients (370 women and 256 men) were divided into three groups: diabetics (61%), hypertensives (23%) and hypertensive diabetic persons (16%). On average, 67.51% of patients regularly use medicinal plants. This proportion is perceptibly the same in all groups and does not depend on sex, age and socio-cultural level. This result shows that phytotherapy is widely adopted in northeastern Morocco. For diabetes, 41 plants were cited, of which the most used were Trigonella foenum-graecum L. (Leguminosae), Globularia alypum L. (Globulariaceae), Artemisia herba-alba Asso. (Compositae), Citrullus colocynthis (L.) Schrad. (Cucurbitaceae) and Tetraclinis articulata Benth. (Cupressaceae). In the hypertension's therapy 18 vegetal species were reported, of which the most used were Allium sativum L. (Liliaceae), Olea europea L. (Oleaceae), Arbutus unedo L. (Ericaceae), Urtica dioica L. (Urticaceae) and Petroselinum crispum A.W. Hill (Apiaceae). Among the 18 species used for hypertension, 14 were also employed for diabetes. Moreover, these two diseases were associated in 41% of hypertensives. These findings suggest that hypertension observed in this region would be in a large part related to diabetes.

Interactions between nitric oxide and prostanoids in isolated perfused kidneys of the rat.

1. The present study was aimed to assess the interaction between nitric oxide (NO) and thromboxane (Tx) A2-prostaglandin (PG) H2 in single-pass perfused isolated kidneys of the rat. 2. Noradrenaline (NA, 63 and 110 nM) dose-dependently elevated the renal vascular resistance (RVR), the glomerular filtration rate (GFR) and the urinary excretion of sodium (UNa V). Infusion of N omega-nitro-L-arginine methyl ester (L-NAME, 100 microM), an inhibitor of NO synthesis, enhanced the effects of NA on RVR and on UNa V, but decreased those on GFR. The TxA2-PGH2 (TP) receptor blockade by GR32191B (10 microM) attenuated this potentiating effect of L-NAME. 3. When renal perfusion pressure was stepwise increased from 90 to 150 mmHg, L-NAME similarly decreased renal perfusion flow rate and GFR. 4. The venous excretion of TxB2 and 6-keto-PGF1 alpha was increased by L-NAME in baseline conditions as well as after NA or increasing renal perfusion pressure (RPP). 5. These results suggest that: (1) TxA2 and PGH2 play an important role in the overall effect of the renal prostanoids, (2) NO strongly interacts with the cyclo-oxygenase pathway and reduces the prostanoid synthesis in the kidney, and (3) the pressor effect of L-NAME partly relies upon the vasoconstrictor effect of TxA2 and PGH2.