Differential expression and activity of matrix metalloproteinases 2 and 9 in canine early placenta.

The zonary and endotheliochorial dog placenta is the most invasive placenta of carnivores. The importance of matrix metalloproteinases (MMP) in placenta invasiveness has been determined in several mammals including species with haemochorial, epitheliochorial and endotheliochorial placentation. Regarding the latter, the expression of MMP enzymes has been studied in the cat and the mature canine placenta. The aim of this study was to analyse the expression and activity of MMP-2 and MMP-9 in the early dog placenta. Placentae from 18 to 30 days of pregnancy were collected from four bitches. Two placentae from each bitch were analysed. Placental tissue from one uterine horn was fixed in formaldehyde for immunohistochemistry, while marginal haematoma, labyrinth, non-implantative and implantative endometrium from the contralateral horn were immediately frozen in dry ice for the analysis of MMP expression (Western blot [WB]) and activity (zymography). MMP-2 and MMP-9 were evidenced in the labyrinth, maternal glands and marginal haematoma; this finding was directly correlated with levels of MMP expression by WB, and with the activity of MMP-2, mainly in the haematoma (the area of major remodelling of tissues). Thus, although MMP-9 is well expressed in the early canine placenta, it is not active. Given the important role of MMPs for invasiveness, maternal-foetal angiogenesis and the establishment of a correct foetal nutrition, the results are consistent with the findings in other species in which the MMP-2 activation precedes the MMP-9 one in early placentation.

IFPA Meeting 2010 Workshops Report II: Placental pathology; trophoblast invasion; fetal sex; parasites and the placenta; decidua and embryonic or fetal loss; trophoblast differentiation and syncytialisation.

Workshops are an important part of the IFPA annual meeting. At IFPA Meeting 2010 diverse topics were discussed in twelve themed workshops, six of which are summarized in this report. 1. The placental pathology workshop focused on clinical correlates of placenta accreta/percreta. 2. Mechanisms of regulation of trophoblast invasion and spiral artery remodeling were discussed in the trophoblast invasion workshop. 3. The fetal sex and intrauterine stress workshop explored recent work on placental sex differences and discussed them in the context of whether boys live dangerously in the womb.4. The workshop on parasites addressed inflammatory responses as a sign of interaction between placental tissue and parasites. 5. The decidua and embryonic/fetal loss workshop focused on key regulatory mediators in the decidua, embryo and fetus and how alterations in expression may contribute to different diseases and adverse conditions of pregnancy. 6. The trophoblast differentiation and syncytialisation workshop addressed the regulation of villous cytotrophoblast differentiation and how variations may lead to placental dysfunction and pregnancy complications.

Preimplantation embryotoxicity after mouse embryo exposition to reactive oxygen species

Exposure of either gametes or embryos to conditions and/or factors that generate oxidative stress has been associated with impaired early embryogenesis. The effects of reactive oxygen species (ROS) on mouse preimplantation development, depending of the ROS-concentration and time of exposition, were studied. Two-cell embryos were incubated with 5, 10, 25 and 50 microM of hydrogen peroxide (H2O2) for 30 and 60 minutes of exposition and allowed to develop for 72 h to study the quality of Development. The incubation with 50 microM H2O2 for 30 or 60 minutes, strongly inhibited the 2-cell embryo development as compared to the control (p < 0.001). Twenty-five microM H2O2 produced inhibition of blastocyst formation (p < 0.001) and 10 microM H2O2 significantly decreased the percentages of expanded and hatchedblastocysts, which resulted morphologically altered (p < 0.05 and p < 0.01, respectively). The higher H2O2 concentrations were able to elicit necrotic morphology in the 2-cell arrested embryos, while 10 microM H2O2 induced moderate damage with the arrested embryos partially fragmented. In conclusion, important causes for defective preimplantation development and for early embryo losses may be due to oxidative stress because early mouse embryos exposed to ROS for short times arrested at the first cellular cycle (2-cell) and/or impaired embryo differentiation and morphogenesis, being these effects ROS-concentration-dependent.

Preimplantation embryotoxicity after mouse embryo exposition to reactive oxygen species

Exposure of either gametes or embryos to conditions and/or factors that generate oxidative stress has been associated with impaired early embryogenesis. The effects of reactive oxygen species (ROS) on mouse preimplantation development, depending of the ROS-concentration and time of exposition, were studied. Two-cell embryos were incubated with 5, 10, 25 and 50 microM of hydrogen peroxide (H2O2) for 30 and 60 minutes of exposition and allowed to develop for 72 h to study the quality of Development. The incubation with 50 microM H2O2 for 30 or 60 minutes, strongly inhibited the 2-cell embryo development as compared to the control (p < 0.001). Twenty-five microM H2O2 produced inhibition of blastocyst formation (p < 0.001) and 10 microM H2O2 significantly decreased the percentages of expanded and hatchedblastocysts, which resulted morphologically altered (p < 0.05 and p < 0.01, respectively). The higher H2O2 concentrations were able to elicit necrotic morphology in the 2-cell arrested embryos, while 10 microM H2O2 induced moderate damage with the arrested embryos partially fragmented. In conclusion, important causes for defective preimplantation development and for early embryo losses may be due to oxidative stress because early mouse embryos exposed to ROS for short times arrested at the first cellular cycle (2-cell) and/or impaired embryo differentiation and morphogenesis, being these effects ROS-concentration-dependent.(AU)

Hydrosalpinx fluid affects murine embryonic development in a coculture system with epithelial endometrial cells.

OBJECTIVE: The aim of the present investigation was to assess whether a coculture system protects from the effect of hydrosalpinx fluid (HF) on murine embryo development, evaluated through blastocyst cell number. DESIGN: Controlled prospective study. SETTING: Academic research center. PATIENT(S): Endometrium and HF from six patients and endometrium from six normal patients. INTERVENTION(S): Murine embryos were exposed to the absence or presence of different concentrations of human HF: 0% HF (control), 50% HF, 70% HF in human tubal fluid, and 100% HF, in a simple culture system (SCS), epithelial coculture system (ECS), and hydrosalpinx epithelial coculture system (HECS). MAIN OUTCOME MEASURE(S): Embryonic development at 72 hours and blastocyst cell number determined by the Tarcowsky method. RESULT(S): In SCS, 91.9% of the embryos reached the blastocyst stage, and no significant differences were shown in the presence of HF. However, significant differences were observed in the blastocyst cell number. Of the embryos cultured in ECS, 97.1% reached the blastocyst stage, and high concentrations of HF caused a decrease in embryonic development. A significant difference was observed between ECS and HECS in embryo development without HF. When HF was added, a significant decrease in blastocyst cell number was seen in embryos exposed to HECS compared with ECS. CONCLUSION(S): Our data suggest that normal and hydrosalpinx endometria do not protect from the deleterious effect of HF on embryo development at the concentrations evaluated. This effect is dose dependent and was determined through the blastocyst cell number.

Impact of chronic low-dose ethanol ingestion during sexual maturation of female mice on in-vitro and in-vivo embryo development.

Little is known of the consequences of ethanol intake prior to fertilization on preimplantation embryo development. Recently we showed that chronic 10 and 5% w/v ethanol intake by young female mice reduces in vitro fertilization (IVF) rates. The purpose of the present work was to investigate whether the adverse effects of preconceptional low-dose chronic ethanol intake by sexually maturing female mice affects preimplantation embryo growth in vitro or in vivo in subsequent pregnancy. Prepubertal female mice were given 5% ethanol in their drinking water for 30 days. On day 27 and 29 of the ethanol treatment, females were superovulated. IVF-derived cultured embryos (in vitro development) or embryos obtained from oviducts and uteri (in vivo development) were evaluated. Whether analyzed on a per embryo or per dam basis, ethanol treatment was associated with a significant decrease in progression through embryo stages during the seven days of in vitro development and with an increase in morphologically abnormal embryos. Progression through embryo stages during four days of in vivo development was also inhibited by ethanol pretreatment of dams At 99 h post-hCG of in vivo development, there were fewer total, hatched, and expanded blastocysts, and a complete absence of implanting blastocysts among females treated with ethanol. In summary, low-dose chronic ethanol consumption of sexually maturing female mice prior to conception has adverse effects on preimplantation embryo development, both under in vitro and in vivo conditions, manifested as retarded development, embryo anormalities, and a reduction in expansion and hatching of the preimplantation blastocyst.

Methylene blue inhibits the increase of inducible nitric oxide synthase activity induced by stress and lipopolysaccharide in the medial basal hypothalamus of rats.

In infection bacterial products such as lipopolysaccharides (LPS) induce inducible nitric oxide synthase (iNOS) that produces large quantities of NO toxic to the invading organisms, but also often has toxic effects on host cells. Therefore, inhibition of iNOS activity might be beneficial in combatting these adverse effects. To determine if methylene blue (MB), an oxidizing agent that inactivates iNOS, would reduce the iNOS levels in the medial basal hypothalami (MBH) of conscious male rats, LPS (5 mg/kg) was injected intravenously (i.v.), and after 3 h they were injected i.v. with either MB (3 mg/kg) or saline and the effects on iNOS in the MBH determined. iNOS was measured by conversion of labeled arginine into citrulline by incubating MBH in the absence of calcium (Ca(2+)) since iNOS does not require Ca(2+) for activation. The results indicate that iNOS was induced by the injection of saline, but the induction by LPS was much greater, an increase of 10-fold above that of control sham-operated animals. Both the induction of iNOS from the stress of saline injections and LPS were completely eliminated by MB indicating that MB might be beneficial in preventing injury to brain tissue following LPS injection. There was no effect of either LPS or MB on the Ca(2+)-dependent constitutive NOS activity.

Successful capacitation and homologous fertilization in vitro in Calomys musculinus and Calomys laucha (Rodentia - sigmodontinae).

Small South American rodents of the genus Calomys have been used extensively for virology and ecological research. Previous studies have demonstrated that Calomys musculinus and Calomys laucha have a relatively short oestrous cycle and that superovulation and parthenogenetic activation can be induced. The purpose of this study was to determine the requirements for in vitro manipulation of the male gamete and in vitro fertilization. Two culture media and different concentrations of spermatozoa were tested for their ability to support sperm motility, hyperactivation and the acrosome reaction. The ability of capacitated Calomys spermatozoa to penetrate zona-free hamster eggs was also evaluated. In vitro fertilization was assessed by examining attachment and binding to the zona pellucida, second polar body extrusion, pronucleus formation and the fertilizing sperm tail. The results of the study showed that: (i) Tyrode's albumin lactate pyruvate (TALP) medium was more effective than T6 medium for maintaining sperm motility in vitro; (ii) hyperactivation was achieved with TALP but not with T6; (iii) the acrosome reaction was easily distinguished by light microscopy and depends on time and sperm concentration; (iv) capacitated spermatozoa are able to penetrate zona-free hamster eggs; and (v) superovulated oocytes can be fertilized in vitro. This is the first report of capacitation and in vitro fertilization for Calomys sp. These results provide opportunities to use C. musculinus and C. laucha as new laboratory animals for research into reproductive biology.

Alterations in preimplantation in vivo development after preconceptional chronic moderate alcohol consumption in female mice.

Although many studies have explored the effects of acute or chronic ethanol exposure during the postimplantation period on embryo/fetal development, few reports have described the ethanol effects on preimplantation embryo development. Little is known about the effects of ethanol consumption prior to gestation on embryo growth. Recently, we have shown that chronic moderate ethanol intake by prepubertal female mice reduces the ovulatory response and impairs in vitro fertilization and in vitro embryo preimplantation development. The purpose of the present work was to evaluate the effects of preconceptional chronic moderate ethanol ingestion on preimplantation embryo morphology and differentiation, the timing of cleavage and embryo growth in vivo, and to determine the time pattern in which alterations appear. Prepubertal female mice were treated with 10% (w/v) ethanol for 30 days prior to conception. After inducing ovulation on day 27 and 29 of the ethanol treatment, females were mated with control males and the day of presence of vaginal plug was day 1. On day 1, a decreased percentage of normal fertilized oocytes, elevated parthenogenetic oocyte activation and unfertilized eggs with abnormal metaphase II were found in ethanol-treated, compared to control females. On day 2, while any differences in the total percentage of 2-cell embryos were observed, the treated females had a significantly higher percentage of morphologically abnormal embryos, compared to control females. On day 3, the preconceptional consumption of ethanol produced significantly reduced percentages of compacted morulae and an increased percentage of uncompacted morulae. The total percentage of morulae in the treated females was lower than in controls. On day 4, ethanol-treated females showed significantly decreased percentages of hatched attached blastocysts and increased early blastocyst and morula percentages, compared to controls. Thus, preconceptional chronic moderate ethanol ingestion by prepubertal female mice produced retarded development, impaired blastocyst hatching, abnormal embryo morphology and embryo loss by fragmentation due to alterations induced in the female gamete.

Deleterious effects of chronic moderate alcohol intake by female mice on preimplantation embryo growth in vitro.

The susceptibility of preimplantation stages of embryo development to preconceptional alcohol ingestion by females has had little investigation. We have recently shown that chronic 10% (w/v) ethanol intake by young female mice reduces the ovulatory response and impairs the quality of the oocytes. The aim of this study was to investigate the effects of 10% ethanol administration for 30 days on immature female mice on the day of in-vitro fertilization (day 1) and on preimplantation embryo development. Female mice were ovulated on days 27 and 29 of ethanol treatment and in-vitro fertilization was performed 16 h post-human chorionic gonadotrophin administration (day 30). The oocytes from the ethanol-treated females inseminated with spermatozoa from control males, showed a significantly higher percentage of parthenogenetic activation compared to the control females. An increased percentage of fragmented oocytes was found after insemination, compared to control females. When the embryos were cultured, the percentage of 2-cell (day 2), 4-cell (day 3) embryos, and compacted morulae (day 4) was significantly reduced in treated females, compared to control females. On day 5, we found a highly significant decreased percentage of early and expanded blastocysts in the ethanol-treated females. The percentage of hatching and hatched (extruded) blastocysts was also reduced significantly in treated females at days 6 and 7 (blastocyst stages). An increased percentage of morphologically abnormal embryos was found on days 5 and 6 in ethanol-treated females compared with controls. We conclude that chronic moderate ethanol ingestion by young female mice results in decreased fertilization, embryo growth retardation, cleavage arrest, and abnormal embryo development in vitro.

Low chronic ethanol consumption affects ovulation and PGE synthesis by the cumulus cell masses in mice.

Central and gonadal function can be affected by chronic consumption of high and moderate doses of ethanol. Few studies have been conducted to determine the effect of ethanol intake at ovarian and gamete level. Previously, we showed that fertilization rates of low chronic ethanol treated female mice were diminished. Also, our recent results indicated that moderate chronic intake of ethanol by immature females could alter the ovulatory quantity and produce morphological alterations in the superovulated oocytes. Furthermore, PGE production by oocyte cumulus complexes (OCCs) was reduced in the females treated with 10% (w/v) ethanol. In the present investigation, we studied the effects of 5% ethanol treatment given to immature mice for 30 days on the quality and quantity of oocytes superovulated at 16 h posthuman chronic gonadotrophin. Treated females had impaired ovulation rates (P < 0.05) as compared to the controls. The percentage of activated and morphologically abnormal oocytes was elevated in the ethanol-treated females (P < 0.05). PGE synthesis by the OCCs was higher than in the controls (P < 0.01). In summary, the administration of long-term ethanol at a relatively low dose to immature females produces decreased ovulation rates, abnormal oocyte morphology with high spontaneous activation and altered levels of PGE production by the oocytes' cumulus complexes. The relationship between the oocyte quality and abnormal synthesis of PGE is discussed.

The kinetics of oocyte activation and dynamics of polar body formation in Calomys musculinus and Calomys laucha (Rodentia-Sigmodontinae).

The objective of this study was to determine whether Calomys laucha and Calomys musculinus superovulated oocytes undergo parthenogenetic activation following activation stimuli. Cumulus-intact or denuded oocytes were treated with medium containing ethanol (7%), medium containing strontium chloride, or medium alone. They were then incubated for 6-8 h to allow for activation. A group of oocytes was fixed immediately after maturation to serve as a control. The nuclear status of the oocytes was examined after staining with Hoechst 33342, to determine the timing of pronuclear progression from metaphase II to anaphase II or telophase II or to the pronuclear stage. The proportion of oocytes that underwent activation was higher for oocytes treated with ethanol or strontium chloride than in those incubated in medium alone, for the two species studied (p < 0.001). There was little evidence of spontaneous activation occurring in oocytes during the treatments. Most of the activated oocytes contained a single haploid pronucleus, but it was possible to find immediate cleavage and two pronuclei. The different classes of activated oocytes were cultured for 5 days. The type of activating treatment had a marked effect on the ability of the resulting C. musculinus and C. laucha parthenogenetic embryos to develop to the preimplantation stages. Incubation with ethanol produced only 8-cell embryos while the embryos induced with strontium chloride reached the blastocyst stage. This is the first report of parthenogenesis in C. musculinus and C. laucha. The ability of strontium ions to induce matured secondary oocytes to initiate parthenogenesis and obtain further development of Calomys provides opportunities to use Calomys oocytes in vitro and, therefore, to study the genetics, cell biology and virology of development.

Superovulation in vesper mice, Calomys laucha--an important biomedical model for hantavirus and arenavirus (Rodentia-Sigmodontinae).

Sigmodontine rodents are poorly studied and have not received much attention as a reproductive model. Renewed interest in the South American rodents has been stimulated by their link to endemic diseases that are transmitted to man. Calomys laucha acts as a reservoir of two dangerous viruses: an arenavirus named 'Junin virus', the aetiological agent of Argentinian haemorrhagic fever, and the hantavirus, both of which constitute serious sanitary problems. The aim of this study was to establish suitable conditions to superovulate the vesper mouse, Calomys laucha. We examined the hormonal doses, the time interval between hormones, the time-course of ovulation, and the effect of female age on the response to exogenous hormone administration. Female mice were injected with 5-5, 8-8 or 12-15 IU of PMSG/hCG, 48 h apart, at different age intervals (from 30 to > 120 days old). The best superovulation rate was obtained with 8-8 IU PMSG/hCG. Ovulation started about 10 h post-hCG and was completed during the next 4-5 h, and was achieved irrespectively from the oestrus cycle stage. The number of oocytes was influenced by the age of the females. The youngest females had only a superovulatory response. Females older than 61 days showed both ovulatory and superovulatory responses, although 91-120-day-old females had a high ovulatory response. Most of the oocytes (96.5%) recovered were morphologically normal. The genus Calomys constitutes a reproductive model completely different from conventional laboratory rodents.

Relationship between mouse uterine contractility, nitric oxide and prostaglandin production in early pregnancy.

Despite the evidence for a functional role of nitric oxide (NO) in the regulation of uterine contractility in several species, there is little information about the effects of this gas on the mouse uterus. The aims of this study were to investigate if the NO relaxation pathway is present in mouse pregnant uterus and the relationship with the uterotonic prostaglandins (PGs E and F2alpha) production. We evaluated the effect of the treatment with a competitive nitric oxide synthase (NOs) inhibitor: N(G)-monomethyl-L-arginine on the spontaneous contractile activity and prostaglandin production on two different days of pregnancy: second day of pregnancy (preimplantation stage) and on the afternoon of the fifth day of pregnancy (postimplantation stage). We found that only on the fifth day of pregnancy did the inhibitor induce a highly significant isometric developed tension (IDT) and that this effect was maintained throughout the experiment. In order to evaluate if the generation of NO was also different between the two days of pregnancy, NOs activity was measured. Total NOs activity was significantly elevated during the postimplantation stage. We studied the interaction between the NO and cyclooxygenase (COX) pathways on the fifth day of pregnancy, and the data show no stimulation of PGs production by endogenous NO. In summary, we found that NO participates in the control of uterine contractility on the fifth day (a postimplantation stage) and that in this condition the NO was not able to elicit an increase in PGs production.

Progesterone enhances prostaglandin E2 production via interaction with nitric oxide in the mouse acrosome reaction.

In the present study we evidenced that progesterone could directly stimulate sperm nitric oxide synthase (NOS) in capacitated mouse spermatozoa. This stimulation led to an increase in sperm prostaglandin E2 (PGE2) production and subsequent acrosomal exocytosis. However, cGMP levels were not modified during the progesterone-induced acrosome reaction under our experimental conditions. These results suggest a functional role for nitric oxide as an intracellular messenger through, at least, stimulation of PGE2 production during the acrosome reaction triggered by progesterone.

Mouse oocyte quality and prostaglandin synthesis by cumulus oocyte complex after moderate chronic ethanol intake.

Chronic ingestion of ethanol produces a variety of effects on female reproductive function, depending on the dose and the exposure time but the mechanism of alcohol-induced ovarian failure has been little studied. Also the effects of chronic ethanol consumption on the oocyte quality in relation to morphological alterations and PGE synthesis by the oocyte cumulus complexes (OCCs) have not been described. In this study, immature female mice were treated with 10% ethanol in drinking water for 30 days. Then they were induced to superovulate, and at 14, 16 and 20 h post-hCG the quality of the ovarian and oviductal oocytes and PGE production by OCC was determined. At 14 h post-hCG, the percentage of oviductal immature oocytes was increased in the ethanol-treated females (P < 0.05). At 16 h post-hCG, the percent of oviductal activated oocytes was higher in the treated females (P < 0.05), and the ovarian immature oocytes were decreased as compared to the control females (P < 0.05). At 20 h post-hCG, the ethanol-treated females had higher percents of activated oocytes in the oviducts and in the ovaries (P < 0.05) with respect to the controls. PGE synthesis by OCCs, assessed by RIA, was decreased in the treated female mice (P < 0.001). In summary, moderate chronic ethanol treatment in immature female mice can produce morphologic abnormalities in the oocytes (high parthenogenetic activated rates) and altered PGE production in the OCCs.

Effects of low chronic ethanol exposure on prostaglandin E synthesis by preimplantation mouse embryos.

Embryo prostaglandin (PG) synthesis plays a role in the modulation of embryo metabolism and viability, and in the beginning of the implantation. The effects of ethanol consumption seem to be mediated at least in part by PGs. Increased PG production of postimplantation embryos is associated with retardation and abnormalities in the gestational period. The aim of this study was to find out the effects of low chronic ethanol ingestion by mice, previous to pregnancy, on the PGE released by in vitro and in vivo derived embryos. Immature females or adult males were treated with 5% ethanol for 30 days. After fertilization and mating, two-cell embryos, morulae and blastocysts were collected. The PGE synthesis and release were measured by radioimmunoassay. PGE production by in vitro derived two-cell embryos from ethanol-treated females was lower than in the control group (P < 0.01). Also, PGE production was reduced when two-cell embryos came from ethanol-treated males (P < 0.01). There were no differences in PGE synthesis by in vitro derived morulae and blastocysts in these groups. Two-cell embryos derived from mating produced lower quantities of PGE when they came from ethanol-treated females mated with control males, as compared to the control group. PGE release by in vivo derived blastocysts from ethanol-treated females was reduced significantly, as compared to the control group (P < 0.01). We conclude that a low concentration of ethanol administered chronically to immature females reduces PGE synthesis and release by two-cell embryos from culture in vitro, and by embryos of days 2 and 4 from in vivo development.

Impaired mouse fertilization by low chronic alcohol treatment.

Little is known about the effects of low chronic alcohol intake on fertility, particularly in females. Recently, we have shown that chronic 10% (w/v) ethanol treatment affects in-vitro fertilization of mouse female gamete. The aim of this study was to solve questions concerning the lowest dose and duration of ethanol treatment required to alter the fertility of immature and adult female and adult male mouse. Mice were treated with 5% and 2.5% (w/v) ethanol in drinking water for 4 weeks. The in-vitro fertilization rates were significantly decreased with the 5% ethanol when oocytes from prepubertal and pubertal ethanol-treated females were inseminated with spermatozoa from adult control males. The in-vitro fertilization rates were not diminished when oocytes from control females were inseminated with spermatozoa from adult ethanol-treated males. Haploid oocytes were increased when oocytes came from immature females treated with ethanol. The in-vitro fertilization rates were not decreased in adult treated females. The in-vivo fertilization rates were not modified when prepubertal ethanol-treated females were mated with adult control males. Fragmented oocytes, in the in-vitro fertilization experiments, were significantly increased when they came from prepubertal and adult treated females inseminated with ethanol-treated males. These results show that there is a threshold of the ethanol dose to produce an effect. Chronic low ethanol ingestion by immature female mice has a deleterious effect on their in-vitro fertilization. Furthermore, acute ethanol ingestion by adult females during the induction of ovulation resulted in high parthenogenetic activation and fragmentation of mouse oocytes.

Prostaglandin synthesis by cumulus-oocyte complexes: effects on in vitro fertilization in mice.

Cumulus-oocyte complexes, obtained from superovulated Balb/C virgin female mice, released to the incubation media significant amounts of PGE1, PGE2 and PGF2 alpha, as estimated by bioassay. Fertilization rates in vitro decreased sharply when cumulus-oocyte complexes were treated with indomethacin (10(-6) M) and then inseminated with 5000 sperm per oocyte. In order to explore if the reduced prostaglandin (PG) concentration was responsible for diminished fertilization rates, PGE1, PGE2 and PGF2 alpha (10(-9) M) were added to the fertilization media of treated oocytes. PGE1 and PGE2 but not PGF2 alpha returned fertilization rates to control levels. Besides, PGE1 (10(-9) M) enhanced fertilization rates with reduced sperm numbers (1000 sperm per oocyte) of untreated cumulus-oocyte complexes. In conclusion, PG synthesis and release of mouse cumulus-oocyte complexes affects fertilization in vitro, and it is suggested that PGs of the E series modulate sperm function at the moment of fertilization.

Ethanol inhibits luteinizing hormone-releasing hormone (LHRH) secretion by blocking the response of LHRH neuronal terminals to nitric oxide.

It has previously been shown that alcohol can suppress reproduction in humans, monkeys, and small rodents by inhibiting release of luteinizing hormone (LH). The principal action is via suppression of the release of LH-releasing hormone (LHRH) both in vivo and in vitro. The present experiments were designed to determine the mechanism by which alcohol inhibits LHRH release. Previous research has indicated that the release of LHRH is controlled by nitric oxide (NO). The proposed pathway is via norepinephrine-induced release of NO from NOergic neurons, which then activates LHRH release. In the present experiments, we further evaluated the details of this mechanism in male rats by incubating medial basal hypothalamic (MBH) explants in vitro and examining the release of NO, prostaglandin E2 (PGE2), conversion of arachidonic acid to prostanoids, and production of cGMP. The results have provided further support for our theory of LHRH control. Norepinephrine increased the release of NO as measured by conversion of [14C]arginine to [14C]citrulline, and this increase was blocked by the alpha 1 receptor blocker prazosin. Furthermore, the release of LHRH induced by nitroprusside (NP), a donor of NO, is related to the activation of soluble guanylate cyclase by NO since NP increased cGMP release from MBHs and cGMP also released LHRH. Ethanol had no effect on the production of NO by MBH explants or the increased release of NO induced by norepinephrine. Therefore, it does not act at that step in the pathway. Ethanol also failed to affect the increase in cGMP induced by NP. On the other hand, as might be expected from previous experiments indicating that LHRH release was brought about by PGE2, NP increased the conversion of [14C]arachidonic acid to its metabolites, particularly PGE2. Ethanol completely blocked the release of LHRH induced by NP and the increase in PGE2 induced by NP. Therefore, the results support the theory that norepinephrine acts to stimulate NO release from NOergic neurons. This NO diffuses to the LHRH terminals where it activates guanylate cyclase, leading to an increase in cGMP. At the same time, it also activates cyclooxygenase. The increase in cGMP increases intracellular free calcium, activating phospholipase A2 to provide arachidonic acid, the substrate for conversion by the activated cyclooxygenase to PGE2, which then activates the release of LHRH. Since alcohol inhibits the conversion of labeled arachidonic acid to PGE2, it must act either directly to inhibit cyclooxygenase or perhaps it may act by blocking the increase in intracellular free calcium induced by cGMP, which is crucial for activation of of both phospholipase A2 and cyclooxygenase.