Distribution of NADPH-diaphorase and nitric oxide synthase (NOS) in different regions of porcine oviduct during the estrous cycle.

Nitric oxide synthase (NOS) is responsible for the biological production of nitric oxide (NO) in several organs, including those of the reproductive tract. We investigated potential changes in NADPH-diaphorase (NADPH-d) activity (marker for NOS activity) and the presence and distribution of NOS in the porcine oviduct. Tissues were obtained from gilts (n=16) on different days of the estrous cycle. One fallopian tube was used for histo- and immunohistochemistry and the other for Western blotting analysis. NADPH-d activity was much higher in the epithelium of the mucosa than in the myosalpinx. The highest activity of NADPH-d was always found in the epithelium of the isthmus. The intensity of the reaction (arbitrary units +/- SEM) in isthmus epithelium increased from the postovulatory period until early proestrus (96.2 +/- 11.2) and then gradually decreased. The lowest intensity of NADPH-d reaction in the epithelium of the isthmus was seen at estrus (58.4 +/- 7.7). The most intense NADPH-d activity in myosalpinx of all parts of the oviduct was observed at the postovulatory stage of the estrous cycle (isthmus 38.3 +/- 2.5; ampulla 35.6 +/- 4.2; infundibulum 24.7 +/- 0.8) and then decreased during the remaining stages of the estrous cycle (p< 0.001). The presence of endothelial NOS (eNOS) was detected in epithelial cells of mucosa and in endothelium of vascular tissues and myosalpinx during all studied days of the estrous cycle. The positive reaction for inducible NOS (iNOS) was restricted only to the endothelium of lymph vessels and some blood vessels. Because our Western blotting analysis revealed that porcine oviduct contains eNOS but not iNOS, we suggest that eNOS is the main isoform of NOS expressed in the porcine oviduct. We concluded that the different activity of NADPH-d in the various regions of the oviduct, accompanied by changes in its activity during the course of the estrous cycle, could indicate an important role of NO in regulation of tubal function.     

Reproductive function in female mice lacking the gene for endothelial nitric oxide synthase.

Nitric oxide (NO) acts as a neuronal messenger in both the central and peripheral nervous systems and has been implicated in reproductive physiology and behavior. Pharmacological inhibition of nitric oxide synthase (NOS) with the nonspecific NOS inhibitor, l-N(G)-nitro-Arg-methyl ester (l-NAME), induced deficits in both the number of ovarian rupture sites and the number of oocytes recovered in the oviducts of mice. Female neuronal NOS knockout (nNOS-/-) mice have normal numbers of rupture sites, but reduced numbers of oocytes recovered following systemic injections of gonadotropins, suggesting that NO produced by nNOS accounts, in part, for deficits in ovulatory efficiency observed after l-NAME administration. Additionally, endothelial NOS knockout (eNOS-/-) mice have reduced numbers of ovulated oocytes after superovulation. Because endothelial NOS has been identified in ovarian follicles, and because of the noted reduced breeding efficiency of eNOS-/- mice, the present study sought to determine the role of NO from eNOS in mediating the number of rupture sites present after ovulation. Estrous cycle length and variability were consistently reduced in eNOS-/- females. The number of rupture sites was normal in eNOS-/- mice under natural conditions and after administration of exogenous GnRH. After exogenous gonadotropin administration, eNOS-/- females displayed a significant reduction in the number of ovarian rupture sites. Female eNOS-/- mice also produced fewer pups/litter compared to WT mice. These data suggest that NO from endothelial sources might play a role in mediating rodent ovulation and may be involved in regulation of the timing of the estrous cycle.     

Sex and estrous cycle-dependent differences in glial fibrillary acidic protein immunoreactivity in the adult rat hippocampus

Sex differences in the morphology and function of the hippocampus have been reported in several species, but it is unknown whether a sexual dimorphism exists in glial fibrillary acidic protein (GFAP) expression in the rat hippocampus. We analyzed GFAP immunoreactivity in the hippocampus of intact adult male rats as well as in females during diestrus and proestrus phases of the estrous cycle. We found that in CA1, CA3, and dentate gyrus, GFAP immunoreactivity was higher in proestrus females as compared with males and diestrus females. In CA1, a similar GFAP immunoreactivity was found in males and in diestrus females, but in dentate gyrus, males presented the lowest GFAP content. Interestingly, differences in astrocyte morphology were also found. Rounded cells with numerous and short processes were mainly observed in the hippocampus during proestrus whereas cells with stellate shape with few and long processes were present in the hippocampus of males and diestrus females. The marked sex and estrous cycle-dependent differences in GFAP immunoreactivity density and in astrocyte number and morphology found in the rat hippocampus, suggest the involvement of sex steroid hormones in the sexually dimorphic functions of the hippocampus, and in the change in its activity during the estrous cycle.     

The preovulatory rise of ovarian ornithine decarboxylase is required for progesterone secretion by the corpus luteum

Ovarian progesterone secretion during the diestrus stage of the estrous cycle is produced by luteal cells derived from granulosa and thecal cells after the differentiation process that follows ovulation. Our results show that blockade of the preovulatory rise of ovarian ornithine decarboxylase (ODC), a key enzyme in polyamine biosynthesis, by treatment with the specific inhibitor alpha-difluoromethylornithine (DFMO) leads to a significant decrease in the ovarian progesterone content and a dramatic fall in the plasma levels of this hormone during the following diestrus. The same inhibition was produced in spite of the fact that both luteinizing and follicle stimulating hormones were given concomitantly with DFMO. On the other hand, the acute rise in the plasma progesterone levels observed after administration of human chorionic gonadotropin to mice at different periods of the estrous cycle was not affected by DFMO administration. Our results indicate that although elevated levels of ODC are not required for acute ovarian steroidogenesis, the preovulatory peak of ovarian ODC activity observed in the evening of proestrus may be critical for the establishment of a constitutive steroidogenic pathway and progesterone secretion by the corpus luteum during the diestrus stage of the murine estrous cycle.     

Serotonin and norepinephrine uptake in discrete brain regions during the pregnant mare serum (PMS) induced estrous cycle in the rat

The role of hypothalamic and limbic aminergic systems in neuroendocrine control in the rat has been investigated by measuring the temporal changes in serotonergic and noradrenergic neuronal uptake in a model of the estrous cycle. Using the pregnant mare serum (PMS) model of induced ovulation and an in vitro uptake system, serotonergic activity reached peak values (p = 0.01) in the suprachiasmatic nuclear region (SNR) and median eminence (ME) (p = 0.007) during the critical period for luteinizing hormone release. 5, 7-dihydroxytryptamine lesions of the SNR or ME regions inhibited PMS induced ovulation suggesting that at least in these two areas a certain amplitude of 5-HT activity may be required at some time prior to the critical period for LH release. Serotonergic uptake during diestrus in the median eminence showed maximum values during the mid-light phase suggesting a role for this region in ovulation control mechanisms prior to the proestrous critical period. Significant changes (p = 0.001) in serotonergic uptake were found in mid to later light diestrus in the amygdala. This pattern was repeated during proestrus and estrus only in the amygdala suggesting a daily pattern of serotonergic uptake during the estrous cycle. Significant changes (p = 0.001) in noradrenergic uptake only occurred in the amygdala during proestrus.     

Localization and correlation between NADPH-diaphorase and nitric oxide synthase isoforms in the porcine uterus during the estrous cycle

Nitric oxide (NO), a highly reactive free radical is involved in vasodilation, neurotransmission, hormone secretion, and reproduction. Since all known nitric oxide synthase (NOS) isoforms possess NADPH-diaphorase (NADPH-d) activity, NADPH-d histochemistry was used as a commonly accepted procedure for NOS identification. The aim of our study was to determine the cellular localization of NADPH-d, eNOS, and iNOS in the porcine uterus and the correlation between NADPH-d and NOS activity in the early, middle, late luteal, and follicular phase of the estrous cycle. Light-microscopic observations of the sections revealed the differential expression of the NADPH-d in the analyzed stages of the estrous cycle. The most intense staining was observed in the luminal epithelium in the late luteal phase and in some groups of the endometrial glands in all studied stages. Positive reaction was also found in the endothelial cells of blood vessels and in the myometrium itself. Immunostaining for eNOS was observed in the luminal and glandular epithelium in all studied stages, but no clear fluctuations were observed. The endothelium of both endometrial and myometrial blood vessels displayed pronounced eNOS immunostaining. Strong iNOS staining was observed in the luminal epithelium in the late luteal and follicular phase and in selected groups of endometrial glands. Thus, only NADPH-d and iNOS undergo cyclic changes in the studied stages of the estrous cycle. The differential expression of NADPH-d/NOS in the porcine uterine horn during the estrous cycle suggests a role for NO in modulating uterine function.     

Nitric oxide is essential for optimal meiotic maturation of murine cumulus-oocyte complexes in vitro

This study was conducted to determine whether endothelial-derived nitric oxide synthase (eNOS) affects meiotic maturation of mouse oocytes in vitro. Cumulus-oocyte complexes (COC) were isolated from ovarian follicles of 27-day-old PMSG-primed wildtype (WT), and eNOS-knockout (eNOS-KO) females, and cultured in drops of medium under oil at 37 degrees C for 16-18 hr. Experiment 1 was carried out to determine effects of eNOS deficiency on the ability of COC to mature in vitro. To determine whether acute synthesis of nitric oxide (NO) was required for oocyte maturation, COC collected from WT mice were cultured in medium without (control) or with different doses of N(omega)-nitro-L-arginine methyl ester (L-NAME), an inhibitor of NOS (exp. 2). To assess effects of NO deficiency on the kinetics of germinal vesicle breakdown (GVBD), COC from WT and eNOS-KO females were observed for 3.5 hr. COC from WT females were also incubated in medium without or with L-NAME (exp. 3 and 4). After the culture period, cumulus cells were removed, and oocytes were counted and classified as metaphase II (M II), metaphase I (M I) or showing atypical (degenerative) morphology. To determine viability and nuclear morphology of oocytes, they were stained with fluorescein diacetate or 4,6-diamidine-2'-phenylindole dihydrochloride, respectively. There were no differences in body weights but ovarian weights were lower in eNOS-KO mice compared with WT mice (P < 0.05). Ovaries from eNOS-KO mice contained fewer COC collected relative to WT mice (P < 0.01). Maturation of COC from eNOS-KO mice or WT oocytes treated with L-NAME resulted in a lower percentage of oocytes at M II stage (P < 0.01 and P < 0.05, respectively) and a higher percentage of oocytes at M I or atypical stages compared with those from WT (P < 0.01 and P < 0.05, respectively). Many oocytes that showed either an arrest in M I stage or abnormal morphology were not viable. Several oocytes in M II stage demonstrated abnormalities in distribution of maternal chromosomes. Our data demonstrate that eNOS-derived NO is a key modulator of oocyte meiotic maturation in vitro. These results support our previous observations in vivo and indicate that eNOS/NO has independent functions in both oocyte maturation and follicular/oocyte development.     

Neuroendocrine disruption is associated to infertility in chronically stressed female rats

Infertility is a growing worldwide public health problem, and stress is a main factor exerting detrimental effects on female reproduction. However, knowledge regarding the neuroendocrine changes caused by chronic stress in females is limited. Therefore, this study assessed the effects of stress on hormones that control female reproduction during the proestrus and diestrus stages of the estrous cycle, as well as its effects on fertility. Adult females were assigned to either a control or a stress group. Stress consisted of exposure, for 15 min, to cold-water immersion daily for 30 days. Estrous cyclicity, female sexual behavior, as well as hypothalamic kisspeptin, gonadotropin releasing hormone (GnRH) content, serum luteinizing hormone (LH), estradiol (E₂), progesterone (P₄), corticosterone (CORT) and fertility were assessed after chronic stress. The results show that chronically stressed females exhibited disrupted estrous cyclicity, decreased receptivity, low pregnancy rates and lower numbers of fetuses. The content of Kisspeptin and GnRH in the Anteroventral Periventricular/medial Preoptic Area decreased during proestrus, while Kisspeptin increased in the Arcuate nucleus in proestrus and diestrus. Serum LH decreased only during proestrus, whereas E₂ and P₄ concentrations decreased during proestrus and diestrus, with a concomitant increase in CORT levels in both stages. As a whole, these results indicate that chronic stress decreases Kisspeptin content in AVPV nucleus and GnRH in POA in females, and might induce disruption of the LH surge, consequently disrupting estrous cyclicity and fertility, leading to lower rates of pregnancy and number of fetuses.     

Effects of thymulin and GnRH on the release of gonadotropins by in vitro pituitary cells obtained from rats in each day of estrous cycle

The effects of thymulin and GnRH on FSH and LH release were studied in suspension cultures of anterior pituitary cells from female adult rats sacrificed on each day of the estrous cycle. The spontaneous release of gonadotropins by pituitaries, as well as their response to GnRH or thymulin addition, fluctuated during the estrous cycle. Adding thymulin to pituitary cells from rats in diestrus 1 increased the concentration of FSH; while in cells from rats in estrus, FSH level decreased. Thymulin had a stimulatory effect on the basal concentration of LH during most days of the estrous cycle. Adding GnRH increased FSH release in cells from rats in diestrus 1, diestrus 2, or proestrus, and resulted in higher LH levels in cells obtained from rats in all days of the estrous cycle. Compared to the GnRH treatment, the simultaneous addition of thymulin and GnRH to cells from rats in diestrus 1, diestrus 2, or proestrus resulted in lower FSH concentrations. Similar results were observed in the LH release by cells from rats in diestrus 1, while in cells from rats in proestrus or estrus, LH concentrations increased. A directly proportional relation between progesterone serum levels and the effects of thymulin on FSH release was observed. These data suggest that thymulin plays a dual role in the release of gonadotropins, and that its effects depend on the hormonal status of the donor's pituitary.     

Modifications des cellules gonadotropes de la préhypophyse chez la ratte injectée de testostérone au cours du cycle oestral

Summary 4-day cyclic adult female Wistar rats were injected subcutaneously with testosterone propionate on diestrus 1 at 16:00 and on diestrus 2 at 10:00 respectively. Non-injected females served as controls. Autopsy was performed on diestrus 2 at 23:00, and on proestrus at 14:00 and 17:00 respectively. The blue Alcian-PAS staining was used to evidence FSH () and LH () pituitary cells.In control animals and in diestrus 2 injected females only a small number of FSH cells could be detected on diestrus 2 at 23:00. This number increased markedly on proestrus at 14:00 and decreased on proestrus at 17:00. A similar evolution was observed in diestrus 1 testosterone injected females, but the number of FSH cells appeared higher at any stage of autopsy in these females than in diestrus 2 injected females and in control rats.In control females, numerous LH cells were observed on diestrus 2 at 23:00. The number of these cells was diminished on proestrus at 14:00 and still more at 17:00. On the contrary few LH cells were detected in testosterone injected females on the evening of diestrus 2. An increase of these cells occurred on proestrus at 14:00, followed at 17:00 by only a weak diminution as established by comparison with control animals.An inhibition of FSH release and a suppression of the proestrus surge of LH were therefore supposed to cause, on one hand, the slowing up of follicular growth observed in diestrus 1 injected females and, on the other hand, the blockage of ovulation noted in both diestrus 1 and diestrus 2 treated animals.

Travail effectué avec l'aide de la D.G.R.S.T. Contrat n^o 72.7.0030.     

Endothelial nitric oxide synthase (NOS3) knockout decreases NOS2 induction, limiting hyperoxygenation and conferring protection in the postischemic heart

Although it has been shown that endothelial nitric oxide synthase (eNOS)-derived nitric oxide downregulates mitochondrial oxygen consumption during early reperfusion, its effects on inducible NOS (iNOS) induction and myocardial injury during late reperfusion are unknown. Wild-type (WT) and eNOS(-/-) mice were subjected to 30 min of coronary ligation followed by reperfusion. Expression of iNOS mRNA and protein levels and peroxynitrite production were lower in postischemic myocardium of eNOS(-/-) mice than levels in WT mice 48 h postreperfusion. Significantly improved hemodynamics (+/-dP/dt, left ventricular systolic pressure, mean arterial pressure), increased rate pressure product, and reduced myocardial infarct size (18 +/- 2.5% vs. 31 +/- 4.6%) were found 48 h after reperfusion in eNOS(-/-) mice compared with WT mice. Myocardial infarct size was also significantly decreased in WT mice treated with the specific iNOS inhibitor 1400W (20.5 +/- 3.4%) compared with WT mice treated with PBS (33.9 +/- 5.3%). A marked reperfusion-induced hyperoxygenation state was observed by electron paramagnetic resonance oximetry in postischemic myocardium, but Po(2) values were significantly lower from 1 to 72 h in eNOS(-/-) than in WT mice. Cytochrome c-oxidase activity and NADH dehydrogenase activity were significantly decreased in postischemic myocardium in WT and eNOS(-/-) mice compared with baseline control, respectively, and NADH dehydrogenase activity was significantly higher in eNOS(-/-) than in WT mice. Thus deficiency of eNOS exerted a sustained beneficial effect on postischemic myocardium 48 h after reperfusion with preserved mitochondrial function, which appears to be due to decreased iNOS induction and decreased iNOS-derived peroxynitrite in postischemic myocardium.     

NO production by cNOS and iNOS reflects blood pressure changes in LPS-challenged mice

Increased nitric oxide (NO) production is the cause of hypotension and shock during sepsis. In the present experiments, we have measured the contribution of endothelial (e) and inducible (i) nitric oxide synthase (NOS) to systemic NO production in mice under baseline conditions and upon LPS treatment (100 microg/10 g ip LPS). NO synthesis was measured by the rate of conversion of l-[guanidino-15N2]arginine to l-[ureido-15N]citrulline, and the contribution of the specific NOS isoforms was evaluated by comparing NO production in eNOS-deficient [(-/-)] and iNOS(-/-) mice with that in wild-type (WT) mice. Under baseline conditions, NO production was similar in WT and iNOS(-/-) mice but lower in eNOS(-/-) mice [WT: 1.2 +/- 0.2; iNOS(-/-): 1.2 +/- 0.2; eNOS(-/-): 0.6 +/- 0.3 nmol. 10 g body wt-1. min-1]. In response to the challenge with LPS (5 h), systemic NO production increased in WT and eNOS(-/-) mice but fell in iNOS(-/-) mice [WT: 2.7 +/- 0.3; eNOS(-/-): 2.2 +/- 0.6; iNOS(-/-): 0.7 +/- 0.1 nmol. 10 g body wt-1. min-1]. After 5 h of LPS treatment, blood pressure had dropped 14 mmHg in WT but not in iNOS(-/-) mice. The present findings provide firm evidence that, upon treatment with bacterial LPS, the increase of NO production is solely dependent on iNOS, whereas that mediated by cNOS is reduced. Furthermore, the data show that the LPS-induced blood pressure response is dependent on iNOS.     

Sepiapterin enhances angiogenesis and functional recovery in mice after myocardial infarction

Uncoupling of nitric oxide synthase (NOS) has been implicated in left ventricular (LV) remodeling and dysfunction after myocardial infarction (MI). We hypothesized that inducible NOS (iNOS) plays a crucial role in LV remodeling after MI, depending on its coupling status. MI was created in wild-type, iNOS-knockout (iNOS(-/-)), endothelial NOS-knockout (eNOS(-/-)), and neuronal NOS-knockout (nNOS(-/-)) mice. iNOS and nNOS expressions were increased after MI associated with an increase in nitrotyrosine formation. The area of myocardial fibrosis and LV end-diastolic volume and ejection fraction were more deteriorated in eNOS(-/-) mice compared with other genotypes of mice 4 wk after MI. The expression of GTP cyclohydrolase was reduced, and tetrahydrobiopterin (BH(4)) was depleted in the heart after MI. Oral administration of sepiapterin after MI increased dihydrobiopterin (BH(2)), BH(4), and BH(4)-to-BH(2) ratio in the infarcted but not sham-operated heart. The increase in BH(4)-to-BH(2) ratio was associated with inhibition of nitrotyrosine formation and an increase in nitrite plus nitrate. However, this inhibition of NOS uncoupling was blunted in iNOS(-/-) mice. Sepiapterin increased capillary density and prevented LV remodeling and dysfunction after MI in wild-type, eNOS(-/-), and nNOS(-/-) but not iNOS(-/-) mice. N(ω)-nitro-L-arginine methyl ester abrogated sepiapterin-induced increase in nitrite plus nitrate and angiogenesis and blocked the beneficial effects of sepiapterin on LV remodeling and function. These results suggest that sepiapterin enhances angiogenesis and functional recovery after MI by activating the salvage pathway for BH(4) synthesis and increasing bioavailable nitric oxide predominantly derived from iNOS.     

Inducible nitric oxide synthase provides protection against injury-induced thrombosis in female mice

Nitric oxide (NO) is an important vasoactive molecule produced by three NO synthase (NOS) enzymes: neuronal (nNOS), inducible (iNOS), and endothelial NOS (eNOS). While eNOS contributes to blood vessel dilation that protects against the development of hypertension, iNOS has been primarily implicated as a disease-promoting isoform during atherogenesis. Despite this, iNOS may play a physiological role via the modulation of cyclooxygenase and thromboregulatory eicosanoid production. Herein, we examined the role of iNOS in a murine model of thrombosis. Blood flow was measured in carotid arteries of male and female wild-type (WT) and iNOS-deficient mice following ferric chloride-induced thrombosis. Female WT mice were more resistant to thrombotic occlusion than male counterparts but became more susceptible upon iNOS deletion. In contrast, male mice (with and without iNOS deletion) were equally susceptible to thrombosis. Deletion of iNOS was not associated with a change in the balance of thromboxane A(2) (TxA(2)) or antithrombotic prostacyclin (PGI(2)). Compared with male counterparts, female WT mice exhibited increased urinary nitrite and nitrate levels and enhanced ex vivo induction of iNOS in hearts and aortas. Our findings suggest that iNOS-derived NO in female WT mice may attenuate the effects of vascular injury. Thus, although iNOS is detrimental during atherogenesis, physiological iNOS levels may contribute to providing protection against thrombotic occlusion, a phenomenon that may be enhanced in female mice.     

The role of eNOS, iNOS, and NF-kappaB in upregulation and activation of cyclooxygenase-2 and infarct size reduction by atorvastatin

Pretreatment with atorvastatin (ATV) reduces infarct size (IS) and increases myocardial expression of phosphorylated endothelial nitric oxide synthase (p-eNOS), inducible NOS (iNOS), and cyclooxygenase-2 (COX2) in the rat. Inhibiting COX2 abolished the ATV-induced IS limitation without affecting p-eNOS and iNOS expression. We investigated 1) whether 3-day ATV pretreatment limits IS in eNOS(-/-) and iNOS(-/-) mice and 2) whether COX2 expression and/or activation by ATV is eNOS, iNOS, and/or NF-kappaB dependent. Male C57BL/6 wild-type (WT), University of North Carolina eNOS(-/-) and iNOS(-/-) mice received ATV (10 mg.kg(-1).day(-1); ATV(+)) or water alone (ATV(-)) for 3 days. Mice underwent 30 min of coronary artery occlusion and 4 h of reperfusion, or hearts were harvested and subjected to ELISA, immunoblotting, biotin switch, and electrophoretic mobility shift assay. As a result, ATV reduced IS only in the WT mice. ATV increased eNOS, p-eNOS, iNOS, and COX2 levels and activated NF-kappaB in WT mice. It also increased myocardial COX2 activity. In eNOS(-/-) mice, ATV increased COX2 expression but not COX2 activity or iNOS expression. NF-kappaB was not activated by ATV in the eNOS(-/-) mice. In the iNOS(-/-) mice, eNOS and p-eNOS levels were increased but not iNOS and COX2 levels; however, NF-kappaB was activated. In conclusion, both eNOS and iNOS are essential for the IS-limiting effect of ATV. The expression of COX2 by ATV is iNOS, but not eNOS or NF-kappaB, dependent. Activation of COX2 is dependent on iNOS.     

Inhibition of estrus and corpora lutea function with Norgestomet

Forty-five nonpregnant, nonlactating, Angus and Brangus cows were utilized to determine how long a Norgestomet ear implant would inhibit estrus when administered at various stages of an estrous cycle. All cows completed a nontreated estrous cycle to ensure normal cyclicity. At the second observed estrus (estrus = Day 1), cows were randomly allotted to be treated at metestrus (Day 3 or Day 4, n = 15); at diestrus (Day 9 or Day 10, n = 14); or at proestrus (Day 15 or Day 16, n = 16). All cows received a 2-ml intramuscular injection of 3 mg of Norgestomet accompanied by a 6-mg Norgestomet ear implant, which remained in situ for 21 days, or until individual cows were observed in estrus. Estrus was inhibited for a mean (+/- SEM) of 18.7 +/- 0.7, 19.9 +/- 0.8, and 17.0 +/- 0.8 days, respectively, when cows were treated at metestrus, diestrus, and proestrus (metestrus and diestrus vs proestrus; P < 0.05). Estrus was inhibited for an entire 21-day implantation period in 27, 50, and 38% of cows treated at metestrus, diestrus, and proestrus, respectively (P > 0.10). Norgestomet inhibited estrus in all cows for 11, 17, and 11 days after implantation when treatment was initiated at metestrus, diestrus, and proestrus, respectively (P > 0.10). These data indicate that a 6-mg Norgestomet ear implant effectively inhibits estrus in all cows for a maximum of 11 days, with some cows exhibiting estrus by Day 12 with the Norgestomet implant in situ.     

Expression of nitric oxide synthase isoforms in the mouse kidney: cellular localization and influence by lipopolysaccharide and Toll-like receptor 4

We determined the cellular mRNA expression of all intrarenal nitric oxide (NO)-producing NO synthase (NOS) isoforms, endothelial NOS (eNOS) and neuronal NOS (nNOS) and inducible NOS (iNOS) in kidneys from wild-type mice (WT) and immune deficient Toll-like receptor 4 (TLR4) mutant mice, during normal physiological conditions and during a short-term (6–16 h) endotoxic condition caused by systemically administered lipopolysaccaride (LPS). Investigations were performed by means of in situ hybridization and polymerase chain reaction amplification techniques. In WT, LPS altered the expression rate of all intrarenal NOS isoforms in a differentiated but NOS-isoform coupled expression pattern, with iNOS induction, and up- and down-regulation of the otherwise constitutively expressed NOS isoforms, e.g. eNOS and nNOS and an iNOS isotype. In TLR4 mutants, LPS caused none or a lowered iNOS induction, but altered the expression rate of the constitutive NOS isoforms. It is concluded that the intrarenal spatial relation of individual NOS-isoforms and their alteration in expression provide the basis for versatile NO-mediated renal actions that may include local interactions between NOS isoforms and their individual NO-target sites, and that the NOS-isoform dependent events are regulated by TLR4 during endotoxic processes. These regulatory mechanisms are likely to participate in different pathophysiological conditions affecting NO-mediated renal functions.     

Gonadotropin-releasing hormone binding sites in ovaries of normal cycling and persistent-estrus rats

The gonadotropin-releasing hormone (GnRH) binding capacity in ovaries and pituitaries of normal cycling rats at different stages of the estrous cycle and in ovaries of persistent-estrus rats was measured using radioligand-receptor assay (RRA). Persistent estrus was induced either by neonatal administration of testosterone propionate (1.25 mg s.c.) on the second day of life or by a hypothalamic suprachiasmatic frontal cut made with Halász' knife. All animals were killed during the critical period (1400-1600 h), and GnRH receptor was assayed. GnRH receptor levels in both ovaries and pituitaries changed during the estrous cycle. The total number of ovarian GnRH binding sites was significantly higher in proestrus than in diestrus 1, the stage in which the lowest level was found. When binding sites were expressed in fmol/mg ovary, the highest level was observed in diestrus 2; however, no changes were observed during the estrous cycle when GnRH binding sites were expressed as fmol/mg protein. Changes noted were very similar to those demonstrated in pituitary GnRH receptors in our present and previous experiments. Higher levels of pituitary binding sites were found in diestrus 2 and proestrus than in estrus and diestrus 1. The changes in the GnRH receptor levels were more striking in the pituitary than in the ovaries. It appears that the total number of ovarian GnRH binding sites was not altered in either of the two persistent-estrus groups, but that their concentration was significantly higher (expressed in fmol/mg ovary or fmol/mg protein) than on any day during the estrous cycle.(ABSTRACT TRUNCATED AT 250 WORDS)     

Studies on the relationship between the estrous cycle of BALB/c mice and their resistance to Paracoccidioides brasiliensis infection

A relationship between the estrous cycle and non-specific host resistance to Paracoccidioides brasiliensis yeast cells was examined by using both sexes of adult BALB/c mice. They were divided into 6 groups, including a male group and females at proestrus, estrus, metestrus-I, metestrus-II and diestrus. The mice received yeast cells through three different inoculation routes; intravenous, intraperitoneal and intratracheal. In all of the inoculation routes, the clearance of the yeast cells was influenced by the estrous cycle. The female mice at estrus, which might have high blood estrogen levels, showed a marked clearance of the yeast cells from the blood, peritoneal cavity and lungs. These results suggested that non-specific host resistance to the yeast cells was enhanced by estrogen. All female groups inoculated by the three routes showed higher clearance of the yeast cells than the male group.     

Messenger RNA levels of estrogen receptors alpha and beta and progesterone receptors in the cyclic and inseminated/early pregnant sow uterus

The aim of the present study was to investigate differences in the expression of mRNAs for ERalpha, ERbeta and PR in the sow uterus at different stages of the estrous cycle as well as in inseminated sows at estrus and during early pregnancy by use of solution hybridization and in relation to plasma levels of estradiol and progesterone. Uterine samples were collected at different stages of the estrous cycle and after insemination/early pregnancy. In the endometrium, the expression of ERalpha mRNA and PR mRNA was similar for cyclic and early pregnant groups. Both were highest at early diestrus/70 h after ovulation and ERalpha mRNA was lowest at late diestrus/d 19 while PR mRNA was lowest at diestrus and late diestrus/d 11 and d 19. The expression of endometrial ERbeta was constantly low during the estrous cycle but higher expression was found in inseminated/early pregnant sows at estrus and 70 h after ovulation. In the myometrium, high expression of ERalpha mRNA and PR mRNA was observed at proestrus and estrus in cyclic sows and at estrus in newly inseminated sows. Higher expression of myometrial ERbeta mRNA was found in inseminated/early pregnant sows compared with cyclic sows, although significant only at estrus. In conclusion, the expression of mRNAs for ERalpha, ERbeta and PR in the sow uterus differed between endometrium and myometrium as well as with stages of the estrous cycle and early pregnancy. In addition to plasma steroid levels, the differences between cyclic and inseminated/early pregnant sows suggest that other factors, e.g. insemination and/or the presence of embryos, influence the expression of these steroid receptor mRNAs in the sow uterus.