Region-specific expression of nitric oxide synthases in the bovine oviduct during the oestrous cycle and in vitro

Nitric oxide synthases (NOS) account for the endogenous production of nitric oxide (NO), a small and permeable bioreactive molecule. NO is known to act as a paracrine mediator during various processes associated with female reproduction. In the present study, the mRNA expression of the endothelial (eNOS) and inducible (iNOS) NO synthases were examined in bovine oviduct epithelial cells (BOEC) during the oestrous cycle. In addition, eNOS and iNOS mRNA and protein were localised by in situ hybridisation and immunocytochemistry respectively. Furthermore, the effects of exogenously applied oestradiol-17β and progesterone on NOS mRNA regulation were studied in a suspension culture of BOEC. The eNOS mRNA abundance was low around ovulation (day 0) and increased significantly until pro-oestrus (day 18) in the ampulla. Immunoreactive protein of eNOS was detected predominantly in endothelial cells as well as in secretory oviduct epithelial cells at pro-oestrus. The iNOS mRNA concentration was significantly reduced in the isthmus at pro-oestrus (day 18) and oestrus (day 0) compared with persistently high levels in the ampulla. By in situ hybridisation, specific iNOS transcripts were additionally demonstrated in the oviduct epithelium. Immunoreactive iNOS protein was localised in secretory epithelial cells as well as in the lamina muscularis. The in vitro stimulation showed that both NOS were stimulated by progesterone, but not by oestradiol-17β. The region-specific modulated expression of eNOS and iNOS provides evidence for an involvement of endogenously produced NO in the regulation of oviductal functions.

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The interaction of fresh and frozen-thawed ram spermatozoa with oviducal epithelial cells in vitro

Reproduction Fertility and Development ◽

10.1071/rd00032 ◽

2000 ◽

Vol 12 (6) ◽

pp. 237 ◽

Cited By ~ 11

Author(s):

L. Gillan ◽

G. Evans ◽

W. M. C. Maxwell

Keyword(s):

Epithelial Cell ◽

Epithelial Cells ◽

Small Proportion ◽

Oestrous Cycle ◽

Cell Monolayers ◽

Oviduct Epithelial Cell ◽

Epithelial Cell Monolayers ◽

Oviduct Epithelial Cells

In order to investigate the interaction of fresh and frozen–thawed spermatozoa with oviduct epithelial cells, spermatozoa were co-incubated with ovine oviduct epithelial cell monolayers (OECM) derived from either complete oviducts, at any stage of the oestrous cycle (Experiments 1 and 2), or from different regions of the oviduct at different stages of the cycle (Experiment 3). Fresh and frozen—thawed spermatozoa displayed different patterns of binding to, and release from, the OECM. Frozen—thawed spermatozoa immediately bound to the complete oviduct OECM and were released after 2 h. A small proportion of fresh spermatozoa bound immediately, increasing to a maximum after 2 h, and were gradually released thereafter. When only the cells that were released from the OECM were observed by chlortetracycline staining in Experiment 2, it was found that the presence of an OECM increased the number of capacitated fresh spermatozoa while decreasing the number of capacitated frozen–thawed spermatozoa. Overall, the OECM advanced the membrane state of both types of spermatozoa from uncapacitated to acrosome-reacted. Fresh and frozen—thawed spermatozoa bound to OECM derived from the cells of the isthmus and the ampulla in similar proportions. However, more spermatozoa were capacitated when incubated with OECM derived from isthmic rather than ampullary cells. Higher proportions of fresh spermatozoa bound to, and were acrosome-reacted following incubation with OECM derived from post- rather than pre-ovulatory tracts. Such differences were not observed for frozen—thawed spermatozoa. The findings reported in this study show that fresh and frozen—thawed spermatozoa behave differently when in contact with oviduct cells in vitro. This may be a consequence of the more advanced membrane state of the frozen spermatozoa upon thawing.

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Suppressive Effect of Quercetin on Nitric Oxide Production from Nasal Epithelial Cells In Vitro

Evidence-based Complementary and Alternative Medicine ◽

10.1155/2018/6097625 ◽

2018 ◽

Vol 2018 ◽

pp. 1-8 ◽

Cited By ~ 4

Author(s):

Nachi Ebihara ◽

Kana Takahashi ◽

Haruka Takemura ◽

Yuko Akanuma ◽

Kazuhito Asano ◽

...

Keyword(s):

Nitric Oxide ◽

Epithelial Cells ◽

Mrna Expression ◽

Suppressive Effect ◽

Inos Mrna ◽

No Production ◽

Minimum Concentration ◽

Nasal Epithelial Cells ◽

Human Nasal Epithelial Cells

Nitric oxide (NO) is known to play pivotal roles as one of the final effector molecules in the development of allergic diseases, including allergic rhinitis (AR). Although quercetin has been reported to attenuate the clinical conditions of AR, its influence on NO production is not well defined. The present study aimed to examine the influence of quercetin on in vitro NO production from nasal epithelial cells after interleukin- (IL-) 4 stimulation. Human nasal epithelial cells (HNEpCs) at a concentration of 1 x 105 cells/ml were stimulated with 10.0 ng/ml of IL-4 in the presence and absence of quercetin. After 48 hours, the culture supernatants were collected and assayed for NO (NO2 and NO3) using the Griess method. The influences of quercetin on the transcription factor, STAT6, activation, and iNOS mRNA expression were also examined using ELISA and real-time quantitative RT-PCR, respectively. Addition of quercetin to cell cultures caused suppression of NO production from HNEpCs after IL-4 stimulation. The minimum concentration of quercetin that caused significant suppression was 1.0 nM. Treatment of cells with quercetin at more than 1.0 nM suppressed STAT6 activation and iNOS mRNA expression induced by IL-4 stimulation. The present results strongly suggested that quercetin favorably modified the clinical condition of AR through the suppression of NO production from nasal epithelial cells after IL-4 stimulation.

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234 REGION SPECIFIC ABUNDANCE OF INDUCIBLE NITRIC OXIDE SYNTHASE (iNOS) IN THE BOVINE OVIDUCT DURING THE ESTROUS CYCLE

Reproduction Fertility and Development ◽

10.1071/rdv17n2ab234 ◽

2005 ◽

Vol 17 (2) ◽

pp. 267

Author(s):

S.E. Ulbrich ◽

S. Rehfeld ◽

R. Rottmayer ◽

S. Hiendleder ◽

E. Wolf ◽

...

Keyword(s):

Nitric Oxide ◽

Epithelial Cells ◽

Estrous Cycle ◽

Mrna Abundance ◽

Inos Mrna ◽

Special Importance ◽

Luminal Epithelium ◽

Inos Protein ◽

Estradiol 17Β

Nitric oxide synthases (NOS) are involved in a large number of biochemical processes. By generating inorganic free NO radicals, which are important modulatory agents, NOS are supposed to contribute to the regulation of mammalian reproduction. We have, therefore, studied the expression and localization of inducible NOS (iNOS) in the bovine oviduct during the estrous cycle. Simmental heifers were synchronized and slaughtered at Days 0, 3.5, 12, or 18 after standing heat. Bovine oviduct epithelial cells (BOEC) of ampulla and isthmus were collected separately for ipsi- and contralateral oviducts, as determined by the ovulatory follicle or the functional corpus luteum. In addition, a BOEC in vitro suspension culture from heifers of day 3.5 was established and stimulated with physiological doses of progesterone and estradiol-17β (10 ng/mL and 10 pg/mL, respectively). Total RNA was extracted and iNOS mRNA was absolutely quantified using real-time RT-PCR. For statistical analysis, the MIXED procedure of the SAS program package (SAS Institute, Inc., Cary, NC, USA) was used to model the experimental design (n = 4). Differences were indicated as statistically significant in the case of P < 0.05. Furthermore, immunoreactive iNOS protein was localized on serial oviductal cryosections. Transcripts of iNOS mRNA were detected in bovine oviducts throughout the estrous cycle. Since the level of 18S rRNA did not differ significantly the data were not normalized. Positive immunohistochemical staining for iNOS in the luminal epithelium gave evidence for the presence of iNOS protein in the bovine oviduct. At estrus (Day 0) and Day 18, the mRNA abundance of iNOS in epithelial cells of both ipsi- and contralateral isthmus was significantly lower than in epithelial cells of the ampulla. At Day 3.5, iNOS expression was significantly reduced in the contralateral but not the ipsilateral isthmus, whereas the abundance in the ipsilateral isthmus reached levels similar to those in the ampulla. At Day 12 the mRNA abundance was low without region specific differences. A down-regulation of iNOS could be associated with an accelerated oviductal transport due to higher contractility and increased ciliary motion in the luminal epithelium of the oviduct. This might be of special importance around ovulation time for gametes as well as for the formation of a homogeneous oviductal fluid. A modulated expression pattern of iNOS during the estrous cycle points towards a possible hormonal regulation. Preliminary in vitro results showed that in BOEC iNOS responded to progesterone, while no such up-regulation was observed after estradiol-17β stimulation. As Day 3.5 coincides with early embryonic development shortly after fertilization, the possible progesterone-dependent increase of iNOS transcripts in the ipsilateral isthmus might lead to a necessary local quiescence. Due to region-specific different transcript abundance, the presence of a local NO-system in the bovine oviduct can be postulated. Therefore, these findings point out a possible important role of iNOS in the oviduct for reproductive success. This work was supported by the DFG Ei 296/10-1 and the Ev. Studienwerk eV.

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Expression of endothelial and inducible nitric oxide synthases is modulated in the endometrium of cyclic and early pregnant mares

Reproduction Fertility and Development ◽

10.1071/rd03103 ◽

2004 ◽

Vol 16 (7) ◽

pp. 689 ◽

Cited By ~ 18

Author(s):

H. Welter ◽

H. Bollwein ◽

F. Weber ◽

S. Rohr ◽

R. Einspanier

Keyword(s):

Nitric Oxide ◽

Nitric Oxide Synthases ◽

Oestrous Cycle ◽

Inos Mrna ◽

Enos Mrna ◽

Intense Labelling ◽

Polymerase Chain ◽

First Time ◽

Enos Protein ◽

Inducible Nitric Oxide

The expression of the endothelial and inducible nitric oxide synthases (eNOS and iNOS, respectively) was examined in the endometrium of cyclic and pregnant mares by real-time polymerase chain reaction and immunohistology. The concentration of eNOS mRNA varied throughout the oestrous cycle, with significantly higher transcripts on Day 5 of the oestrous cycle (P < 0.05), whereas iNOS transcription did not change significantly over time (P > 0.05). In early pregnant mares both eNOS and iNOS mRNA increased between Days 12 and 15 (P < 0.05). In cyclic mares, eNOS protein was detected immunocytochemically in endometrial epithelia, the basement membrane, the endothelial layer and smooth muscle cells of the vasculature. Using immunocytochemical methods, iNOS protein was undetectable in the endometrium of cyclic mares but could be demonstrated in pregnant mares. Endometrial epithelia of pregnant mares were immunopositive for both proteins with a more intense labelling for iNOS. Thus, the present study describes for the first time the modulation and spatial distribution of eNOS and iNOS expression during the oestrous cycle and early pregnancy, suggesting that ovarian steroids are differently involved in the regulation of each NOS. Localisation of eNOS protein in endometrial epithelia and various vascular components indicates that this isoform may be involved in the regulation of endometrial cyclicity. The presence and increase of both forms of NOS during early gestation suggest a role for them in the control of endometrial vascular bed and glandular activity to provide a suitable microenvironment for successful pregnancy.

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Recombinant TIMP-1 and -2 enhance the proliferation of rabbit corneal epithelial cells in vitro and the spreading of rabbit corneal epithelium in situ

Current Eye Research ◽

10.1076/ceyr.17.1.47.5247 ◽

1998 ◽

Vol 17 (1) ◽

pp. 47-52 ◽

Cited By ~ 16

Author(s):

Shizuya Saika ◽

Yoshiji Kawashima ◽

Yuka Okada ◽

Sai-Ichi Tanaka ◽

Osamu Yamanaka ◽

...

Keyword(s):

Epithelial Cells ◽

Corneal Epithelium ◽

Corneal Epithelial Cells ◽

Corneal Epithelial

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Pro-inflammatory cytokine-induced nitric oxide production and iNOS mRNA expression in human colonic epithelial cells

Gastroenterology ◽

10.1016/0016-5085(95)27712-9 ◽

1995 ◽

Vol 108 (4) ◽

pp. A851

Keyword(s):

Nitric Oxide ◽

Epithelial Cells ◽

Mrna Expression ◽

Inflammatory Cytokine ◽

Inos Mrna ◽

Nitric Oxide Production ◽

Colonic Epithelial Cells ◽

Oxide Production

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In vitro optimization of the Gallus domesticus oviduct epithelial cells culture

Theriogenology ◽

10.1016/j.theriogenology.2011.12.029 ◽

2012 ◽

Vol 77 (9) ◽

pp. 1834-1845 ◽

Cited By ~ 8

Author(s):

K. Kasperczyk ◽

A. Bajek ◽

R. Joachimiak ◽

K. Walasik ◽

A. Marszalek ◽

...

Keyword(s):

Epithelial Cells ◽

Gallus Domesticus ◽

Oviduct Epithelial Cells

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Gene and protein expressions of nitric oxide synthases in ischemia-reperfused peripheral nerve of the rat

AJP Cell Physiology ◽

10.1152/ajpcell.2001.281.3.c849 ◽

2001 ◽

Vol 281 (3) ◽

pp. C849-C856 ◽

Cited By ~ 35

Author(s):

Wen-Ning Qi ◽

Zuo-Qin Yan ◽

Peter G. Whang ◽

Qi Zhou ◽

Long-En Chen ◽

...

Keyword(s):

Nitric Oxide ◽

Peripheral Nerve ◽

Ischemia Reperfusion ◽

Nitric Oxide Synthases ◽

Inos Mrna ◽

Mrna Levels ◽

Protein Expressions ◽

Enos Mrna ◽

Nos Isoforms ◽

Endothelial Nitric Oxide

This study examined mRNA and protein expressions of neuronal (nNOS), inducible (iNOS), and endothelial nitric oxide synthases (eNOS) in peripheral nerve after ischemia-reperfusion (I/R). Sixty-six rats were divided into the ischemia only and I/R groups. One sciatic nerve of each animal was used as the experimental side and the opposite untreated nerve as the control. mRNA levels in the nerve were quantitatively measured by competitive PCR, and protein was determined by Western blotting and immunohistochemical staining. The results showed that, after ischemia (2 h), both nNOS and eNOS protein expressions decreased. After I/R (2 h of ischemia followed by 3 h of reperfusion), expression of both nNOS and eNOS mRNA and protein decreased further. In contrast, iNOS mRNA significantly increased after ischemia and was further upregulated (14-fold) after I/R, while iNOS protein was not detected. The results reveal the dynamic expression of individual NOS isoforms during the course of I/R injury. An understanding of this modulation on a cellular and molecular level may lead to understanding the mechanisms of I/R injury and to methods of ameliorating peripheral nerve injury.

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