Blood nitric oxide and ovarian steroids levels during the cycle stages
in Brown Swiss cows
Aziz BÜLBÜL1,H. Ahmet ÇELİK2, Meltem ŞİRELİ3, Gülcan AVCI4, Turan CİVELEK5
1 Afyon Kocatepe University, Faculty of Veterinary Medicine, Department of Physiology; 2Department of Obstetrics and
Gynecology, Afyonkarahisar; 3 Ankara University, Faculty of Veterinary Medicine, Department of Physiology, Ankara; 4 Afyon
Kocatepe University, Faculty of Veterinary Medicine, Department of Biochemistry; 5 Department of Internal Medicine,
Afyonkarahisar, Turkey.
Summary: The purpose of this study was to examine the fluctuations of the nitric oxide (NO) levels during the estrus cycle stages of cows and the association of these variations with ovarian steroids. In this study eighteen Brown Swiss cows were used. The ovarian activities of the cows were determined by rectal and ultrasound examinations. The estruses of the cows were synchronized by double dose prostaglandin F2α (PGF2α) administered with 11-day intervals. The blood samples of the cows in the cycle stages were
taken, and NO, estradiol and progesterone values were measured. Highest estradiol concentration was determined during estrus stage (p<0.05). Progesterone was the lowest in estrus, whereas it was the highest in midcycle stage (p<0.05). The highest NO value was measured during the estrus (p<0.05) and the lowest NO value in the metestrus (p<0.05). In conclusion, the variations was observed in the NO level in different cycle periods in Brown Swiss cows, and the NO was found to be in highest concentration in proestrus and estrus periods like 17β-estradiol
Keywords: Cow, estradiol, estrus cycle, nitric oxide, progesterone
İsviçre Esmeri ineklerin siklus dönemlerinde kan nitrik oksit ve ovaryum steroid düzeyleri
Özet: Çalışmada İsviçre Esmeri ineklerde östrüs siklusunun dönemlerinde kan nitrik oksit ve ovaryum steroidlerinin düzeylerinin belirlenmesi amaçlanmıştır. Çalışmada 18 İsviçre Esmeri inek kullanıldı. Rektal ve ultrason muayeneleri ile ineklerin ovaryum aktiviteleri belirlendi. Hayvanların östrüsleri 11 gün ara ile çift doz PGF2α uygulanarak senkronize edildi. Siklusun dönemlerinde kan örnekleri alınarak nitrik oksit (NO), östradiol ve progesteron analizleri gerçekleştirildi. En yüksek östradiol düzeyi östrüs döneminde belirlendi (p<0.05). Progesteron düzeyi östrüs döneminde en düşük; erken, orta ve geç diöstrus dönemlerinde ise en yüksek ölçüldü (p<0.05). Nitrik oksit en yüksek östrüs döneminde, en düşük ise metöstrüs döneminde belirlendi. Sonuç olarak İsviçre Esmeri ineklerde kan NO düzeyinin siklusun dönemlerinde değiştiği ve 17β-östradiolün en yüksek düzeyde bulunduğu proöstrus ve östrus dönemlerinde NO’nun da yüksek olduğu belirlenmiştir.
Anahtar sözcükler: İnek, nitrik oksit, östradiol, östrüs siklusu, progesteron.
Introduction
Nitric oxide (NO) is an inorganic, short lived (a few second) free radical gas that due to its high solubility, freely diffuses through biological membranes. It is synthesized from L-arginine. The involvement of NO in the modulation of ovarian function is documented by several studies aimed at demonstrating its production within the ovary and at clarifying its role in the regulation of steroidogenesis, follicle development, oocyte maturation, ovulation, luteal function, and luteal regression (5,6,16,17). It is now well known that both folliculogenesis and ovulation are regulated by a variety of factors, such as cytokines, growth factors, and locally produced substances, among which NO seems to play an important role. Nitric oxide levels have been shown to change during follicular growth (30). Several studies indicate that there are interactions between NO and
ovariansteroidogenesis (14,16,17). While NO contributes to the development of corpus luteum by increasing the flow of blood with its vein enlarging effect, it also controls the PGE2 level. Hurwitz et al. (15) reported a decrease in the level of NO, PGE2 and progesterone, after the inhibition of NO synthesis (NOS) enzymes. It has been indicated that NO, which plays a role in the optimal formation of corpus luteum in the early and middle periods of luteal phase, participates in the regulation of functional luteolysis at the end of the luteal phase (32). Likewise, Jaroszewski and Hansel (20) have reported that NO plays a role in the beginning of luteal regression in cows.
The aim of this study was to reveal the occurrence of relationship between NO, 17β-estradiol and progesterone concentration at cycle stages in Brown Swiss cows.
Material and Methods
Animals
Eighteen Brown Swiss cows of 4–5 years of age that were kept and fed under identical conditions in Konya International Bahri Dagdas Agricultural Research Institute were used for the study. These animals were selected among those that had calving normally, and those had average 65 ± 10 days and encountered no problems in postpartum period.
Experimental procedure
In the first place, the ovarian activities of the animals were determined based on the presence of a functional corpus luteum at palpation per rectum, ultrasonographic examinations (VetScanner480; Pie Medical, Maastricht, The Netherlands) and analysis of progesterone. Rectal palpation and ultrasonographic examinations were performed on restrained animals after evacuating the rectum. No physical or chemical methods were used to restrain the animal during the procedure.
Animals that determined ovarian activities were synchronized with double dosage of prostaglandin F2α (500 µg Cloprostenol) injections administered intramuscularly with intervals of 11 days. The estruses of animals were determined with person’s observation and the experimental procedure was started after determination of estrus. The animals were not inseminated after estrus.
The examination of ovaries were done with rectal and ultrasonographic examinations at estrus (day=0), metestrus (days= 2 to 3), early diestrus (days= 5 to 6), midcycle stage (days= 8 to 10), late diestrus (days= 14 to 16) and proestrus (days= 19 to 21) stages of cycle for determination follicles and corpus luteum diameter. In the transrectal applications, the probe of the ultrasonography device was places on ovaries and they were scanned as separate slices. The locations of the follicles and corpus luteum in both ovaries were determined and recorded during ultrasound examinations. Diameter changes in these structures were also determined.
Blood samples were collected from the animals on the same days with the rectal and ultrasonographic examinations. Ten milliliters of blood taken from jugular vein were transferred to non-heparinized and containing an anticoagulant (heparin sulphate) glass tubes. Samples were centrifuged for 10 min with 1620 g. Plasma (for hormones) and serum (for NO) samples were stored at -20°C until 17β-estradiol , progesterone and NO assays were done.
Analytical techniques
Measurement of NO levels: In order to measure the amount of NO in serum, a method (Griess reaction) based on the principle of forming a colorful azo
derivative with a primary aromatic amine (sulphanilamide) diazotization and N-(1-naphthyl) ethylenediamine (NEDD) in nitrite acidic setting. According to the procedure, sulfanilamide was dissolved in HCl to the concentration 1%, and N-(1-naphtile) ethylenediamine (NEDD) was dissolved in the same solvent to the concentration 1% and vanadium chloride was added to convert nitrate to nitrite (23).
Hormone assay: Hormone analyses were performed with EIA on plasma samples according to Jabri (19) for 17β-estradiol and Prakash et al. (28) for Progesterone. The plasma hormone concentrations were assayed by a double antibody microtitration plate enzyme immunoassay (EIA) method, and in every experiment series, two control plasma samples were used to control the accuracy of the assay.
Statistical analysis
Variance analyses was conducted in order to find out whether the difference of NO, 17β-estradiol and progesterone values between cycle periods are significant, and Duncan test were used to determine from which groups this variance stemmed from. Group differences were declared significant at p< 0.05.
Results
Follicle and CL measurements, serum NO, plasma 17β-estradiol and progesterone values were given table 1. The diameters of follicles were found differently at various cycle stages. Diameters of follicle were the highest at estrus. The differenciency can not be determined among metestrus and early diestrus also midcycle stage, late diestrus and proestrus respectively.
Highest 17β-estradiol concentration was determined during estrus stage. The lower concentration of 17β-estradiol was observed at other cycle stages.
Likewise follicles, the diameters of corpus luteum were observed differently. Progesterone were also determined various concentration. While progesterone level in estrus was lower than in metestrus and proestrus, it in early diestrus, midcycle stage and late diestrus was higher than other cycle stages.
The highest NO concentration occurred in the estrus and the lowest in the metestrus and late diestrus periods. The NO concentration was decreased preceding the estrus to metestrus. The concentration increased during early diestrus. The level of NO at early diestrus similarly the same concentration in midcycle stage but then decrease at late diestrus. The level of NO start increased at proestrus stage. The NO values at proestrus statistically same the estrus. Likewise, metestrus value was found same late diestrus also early diestrus and midcycle NO values were the same concentration.
Discussion and Conclusion
The fact shown by the study that the NO levels and follicle diameters of cows in the estrus and proestrus periods were bigger than those in other periods, increase in the nitrite/nitrate levels in the circulating blood and follicle diameter enlargement in the follicular phase of the cycle, similar finding in rat was reported by Jablonka-Shariff et al. (18). Al-Hijji et al. (3) and Dixid and Parvizi (10) show that in similarly to the findings of the experiments conducted on animals that estrogen increases NO production. Nitric oxide is reported as a key mediator of estrogen effect in body by many researches (14,16,17). Also it has been proven that 17β-estradiol modulates NOS activity and its gene expression both in reproductive and nonreproductive tissues. This effect can be positively or negatively and final effect is determined by the tissue type and NOS isoform (14). Concerning the ovary, the vascular changes in the ovary changes upon estrogen during follicular development and it is attributed to NO (1). Due to stimulation of endothelial NO synthesis, estrogen can act as a vasodilator as reported. In this reason it can be interpreted that this could have stemmed from the synthesis-increasing effect of estrogen in periods when estrogen hormone is high. As a matter of fact, the decrease in the level of estrogen, the main source of which is Graaf follicle (13), as a result of the ovulation of the follicle will affect the NO level too.
In cows, ovulation occurs at metestrus and consequently estrogen concentration decrease (13). So the metestrus stage is characterized by low estrogen levels related to this. We found that the serum samples which were taken during metestrus had low estrogen levels. The finding of the study that the NO level is low in the metestrus period, during which estrogen level is low in cows, shows that it comes out after the decrease in the estrogen-origin stimulation. Today it is not clear the exact mechanism of possible 17β-estradiol induction to stimulate NO release. It is can be taught that NO release
activity is stimulated, NOS protein is increased, and/or the levels of essential cofactors for NOS are increased by estrogen through a receptor operated mechanism. Besides, the levels of NO collectors, such as superoxide anions and factors which accelerate the inactivation and breakdown of intracellular NO might be decreased by 17β-estradiol (4,8).
The increase observed in the 17β-estradiol, progesterone and NO level in the early diestrus stage. The reasons of rise may be increasing follicular diameters at 17β-estradiol levels, starting corpus luteum development at progesterone levels. The finding of increase NO levels supports the findings of other studies (22,26) which report that as the cycle progresses, sensitivity to NO increases and more NO is synthesized in the luteinized ovarium. In the luteal period, increase in the flow of blood ensures the formation of corpus luteum and maintains its functional characteristics (21). The evidence that NO, which decreases vein resistance and increases the flow of blood, has increased in the mentioned period is in compliance with the said physiological mechanism. Much evidence suggests that NO is involved in the regulation of CL function and lifespan, but opposing actions have been reported, depending on the stage of CL development. Motta et al. (26) observed that in the midcycle stage CL in the rat, NO favoring the maintenance of CL. Dong et al. (11); speculated that NO could reduce or prevent luteolytic effects of prostaglandins on newly development CL, thus maintaining adequate progesterone. Nitric oxide is also possibly involved in the control of luteal vascularization. In fact, Nitric oxide produced by endothelial luteal cells increases blood flow by stimulating arteriolar smooth muscle relaxation and favors angiogenesis through an increase in vascular endothelial growth factor production by capillary pericytes (29). Increase in the level of progesterone in the metestrus to early diestrus period may lead to the beginning of the development of corpus luteum. While a significant increase is observed in the
Table 1. Follicle and corpus luteal size, serum nitric oxide, plasma 17β-estradiol and progesterone levels of animals during their estrus cycle periods (n=18). Data were given as mean ± STD.
Tablo 1. Östrus siklusu süresince hayvanlardaki follikül ve corpus luteum büyüklükleri ile serum nitrik oksit, plazma östradiol ve progesteron düzeyleri.
Estrus Metestrus Early diestrus Midcycle stage Late diestrus Proestrus Day of the estrus cycle
0 2 to 3 5 to 6 8 to 10 14 to 16 19 to 21
Follicle (cm) 1.54±0.19a 0.70 ±0.11c 0.74 ± 0.09c 1.13 ± 0.10b 1.17 ±0.06b 1.19± 0.06b
Corpus luteum (cm) --- 1.25 ± 0.08d 1.56 ± 0.20c 2.13 ± 0.27a 1.87±0.16b 1.34± 0.09d
Nitric oxide (µmol/L) 16.44±4.20a 7.69±1.35c 11.35±1.16b 11.34±1.29b 7.97±1.19c 15.42±2.91a
17β-estradiol (pg/ml) 16.20±3.80a 3.50±0.95b 4.30±0.72b 8.23±1.56b 7.76±2.10b 14.40±3.10a
Progesterone (ng/ml) 0.34±0.17a 1.70±0.85b 3.09±0.97a 4.30±1.64a 3.32±1.29a 1.28±0.10b
progesterone level owing to the acceleration of the development of corpus luteum in the early luteal period, a rise is also observed in the level of NO which takes part in supporting the development of corpus luteum (13).
The NO level in the mid-cycle period when corpus luteum completes it development has been found to be similar to the concentration in the early luteal period. While the NO level in the late luteal period was found to be at a similar level with the metestrus period, it has been observed that the decrease which started in the mid-cycle continued through the late diestrus too. Within the framework of the findings of the study, a decrease has been observed in the late luteal period in both progesterone and NO levels (p<0.05). Decrease in the progesterone level may be associated with the beginning of lysis in corpus luteum, and the decrease in the NO level with the increase in the level of PGF2α which is responsible for luteolysis. The level of PGF2α must increase in order to corpus luteum to regress structurally and functionally. High levels of NO in the early luteal period and in the mid-cycle period may lead to the increased secretion of PGF2α, which results in the beginning of luteolysis, and also due to the negative effect of this molecule (26, 32) may lead to a decrease in its own level.
The expression of eNOS, as well as total NOS activity, diminishes with CL aging sheep, (29), rabbit, (7,13), rat, (24), even though different findings have been reported in humans (9). Nitric oxide is also involved in luteolysis, which depends on an oxytocin-mediated prostaglandin release. Evidence exists that shows that oxytocin acts by enhancing NOS activity (24,25) and NO stimulates the synthesis of PGF2α on human, (12), bovine, (32), which in turn increases NOS activity, thus activating a positive feedback mechanism (7,26). At the same time, Nitric oxide decreases progesterone production (31). Alternative mechanisms by which NO participates in luteal regression involve lowering 17β-estradiol production, resulting in the subsequent demise of the CL (27), and increasing apoptosis (33). In fact, the large amounts of NO induced by iNOS during the late stage of CL are likely to exert a proapoptotic specify. Estrogen induced NO production may be attenuated by progestins. Even in presence of high concentration of 17β-estradiol, serum nitrit/nitrate levels is decreased by luteal increases in progesterone levels in hormonally stimulated menstrual cycles in human (9). Nevertheless nitric oxide synthesis is not interrupted by progesterone concentrations during follicular phase. Cytocine induced NO synthesis in cultured endothelial cells can not be inhibited during progesterone inhibits estrogen induced endothelium dependent responses associated with the production of NO.
In the proestrus period, while the decrease in the progesterone level continued, the NO level was observed to be increasing. It is known that in cows, after the luteal period, the development of the follicle that will ovulate in the proestrus period gains impetus and the level of estrogen in circulation increases. An increase may also be expected in the positive effect (2) of the increased level of estrogen on NO synthesis. Therefore, in the proestrus period, when the estrogen level start increases, the level of NO will be increase. As a matter of fact, the fact that NO level increases while the decrease in the progesterone level continues in the late luteal phase confirms these considerations.
In the light of obtained data, it is thought that, NO may be mediated to estrogene and progesterone in the follicular development and CL regression in cows
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Address for correspondance
Aziz Bülbül
Afyonkarahisar Kocatepe University Faculty of Veterinary Medicine, Department of Physiology
ANS Campus 03200 Afyonkarahisar - Turkey e-mail:[email protected]
