Praca oryginalna Original paper
Ruminant cumulus-oocyte complexes (COC) in vitro are exceptionally sensitive to nutrient imbalance and abnormalities in the surrounding environment (16) owing to the influences e.g. of different phytoestro-gens (PEs) (17). PEs comprising different classes of polyphenolic, non-steroidal plant compounds with estrogenic (7) and antyestrogenic properties (19) or pro-perties independent of their estrogenicity (11) occur ubiquitously in fodder and exert the following well known adverse effects on the ovary: they suppress follicular development (especially in its early stages) and inhibit oestradiol action, disrupt normal endo-crine function, (they are referred to as endoendo-crine dis-rupters), increase androgen levels, decrease oestrogen levels and induce multioocyte follicles in the matu-ring mouse ovary.
Despite these influences on follicle growth, the effect of PEs on oocyte maturation remains to a large degree unknown. With respect to the influence of PEs on oocyte maturation, it is only known that in vitro genistein is able to block oocyte growth and disrupt follicle morphology (17, 22). However, considerably less is known about the influence of individual PEs on the maturation process of cow oocytes. The pro-liferative responses of PEs are strongly dependent on doses (19). At low doses they induce proliferative actions but at high doses they take advantage of anti-proliferative and anti-estrogenic actions of PE.
The aim of the study was to elucidate the influence of different phytoestrogens (izoflavons represented by genistein, stilbens by resveratrol (RES), lignans by enterodiol and coumestans by coumstrol) on the
Disturbances of cow oocyte maturation
by phytoestrogens
EWA BORZYM, RYSZARD BOBOWIEC, URSZULA KOSIOR-KORZECKA, FRANCO MARTELLI*, ARTUR BURMAÑCZUK**
Department of Pathophysiology, Chair of Preclinical Veterinary Sciences, Faculty of Veterinary Medicine, University of Life Sciences, Akademicka 12, 20-033 Lublin, Poland
**Department of Pharmacology Chair of Preclinical Veterinary Sciences, Faculty of Veterinary Medicine, University of Life Sciences, Akademicka 12, 20-033 Lublin, Poland
*Department of Anatomy, Biochemistry and Veterinary Physiology, University of Pisa, Viale delle Piagge 2, I-56124 Pisa, Italy
Borzym E., Bobowiec R., Kosior-Korzecka U., Martelli F., Burmañczuk A.
Disturbances of cow oocyte maturation by phytoestrogens Summary
The aim of the study was to elucidate the influence of different phytoestrogens (PEs) such as izoflavons (represented by genistein), stilbens (represented by resveratrol (RES)), lignans (represented by enterodiol) and coumestans (represented by coumstrol) on the maturation process of isolated bovine oocytes. We assumed that their influence on the rate of oocyte growth and maturation would be related primarily to their estrogenic activity (isoflavones and coumestans are similar to the 17â estradiol E2, the most potent oestrogen). Eighty to one hundred oocytes were cultured in 4 ml of the medium in six-well multidishes. Cumulus oocyte complexes (COCs) were matured at 39°C in humidified air containing 5% CO2 for 24 hours. Four phytoestrogens were tested at two concentrations: 10 µg/ml and 100 µg/ml. These results clearly show that there is no significant relation between PEs and the survival of cow oocytes. However, under the influence of different PEs the expansion of oocytes was suppressed, especially when genistein and coumestrol were introduced into the culture medium. It was noted that the addition of the phytoestrogens into the medium decreased the rate of oocyte maturation. The highest increase of immature oocytes (p £ 0.05) was noted after the addition of coumestrol (30.77%) and genistein (20%) (fig. 4). The most pronounced suppression of oocyte maturation by genistein and coumestrol observed in our research indicates, therefore, that there is a direct relationship between the potency of the estrogenic activity of particular PEs and the inhibition of oocyte maturation. We concluded from our studies that PEs may disturb the conception and fertilisation rate in the cow by prolonging cow oocyte maturation.
maturation process of isolated bovine oocytes. It was assumed that their influence on the rate of oocyte growth and maturation would be related primarily to their estrogenic activity (isoflavones and coumestans are similar to the 17â estradiol E2, the most potent oestrogen). Furthermore, since the biological effects of PEs vary depending on their chemical structures, we sought to determine how different phytoestrogens affect the maturation of cow oocytes. In the course of the present study, another aim was to evaluate the concentration-response relationship by using different doses of PEs.
Material and methods
Source of oocytes and culture conditions. Ovaries were collected from 43 cows at a local slaughterhouse and trans-ported to the laboratory in 0.9% saline at room tempera-ture. Within 2 h of slaughter the ovaries were washed three times in PBS containing 50 µg/ml of gentamicin (9). Oocy-tes from the ovary, placed on a petri plate heated to 37°C and filled with TCM-199 culture medium and 60 µl hepa-rin were obtained by slicing the ovary with a razor blade and flushing the created surfaces with a fluid medium. Oocytes were selected under a stereomicroscope (ZOOM, MSZ 200). Next the oocytes were classified following the criteria described by Pujol et al. (14) according to the cumulus oophorus morphology, corona radiata and the cytoplasmic aspect. All oocytes used in this study were pre-viously classified as Grade 1. Oocytes from cows classi-fied as Grade 2-4 were excluded. Examined Grade 1 oocy-tes were washed and cultured in TCM-199 (with Hanks salts; Sigma) supplemented with 3 mg/ml BSA (Sigma), 50 µg/ml gentamicin, 0.02 mg/mg glucose, 0.5 ml/10 ml amino acids (Sigma), ethyl alcohol. Eighty to one hundred oocytes were cultured in 4 ml of the medium in six-well multidishes (Nunc). Cumulus oocyte complexes (COCs) were matured at 39°C in humidified air containing 5% CO2 for 24 hours (Gilchrist, 2007; Suzuki, 2006; Wehrend, 2001). Four phytoestrogens were tested at two concentra-tions, 10 µg/ml and 100 µg/ml. Phytoestrogens (2 mg) were diluted in 10 µl ethyl alcohol and 2.5 ml medium.
Trypan Blue test. Trypan blue is a vital stain used to colour selectively dead tissues or blue cells. Live cells or tissues with intact cell membranes are not coloured. Since the cells are very selective in the compounds that pass through the membrane, in a viable cell Trypan blue is not absorbed; however, it traverses the membrane in a dead cell. Hence, dead cells are shown as a distinctive blue colour under a microscope. The oocytes were tested before and after culturing.
Evolution and meiotic stages. Oocytes removed from the culture were denuded, fixed in acetic acid/ethanol (1 : 3) for 24 hours, stained with 1% aceto-orcein and examined under a phase-contrast microscope at 400 × magnification (1). The nuclear maturation stages of bovine oocytes were classified into GVBD (germinal vesicle breakdown), M I and MII according to the method of K¹tska et al. (6).
Brilliant cresyl blue (BCB) test. Brilliant cresyl blue (BCB) is a vital blue dye which determines the intercellu-lar activity of glucose-6-phosphate dehydrogenase (G6PD),
an enzyme synthesized during the oocyte growth phase, but with decreased activity in oocytes that have finished their growth phase. The cytoplasm of these oocytes turns blue because they do not reduce BCB to a colourless com-pound (Pujol 2004; Rodriguez-Gonzalez 2002). After cul-turing, the cells were washed three times in PBS modified by the addition of 36 mg/l pyruvate (Sigma), 1000 mg/l of glucose (Sigma), 0.5 g/l of bovine serum albumin (BSA) (Sigma Fraction V), and 50 µg/ml of gentamicin (mPBS). Next the oocytes were exposed to BCB diluted in mPBS at the concentration of 26 µM for 90 min at 38.5°C in a humi-dified atmosphere. After exposure to BCB, the oocytes were washed three times in mPBS and classified into two groups, depending on their cytoplasm colouration: oocytes with blue cytoplasm and oocytes without blue cytoplasm coloration.
Statistical analysis. All experiments were repeated at least three times. Statistical analysis of the obtained results was performed using Excel 2003. The results are expres-sed as a mean and standard deviation (x ± SD). Compari-sons between the series of the results were performed using the Students t-test (p £ 0.05 and p £ 0.01) (Statistica 5.0.).
Results and discussion
Cell culture characteristics. Just before the selec-tion of oocytes (control 0 h), 44 oocytes were found alive (91.67%) and 4 dead. After 24 hours of incuba-tion, 18 oocytes were alive (90%) and 2 dead. How-ever, during the incubation with phytoestrogens the survival of the oocytes was as follows: 95% of the living cells were obtained with resveratrol and coume-strol at the concentrations of 10 µg/ml and 100 µg/ml, whereas 90% with the genistein (10 µg/ml) and 85% with genistein (100 µg/ml). When enterodiol at the concentration of 10 µg/ml was added into the culture, there was no significant difference (90%) in the sur-vival of the oocytes. However, the 100 µg/ml concen-tration of this hormone decreased their survival to 80%. These results clearly demonstrate that there is no significant relation between hormones and the sur-vival of cow oocytes.
The expansion of the cumulus oophorus and corona radiata after 24 hours of incubation was also observed. Suzuki reported that this expansion happens in the grown-up oocytes. The number of these grown-up cells depends on the kind of phytoestrogens and their con-centration. After 24 hours of incubation 89 grown-up cells were observed out of 116 in total (fig. 2). After the addition of resveratrol, genistein, coumestrol and enterodiol at the concentration of 10 µg/ml, the num-ber of the expanded cells varied between 52 and 47, out of 70. The changes were observed after the addi-tion of these four phytoestrogens at the concentraaddi-tion of 100 µg/ml, however, with genistein and coumestrol fewer cells were changed (fig. 1).
Nuclear maturation of bovine oocytes. The divi-sion activity of cow oocyte nucleus was measured using orcein. After dyeing the cells the phase of meiosis divisions could be seen (tab. 1). Before starting up
a culture, 93.33% of the oocytes were in the phase of dictioten, and only 4% of the cells were mature. This proportion was reversed after 24 hours of incubation, so that 4.31% of the cells stopped growing, while 76.72% reached metaphase II.
It was observed that the addition of phytoestrogens into the medium decreased the rate of oocyte matura-tion. A significant inhibitor seemed to be coumestrol
which stopped the maturation of the oocytes at the concentration of 10 µg/ ml. The genistein at the concentration of 100 µg/ml (p £ 0.05) also inhibi-ted maturation which was decreased to 47.15%. In contrast to all remaining phytoestrogens, resveratrol increased the growth of oocytes after the addi-tion of a higher dose. However, ene-terodiol and coumestrol at the con-centration of 100 µg/ml caused a de-crease in the number of matured oocy-tes, but much lower than with the genistein (fig. 3).
Maturation of the oocytes in the medium sup-plemented with phytoestrogens (BCB test). It was observed that just after isolation 95.23% of the oocy-tes were in the stage of dictioten (p £ 0.01). However, after 24 h in the culture medium without gens 92% matured (p £ 0.01). When the phytoestro-gens at the concentration of 10 µg/ml were added into the culture medium an increase of immature oocytes was noted. The highest increase in the number of immature oocytes (p £ 0.05) was shown after the addition of coumestrol (30.77%) and genistein (20%) (fig. 4). After the addition of genistein at the concentration of 100 µg/ml the number of immature cells was the highest. However, the lowest percentage of immature cells was observed after the addition of resveratrol at the same concentra-tion. In the case of enterodiol and cumestrol the number of oocytes (p £ 0.05) was the same or even higher, compared to resveratrol (fig. 5).
Cow forage contains several plant-derived, nonsteroidal weakly
s e t y c o O Co0nhrtol C2o4nrthol l o rt a r e v s e R )l m / g µ ( G(eµngi/smte)lin En(µtegr/omdi)lol Co(uµmg/ems)lrtol 0 1 100 10 100 10 100 10 100 r e b m u n e h T d e t s e t s ll e c f o 90 116 70 70 70 70 70 70 70 70 ) % ( ) D B V G ( (938.433) (4.531) 14 15 17 20 14 13 15 16 I e s a h p a t e M ) % ( )I M ( (2.222) (182.296) 14 12 13 17 16 19 18 19 II e s a h p a t e M ) % ( )I I M ( (4.444) (768.972) 52 53 50 33 51 48 47 45
Tab. 1. The influence of phytoestrogens on the number of oocytes in different stages of growing. GVBD germinal vesicle breakdown
74,095 75,745 71,495 47,15 72,75 68,44 67,345 64,195 76,73333333 0 10 20 30 40 50 60 70 80 90 % of maturity 100 µg/ml 10 µg/ml * * p£0.05 resveratrol genistein enterodiol coumestrol control 24 h
Fig. 3. The percentage of oocytes in metaphase II (M II), under the influence of res-veratrol, genistein, coumestrol and enterodiol at the concentrations of 10 µg/ml and 100 µg/ml (n = 70; average ± SD; p £ 0.05)
Fig. 1. Oocytes before culture, the compact layer of cumulus oophorus and corona radiata (phase contrast microscope, × 100)
Fig. 2. Oocytes after culturing, the expansion of cumulus oophorus complex and corona radiata (phase contrast micro-scope, × 100)
estrogenic PEs. The influence of the representatives of four main PE classes, namely flavonoids (genistein), stilbenes (resveratrol (RES), coumestans (coumestrol) and lignans (enterodiol), on oocyte maturation was analysed in this work. The targets of phytoestrogens comprise steroid receptors (19), steroid metabolising enzymes (13), elements of signal transduction (5) and even the DNA processing machinery (22). As has been found in our studies, PEs, apart from provoking anestrus (18), also exert a suppressive effect on the maturation of cow oocytes.
In the studies cumulus-oocyte complexes (COC) isolated from both ovaries of adult cows were used. Such complexes of oocytes are particularly vulnerable when they are enclosed in a growing, hormone-depen-dent follicle (15). During later stages of maturation, oocytes are under a strong influence of estrogens and
androgens (1, 2) which probably do not act on the occytes directly but through the influence on the somatic granulosa cells attached to the oocytes, called cu-mulus cells (10, 16). The resumption of meiosis ensures a local production of the meiosis activating sterols (MAS) by the cumulus cells (15, 16). Thus, the most vital influence on the oocytes occurs through the cumulus cells. In the cow, the oocytes pass through the stages of the first meiotic prophase, leptotene, zygotene, pachytene and diplotene (3). When the oocytes reach the diplotene stage they are enclosed in follicles, a process necessary for oocyte survival. By entering meiosis, the cells lose their ability to prolifera-te, preventing renewal of the naturally decreasing pool oocytes. When rele-ased, oocytes in vitro, in contrast to those confined in the follicular fluid of antral follicles and subjected to a suitable culture medium, undergo undisturbed progression of meiosis. Oocyte meiotic maturation requires a complex network of pathways with the protein phosphorylation and de-phosphorylation by ser/thr kinases (18). It seems that tyrosine phospho-rylation is implicated in the positive regulation of oocyte maturation. As has been documented by Van Cauwenber-ge (17) and also seen in our studies, GVBD is especially inhibited by geni-stein (27% of oocytes remain in the GVBD phase under the influence of 100 µg/ml genistein). Such action of some PEs results probably from the inhibitory action on tyrosine kinase (PTK). Among PEs used in our study genistein appears to be the greatest inhibitor of oocyte maturation. At the dose 100 µg/ml about half of the oocytes remain immature after a 24-hour cultivation period. Some data suggest that the inhibitory effect of PEs on meiosis reinitiation is a consequence of tyro-sine kinase (PTK) inhibition (9, 12). The most pro-nounced inhibitory effect of genistein on meiosis reinitiation in cow oocytes may also derive from a uni-que ability of this flavonoid to arrest the G2/M phase of the cell cycle. This effect may be partly due to the inhibition of kinase activities of cdc2 and cdk2 and a decrease in cyclin B1 expression. Genistein was also shown to inhibit mammalian type II topoisomerase and to stabilise the topoisomerase-DNA cleaved complex (22).
In comparison to estradiol (E2), the relative estroge-nic effects of different PEs are as follow: coumestrol:
Fig. 5. Rate of cow oocyte maturation under the influence of phytoestrogens (at the concentration of 100 µg/ml), the number of oocytes before culture (n = 63), post culture (n = 75), and with the addition of phytoestrogens (n = 65; average ± SD; p £ 0.01, p £ 0.05) 0 20 40 60 80 100 120
Control 0h Control 24 h Resveratrol Genistein Enterodiol Coumestrol ** ** * * * ** % of maturation * p£0.05 ** p£0.01 immature mature ** ** * * ** 0 20 40 60 80 100 120
Control 0h Control 24 h Resveratrol Genistein Enterodiol Coumestrol
% of maturation * p£0.05 ** p£0.01 mature immature Fig. 4. Rate of cow oocyte maturation under the influence of different phyto-estrogens (at the concentration of 10 µg/ml); the number of oocytes before culture (n = 63), post culture (n = 75), and with the addition of phytoestrogens (n = 65; average ± SD; p £ 0.01, p £ 0.05)
0.202, genistein: 0.084, equol: 0.061, diadzein: 0.013 and biochanin A: < 0.006 (9). The most pronounced suppression of oocyte maturation caused by genistein and coumestrol indicates, therefore, that there is a di-rect relationship between the potency of the estro-genic activity of particular PEs and the inhibition of oocyte maturation.
It is also suggested that the suppressive effect of dif-ferent PEs on cow oocytes may derive from keeping the MPF (maturation promoting factor) in its inactive state (17). This initial hypothesis needs further proof, but it may explain why different PEs exert a more or less inhibitory influence on the maturation processes of oocytes. Another way by which PEs induce inhibi-tory effects in the maturation of cow oocytes may result from the stabilisation of the perinuclear micro-tubule network centred on a micromicro-tubule organiser centre (MTOC) closely associated with the nucleus (germinal vesicle GV). As has been found in another species, the peripheral migration of GV starts the morphological events initiating oocyte maturation (17). However, under the influence of PEs, especially genistein and cumestrol, central nuclear positioning may be responsible for delaying oocyte maturation.
An additional putative mechanism of the suppres-sive action of PEs on the maturation of oocytes may result from the action of PE metabolites to which, in the case of genistein, p-ethyl phenol belongs. As has been postulated by Woclawek-Potocka et al. (20), infertility caused by PEs may arise from the action of these active metabolites.
The remaining PEs used in our studies (stilbene -resveratrol and lignan - enterodiol) have not yet been studied with respect to the maturation of oocytes. Un-like the three other PEs used in the studies, resveratrol (RES) does not remarkably influence the maturation process of cow oocytes. The reason for such different effects may derive from the simultaneous action of RES as the agonist/antagonist of ERs (4). The lowest sup-pressive effects of RES on the maturation process of cow oocytes may also result from the weak affinity of RES to ER. Furthermore, RES may be capable of dif-ferentially affecting gene transcription in estrogen--sensitive oocytes.
Given that isolated oocytes in culture were de-prived of high concentrations of E2, which occur in follicle, the inhibitory response evoked especially by genistein and coumestrol would be, in experimental conditions, exaggerated because both these PEs have a high affinity to ER (19). Therefore, further studies are needed in which the actions of PEs will be compa-red in relation to E2 levels in the cultured medium.
In conclusion, PEs may disturb the conception and fertilisation rate in the cow by prolonging cow oocyte maturation. This conclusion is strengthened by the fact that the latening of oocyte maturation in PE-treated oocytes was dose dependent.
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Authors address: prof. dr hab. Ryszard Bobowiec, Akademicka 12, 20-033 Lublin, Poland; e-mail: ryszard.bobowiec@up.lublin.pl
