Interrelationships between ovarian follicles grown in culture and possible mediators

G Model REPBIO 221 No. of Pages 8 Reproductive Biology xxx (2016) xxx–xxx Contents lists available at ScienceDirect Reproductive Biology journal homepage: www.elsevier.com/locate/repbio Original article Interrelationships between ovarian follicles grown in culture and possible mediators Alexander V. Sirotkina,b,* , Iveta Florkovi9cová (Koni9 cková)b , Hans-Jorg Schaefferc, a d Jozef Laurincik , Abdel Halim Harrath a Dept. Zoology and Anthropology, Constantine the Philosopher University, 949 74 Nitra, Slovakia Dept. Genetics and Reproduction, Research Institute of Animal Production, 949 59 Lužianky, Slovakia Universitäts-Frauenklinik, 50931 Köln, Germany d Zoology Department, College of Science, King Saud University, Riyadh, Saudi Arabia b c A R T I C L E I N F O Article history: Received 8 December 2016 Received in revised form 15 January 2017 Accepted 26 January 2017 Available online xxx Keywords: Ovary Follicle Growth Insulin-like growth factor-I (IGF-I) Progesterone Estradiol A B S T R A C T Dominance or cooperation between ovarian follicles can determine the number of ovulations and fecundity, but interrelationships between follicles in mono- and poly-ovulatory species and their mechanisms are poorly understood. The goals of this work were to determine the existence and compare the character of mutual influence of cultured ovarian follicles from a mono-ovulatory species (cow) with established follicular dominance with those from a poly-ovulatory species (pig), in which interrelationship between follicles remain unknown, and to examine the role of ovarian cell proliferation, the insulinlike growth factor I (IGF-I)- oxytocin (OT) system, and steroid hormones in mediating interrelationships among ovarian follicles. Bovine and porcine ovarian follicles were isolated and cultured alone and in pairs, and the percentage of growing follicles was calculated. Porcine follicles were cultured alone and in pairs after addition of exogenous OT and IGF-I (100 ng mL 1) or inactivation of endogenous OT and IGF-I by antisera against these hormones (1%). Proliferation of porcine follicular cells was assessed by SDS PAGE-Western immunoblotting, the release of IGF-I, progesterone, androstenedione and estradiol by cultured porcine ovarian follicles was analyzed by RIA/EIA. Overall, our observations suggest (1) competition/dominance (mutual suppression of growth) in bovine ovarian follicles, (2) cooperation (mutual support of growth) in porcine ovarian follicles, (3) that this mutual growth of porcine ovarian follicles was caused by the promotion of cell proliferation, (4) that this mechanism was probably not involved in bovine follicular dominance, (5) that communication between both porcine and bovine follicles affects their secretory activity, and (6) that both follicular dominance in cows and cooperation of follicles in pigs can be mediated by either down- or up-regulation of the IGF-I-OT system, which in turn affects follicular steroidogenesis and promotes follicular cell proliferation and follicular growth. © 2017 Society for Biology of Reproduction & the Institute of Animal Reproduction and Food Research of Polish Academy of Sciences in Olsztyn. Published by Elsevier Sp. z o.o. All rights reserved. 1. Introduction Interrelationships between follicles within the ovary can determine both individual and species fecundity. In monoovulatory species (e.g., cow, sheep, goat, buffalo, and horse) usually only one dominant follicle grows and develops until ovulation and can result in the generation of offspring, whereas development of subordinate follicles is suppressed by this follicle, and they cease growth and development, and undergo atresia via * Corresponding author at: Dept. Zoology and Anthropology, Constantine the Philosopher University, 949 74 Nitra, Slovakia. E-mail address: asirotkin@ukf.sk (A.V. Sirotkin). apoptosis [1–3]. In poly-ovulatory species (e.g., rodents), several follicles are developed and ovulated during one cycle, and both follicular dominance and cooperation have been observed. Large murine follicles can reversibly induce apoptosis and inhibit growth of co-cultured smaller follicles [4,5]. Conversely, the coculture of ovarian follicles of similar size as that of rats [6] and mice [4] can produce unknown peptide hormone(s), which can promote growth of neighboring follicles. The interrelationships between ovarian follicles in other poly-ovulatory species than rodents (e.g., pigs) have not yet been studied. The intra- and extracellular mechanisms of inter-ovarian communication remain virtually unknown. Results of in vivo studies have shown that dominant follicles in mono-ovulatory http://dx.doi.org/10.1016/j.repbio.2017.01.005 1642-431X/© 2017 Society for Biology of Reproduction & the Institute of Animal Reproduction and Food Research of Polish Academy of Sciences in Olsztyn. Published by Elsevier Sp. z o.o. All rights reserved. Please cite this article in press as: A.V. Sirotkin, et al., Interrelationships between ovarian follicles grown in culture and possible mediators, Reprod Biol (2017), http://dx.doi.org/10.1016/j.repbio.2017.01.005 G Model REPBIO 221 No. of Pages 8 2 A.V. Sirotkin et al. / Reproductive Biology xxx (2016) xxx–xxx species can suppress the growth of subordinate follicles via the promotion of their atresia via apoptosis [1,2], but it remains unknown whether follicular dominance can be mediated with changes in ovarian cell proliferation as well. One study failed to demonstrate follicular dominance in vitro [7]. It is unknown whether mutual support of follicles in poly-ovulatory species is caused by follicular atresia via apoptosis or proliferation. The hormonal mediators of inter-follicular interrelationships have also been insufficiently studied. Local ovarian hormones appear to affect follicular interrelationships more than ovarian cycle-dependent changes in pituitary or exogenous follicle stimulating hormone (FSH) [5,7,8]. Possible candidates for hormonal mediators of follicular selection and dominance are insulin-like growth factor I (IGF-I) and IGF-binding proteins [2,8,9], which, in close collaboration with oxytocin (OT) [10,11], can control ovarian follicular growth, development, and selection. OT-IGF-I axis which can mutually stimulate both OT and IGF-I output can promote growth of ovarian follicles both in vivo [2,8,9,11] and in vitro [10,12,13]. OT-IGF-I axis promoted porcine ovarian follicular cell proliferation (accumulation of promoter and marker of proliferation, proliferating cell nuclear antigene, PCNA [14]) and growth of isolated porcine ovarian follicles [10], as well as the release of ovarian steroid and peptide hormones-regulators of ovarian folliculogenesis in different mammalian species [10,11]. Nevertheless, it remains unknown whether the OT-IGF-I axis can mediate inter-follicular communication and related follicular cell proliferation in both poly- and mono-ovulatory species. Therefore, the interrelationships between follicles in poly-ovulatory species, as well as the mediators of such interrelationship in both poly- and mono-ovulatory species require further elucidation. The first goal of our study was to determine the existence and compare the character of mutual influence of cultured ovarian follicles of a mono-ovulatory species (cow) with established follicular dominance of a poly-ovulatory species (pig), in which interrelationships between follicles remain unknown. The second goal was to examine the role of ovarian cell proliferation and the oxytocin-IGF-I system in mediating the interrelationships among porcine ovarian follicles. To accomplish our goals, isolated similarsized bovine and porcine ovarian follicles were cultured alone and in pairs, whereas the percentage of growing follicles in each group was calculated. In addition, porcine follicles were cultured alone and in pairs after the addition of exogenous OT and IGF-I, or inactivation of endogenous OT and IGF-I by antisera against these hormones. In both bovine and porcine follicular cell proliferation (accumulation of proliferating cell nuclear antigen, PCNA), and release of some key ovarian hormones (IGF-I, progesterone, androstenedione, and estradiol) were analyzed. 2. Materials and methods 2.1. Isolation, culture, and processing of ovarian follicles The ovaries were collected from non-cycling Slovakian white gilts, 180 days of age (weight 85–100 kg) and from cows 2–4 years old (weight 600–700 kg) at follicular stage of the estrous cycle without visible reproductive abnormalities, killed at a local slaughterhouse in accordance with the corresponding European and Slovak regulations. Ovarian follicles (2.5–3.5-mm diameter, representing the majority of visible growing but not ovulating follicles) were collected, processed, and cultured in a DME/F-12 1:1 mixture (Sigma, St. Louis, MO, USA) supplemented with 10% heatinactivated fetal calf serum (Sigma), and 1% antibiotic-antimycotic solution (Sigma), as it was validated and described previously [10,15], although in these experiments only the whole, but not dissected follicles were cultured. Randomly selected whole ovarian follicles were cultured individually or in pairs for 10 d in the medium described above. Bovine follicles were cultured without additions, whereas porcine follicles were cultured with and without recombinant IGF-I (100 ng mL 1 medium; Calbiochem, Lucerne, Switzerland) or synthetic OT (100 ng mL 1; Sigma), sheep antiserum against OT (1%; kindly provided by Prof. A.P.F. Flint, University of Nottingham, Sutton Bonington, U.K.) or rabbit antiserum against human IGF-I (1%; kindly provided by Dr. A.F. Parlow, National Hormone & Pituitary Program, Harbor-UCLA Medical Center, Torrance, CA, USA). This concentration of antisera binds 85–100% of OT or IGF-I produced by follicular cells during culture. Previous studies [10,15] demonstrated that hormones and antisera at these concentrations have maximal effects on both the secretory activity and the size of cultured porcine follicles by the 10th day of culture. The hormones and antisera were of biological grade. They were dissolved in the incubation medium immediately before the experiments. Control tissues were cultured in medium without additions of hormones or antisera. The blank control was represented by medium cultured without follicular tissue. Before and after culture, follicles were weighed and their diameters were measured using a micrometer to  0.1 mm. Culture medium and follicular tissue were frozen at 18  C until radioimmunoassay/enzymatic immunoassay (RIA/ EIA) and SDS PAGE-Western blotting, respectively. The number and viability of cells within the follicles were not determined, although PCNA was detected by western blotting of follicular lysates after culture (see below). 2.2. Immunoassay Concentrations of progesterone, androstenedione, estradiol, and IGF-I were determined in 25–100 mL of incubation medium by RIAs or EIA. Progesterone and androstenedione concentrations were determined using RIA and EIA kits from DSL (Webster, TX, USA). Estradiol was assayed using an RIA kit (BioChem Immuno Systems Italia S.P.A., Rome, Italy), and IGF-I was extracted from the samples and concentrations determined using RIA as described previously [16] using rhIGF-I from Sigma as standard and anti-IGF-I antiserum (dilution 1:10.000) provided by Dr. A.F. Parlow (National Hormone and Pituitary Program, Torrance, USA). All procedures followed the manufacturer’s instructions. All assays were validated for our culture medium. The characteristics of these assays are presented in Table 1. 2.3. Western blotting The separation of PCNA was performed using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE, [17]). Subsequent visualization was by western immunoblotting by using mouse monoclonal antibodies against human PCNA (36 kDa protein; Santa Cruz, Santa Cruz, CA, USA, dilution 1:500 which binds either human, mouse, rat, chicken porcine or bovine PCNA), secondary HRP-conjugated anti-mouse rabbit IgG antibodies (DAKO, Carpinteria, CA, USA; dilution 1:1000), ECL detection reagents, and ECL hyper-film (Amersham International) according to the manufacturer’s instructions and quantified by fraction densitometry normalized for the housekeeping protein fraction (see below). The primary antibody against PCNA is specific for proliferating mouse, rat, human, insect, yeast, porcine, and bovine cells at G1 and S phases of the cell cycle. Incubation medium without cells, or samples processed in the absence of the primary antibody, were used as negative controls. As housekeeping protein, Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH) and the corresponding antibody (BD Trans Lab, dilution 1:500) were used (not shown). The molecular weights of fractions were evaluated using a molecular weight calibration set (18, 24, 45, and 67 kDa; Please cite this article in press as: A.V. Sirotkin, et al., Interrelationships between ovarian follicles grown in culture and possible mediators, Reprod Biol (2017), http://dx.doi.org/10.1016/j.repbio.2017.01.005 G Model REPBIO 221 No. of Pages 8 3 A.V. Sirotkin et al. / Reproductive Biology xxx (2016) xxx–xxx Table 1 Characteristics of immunoassays used in experiments. Assay Specificity (cross-reactivity of antiserum) Progesterone 4.8% to 5-pregnane-3,20-dione 2.2% to 20-dihydroprogesterone 0.8% to 17-hydroxyprogesterone <0.08% to 5-pregnane-3,20-dione, 11-desoxycortisol, pregnone, androstendiol, testosterone, estradiol Androstenedione <0.1% to progesterone, desoxycortisol, pregnone, testosterone, estradiol 0.47% to estriol Estradiol 1.77% to estrone <0.0001 to progesterone, androstenedione, and estradiol metabolites <1% to IGF-II, insulin, oxytocin IGF-I Sensitivity (ng mL 1) Intra-assay coefficient of variation (%) Inter-assay coefficient of variation (%) 0.12 <8.0 <13.1 0.03 <9.2 <10.3 0.06 <1.0 <2.0 0.10 <11.0 <17.0 ICN Biomedicals, Inc., Irvine, CA, USA). The data of densitometry normalized to GAPDH values were expressed in arbitrary units. Duncan’s multiple range test to detect significant (P < 0.05) differences between treatment and control groups. 2.4. Statistics 3. Results Each experimental group was represented by 10–22 cultures containing one or two whole follicle each. The data from each series of experiments are presented as means obtained from at least eight experiments using separate pools of ovaries, each obtained from 20 to 40 animals. Due to substantial variation in follicle growth between the experiments (detected by ANOVA, see below), the data concerning follicular growth obtained in different experiments were pooled and processed together without S.E.M. calculation. Samples of the corresponding groups intended for SDS PAGE-western immunoblotting were pooled before processing, whereas samples of incubation medium intended for RIA/EIA were processed separately and assayed in duplicate. The values of blank controls (serum-supplemented medium incubated without follicles) were subtracted from the specific values determined by RIA in follicles-conditioned medium to exclude any non-specific background (less than 10% of total values). Rates of substance secretion were expressed as mg tissue day 1. Significant differences between the experiments were determined using a one-way analysis of variance (ANOVA), followed by either Chi-square or The majority of bovine and porcine follicles cultured alone decreased or did not change in diameter during culture, although some increased. Usually only one bovine follicle in a pair increased in size, whereas in porcine follicles occasionally only one, but sometimes both follicles in the pairs increased in size The co-culture of two follicles resulted in significant decreases in the percentage of growing bovine follicles (Fig. 1A). These changes were not associated with significant changes in the accumulation of proliferation marker PCNA, although a trend in its reduction occurred (Fig. 2). The co-culture of bovine ovarian follicles significantly reduced the release of IGF-I, progesterone, androstenedione, but not estradiol (Fig. 3A). The co-culture resulted in a dramatic increase in the proportion of growing porcine follicles (Figs. 1 B, Figs. 4–6 ). When porcine follicles were cultured alone in the presence of either IGF-I (Fig. 4) or OT (Fig. 5), their growth rate increased to a level equal to that of follicles cultured in pairs. Conversely, pairs of follicles cultured in the presence of antisera against IGF-I (Fig. 4) or OT (Fig. 5) showed reduced growth to a level equal to that of single follicles (Figs.1,4,5). A. B. 60 50 * 50 30 * 20 % growing follicles % growing follicles 40 40 * 30 20 10 10 0 0 Alone Pairs Alone Pairs Fig. 1. Effects of co-culture on the growth of bovine (A) and porcine (B) ovarian follicles. Values are means. * – significant differences (P < 0.05) between the corresponding groups of follicles cultured alone and in pairs. Please cite this article in press as: A.V. Sirotkin, et al., Interrelationships between ovarian follicles grown in culture and possible mediators, Reprod Biol (2017), http://dx.doi.org/10.1016/j.repbio.2017.01.005 G Model REPBIO 221 No. of Pages 8 4 A.V. Sirotkin et al. / Reproductive Biology xxx (2016) xxx–xxx Fig. 2. Effect of co-culture on the accumulation of proliferating cell nuclear antigen (PCNA) in cultured bovine ovarian follicles. Molecular weight of the fraction is indicated on the left. Results of densitometry (in arbitrary units) are shown below the corresponding fraction. Values are means. A. B. Hormone release (ng or pg/mg tissue) 80 70 60 50 * 40 30 * 20 * 10 * 0 alone pairs IGF-I alone pairs alone pairs alone pairs P A E Hormone release (ng or pg/mg tissue) 250 * 200 150 100 50 * 0 alone pairs IGF-I alone pairs alone pairs alone pairs P A E Fig. 3. Effects of co-culture on the release of IGF-I, progesterone, androstenedione, and estradiol by cultured bovine (A) and porcine ovaria (B) ovarian follicles Values are means  SEM. * – significant differences (P < 0.05) between follicles cultured alone and in pairs. The co-culture of porcine follicles promoted not only their growth, but also the accumulation of PCNA (Figs. 6 and 7). The co-culture and hormone-induced changes in growth rate were associated with corresponding changes in PCNA accumulation. Both IGF-I (Fig. 6) and OT (Fig. 7) increased the expression of this proliferation marker above values that occurred in follicles cultured alone or in pairs, whereas immunoblockage of either IGF-I (Fig. 6) or OT (Fig. 7) reduced PCNA levels in follicles cultured in pairs below the level of follicles cultured alone. Comparison of secretory activity of porcine ovarian follicles cultured alone or in pairs showed that Please cite this article in press as: A.V. Sirotkin, et al., Interrelationships between ovarian follicles grown in culture and possible mediators, Reprod Biol (2017), http://dx.doi.org/10.1016/j.repbio.2017.01.005 G Model REPBIO 221 No. of Pages 8 5 A.V. Sirotkin et al. / Reproductive Biology xxx (2016) xxx–xxx 60 % growing follicles 50 * * 40 30 20 * 10 0 Alone Pairs Alone+ IGF-I Pairs+ AS against IGF-I Fig. 4. Effects of co-culture, addition of IGF-I (100 ng mL 1) and immunoblockage of IGF-I by antiserum (AS) against IGF-I (1%) on the growth of porcine ovarian follicles. Values are means. * – significant differences (P < 0.05) between the corresponding groups cultured with and without additions of hormone or antiserum. 60 * % growing follicles 50 * * 40 30 20 10 0 Alone Pairs Alone+ OT Pairs+ AS against OT Fig. 5. Effects of co-culture, addition of OT (100 ng mL 1) and immunoblockage of OT by antiserum (AS) against oxytocin (1%) on the growth of porcine ovarian follicles. Values are means. * – significant differences (P < 0.05) between the corresponding groups cultured with and without additions of hormone or antiserum. follicles cultured together released significantly more IGF-I and lesser progesterone than did follicles cultured alone, whereas no differences in the release of androstenedione and estradiol release were found (Fig. 3B). 4. Discussion This study was the first to use cultures of bovine and porcine follicles to determine follicular interrelationships. In our cultures, some follicles decreased and some increased in diameter. The reduction in follicular size during long-term culture may be caused by tissue shrinkage because of minor osmotic imbalance between the follicular fluid and the culture medium, or dominance of apoptosis over cell proliferation and follicular growth. Conversely, increases in follicular diameter could be the result of ovarian cell proliferation. Changes in diameter of porcine follicles were always associated with the corresponding changes in accumulation of PCNA, a marker of cell proliferation. Please cite this article in press as: A.V. Sirotkin, et al., Interrelationships between ovarian follicles grown in culture and possible mediators, Reprod Biol (2017), http://dx.doi.org/10.1016/j.repbio.2017.01.005 G Model REPBIO 221 No. of Pages 8 6 A.V. Sirotkin et al. / Reproductive Biology xxx (2016) xxx–xxx PCNA 36kD- 10 * PCNA, arbitrary units 8 * 6 4 2 0 Alone Pairs Alone+ IGF-I Pairs+ antiserum against IGF-I Fig. 6. Effects of co-culture, addition of IGF-I (100 ng mL 1) and immunoblockage of IGF-I by antiserum against IGF-I (1%) on the accumulation of proliferating cell nuclear antigen (PCNA) in cultured porcine ovarian follicles. Results of densitometry are shown below the corresponding fraction. Values are means. * – significant differences (P < 0.05) between the corresponding groups cultured with and without additions of hormone or antiserum. Co-culture of bovine ovarian follicles suppressed their growth. This suppression likely reflected follicular selection and dominance previously described in bovine ovaries in vivo [1–3]. Such follicular competition and selection could prevent the growth and ovulation of multiple follicles in mono-ovulatory species, which is not able to rear numerous offspring. To the best of our knowledge, we are the first to demonstrate follicular dominance in monoovulatory species in vitro. Furthermore, this study is likely the first to demonstrate the lack of co-culture influence on PCNA changes in bovine follicles. The dominant follicle suppresses the subordinate ones rather via promotion of their apoptosis via atresia, as noted previously [1–3], but not via reduction in their cell proliferation. Therefore, atresia of subordinate follicles in cows could be caused by an increase in the apoptosis/proliferation rate because of an increase in apoptosis, but not of a decrease in proliferation. Comparison of secretory activity of bovine ovarian follicles cultured alone and in pairs showed that co-culture suppressed the release of IGF-I, progesterone, and androstenedione, but not of estradiol. These observations suggest that suppression of subordinate follicle growth and steroidogenesis could be caused by follicular atresia via apoptosis induced by reduction in release of IGF-I, a known inhibitor of follicular apoptosis and promoter of steroidogenesis [2,8–13]. It remains unknown why reduction in IGF-I and steroid hormones in our experiments did not influence their secondary target PCNA, which is a promoter and marker of follicular cell proliferation [2,8–13]. We cannot exclude the possibility that they influence other regulators of the cell cycle, which we did not measure in our study. Mechanisms of follicular dominance in mono-ovulatory species could be a subject of further studies. The present in vitro approach could be useful for further studies of interrelationships between follicles and their mechanisms in mono-ovulatory species. In contrast to bovine follicles, porcine follicles exerted mutual support in their growth. This suggests that in poly-ovulatory pigs, follicular selection and dominance of one follicle is less prominent than follicular cooperation, which promotes development and ovulation of several follicles during one cycle. Our observations are the first line of evidence for follicular cooperation in porcine ovaries. It is in agreement with previous observations of such cooperation between cultured follicles in other poly-ovulatory species, such as the rat [6] and mouse [4], although in these species the ovarian selection could occur too [4,5]. Our observations is the first demonstration that the cross-talk between ovarian follicles can induce not only apoptosis, but that mutual support of follicles can be due to alternative mechanism – mutual stimulation of follicular cell proliferation via PCNA promoter of S-phase of the cell cycle [14]. Our observations provide new knowledge concerning hormones that can mediate follicular cross-talking. Previous in vivo studies showed that IGF-I and steroid hormones could be mediators of follicular dominance in the cow, a mono-ovulatory species [1–3]. In vitro studies showed that both IGF-I and OT promoted porcine follicular hormone release, cell proliferation, and growth. Moreover, the reciprocal stimulation of IGF-I and OT, similarity of their effects, and the prevention of the majority of IGFI effects by OT blockade suggest the existence of a self-stimulating intra-ovarian IGF-I-OT system [10,15]. The present experiments showed that co-culture of porcine ovarian follicles stimulated not only cell proliferation and growth, but also IGF-I release and reduction of progesterone output. These hormones are involved in the control of ovarian cell proliferation, follicular growth, luteinization, and atresia via apoptosis, and IGF-I is a stimulator of OT and a regulator of steroidogenesis [2,3,10,15]. In our experiments, the co-culture of porcine ovarian follicles promoted Please cite this article in press as: A.V. Sirotkin, et al., Interrelationships between ovarian follicles grown in culture and possible mediators, Reprod Biol (2017), http://dx.doi.org/10.1016/j.repbio.2017.01.005 G Model REPBIO 221 No. of Pages 8 7 A.V. Sirotkin et al. / Reproductive Biology xxx (2016) xxx–xxx PCNA 36kD- 10 * PCNA, arbitrary units 8 * 6 4 * 2 0 Alone Pairs Alone+ OT Pairs+ antiserum against OT Fig. 7. Effects of co-culture, addition of OT (100 ng/mL 1), and immunoblockage of OT by antiserum (AS) against oxytocin (1%) on the accumulation of proliferating cell nuclear antigen (PCNA) in cultured porcine ovarian follicles. IGF-I release, whereas both OT and IGF-I promoted basal and coculture-induced ovarian cell proliferation and follicular growth, as well as mimicked the effect of co-culture. Moreover, the immunoblockage of endogenous IGF-I and OT suppressed these processes and prevented the effect of co-culture. All these observations suggest that IGF-I and OT mediate the stimulatory action of follicular co-culture on cell proliferation and follicular growth, and can be hormonal mediators of follicular cooperation in the porcine ovary. We hypothesize that the presence of neighboring follicles could stimulate the release of IGF-I, which in turn promotes OT release, and changes progesterone output. These hormonal changes promote the production of intracellular PCNA, PCNA-induced follicular cell proliferation, and follicular growth. We cannot exclude the possibility that increased IGF-I release also suppresses ovarian follicular cell apoptosis. Identification of hormonal and intracellular mediators of interfollicular communication is important both to understanding the mechanisms of ovarian folliculogenesis and to improving current methods of hormonal regulation of fecundity. Therefore, these mediators and their interrelationships in both mono- and polyovulatory species require further studies. Such studies could include not only in-vitro, but also in-vivo experiments, and not only middle-sized, but also large follicles presented in both bovine and porcine follicular waves. Nevertheless, our observations demonstrated (1) follicular competition/dominance (mutual suppression of growth) in the bovine ovary, (2) cooperation (mutual support of growth) in porcine ovarian follicles, (3) that the mutual support of growth of porcine ovarian follicles are caused by promotion of PCNA/cell proliferation, (4) that this mechanism is probably not involved in bovine follicular dominance, (5) that communication between both porcine and bovine follicles affects their secretory activity, and (6) that both follicular dominance/ suppression of the subordinate follicles in cows and cooperation/ mutual support of follicles in pigs can be mediated by either downor up-regulation of the IGF-I-OT system, which in turn affects follicular steroidogenesis, and promotes follicular cell proliferation and follicular growth. Acknowledgments The authors thank Prof. A.P.F. Flint (University of Nottingham, Sutton Bonington, UK) for kind gifts of antiserum against OT, Dr. A. F. Parlow (National Hormone & Pituitary Program, Harbor-UCLA Medical Center, Torrance, CA, USA) for donation of anti-IGF-I antiserum, and Ms. K. Tothová and Ž. Kuklová for technical assistance. This work was supported by Slovak Research and Development Agency under the contract No. APVV-14-0001 and was co-funded by the projects VEGA1/0022/15, VEGA1/0327/16, KEGA001UKF-4/2016, and the Ministry of Agriculture of the Slovak Republic, project RUVVR 07-13. The authors would also like to extend their sincere appreciation to the International Scientific Partnership Program ISPP at King Saud University for funding this research work through ISPP# 0013. References [1] Fortune JE. Ovarian follicular growth and development in mammals. Biol Reprod 1994;50:225–32. [2] Webb R, Campbell BK. Development of the dominant follicle: mechanisms of selection and maintenance of oocyte quality. Soc Reprod Fertil Supp 2007;64:141–63. [3] Aerts JM, Bols PE. 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