# Oxytocin mediates some effects of insulin-like growth factor-I on porcine ovarian follicles

A.V. Sirotkin, I. Florkovicova, A.V. Makarevich, H.J. Schaeffer, J. Kotwica,

Pierre-Guy Marnet, P. Sanislo

► To cite this version:

A.V. Sirotkin, I. Florkovicova, A.V. Makarevich, H.J. Schaeffer, J. Kotwica, et al.. Oxytocin mediates some effects of insulin-like growth factor-I on porcine ovarian follicles. Journal of Reproduction and Development, 2003, 49 (2), pp.141-149. 10.1262/jrd.49.141. hal-02669427

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<figure><img src="image_1.png" /></figure>
—Original—

# Oxytocin Mediates Some Effects of Insulin-Like Growth Factor-I on Porcine Ovarian Follicles

Alexander V. SIROTKIN¹), Iveta FLORKOVIČOVÁ¹),

Alexander V. MAKAREVICH¹), Hans-Jörg SCHAEFFER²), Ján KOTWICA³),

Pierre-Guy MARNET⁴) and Pavel SANISLO¹)

¹)Research Institute of Animal Production, 949 92 Nitra, Slovakia, ²)Universitäts-Frauenklinik, 50931 Köln, Germany, ³)Institute of Animal Production and Food Research, 10–718 Olsztyn-Kortowo, Poland, and ⁴)INRA/ENSAR, 35042 Rennes Cedex, France

**Abstract.** The aims of the present study were (1) to investigate the influence of insulin-like growth factor-I (IGF-I) on follicular size, on the secretion of oxytocin (OT), progesterone (P), estradiol (E), IGF binding protein-3 (IGFBP-3), inhibin A, inhibin B and cAMP and on the expression of proliferation-associated peptide PCNA, ERK-related mitogen activated protein kinase (MAPK/ERK1, 2) and protein kinase A (PKA) in cultured porcine ovarian follicles; (2) to examine the effects of OT on IGF-I and on these functions; and (3) to determine whether the effects of IGF-I can be mediated by OT. To define the involvement of OT in mediating IGF-I action, we compared responses of porcine ovarian follicles to IGF-I and OT and examined whether blockade of endogenous OT by specific antiserum can affect IGF-I action. It was observed that IGF-I (1, 10 or 100 ng/ml) was able to prevent a decrease in the size of ovarian follicles during culture and caused an increase in the diameter of some follicles. It also stimulated the secretion of OT, P, IGFBP-3, inhibin A and cAMP, decreased the secretion of E and inhibin B (RIA/EIA/ELISA), and induced the expression of PCNA, PKA, MAPK/ERK1, but not MAPK/ERK2 (Western blotting). Like IGF-1, OT (100 ng/ml) prevented decrease in follicular size and increased the diameter of some follicles. It also stimulated the secretion of P and IGF-I, but not E. Antiserum against OT (1%), when given alone, did not affect the reduction of follicular size but slightly increased the percentage of follicles increasing their diameter during culture. The antiserum also inhibited secretion of OT and cAMP but not the secretion of P, E, IGFBP-3 or the expression of PKA, MAPK/ERK1 or 2. When given together with IGF-I, the antiserum prevented the stimulatory action of IGF-I on the proportion of enlarged follicles and on OT, IGFBP-3 and MAPK/ERK1. It augmented the effect of IGF-I on P, but not the effect on E, cAMP, PKA or MAPK/ERK2. These observations demonstrate the involvement of IGF-I and OT in the control of ovarian follicular size and follicular cell proliferation, progestagen, estrogen, IGFBP-3, inhibin A and B secretion and in cAMP/PKA- and MAPK/ERK1-dependent intracellular mechanisms. Furthermore, the reciprocal stimulation of IGF-I and OT and the similarity of some their effects, together with the prevention or augmentation of some IGF-I effects after OT blockade, suggest that IGF-I action can be mediated by OT.

**Key words:** Insulin-like growth factor-I (IGF-I), Oxytocin, Insulin-like growth factor binding protein-3 (IGFBP-3), Progesterone, Estradiol, Cyclic adenosine monophosphate (cAMP), Inhibin, Mitogen-activated protein kinase (MAPK, ERK1, ERK2), Protein kinase A

(J. Reprod. Dev. 49: 141–149, 2003)

Insulin-like growth factor I (IGF-I), of extra- and intraovarian origin, is a well-known regulator of ovarian functions including cell proliferation,

steroidogenesis [1–5] and oxytocin (OT) secretion
[2, 5]. It also regulates prostaglandin F, but not prostaglandin E or insulin-like growth factor binding protein 3 (IGFBP-3) secretion [2]. The effects of IGF-I on other ovarian functions, in particular on growth and the secretion of inhibins by isolated ovarian follicles are as yet unknown. The mechanisms of IGF-I action, as well as the interrelationships between IGF-I and other substances in the ovary, have not been fully studied. Ovarian function is regulated by cAMP/protein kinase A (PKA [5-7]), cGMP/protein kinase G [8] and mitogen-activated protein kinases (MAPK/ERK1 and 2 [5]). Furthermore, cyclic nucleotides, PKA and MAPK mediate IGF-I action on rabbit ovarian cells and embryos [5] and porcine oocytes [7]. It is not known whether cyclic nucleotides, PKA and MAP kinase are regulated by IGF-I in porcine ovarian follicular cells. It is also unclear, whether IGF-I affects ovarian function directly or through other hormones. We have shown that IGF-I stimulates OT secretion and that IGF-I and OT have similar effects on porcine ovarian follicles [2], but sound evidence of indirect action is absent. Such evidence could be obtained by inactivating OT.

The aim of the present investigation were (1) to define the influence of IGF-I on ovarian cell proliferation, OT, P, E, inhibin A, inhibin B and IGFBP-3 secretion; (2) to investigate the effect of IGF-I on the size of cultured follicles and their expression of cAMP, PKA and MAPK/ERK1, 2; and (3) to determine whether IGF-I effects can be mediated by OT. In these experiments we compared the effects of IGF-I and OT and determined whether inactivation of endogenous OT with specific antiserum affects the action of IGF-I on porcine ovarian follicles.

## Materials and Methods

### Isolation, culture, and processing of ovarian follicles

Non-cycling Slovakian white gilts, 180 days of age and without visible reproductive abnormalities, were killed at a local slaughterhouse. Ovarian follicles (2.5-3.5 mm in diameter) were collected, processed and cultured in DME/F-12 1:1 mixture (Sigma, St. Louis, MO, USA) supplemented with 10% heat-inactivated fetal calf serum (FCS, Sigma), and 1% antibiotic-antimycotic solution (Sigma) as described previously [2]. In the

first series of experiments, randomly selected whole ovarian follicles were cultured individually for 10 days in the medium described above, with or without recombinant IGF-I (100 ng/ml medium; Calbiochem, Lucerne, Switzerland) or synthetic OT (100 ng/ml; Sigma). Preliminary studies demonstrated that these concentrations of IGF-I and OT control both the secretory activity [2] and the size (data not shown) of cultured porcine follicles. Before and after culture, the follicles were weighed and their diameters were measured by micrometer to ± 0.1 mm. Culture medium and follicular tissue were frozen at -18 C to await RIA/EIA and SDS PAGE-Western blotting.

In the second series of experiments, whole follicles were cultured individually for 10 days with or without recombinant IGF-I (100 ng/ml medium; Calbiochem), sheep antiserum against OT (1%), kindly provided by Prof. A.P.F. Flint (University of Nottingham, Sutton Bonington, UK), or a combination of IGF-I and anti-OT antiserum. This concentration of antiserum binds 85-100% of OT produced by follicular cells during culture. The follicular diameter was measured before and after culture, as described above.

In the third series of experiments, quarters of follicles were cultured (3 per culture well) for 2 days in medium without serum, with or without recombinant IGF-I (0, 1, 10 or 100 ng/ml; Calbiochem) and antiserum against OT (1%). After culture, follicle quarters were weighed and the tissues intended for SDS PAGE-Western blotting were lysed as described previously [2]. Culture medium was stored at -20 C until analysis.

In all experiments, IGF-I, OT and the anti-OT antiserum were of the purest grade available. These substances were dissolved in incubation medium immediately before the experiments. Control tissues were cultured in medium without additions of IGF-I, OT or anti-OT antiserum. The blank control was represented by medium cultured without follicular tissue. The number and viability of cells within the follicles were not determined, although proliferating cell nuclear antigen (PCNA), mitogen-activated protein kinase and PKA were detected by Western blotting of follicular lysates after culture (see below).

### Immunoassay

Concentrations of P, E, OT, IGF-I, IGFBP-3, inhibin A, inhibin B and cAMP were determined in
25-100 µl of incubation medium by RIAs, EIA or ELISA. P concentration was determined using RIA kit (DSL, Webster, TX, USA), E was assayed using an RIA kit (BioChem Immuno Systems Italia, Rome, Italy), IGFBP-3 was determined after extraction using an RIA kit (Mediagnost, Tuebingen, Germany), cAMP was measured using an RIA kit (Immunotech, Marseille, France), whilst inhibins A and B were measured using ELISA kits (Serotec, Kidlington, Oxford, UK). All procedures followed the manufacturers' directions. Concentrations of IGF-I were determined, after extraction, by RIA [9]. Rabbit antiserum against IGF-I was kindly provided by Dr. A.F. Parlow (National Hormone & Peptide Program, Harbor-UCLA Medical Center, Torrance, CA, USA). Oxytocin concentrations were measured by RIA and EIA as described previously [10, 11] using rabbit antiserum against OT kindly provided by Dr. G. Kotwica (University of Agriculture and Technology, Olsztyn, Poland). All assays were validated for our culture medium. The characteristics of these assays are presented in

Table 1.

Western blotting

The separation of PCNA and kinases was performed using sodium dodecyl sulphate polyacrylamide gel electrophoresis [12]. Subsequent visualization was by Western immunoblotting as described previously [16] using mouse monoclonal antibody against PCNA (1:500 dilution; 36-kD protein; Santa Cruz, Santa Cruz, CA, USA), rabbit polyclonal antisera against ERK-1 (44-kD protein) and ERK-2 (42-kD protein) isoforms of MAP kinase (1:2.000 dilution; Sigma), or against the catalytic subunit of PKA (1:100 dilution; Santa Cruz), secondary HRP-conjugated anti-mouse and anti-rabbit IG antibodies, ECL detection reagents and ECL Hyper-film (Amersham International). The primary antiserum against PCNA is specific for proliferating mouse, rat, human, insect, yeast porcine and bovine cells at the G1 and S phases of the cell cycle. The anti-PKA antiserum is specific for alpha, beta and gamma catalytic PKA subunits of mouse, rat, human,

<table><caption>**Table 1.** Characteristics of immunoassays used in experiments</caption><thead><tr><th rowspan="2">Assay</th><th rowspan="2">Specificity<br>(cross-reactivity of antiserum)</th><th rowspan="2">Sensitivity<br>(ng/ml)</th><th colspan="2">Coefficient of variation (%)</th></tr><tr><th>Intra-assay</th><th>Inter-assay</th></tr></thead><tbody><tr><td>Progesterone</td><td>4.8% to 5-pregnane-3,20-dione<br>2.2% to 20-dihydroP<br>0.8% to 17-hydroxyP<br>&lt;0.08% to 5-pregnane-3,20-dione, 11-desoxycortisol, pregnone, androstendiol, testosterone, estradiol</td><td>0.12</td><td>&lt;8.0</td><td>&lt;13.1</td></tr><tr><td>Estradiol</td><td>0.47% to estriol<br>1.77% to estrone<br>&lt;0.0001 to progesterone, androstenedione and estradiol metabolites</td><td>0.06</td><td>&lt;1.0</td><td>&lt;2.0</td></tr><tr><td>IGF-I</td><td>&lt;1.9% to IGF-II,<br>&lt; 0.01% to insulin, proinsulin<br>&lt; 0.001% to oxytocin, progesterone and estradiol</td><td>0.34</td><td>&lt;10</td><td>&lt;18</td></tr><tr><td>Oxytocin</td><td>&lt;0.01% to arg-vasopressin, lys-vasopressin, arg-vasotocin, IGF-I and IGFBP-3</td><td>0.0035</td><td>&lt;5.0</td><td>&lt;9.0</td></tr><tr><td>IGFBP-3</td><td>&lt;0.001% to IGFBP-I, IGFBP-2, IGFBP-4, IGF-I, IGF-II and oxytocin</td><td>0.5</td><td>&lt;6.2</td><td>&lt;8.3</td></tr><tr><td>Inhibin A</td><td>&lt;0.01% to inhibin B, pro-alpha C subunit of inhibin A, activins and oxytocin, IGF-I</td><td>3.9</td><td>&lt;10</td><td>&lt;10</td></tr><tr><td>Inhibin B</td><td>&lt;1% to inhibin A,<br>&lt;0.01% to pro-alpha C subunit of inhibin B, activins, oxytocin, and IGF-I</td><td>15.6</td><td>&lt;7.0</td><td>&lt;7.0</td></tr><tr><td>Cyclic AMP</td><td>100% to ScAMP,<br>&lt;1500% to cAMP<br>&lt;0.4% to cGMP,<br>&lt;0.7% to 5'AMP,<br>&lt;7.0% to ATP,<br>&lt;0.2% to ScGMP,<br>100% to ScAMP,<br>&lt;1500% to cAMP</td><td>0.2</td><td>&lt;11.0</td><td>&lt;16.0</td></tr></tbody></table>
bovine and porcine origin. The primary antiserum against MAPK/ERK1 and 2 binds mouse, rat, human and porcine ERK1 and ERK2, but not ERK3 or MAP kinase (MEK). Incubation medium without cells, or samples processed in the absence of primary antibody, were used as negative controls. The molecular weights of fractions were evaluated using a molecular weight calibration set (18, 24, 45 and 67 kD; ICN Biomedicals, Irvine, CA, USA).

## Statistics

In the first and second series of experiments, each experimental group was represented by 10–22 cultures using one whole follicle each. In the third series of experiments, each group was represented by 6 cultures containing three randomly selected quarters of follicles. The data from each series of experiments are presented as means obtained in three experiments using separate pools of ovaries, each obtained from 20–40 animals. In each experiment, the value of the blank control was subtracted from the values determined in follicle-conditioned medium to exclude non-specific effects. Rates of substance secretion are expressed per mg tissue/day. Significant differences between the experiments were determined using two-way analysis of variance (ANOVA), followed by Duncan's multiple range test to detect significant differences between treatment and control groups.

## Results

### Effect of IGF-I and OT on size, proliferation and secretory activity of follicles in long-term culture in serum-supplemented medium

In the first series of experiments, more than 90% of follicles in the control group decreased or did not

change in diameter, while a few showed an increase. Lysates of these control follicles contained no, or poorly visible PCNA. All follicles secreted substantial amounts of P, E, OT, IGFBP-3, inhibin A and inhibin B. Addition of IGF-I (100 ng/ml) partially prevented follicles from decreasing in size during culture (follicular diameter after culture with IGF-I was 2.31 ± 0.08 mm compared with 1.62 ± 0.14 mm in controls) and resulted in an increase in the diameters of a significant number (44 versus 3% in control) of follicles. This was associated with an increase in expression of PCNA in follicles cultured with IGF-I. IGF-I also significantly increased P, OT, IGFBP-3, inhibin A secretion and decreased E and inhibin B secretion (Table 2 and Fig.1). Similarly, OT (100 ng/ml) partially prevented the reduction of follicular size during culture (2.73 ± 0.13 mm compared with 1.92 ± 0.25 mm in control), increased follicular diameter in 19% of follicles (control follicles did not change), stimulated expression of PCNA and enhanced the secretion of P and IGF-I, but not of E. The effects of OT on IGFBP-3 and inhibins were not studied in these experiments (Table 3 and Fig.1).

### Effect of IGF-I and antiserum against OT on the size of follicles in long-term culture in a serum-supplemented medium

In the second series of experiments, as in the first, most follicles cultured without treatment decreased in diameter during culture (from 3.03 ± 0.12 to 2.62 ± 0.08 mm) whilst an insignificant proportion (less than 2%) of follicles showed an increase in size. Addition of IGF-I partially prevented this from occurring (mean follicular diameter of IGF-I-treated follicles was 2.88 ± 0.16 mm) and significantly increased the proportion of follicles (up to 42%) whose diameter increased during culture. Antiserum against OT, when given alone,

<table><caption>**Table 2.** Effect of IGF-I treatment on the size and secretory activity of porcine ovarian follicles cultured 10 days in a serum-supplemented medium</caption><thead><tr><th>Treatment</th><th>Number of follicles analysed</th><th>Follicular diameter (mm) before culture</th><th>Follicular diameter (mm) after culture</th><th>Proportion of enlarged follicles during culture (%)</th><th>Secretion of progesterone (ng/mg tissue/day)</th><th>Secretion of estradiol (ng/mg tissue/day)</th><th>Secretion of oxytocin (ng/mg tissue/day)</th><th>Secretion of IGFBP-3 (ng/mg tissue/day)</th><th>Secretion of inhibin A (ng/mg tissue/day)</th><th>Secretion of inhibin B (ng/mg tissue/day)</th></tr></thead><tbody><tr><td>None (control)</td><td>30</td><td>2.68 ± 0.18</td><td>1.62 ± 0.14</td><td>3.3 ± 3.3</td><td>19.6 ± 3.4</td><td>277 ± 109</td><td>36.4 ± 6.6</td><td>6.2 ± 0.2</td><td>315 ± 50</td><td>381 ± 62</td></tr><tr><td>IGF-I (100 ng/ml)</td><td>64</td><td>2.46 ± 0.10</td><td>2.31 ± 0.08*</td><td>43.8 ± 4.0*</td><td>34.5 ± 6.8*</td><td>137 ± 27*</td><td>79.9 ± 4.7*</td><td>7.2 ± 0.2*</td><td>956 ± 189*</td><td>161 ± 38*</td></tr></tbody></table>

Values are means ± S.E.M., *: significant (P<0.05) differences with control.
<figure><img src="image_2.png" /></figure>

Control IGF-I Control Oxytocin

<caption>**Fig. 1.** Effect of IGF-I and OT on the presence of proliferating cell nuclear antigen (PCNA) in cultured porcine ovarian follicles. Whole follicles were cultured for 10 days in serum-supplemented DME/F12 mixture with or without recombinant IGF-I (100 ng/ml) or synthetic OT (100 ng/ml). Lysed follicular tissue was subjected to SDS-PAGE and immunoblotting with monoclonal antiserum against PCNA, horseradish peroxidase-conjugated anti-mouse IgG antibody and ECL visualization system. The IGF-I and OT treatments (with their respective controls) are presented in one panel but were processed in separate batches. Results in each series are a summary of three independent experiments.</caption>

did not affect the spontaneous reduction of follicular size but slightly increased the proportion of enlarged follicles (up to 9%). The anti-OT antiserum partially prevented the effect of IGF-I on the proportion (17%) of enlarged follicles, but not

that on follicular size (2.98 ± 0.22 mm) (Table 4).

### Effects of IGF-I and antiserum against OT on secretory activity and expression of MAPK and PKA in follicles in short-term serum-free culture

In the third series of experiments, addition of IGF-I at 10 or 100 ng/ml increased OT secretion in a manner suggesting a concentration-dependent effect. In medium supplemented with OT antiserum, the concentration of OT was below the limit of sensitivity of the assay, in both control and IGF-I-treated groups (Fig. 1a). Treatment with 10 ng/ml IGF-I increased P secretion, with a lesser effect at 100 ng/ml. Addition of OT antiserum did not influence basal P secretion, but dramatically increased the IGF-I-stimulated P secretion in a concentration-dependent manner. The effects of IGF-I and OT antiserum were additive (Fig. 1b). The highest concentration (100 ng/ml) of IGF-I significantly increased cAMP secretion. IGFBP-3 secretion was stimulated by IGF-I at 1 ng/ml, with a lesser effect at 10 ng/ml. Addition of OT antiserum enhanced basal, but not IGF-I-induced cAMP secretion (Fig. 1c). The antiserum did not affect basal IGFBP-3 secretion but completely prevented the stimulatory action of IGF-I on IGFBP-3 (Fig. 1d). IGF-I decreased E secretion at 1

<table><caption>**Table 3.** Effect of oxytocin treatment on the size and secretory activity of porcine ovarian follicles cultured 10 days in a serum-supplemented medium</caption><thead><tr><th>Treatment</th><th>Number of follicles analyzed</th><th>Follicular diameter (mm) before culture</th><th>Follicular diameter (mm) after culture</th><th>Proportion of enlarged follicles (%)</th><th>Secretion of progesterone (ng /mg tissue/day)</th><th>Secretion of estradiol (ng /mg tissue/day)</th><th>Secretion of IGF-I (ng/mg tissue/day)</th></tr></thead><tbody><tr><td>None (control)</td><td>30</td><td>3.15 ± 0.29</td><td>1.92 ± 0.25</td><td>0</td><td>21.5 ± 3.5</td><td>735 ± 284</td><td>28.1 ± 6.6</td></tr><tr><td>Oxytocin (100 ng/ml)</td><td>66</td><td>3.27 ± 0.13</td><td>2.73 ± 0.13*</td><td>18.7 ± 4.3*</td><td>40.5 ± 8.4*</td><td>849 ± 166</td><td>43.9 ± 8.7*</td></tr></tbody></table>

Values are means ± S.E.M., *: significant (P<0.05) differences with control.

<table><caption>**Table 4.** Effect of IGF-I and antiserum against oxytocin on the size of porcine ovarian follicles cultured for 10 days in a serum-supplemented medium</caption><thead><tr><th>Treatment</th><th>Number of follicles analysed</th><th>Follicular diameter (mm) before culture</th><th>Follicular diameter (mm) after culture</th><th>Proportion of enlarged follicles during culture (%)</th></tr></thead><tbody><tr><td>None (control)</td><td>60</td><td>3.03 ± 0.12</td><td>2.62 ± 0.08</td><td>1.8 ± 1.7</td></tr><tr><td>IGF-I (100 ng/ml)</td><td>40</td><td>3.10 ± 0.13</td><td>2.88 ± 0.16*</td><td>42.0 ± 3.3*</td></tr><tr><td>Antiserum against OT (1%)</td><td>30</td><td>3.40 ± 0.14</td><td>2.68 ± 0.14</td><td>9.3 ± 1.2*</td></tr><tr><td>IGF-I (100 ng/ml) + antiserum against OT(1%)</td><td>30</td><td>3.17 ± 0.21</td><td>2.98 ± 0.22</td><td>16.6 ± 7.2*</td></tr></tbody></table>

Values are means ± S.E.M., *: significant (P<0.05) differences with control.
ng/ml, but not at other concentrations. The
presence of OT antiserum did not influence basal or
IGF-I-inhibited E secretion (Fig. 1f).

The 42-kD ERK2 isoform of MAPK, but not the
44-kD ERK1 isoform, was detected in follicles
cultured without treatment. Addition of IGF-I did
not substantially affect ERK2 but resulted in the
appearance of a 44-kD ERK1 band. OT antiserum
did not affect MAP kinase but when given in
combination with IGF-I reduced the expression of
the IGF-I-induced ERK-1 fraction.

Catalytic subunits of PKA with approx. 47 kD M.W. were observed in follicular lysates. Addition of either IGF-I, antiserum against OT or a combination of these increased the expression of PKA over the control, whilst no visible differences between treatments were found (Fig. 2).

Discussion

The present observations of IGF-I action on P, E, OT, IGFBP-3 and cAMP secretion confirm previous evidence of the important role of IGF-I in the control of ovarian steroidogenesis [1–3], OT [2, 4], IGFBP-3 [2] cAMP and MAPK [5] production. This seems to be the first report of a direct influence of IGF-I on follicular size and on inhibin A and inhibin B secretion. The reduction in follicular size during long-term culture may be due to tissue shrinkage (caused by minor osmotic imbalance between the follicular fluid and the culture medium), or to physiological effects (dominance of apoptosis over cell proliferation and growth). The observation that IGF-I prevents and sometimes reverses the reduction of follicular size may suggest that it can stimulate the secretion of follicular fluid and/or encourage cell and tissue development. The latter effect is consistent with previous reports [1, 3–5] of an anti-apoptotic and/or proliferative action of IGF-I on ovarian cells. This hypothesis is also supported by the marked stimulation of cell proliferation (PCNA accumulation) observed in our experiments in response to IGF-I treatment.

The IGF-I-induced accumulation of cAMP in culture medium, stimulation in PKA expression and appearance of the ERK2 isoform of MAPK in follicular cells is indirect evidence for the involvement of cAMP/PKA- and MAPK-dependent intracellular mechanisms in the mediation of IGF-I effects on porcine ovarian

follicles. This agrees with previous data on the
involvement of these systems in the action of IGF-I
action on rabbit ovarian cells and embryos [5] and
on porcine oocytes [7].

The action of OT on the secretion of P, E and IGF-I, as well as the influence of antiserum against OT on cAMP observed in our experiments, confirms previous evidence an involvement of OT in the control of porcine ovarian secretory activity [2, 13]. Moreover, the influence of anti-OT antiserum on cAMP and PKA suggests that the cAMP/PKA intracellular cascade mediates the effects of OT on the ovary. On the other hand, the lack of effect of OT blockade on the expression of MAPK suggests no interrelationship between OT and MAPK. The observed ability of OT to prevent the spontaneous reduction of follicular diameter during culture and to increase it in some follicles suggests that OT, like IGF-I, influences osmotic or tissue characteristics of isolated follicles, inhibits cell death and/or promotes proliferation or growth. This hypothesis is supported by OT-induced PCNA accumulation within the follicles. However, it is not consistent with the lack of influence of OT antiserum on the reduction of follicular diameter and the stimulatory action of this antiserum on follicular enlargement which we observed. A proliferative action of OT has been observed in non-reproductive cells [14], but not previously on ovarian cells and requires further investigation.

Some of the effects of OT (on growth and P
secretion) mimicked those of IGF-I although the
actions of the two hormones did not entirely
coincide. Nevertheless, the reciprocal stimulation
of OT and IGF-I, as well as the coincidence of some
of their effects is indirect evidence of a functional
coupling between these substances. The ability of
the antiserum against OT to modify the majority of
the IGF-I actions supports this hypothesis. Anti-OT
antiserum blocked not only OT secretion, but also
the effects of IGF-I on follicular size, and on IGFBP-
3 and ERK2. It augmented the effect of IGF-I on P
but did not alter the effect of IGF-I on E, cAMP,
PKA or ERK1. This suggests that IGF-I affects
follicular size, IGFBP-3 and MAPK/ERK2 through
stimulation of OT but affects P through inhibition
of OT. The actions of IGF-I on E, cAMP/PKA and
MAPK/ERK1 are probably not OT-dependent.

Taken together, our observations demonstrate
important roles for IGF-I and OT in controlling
ovarian follicular cell proliferation, follicular size,
<figure><img src="image_3.png" /><figcaption>**Fig. 2.** Effect of IGF-I (white circles) and IGF-I in combination with antiserum against OT (black circles) on secretion of OT (a), P (b), cAMP (c), IGFBP-3 (d), and E (e) secretion by cultured porcine ovarian follicles. Quarters of follicles were cultured for 2 days in serum-free DME/F12 mixture with recombinant IGF-I (0, 1, 10 or 100 ng/ml) alone or in combination with antiserum against OT (1%). Concentrations of substances in the medium were measured by RIA/EIA/ELISA. Values are means ± S.E.M., *: significant (p<0.05) difference compared with control (medium without IGF-I). Results are representative of three independent experiments.</figcaption></figure>
<figure><img src="image_4.png" /><figcaption>**Fig. 3.** Effect of IGF-I and antiserum against OT on the presence of ERK1 and ERK2 isoforms of MAP kinase and of protein kinase A in cultured porcine ovarian follicles. Quarters of follicles were cultured for 2 days in serum-free DME/F12 mixture with or without recombinant IGF-I (10 ng/ml) and antiserum against OT (1%). Lysed follicular tissue was subjected to SDS PAGE and immunoblotting with polyclonal antiserum against ERK-1 and ERK-2 isoforms of MAP kinase or polyclonal antiserum against catalytic subunits of PKA, horseradish peroxidase-conjugated anti-rabbit IgG antibody and ECL visualization system. Results are a summary of three independent experiments.</figcaption></figure>

progestagen, estrogen, IGFBP-3, inhibin A, inhibin B, cAMP/PKA- and MAPK-dependent intracellular mechanisms. Furthermore, the reciprocal stimulation of IGF-I and OT and the coincidence of some their effects, together with the prevention or augmentation of the majority of IGF-I effects by OT blockade, suggest that these IGF-I effects can be mediated by OT.

**Acknowledgements**

The authors thank Prof. A.P.F. Flint (University

of Nottingham, Sutton Bonington, U.K.) and Dr. G. Kotwica (University of Agriculture and Technology, Olsztyn, Poland) for kind gifts of antiserum against OT, Dr. A.F. Parlow (National Hormone & Peptide Program, Harbor-UCLA Medical Center, Torrance, CA, USA) for donation of anti-IGF-I antiserum, Prof. J. Bulla and Prof. L. Hetényi for permanent support of these studies, K. Tothová, M. Blahová, Ž. Kuklová and B Ustianovska for technical assistance, and Dr. M.R. Luck (University of Nottingham, Sutton Bonington, U.K.) for editing the manuscript and helpful discussion.

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