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SDontaneous Maturation of A

Primary Follicle Effects of Some Peptidergic Ovarian

Regulators

P. FRANCHIMONT,”A. MATHIEU,”M. T. HAZEE- HAGELSTEIN,” CH. CHARLET-RENARD,” AND

U. GASPARDb aDepmmcnt ofEndarrinology Radiuimmummsay hbowtory Unim‘zy of LGje C.H. U. B23

B-4o00, L&e, Bebium bD~rtment of GytltGDgy

Universizy of LGje C.H. U. B35 B-POOO, L&e, Bebium

Peptidic ovarian regulators are substances locally produced within the follicle and/or the corpus luteum (and the stroma?) and displaying paracrine and/or autocrine actions by specific receptor activation; their production is modulated by hormone(s) and their interaction^.^^ Peptidic regulators are implied in several ovarian processes of reproduction: follicular growth, follicular differentiation with estrogens (E) and in- hibin (INH) production, luteinization of granulosa cells (GC), meiotic competence acquisition (MCA), and meiotic resumption inhibition (MRI) of the oocytes.

The sequence of events may be followed by assaying peptidergic regulators in fluid of follicles at different stages of maturation and by determining mRNA for these substances in cells of constituting compartments of the follicles: GC, theca cells (TC), cumulus oophorus, and oocytes. Another approach consists in investigating the effects of ovarian regulators on GC or TC in primary monolayer culture. The spontaneous maturation of rat primary follicle is also a model that permits the precise measurement of the interactions of regulators on follicular differentiation and on oocyte MCA.

MODELS AND ASSAYS

Desviption of the Model

Primary follicles are collected from 13-day-old female rats and cultured in serum- free medium in absence of hormones for 12 days.1.2 Ovaries were dissected, freed of fat and oviducts, and submitted to enzymatic digestion by collagenase and DNAse (4mgml and 10 pg/ml, respectively). Primary follicles were collected and cultured in 35-mm plastic dishes containing 3 ml minimum essential medium (MEM), supple- mented with crystallized bovine serum albumin (3g/ml), uridine (0,25 mM), pyruvate (1 mM), insulin (5 pg/ml), iron-saturated transferrin ( 5 pglrnl), selenium (5 rig/ ml), and penicillin-streptomycin (100 IU/ml; 100 pg/ml) at 37°C in a 5%

46

FRANCHIMONT et al.: MATURATION OF PRIMARY FOLLICLE 47

C02-95% air gazed incubator. Two hundred follicles were cultured in each dish. Medium was replenished every two days by removing 1 ml of medium and replacing 1 ml with fresh medium.

WmutMn of MkociG Cumpei%nce Aqui3zbn. At the end of each culture period, follicles were dislodged from the petri dishes, and oocytes were denuded from adherent GC by repeated pipetting. After two washes in modified MEM, oocytes were incubated for 6 hours in the same medium. The breakdown of germinal vesicle (GVBd) and the disappearance of the nucleolus were the criteria for the meiosis resumption indicating the MCA. Normarski optical microscopy was used for determining the percentage of oocytes with GVBd and the diameter of denuded oocytes. The diameter did not include the zona pellucida.

Waimmunoassay (RIA) of U p Estradwl (EZ), Androstenea'k (A), and Prgesterone (P). This has been described in detail el~ewhere.~

Wioimmutwassay of a Immunurmtive Inhibin (a Ir Inh). a Ir inhibin in the culture medium was determined with a solid phase two-side immunoenzymatic assay kit (Medgenix, Fleurus, Belgium). A goat polyclonal antibody directed against a 15 - 32 subunit was designated as the capture antibody and coated on the wells of a 96- well microtiter plate. A mouse monoclonal antibody against the sequence 1-17 of a subunit was conjugated to peroxydase and designated as the tracer antibody. The method has been described in detail.4 Results were expressed in U/ml of human inhibin extracted from human follicular fluid by affinity chromatography. There was a complete cross-reaction between rat and human inhibin in this assay, whereas no cross-reaction was observed for pituitary hormones (FSH, LH, etc.), TGF-a, TGF- p , IGF-1 and IGF-2, and human seminal or prostatic inhibin-like peptides. The sensitiv- ity is 0.1 U/ml; the intra- and interassay coefficients of variations (CV) were 2.4 and 7.9%, respectively. As the Elisa measured substances containing a Inh subunit, it quantified inhibin, its free a subunit, and the precursors containing the a subunit; the amount determined therefore represented the sum of all these immunoreactive forms, rather than inhibin only. Such a lack of specificity for inhibin alone is also shared by other RIA based on the use of antibody against the a subunit5-' and by RIA based on heterologous bovine inhibin, since the polyclonal antibodies employed are raised against epitopes of the a s u b ~ n i t . ~ ? ~ Presently, no sandwich assay using a couple of monoclonal antibodies, one directed against an epitope of the a subunit and the other against an epitope o f p subunit, is available. Indeed, PA and PB subunits are bound to binding proteins (one of them being follistatin) that inhibit any reaction between the antibody and its /3 epitope.'O.ll

IGF Assays

In plasma, almost all body fluids, and conditioned cell culture media, the insulin- like growth factors (IGFs) are bound to large molecular weight proteins. These IGF- binding protein complexes can be dissociated by acid-ethanol precipitation. l Z Quantita- tive measurements of IGFs in neutralized ethanol-acid-extracted media were performed using the IGF-1 extraction RIA kit (Nichols Institute Diagnostics, CA) and our previously described IGF-2 RIA,13 respectively.

According to the manufacturer, the antiserum for IGF-1 shows virtually no cross- reaction with other peptide hormones. Studies have shown <0.01% reactivity with human growth hormone (hGH), human thyroid-stimulating hormone (hTSH), hu- man luteinizing hormone (hLH); < 0.02% with prolactin, porcine insulin; < 0.5% with IGF-2. IGF-1 RIA has a calculated sensitivity of 0.06 ng/ml, and the intra- and interassay CVs were less than 2.9% and 11.4%, respectively. The monoclonal antibody

48 Ah'NALS NEW YORK ACADEMY OF SCIENCES

against rat IGF-2 (Lot 05522N, Ammo, Nagoya, Japan) did not cross-react with human insulin. The cross-reaction of human recombinant IGF-1 was not parallel and was estimated to be 8.3% at 50% displacement of tracer. The IGF-2 IUA sensitivity was 0.01 ng/assay; intra- and interassay CVs were 6% and 12%, respectively.

Statistical Analysis

Comparison of mean values was performed using the Mann-Whitney U-test at each period of renewal of the culture medium. Response curves for the control and the dose-levels of each of the four ovarian regulators were compared by means of Zerbe's randomization test." This method permits the comparison of response curves not only for each culture period but for any time interval as well, thereby providing a global assessment of dose difference. It is based on an approximate F test with degrees of freedom that depend on both the observations and the time interval considered. Differences were considered statistically significant when p < 0.05.

SPONTANEOUS MATURATION AND MEIOTIC COMPETENCE ACQUISITION

In qbsence of hormones and ovarian regulators, differentiation oSthe follicles was observed by the increase of A, E2, and a Ir Inh in the culture medium. A levels increased from day 2, reached their maximum at day 6, and then declined until the end of the culture. E2 was very weak until day 6, then increased to a maximum at day 10-12. a Ir Inh increased slowly from day 2 to day 6 and more rapidly from day 8 to day 12. No progesterone was detected in the culture medium for the duration of the experiment.

IGF-1 and IGF-2 were detectable from day 4 (already in day 2 for IGF-2) of culture and progressively increased to reach their maximum between days 10-12.

No oocyte displayed MCA, as determined by GVBd, at day 2 and 4 of culture. Then, MCA progressively increased with the culture duration, and 45 to 50% of denuded oocytes had resumed meiosis at day 12 of culture.

E14ect of Activin A

All the tested recombinant activin A (r Act A) concentrations significantly @ < 0.001) reduced A levels, whereas the highest dose (100 ng/ml) significantly stimulated E2 production into the culture medium of primary follicles (FIG. 1).

Ten, 50, and 100 pg/ml r Act A significantly increased a Ir Inh levels. IGF-1 and IGF-2 curves of production were reduced by 10 (for IGF-l), 50, and

No significant effect of the three doses of r Act A was observed on MCA. This experiment confirms results obtained with other models (rat and bovine GC),

demonstrating the activin stimulates follicle-stimulating hormone (FSH)-induced aromatax activity and immunoreactive inhibin release into the culture m e d i ~ m . l ~ - ' ~ Similarly, the inhibitory effect ofactivin on luteinizing hormone (LH)-induced andro- gen production by rat16 and adult human theca interstitial cells,2O was also assessed. The largest effect of c Act A at all the doses on inhibition of A levels should indicate either a preferential effect of this regulator on theca cell-produced androgen, or a more evident effect related both to an inhibition of androgen production and to their consumption by aromatase activity to generate estrogens.

100 pg/ml r Act A, compared to their control curves, respectively.

PRANCHIMONT er a/.: MATURATION OF PRIMARY FOLLICLE 49

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FIGURE 1. Evolution of estradiol (upper panel) and androstendione (lower panel) dur- ing the culture period of rat primary follicles in the absence (+) or presence of activin A: 10 ng/ml (O), 50 ng/ml (H), and 100 n g / d ( A ). **: p < 0.001.

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Most interesting is the observation of an IGF-1 and IGF-2 release inhibition by addition of 50 and 100 pg/ml of r Act A. Is activin negative action on IGFs implied in its inhibitory effect on DNA synthesis of GC?21 Experiments are being carried out to verify this hypothesis.

Finally, r Act A did not modify MCA at the end of the culture. Such observations lead one to consider that differentiation of GC and maturation of oocytes could be processes regulated, to some extent, by different mechanisms.

Effect of TGF-Pl

Results obtained with 0.1, 1, and 10 ng transforming growth factor (TGF-Pl/ ml) were similar to those of r Act A: significant reduction of A at all doses; significant increase of E2 at 10 ng TGF-Pl/ml; and significant augmentation of Ir Inh for 1 and 10 ng TGF-Pl/rnl. IGF-1 levels were significantly inhibited at 1 ng/rnl, whereas 0.1 n g / d and 10 ng/ml were without significant effect. IGF-2 levels in culture medium were significantly decreased for the three investigated doses (FIG. 2). TGF-/3 1 did not modulate MCA. Our experiments confirm that TGF-P1 is implied in the regulation of follicular cell differentiation: it stimulates the FSH-induced aromatase activity and inhibin production of GC and reduces LH-induced androgen release of theca cells. 1 7 ~ w . 2 3

50 ANNALS NEW YORK ACADEMY OF SCIENCES

FIGURE 2. Evolution of IGF-1 (upper panel) and IGF-2 (lower panel) during the culture period of rat primary follicles in the absence (+) or presence of TGF-fll: 0.1 ng/ ml (O), 1 n g / d (m), and 10 nu&( A ). *: p < 0.01; " * : p < 0.001.

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TGF-P1 inhibited IGF-1 in a bell-shaped curve and IGF-2 in a dose-dependent manner. It is known that theca cells produce TGF-P, which inhibits the ability of TGF-a to stimulate either GC or TC As TGF-/3 decreases DNA synthesis of rat ovarian cells,2l such an effect might be mediated by the decrease of IGF-1 and IGF-2 production observed in our experiments. Finally, as for activin A, there is a dissociation between differentiation of GC, IGFs production (perhaps implied in growth of GC and TC), and maturation of oocytes. These three processes could be differently regulated.

E&+c~s Of IGF-1

In our model, IGF-1 did not significantly modify A, whereas 30 n g / d increased E2 levels in culture medium of primary follicles. IGF-1 (3 and 30 n g / d ) also stimu- lated a Ir Inh production in a dose-dependent manner. IGF-1 sigtllficantly reduced IGF-2 levels in the culture medium at 3 and 30 ng/ml. A stimulatory effect of 0.3 n g / d was observed at day 10 and 12, compared to the control values on the same days, but the curves are not significantly different. IGF-1 did not modulate MCA of oocytes.

Granulosa cells are a site of action of IGF-l.25 It enhances DNA synthesis and the growth of cellular components of rat follicles. Furthermore, IGF-1 is capable of

FRANCHIMONT et a/.: MATURATION OF PRIMARY FOLLICLE 5 1

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FIGURE 3. Evolution of a Ir inhibin during the culture period of rat primary follicles in the absence (+) or presence of TGF-a: 0.1 ng/ml (O), 1 ng/ml (U), and 10 ng/ml ( A ). ***: p < 0.0001.

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increasing FSH-induced biosynthesis of E226 and inhibin. 5 ~ 1 5 Our results confirm these previous data. On the contrary, a stimulatory effect on androgensz7 is not evidenced in our model.

The inhibitory effect of IGF-1 on IGF-2 is not explained. A local feedback mecha- nism could be responsible for the reduction of IGF-2 levels. Absence of effect of IGF- 1 on MCA might indicate that this local growth factor possesses a growth promoting effect on the follicles, but no effect on oocyte maturation.

Effect of TGF-a

The three amounts ofTGF-a significantly @ < 0.0001) decreased both E2 and A. They significantly increased inhibin production by primary follicle in culture medium. Curves obtained with 0.1 and 1 n g / d did not differ between them but were very significantly (p < 0.0001) different from the control curve (FIG. 3).

TGF-a significantly stimulated both IGF-1 and IGF-2 release into the culture medium at the three concentrations. Maximum effect on IGF-1 was obtained with 1 n g / d , which was significantly higher than that observed with 0.1 and 10 ng/ml.

At all doses, TGF-a did significantly increase the MCA of oocytes (TABLE 1). Theca cells, but not granulosa cells, produce T G F a Z 8 It stimulates GC and TC

growth participating in cell growth of the follicle.29 The effect of TGF-a on growth of the follicle could be mediated by the increase of IGF-1 and IGF-2 production.

Epidermal growth factor (EGF) and TGF-a share the same receptors. Both growth factors inhibit FSH and CAMP stimulation of estrogens and progesterone production and LH receptor i nd~c t ion .~O-~~ EGF inhibits inhibin production by bovine GC,33 whereas in rat Sertoli cells EGF stimulates inhibin production. This effect of EGF was additive to the similar effect of FSH.34 We also observed inhibition of GC differentia- tion and a stimulatory effect on inhibin production by rat GC.

TGF-a acts on oocyte maturation by augmenting the MCA. The effect on MCA seems to be independent of IGF-1 production, as it does not affect MCA at the three doses investigated. The possible role of IGF-2 is not excluded in these experiments with TGF-a. It may be speculated that maturation of oocytes would be, to some extent, independent of differentiation of GC and of follicular growth.

52 ANNALS NEW YORK ACADEMY OF SCIENCES

TABLE 1. Meiotic Competence Acquisition by TGF-a

Percenr of Meiosis Resumpaon (GVBd) in the Presence of: Days of

Culture 0 n n / d TGF-a 0.1 n g / d TGF-a 1 n g / d TGF-a 10 n g / d TGF-a

2 0 0 0 0 4 0 2 2 3 6 10 f 0.3” 16 2 0.2 15 f 0.3 15 f 0.4 8 17 f 0.5 21 5 0.5 26 f 0.7 26 2 0.6

10 40 2 1.6 38 f 1.5 56 2 2.2 42 f 1.4 12 45 f 1.8 55 f 1.4 54 f 1.9 55 5 1.5

Significance of curves compared to the control curve (Zerbe’s method) p < 0.01 p < 0.001 p < o.Ooo1

“Mean f SEM.

DISCUSSION AND CONCLUSIONS

1. It is generally accepted that local regulators modulate the effects of hormones such as FSH and LH. They could act in the absence of hormones (e.g., IGF-1 on inhibinX), but the effects are less marked than with FSH. In our model, no hormone was added, except insulin in the culture medium. This means that follicular and oocyte maturation might develop in the absence of hormones with production of local growth factors, such as IGF-1 and IGF-2. Alternatively, the structure of primary follicle would be primed by gonadotropins in the last period of gestation or in the neonatal period, when the level of gonadotropins is elevated.

2. There is a dissociation between E2 and A production on the one hand and Ir Inh production on the other hand when TGF-a is added to the culture medium. Inhibin would not be just a criterion of GC differentiation, but might act by itself in local regulation of follicular growth or in modulation of oocyte maturation. This hypothesis is supported by the article of Woodruff cta1.,j5 which showed that inhibin acts in an autocrine arid paracrine manner to stimulate recruited growing follicles and proliferation of GC.

3. There is a dissociation between E2 and A production on the one hand and IGFs production on the other hand; increase in A and E2 by r Act A and TGF-fl is associated with a decrease of IGFs levels. On the contrary, decrease of E2 and A production is associated with an increase of IGFs release when TGF-a is added. It may be speculated that IGFs would be more involved in promoting growth of follicles than in GC differentiation.

4. Dissociation between GC differentiation, IGFs production, and oocyte matura- tion would suggest that the three processes progress more or less independently of each other. Such a possibility was recently proposed by Schoot eta1.,j6 who observed that recombinant FSH alone can induce growth of preovulatory follicles in the presence of subnormal E2 levels. Similarly, Webb e t ~ l . ~ ’ showed that epostane, a 3fl hydroxyste- roid dehydrogenase inhibitor, orally given in the ewe, reduced ovarian steroid produc- tion, but increased ovulation rate.

PRANCHIhlONT et d.: MATURATION OF PRIMARY FOLLICLE 53

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