LH reduces proliferative activity of cumulus cells and accelerates GVBD of porcine oocytes

Molecular and Cellular Endocrinology 209 (2003) 43–50

LH reduces proliferative activity of cumulus cells and accelerates GVBD of porcine oocytes Tetsuji Okazaki a , Masahide Nishibori b , Yasuhisa Yamashita a , Masayuki Shimada a,∗ a

Laboratory of Animal Reproduction, Graduate School of Biosphere Science, Hiroshima University, Higashi-Hiroshima, Hiroshima 739-8528, Japan b Laboratory of Animal Breading and Genetics, Graduate School of Biosphere Science, Higashi-Hiroshima, Hiroshima 739-8528, Japan Received 6 May 2003; accepted 5 August 2003

Abstract It has been reported that LH receptor (LHR) mRNA is not detected in cumulus cells of porcine cumulus–oocyte complexes (COCs) just after collection from small antral follicles. The present study showed that the formation of LHR in cumulus cells was up-regulated by the cultivation with 20 ng/ml FSH. When the newly synthesized receptors were stimulated by 1.0 ␮g/ml LH, significantly higher levels of cAMP and progesterone production in cumulus cells were observed as compared with those of COCs cultured with FSH. A loss of proliferative activity of cumulus cells was induced by the additional LH to FSH-containing medium; however, the inhibitory effect was overcome by progesterone receptor antagonist RU486. Furthermore, the addition of LH also accelerated ongoing GVBD in cumulus cells-enclosed oocytes. These results revealed that during in vitro meiotic maturation of porcine COCs, progesterone secreted by FSH- and LH-stimulated cumulus cells reduced proliferative activity of cumulus cells; the changes of cumulus cells might be involved in inducing meiotic resumption of porcine oocytes. © 2003 Elsevier Ireland Ltd. All rights reserved. Keywords: Cumulus cells; LH; LH receptor; Progesterone; Meiotic resumption; Oocytes

1. Introduction In vivo, LH surge stimulates pre-ovulatory follicles in which LH receptor (LHR) has been expressed in cumulus cells and granulosa cells, that is essential for both meiotic resumption of oocytes and ovulation (Mattioli, 1994). The LH stimulation in vivo acted to inhibit cumulus cells proliferation, and the loss of proliferative activity appeared to be closely associated with LH-regulation of cumulus cell functions (Schuetz et al., 1996). Cytochrome P450scc that cleaved the side chain of cholesterol, was expressed in LH-stimulated cumulus cells and granulosa cells, which resulted in a secretion of progesterone into follicular fluid (Goldschmit et al., 1989). Osborn et al. (1986) reported that when the ovine follicles were cultured with FSH, LH, and P450scc inhibitor, aminoglutethimide, the results was an almost complete inhibition of progesterone production and meiotic progression to the MII stage in oocytes. Additionally, Morgan et al. (1990) reported that a high concentration ∗ Corresponding author. Tel.: +81-824-24-7899; fax: +81-824-24-7988. E-mail address: [email protected] (M. Shimada).

of progesterone in follicular fluid promoted meiotic maturation of rhesus monkey oocytes. These reports indicate that LH surge stimulates both a loss of proliferative activity and an increase of progesterone production in cumulus cells, and thus induces meiotic resumption of oocytes in vivo. Porcine oocytes for in vitro maturation are usually collected from early antral follicles (3–5 mm) of prepubertal gilts without gonadotropin priming. When porcine cumulus oocyte complexes (COCs) were cultured with highly purified LH, the level of progesterone was not increased in the medium, whereas FSH had the ability to increase progesterone production in cumulus cells of COCs (Shimada et al., 2002a). Reverse transcription-polymerase chain reaction (RT-PCR) on mRNA of bovine cumulus cells revealed that in spite of no expression of LHR gene, mRNA for FSH receptor was present in cumulus cells (Van Tol et al., 1996). In mouse COCs, Chen et al. (1994) reported that the functional LHR was slightly detected on cumulus cells immediately recovered from their follicles, however, the formation of the receptors was up-regulated by FSH. Recently, we also showed that the expression of LHR gene was up-regulated by 20-h cultivation with FSH, and the stimulation of LH to its newly synthesized receptors significantly improved

0303-7207/$ – see front matter © 2003 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.mce.2003.08.002

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the developmental competence of oocytes (Shimada et al., 2003). These results suggested that the binding of LH to the resultant receptors might be essential for in vitro meiotic maturation of oocytes as well as those of in vivo. However, the optimal conditions for LHR formation and the critical roles of synthesized LHR in cumulus cells of COCs during in vitro maturation have been remained unclear. The LHR is coupled to the adenylyl cyclase, inducing the production of cyclic AMP (cAMP) (Marsh, 1970), and the increase in cAMP in cumulus cells resulted in the synthesis of many potential factors that stimulate cumulus cell expansion and oocytes maturation (Byskov et al., 1997; Prochazka et al., 1998; Xia et al., 1994, 2000). Progesterone was secreted by forskolin-stimulated cumulus cells of porcine COCs through cAMP-dependent pathway (Racowsky, 1985). In our previous study (Yamashita et al., 2003), we showed that there was a significant positive correlation between the GVBD rate of oocytes and the progesterone concentration in each well. When porcine COCs were cultured with P450scc inhibitor, progesterone production was almost completely suppressed in cumulus cells, and the reduction of GVBD rate was also observed (Shimada and Terada, 2002a). These reports including our previous studies permitted us to speculate that the addition of LH to FSH-containing medium induced a further increase of progesterone production in cumulus cells, which resulted in an acceleration of meiotic resumption of porcine oocytes as compared with those of COCs cultured with FSH. There is little information describing the dose effects of additional LH to FSH-containing medium on the function of cumulus cells and the time period of oocytes exhibiting GVBD. In this study, we investigated the dose responses of LH on cAMP level, progesterone production, and proliferative activity of cumulus cells of porcine COCs. The results showed that the addition of 1.0 ␮g/ml LH to FSH-containing medium increased both cAMP level and progesterone production, and decreased proliferative activity of cumulus cells. The addition of LH also accelerated ongoing GVBD in cumulus cells-enclosed oocytes. Additionally, we also examined the role of large amount of progesterone secreted by cumulus cells in both proliferative activity of cumulus cells and meiotic resumption of cumulus-enclosed oocytes, using RU486.

2. Materials and methods 2.1. General protocol Isolation of porcine COCs was described previously (Shimada and Terada, 2001). The COCs were cultured in the maturation medium supplemented with or without highly purified porcine FSH (NIDDK, Torrance, CA, USA) and/or porcine LH (NIDDK). The maturation medium was modified NCSU37 (Petters and Reed, 1991) supplemented with 10% (v/v) FCS (Gibco BRL, Grand Island, NY, USA), 7 mM Taurine (Sigma Chemical Co., St. Louis, MO,

USA) and 4 mM hypoxanthine (Sigma). After cultivation of COCs, the level of cAMP in cumulus cells was analyzed by HPLC-UV according to our previous study (Shimada and Terada, 2002b). Quantification of progesterone in the medium by HPLC-UV was based on the procedures reported by our previous study (Shimada and Terada, 2002a). The oocytes were fixed with acetic acid/ethanol (1:3) for 48 h, and stained with aceto-lacmoid before examination under a phase-contrast microscope (400×) for evaluation of their chromatin configuration. 2.2. RNA isolation After cumulus cells were separated from 20 COCs, they were washed three times in PBS. Total RNA was extracted from cumulus cells using the SV Total RNA Isolation System (Promega, Madison, WI, USA), according to the instruction manual, and dissolved in 20 ␮l nuclease-free water. 2.3. RT-PCR for LHR mRNA Oligonucleotide primers used for amplification of the LHR were designed from known cDNA sequences of four porcine LHR isoforms (GenBank accession nnumber: M29525; Loosfelt et al., 1989). The upstream primer (5 -CCAATCTCCTAGATGCCACATT GAC-3 ) is identical to nucleotides 861–885 of the porcine cDNA, and the downstream primer (5 -GCTCAGCAACAGAAAGAAATCCC-3 ) represents the reverse complement of nucleotides 1959–1981. This primer pair predicts 185-, 411-, 855- and 1121-base pair (bp) DNA fragments. ␤-actin was used as a control for reaction efficiency and variations in concentrations of mRNA in the original RT reaction. The ␤-actin primers were based on the mouse sequence (GenBank accession number: NM009609; Tokunaga et al., 1986). The upstream primer (5 -CTACAATGAGCTGCGTGTG G-3 ) is identical to nucleotides 192–211 of the mouse cDNA, and the downstream primer (5 -TAGCTCTTCTCCAGGGAG GA-3 ) represents the reverse complement of nucleotides 622–641. The primer pair predicts a 450 bp DNA fragment. RT-PCR was performed according to our previous study (Shimada et al., 2003). Briefly, total RNA was reverse transcribed at 48 ◦ C for 45 min, denatured at 94 ◦ C for 2 min, and amplified for 35 cycles of denaturation at 94 ◦ C for 30 s, primer annealing at 60 ◦ C (LH receptor) or 58 ◦ C (␤-actin) for 1 min, and extension at 68 ◦ C for 2 min, with a final extension step of 7 min at 68 ◦ C. The amplified products were analyzed by electrophoresis on 2% agarose gels. 2.4. DNA sequence Direct sequence was carried out to identify RT-PCR products using a fluorescent dye terminator (DYEnamic ET terminator cycle sequence kit, Amersham, Pisxataway, NJ, USA) and the ABI PRISM 377XL DNA Sequencer

T. Okazaki et al. / Molecular and Cellular Endocrinology 209 (2003) 43–50

2.5. Binding of biotinylated hCG to cumulus cells surrounding oocytes Biotinylated hCG binding assay was based on the procedures reported by our previous study (Shimada et al., 2003) with some modifications. After COCs were incubated for 3 min in ice-cold ethanol, cumulus cells were stripped from COCs. The 1 × 104 cells were incubated in the maturation medium supplemented with 5 ␮g/ml biotinylated hCG at 39 ◦ C for 1 h, and then further cultured in the maturation medium supplemented with 0.67 ␮g/ml of HRP-conjugated avidin (Sigma) for 30 min at 39 ◦ C. Peroxidase activity was determined at 25 ◦ C using o-phenylenediamine (MBL, Nagoya, Japan) in 0.1 mM phosphate buffer (pH 5.1) and 0.015% (v/v) H2 O2 . After the addition of 20% (v/v) H2 SO4 solution, the absorbance at 492 nm was measured on a spectrophotometer. This assay is based on the ability of biotinylated hCG and LH to compete for binding to LHR in cumulus cells of porcine COCs. With this method, the rate of nonspecific binding of biotinylated hCG to cumulus cells was 5.2%. 2.6. 5-Bromo-2-deoxyuridine (BrdU) labeling for cell proliferation The proliferative response of cumulus cells was measured with a BrdU proliferation kit (Roche Molecular Biochemicals, Basel, Switzerland). In short, the 10 COCs in each well were cultured with a 100 ␮M 5-bromo-2-deoxyuridine (BrdU) for 10-h period. At the end of cultivation, cumulus cells were separated from COCs, then washed three times with PBS. They were suspended at 5 × 103 cells per well in the 96-well culture plates, and then the plates were dried for 1 h at 60 ◦ C. A FixDenat solution (200 ␮l) was added to each well, and the plates were incubated for 30 min at 25 ◦ C. The FixDenat solution was removed and peroxidase-labeled anti-BrdU monoclonal antibody (100 ␮l per well) that had been diluted 100-fold with the Antibody dilution solution was added, before the plates were incubated for 1 h at 25 ◦ C. The peroxidase-labeled anti-BrdU monoclonal antibody solution was removed, and each well was washed three times with PBS (200 ␮l each). A tetramethylbenzidine solution (100 ␮l per well) was added, and the plates were incubated for 15 min. After adding 1 M H2 SO4 (25 ␮l per well) to stop the enzymatic reaction, the absorbance at 450 nm was measured with microplate reader.

2.7. Statistical analysis Statistical analyses of all data from three or four replicates for comparison were carried out by one-way ANOVA followed by Duncan’s multiple-range test (Statview; Abacus Concepts Inc., Berkeley, CA). All percentage data were subjected to arcsine transformation before analysis. Differences were considered significant when P < 0.05.

3. Results 3.1. LHR formation in cumulus cells of COCs To examine the dose effects of FSH on the expression of LHR gene in cumulus cells of COCs, COCs were cultured with 0, 2, 20, or 200 ng/ml FSH for 20 h. After cultivation of COCs, the expression of LHR mRNA was analyzed by RT-PCR, and hCG binding level was determined by using the biotinylated hCG for a probe ligand. Four types of amplified product (1121, 855, 411, and 185 bp; LHR1–4, respectively) were detected in cumulus cells. Sequence of the large form (LHR1) was identified as a part of the porcine full-length LHR cDNA (DDBJ/EMBL/GeneBank accession number: M29525), by using basic local alignment search tool (BLAST, Altschul et al., 1990). We also observed that the sequences of three splice variants (LHR2–4) were absent of the putative transmembrane domain (accession numbers: M29526, M29527, and M29528, respectively). The cultivation for 20 h in the medium without FSH did not promote the LHR production (Fig. 1). However, biotinylated hCG binding levels in cumulus cells of COCs was increased with the increasing concentration of FSH in an approximately linear fashion, with maximal stimulation at 20 ng/ml (3.5-fold increased) (Fig. 1). The addition of 200 ng/ml FSH slightly decreased the level of hCG binding

4

c bc

Fold strength *

(Applied Biosystems, Foster City, CA, USA). According to the instructions supplied with the sequencing kit, the cDNA fragments were mixed with four dye-labeled di-deoxynucleotides as terminators, a thermally stable AmpliTaq DNA polymerase, and upstream or downstream primer, then set on the thermal cycler for the sequencing reaction. Extended products were analyzed on the DNA sequencer.

45

3

b

2 a a

1 0 0 hr

0

2

20

200

FSH (ng/ml) 20 hr Fig. 1. Dose-dependent effects of FSH on the level of LH receptor on cumulus cells of porcine COCs cultured for 20 h. (∗) Data are expressed as fold strength of the level of biotinylated hCG binding on cumulus cells immediately recovered from their follicles, defined as 1. (a–c) No common superscripts were significant (P < 0.05).

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3.2. cAMP level in cumulus cells of COCs After 10 h cultivation, the level of cAMP in cumulus cells of COCs cultured without any gonadotropin was 0.37 ± 0.01 pmol/104 cells (Fig. 3). The cultivation for 10 h with 20 ng/ml FSH induced a significant increase of cAMP level in cumulus cells; however, there was no significant effect of LH stimulation on cAMP level (Fig. 3). When COCs were cultured for 20 h, the level of cAMP in cumulus cells was significantly increased by additional 0.1, 1.0, or 10 ␮g/ml LH to the FSH-containing medium in a dose-dependent fashion (Fig. 3). 3.3. Progesterone production by cumulus cells After 10-h cultivation of COCs without any gonadotropin, the low level of progesterone in the medium was observed (3.01 ± 0.43 ng/ml). The addition of 20 ng/ml FSH or 20 ng/ml FSH + 1.0 ␮g/ml LH significantly increased the level of progesterone in the medium, whereas no significant difference was observed between FSH treatment group and FSH + LH group (Fig. 4). When COCs were cultured for 20 h without FSH, the level of progesterone in the medium was 6.70 ± 0.77 ng/ml. The addition of 20 ng/ml FSH significantly increased the progesterone level in the medium (Fig. 4). When COCs were cultured with both 20 ng/ml FSH and 1.0 ␮g/ml LH, the maximal level of progesterone (28.4 ± 2.87 ng/ml) was detected (Fig. 4). The concentration was significantly higher as compared with that in the medium where COCs had been cultured with or without FSH (Fig. 4).

Fig. 2. Dose-dependent effects of FSH on the expression of LHR gene in cumulus cells of COCs. ␤-actin was used as a control for reaction efficiency and variations in concentrations of mRNA in the original RT reaction. RT-PCR was performed for porcine LHR cDNA in 35 cycles of PCR.

as compared with that in cumulus cells of COCs cultured with 20 ng/ml FSH (Fig. 1). The intensity of full length of LHR mRNA (LHR1) in cumulus cells of COCs which were cultured with 200 ng/ml FSH, was also lower than that in COCs cultured with 20 ng/ml FSH (Fig. 2).

e

cAMP level (pmol/104 cells)

2

e de

1.5 d

1 c b

0.5

b

b

b

a

0 Free

0

0.1 1.0 LH (µg/ml)

10.0

Free

0

0.1 1.0 LH (µg/ml)

20 ng/ml FSH

20 ng/ml FSH

10 hr

20 hr

10.0

Fig. 3. The level of cAMP in cumulus cells of COCs which were cultured for 10 h or 20 h in the 20 ng/ml FSH-containing medium supplemented with 0.1, 1.0, or 10.0 ␮g/ml LH. Free: COCs were cultured in the maturation medium without any gonadotropin for 10 h or 20 h. (a–e) No common superscripts were significantly different between treatment groups at the same time points (P < 0.05). Values are mean ± S.E.M. of four replicates.

T. Okazaki et al. / Molecular and Cellular Endocrinology 209 (2003) 43–50

80 e

30 d

20 b

c

40

0

a

FSH

0 free

60

20

b

10

* (% oocyte)

Progesterone level (ng/ml)

40

47

FSH

FSH+LH

free

10 hr

FSH

FSH+LH

20 hr

Fig. 4. The level of progesterone in the medium in which COCs had been cultured for 10 h or 20 h with FSH or FSH + LH. Free: COCs were cultured in the maturation medium without any gonadotropin for 10 h or 20 h; FSH: COCs were cultured with 20 ng/ml FSH for 10 h or 20 h; FSH +LH: COCs were cultured with 20 ng/ml FSH and 1.0 ␮g/ml LH for 10 h or 20 h. (a–e) No common superscripts were significant difference between treatment groups at the same time points (P < 0.05). Values are mean ± S.E.M. of four replicates.

3.4. Proliferative activity of cumulus cells During the first 10-h cultivation period, the incorporation of BrdU in cumulus cells cultured with 20 ng/ml FSH or 20 ng/ml FSH + 1.0 ␮g/ml LH was significantly lower than that in cumulus cells of COCs which were cultured without any gonadotropin (Fig. 5). The addition of LH to FSH-containing medium did not significantly affect the proliferative activity (Fig. 5). To investigate the proliferative activity of cumulus cells during the latter 10-h cultivation period (10–20 h), COCs were cultured with 20 ng/ml FSH or 20 ng/ml FSH + 1.0 ␮g/ml LH for 10 h, followed by a further cul-

FSH+LH

20 hr

FSH

FSH+LH

28 hr

Fig. 6. The additional effects of LH on the rate of GVBD in oocytes at 20-h or 28-h cultivation periods. FSH: COCs were cultured with 20 ng/ml FSH for 20 h or 28 h; FSH + LH: COCs were cultured with 20 ng/ml FSH and 1.0 ␮g/ml LH for 20 h or 28 h. An asterisk (∗) indicates a significant difference between treatment groups at 20 h cultivation (P < 0.05). Values are mean ± S.E.M. of three replicates.

tivation with additional BrdU for 10 h. The incorporation of BrdU in cumulus cells of COCs which were cultured with 20 ng/ml FSH was lower than that of COCs cultured without any gonadotropin; however, the difference was not significant (Fig. 5). The addition of 1.0 ␮g/ml LH to 20 ng/ml FSH-containing medium significantly suppressed the incorporation of BrdU in cumulus cells as compared with that in other groups (Fig. 5). 3.5. The time period of oocyte exhibiting GVBD After COCs were cultured with 20 ng/ml FSH+1.0 ␮g/ml LH for 20 h, over 50% of oocytes were undergoing GVBD. The rate was significantly higher than that of COCs which were cultured with only 20 ng/ml FSH (Fig. 6). When COCs were cultured with FSH or FSH + LH for 28 h, about 70% of oocytes exhibited GVBD (Fig. 6). No significant

Fig. 5. The level of BrdU incorporation into cumulus cells of COCs which were cultured for 10 h or 20 h in the medium supplemented with FSH or FSH + LH. Free: COCs were cultured in the maturation medium without any gonadotropin for 10 h or 20 h; FSH: COCs were cultured with 20 ng/ml FSH for 10 h or 20 h; FSH + LH: COCs were cultured with 20 ng/ml FSH and 1.0 ␮g/ml LH for 10 h or 20 h. (1) Data are expressed as percentage of the absorbance value at 450 nm in cumulus cells of COCs which were cultured for 10 h in the basic medium with 100 ␮M BrdU. (a–d) No common superscripts indicates significant difference between treatment groups at the same time points (P < 0.05). Values are mean ± S.E.M. of three replicates.

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4. Discussion

Fig. 7. The effects of RU486 on the proliferative activity of cumulus cells (a) and the proportion of oocytes exhibiting GVBD (b) when COCs were cultured for 10 h with FSH and LH, and further cultured for 10 h with additional 2.5 ␮M or 25 ␮M RU486. (1) Data are expressed as percentage of the absorbance value at 450 nm in cumulus cells of COCs which were cultured for 10 h in the basic medium with 100 ␮M BrdU. Free: COCs were cultured in the maturation medium without any gonadotropin for 10 h and then further cultured for 10 h with 100 ␮M BrdU. (a–f) No common superscripts were significant difference between treatment groups (P < 0.05). Values are mean ± S.E.M. of three replicates.

differences in GVBD rate was observed between both treatment groups. 3.6. Effects of RU486 on proliferative activity of cumulus cells To investigate the role of large amount of progesterone produced by FSH + LH stimulating cumulus cells in a decrease of proliferative activity, COCs were pre-cultured with 20 ng/ml FSH + 1.0 ␮g/ml LH for 10 h and then further cultured with FSH, LH, and either 2.5 ␮M or 25 ␮M RU486 for 10 h. The proliferative activity in cumulus cells during the latter 10-h cultivation period was increased by RU486 in a dose-dependent fashion (Fig. 7a). The rate of proliferative activity in cumulus cells cultured with FSH+LH and 25 ␮M RU486 after 10 h cultivation with FSH + LH, was significantly higher than that in cumulus cells which were cultured with FSH + LH for 20 h (Fig. 7a). Moreover, the proportion of oocytes exhibiting GVBD was significantly decreased with the increasing concentration of RU486 (Fig. 7b).

It has been reported that highly purified FSH (which is contaminated with less than 1% LH) is able to elicit some cumulus expansion by itself, whereas it is still inadequate in stimulating the optimal expansion induced by crude porcine FSH, which is normally contaminated with about 10% LH bioactivity (Chen et al., 1994; Reichert et al., 1968). In our previous study, the expression of LHR gene was not detected in cumulus cells just after collection from their follicles, whereas the expression was significantly increased when porcine COCs were cultured with FSH for 20 h (Shimada et al., 2003). The results suggested that the action of LH in cumulus cells was dependent on the presence of FSH; however, the optimal level of FSH for the combined treatment with LH and the dose effects of additional LH to FSH containing medium have been remained unclear. In the present study, the 20-h cultivation with 20 ng/ml of FSH resulted in a high level of full-length LHR gene expression. Loosfelt et al. (1989) reported that when the cDNA containing the full-length open reading frame of LHR was transfected into COS-7 cells, hCG bound to its expressed receptors. Moreover, biotinylated hCG binding analysis also revealed that the addition of 20 ng/ml FSH to the maturation medium induced a significant increase in the binding level of hCG to COCs. These results showed that 20 ng/ml was the optimal level of additional FSH to the maturation medium for LHR formation in cumulus cells. When COCs were cultured in 20 ng/ml FSH-containing medium with various concentration of LH, cAMP level in cumulus cells was significantly increased with increasing concentration of LH in an approximately linear response, with maximal stimulation at 1.0 ␮g/ml. Several researchers have shown that the increase of cAMP in cumulus cells stimulated by FSH and/or LH is related in the production of many potential factors which induce oocytes maturation in mouse and pig (Byskov et al., 1997; Xia et al., 1994, 2000). It was also reported that progesterone was secreted by forskolin-stimulated cumulus cells of porcine COCs through cAMP-dependent pathway (Racowsky, 1985). In the present study, when COCs were cultured with both 20 ng/ml FSH and 1.0 ␮g/ml LH, the maximum level of progesterone in the medium and an acceleration of GVBD in oocytes were observed. Thus, the additional LH bound to the newly synthesized LHR on cumulus cells, which might be involved in meiotic resumption of oocytes. Although we have already showed that the high level of progesterone produced by cumulus cells is responsible for an acceleration of GVBD in porcine oocytes (Shimada et al., 2002b; Yamashita et al., 2003), there is little information about the critical role of progesterone in cumulus cells during meiotic maturation of porcine oocytes. Progesterone was a potent antagonist of proliferation of uterine epithelium (Lydon et al., 1995), and the loss of proliferative activity was regulated by the expression of cyclin-dependent kinase (CDK) inhibitor genes (p21waf and

T. Okazaki et al. / Molecular and Cellular Endocrinology 209 (2003) 43–50

p27kip1 ) (Owen et al., 1998; Shiozawa et al., 2001). The elevated CDK inhibitor levels by progesterone suppressed the proliferation but promoted the differentiation of cells (Shiozawa et al., 2001; De Vivo et al., 2002). In the present study, after 20-h cultivation of COCs with FSH and LH, the maximum level of progesterone in the medium and a significantly lower proliferative activity of cumulus cells were observed. The addition of RU486 which bound with high affinity to both progesterone and glucocorticosteroid receptors, overcame the inhibitory effects on proliferative activity, and the level was comparative with that in cumulus cells of COCs which were cultured without gonadotropin. Schuetz et al. (1996) reported that during in vivo ovulation, the loss of proliferative activity appears to be closely associated with LH-regulation of cumulus cell functions. Additionally, the present study showed that FSH and LH also accelerated ongoing GVBD in oocytes; however, the acceleration was suppressed by RU486. Moreover, when COCs were cultured with aminoglutethimide, almost complete inhibitions of both progesterone production and meiotic resumption were observed (Shimada and Terada, 2002a). The results provided the first evidence that during in vitro meiotic maturation of porcine COCs, the large amount of progesterone secreted by FSH- and LH-stimulated cumulus cells induced a reduction of proliferative activity of cumulus cells; in the COCs, meiotic resumption of cumulus-enclosed oocytes was accelerated. In conclusion, the expression of LHR gene in cumulus cells was up-regulated by the cultivation with 20 ng/ml FSH. The addition of 1.0 ␮g/ml LH into FSH-containing medium increased cAMP level in cumulus cells, which stimulated progesterone production. The secreted progesterone induced both a loss of proliferative activity and meiotic resumption of oocytes in vitro.

Acknowledgements Porcine FSH, LH, and hCG were kindly provided by Dr. A.F. Parlow, the National Hormone and Pituitary Program, the National Institute of Diabetes and Digestive and Kidney Disease, USA. Thanks to Dr. A. Barua for insightful comments and suggestions concerning the manuscript, and Dr. M. Fujita for technical advice on the use of HPLC-UV analysis. We thank the staff of the Meat Inspection Office in Hiroshima City for supplying the porcine ovaries. This work was partly supported by Grant-in-Aid for Scientific Research M.S. No. 14760179 from the Japan Society for the Promotion of Science.

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