Molecular Human Reproduction

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Molecular Human Reproduction, Vol. 7, No. 4, 319-323, April 2001
© 2001 European Society of Human Reproduction and Embryology

Gonadotrophins inhibit and activin induces expression of inhibin/activin ß_B subunit mRNA in cultured human granulosa-luteal cells

J. Liu^1,2,4, C. Hydèn-Granskog³ and R. Voutilainen^1,2

¹ Department of Pathology, P.O.Box 21, University of Helsinki, FIN-00014 Helsinki, ² Department of Pediatrics, P.O.Box 1777, Kuopio University Hospital, FIN-70211 Kuopio and ³ Department of Obstetrics and Gynecology, Helsinki University Central Hospital, FIN-00290 Helsinki, Finland

	Abstract

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During the human menstrual cycle, serum inhibin concentrations fluctuate in a cyclic fashion. To examine the regulation of inhibin/activin ß_B subunit gene expression in ovarian granulosa–luteal cells, the levels of ß_B subunit mRNA were determined in primary cultures of human granulosa–luteal cells treated with gonadotrophins and protein kinase modulators. Granulosa cells were obtained from women undergoing an IVF programme. The cells were enzymatically dispersed, separated from red blood cells, and maintained in culture for 5–10 days before addition of different agents. Northern blot analysis with specific oligonucleotide probes was performed to study inhibin/activin ß_B subunit mRNA levels. Both LH and FSH reduced the accumulation of ß_B subunit mRNA in a dose-dependent manner. The protein kinase A activator, (Bu)₂cAMP, and the protein kinase inhibitor staurosporine also inhibited ß_B subunit mRNA expression dose-dependently. Activin A increased dose-dependently ß_B subunit mRNA expression. Our study suggests that activin-induced and gonadotrophin-inhibited ß_B subunit expression in granulosa cells might be key factors in the transition from inhibin B to inhibin A dominance during the menstrual cycle.

activin/cAMP/gonadotrophin/granulosa cells/inhibin

	Introduction

Top Abstract Introduction Materials and methods Results Discussion Acknowledgements References

 
Inhibins and activins are structurally related dimeric glycoprotein hormones initially characterized by their ability to suppress and stimulate, respectively, FSH secretion by the pituitary gland. Inhibins are heterodimers of a common subunit and one of the two distinct, but homologous, ß chains (ß_A or ß_B), whereas activins are heteroor homodimers consisting of two ß subunits. Recent studies have indicated that inhibins/activins may also act as local regulators of cell growth and steroidogenesis in the human ovary (Fraser and Lunn, 1993; Peng et al., 1996). Activin-A has been reported to induce proliferation of human granulosa cells in culture (Rabinovici et al., 1990). Inhibin has been shown to stimulate theca cell androgen synthesis, whereas activin has an inhibitory effect on it (Hillier, 1991). Moreover, in human granulosa–luteal cell cultures, activin has been shown to decrease cholesterol side-chain cleavage enzyme (P450scc) mRNA accumulation (Erämaa et al., 1995) and progesterone secretion (Li et al., 1992).

During the human menstrual cycle, serum inhibin concentrations fluctuate in a cyclic fashion with a different profile for inhibin A and inhibin B. Generally, in the early follicular phase, inhibin A concentrations are low and inhibin B concentrations high, whereas in the mid-luteal phase, inhibin A concentrations are high but inhibin B concentrations low (Groome et al., 1994, 1996). This fluctuation may be determined by different expression levels of the three inhibin/activin subunits modulated by endocrine and locally produced factors. All three inhibin subunit mRNAs and the corresponding peptides are expressed in human follicles and corpus luteum (Roberts et al., 1993), and the mRNAs are also detected in cultured human granulosa–luteal cells (Erämaa et al., 1995). In human ovaries, ß_B subunit mRNA is expressed in excess over mRNA and ß_A subunits in small antral follicles. As the follicle matures under the influence of gonadotrophins to a dominant pre-ovulatory follicle, an increase in and ß_A subunit concentrations is observed, whereas the level of ß_B subunit mRNA decreases (Roberts et al., 1993). In cultured human granulosa–luteal cells, the and ß_A subunit mRNAs are readily detectable, whereas the ß_B subunit mRNA is expressed at very low levels. In a previous study, gonadotrophins increased the and ß_A subunit mRNA content, but they did not affect the low levels of the ß_B subunit mRNA (Erämaa et al., 1994). In contrast, activin A stimulated ß_B subunit mRNA expression, but inhibited the gonadotrophin-induced inhibin subunit mRNA accumulation (Erämaa et al., 1995). These studies indicated that the regulation of the ß_B subunit expression in human granulosa–luteal cells is distinct from that of the and ß_A subunits. Although the regulation of the and ß_A subunit expression has been extensively studied in cultured granulosa–luteal cells, much less is known about the regulation of ß_B subunit expression, at least partly due to its low expression levels. To clarify the ovarian ß_B subunit expression in more detail, we studied the effects of different hormones and protein kinase modulators on the ß_B subunit mRNA levels in cultured human granulosa–luteal cells.

	Materials and methods

Top Abstract Introduction Materials and methods Results Discussion Acknowledgements References

 
Cell cultures
Human granulosa–luteal cells were obtained by follicular aspiration from women undergoing oocyte retrieval for IVF. A total of 133 unselected women with different infertility reasons, including infertility of the spouse (24 cases), tubal obstruction (22 cases), endometriosis (18 cases), ovulatory disorder (nine cases), ovulatory disorder and endometriosis (three cases), polycystic ovaries (two cases), combined (male and female reason, 13 cases), and unexplained (42 cases), donated cells for the study. The age range of the women was 24–41 years (mean 33). The development of multiple follicles was induced by combining a gonadotrophin-releasing hormone (GnRH) analogue (Synarela: Searle, Bretigny-Sur-Orge, France; Suprecur: Hoechst Marion Roussel, Frankfurt am Main, Germany; or Zoladex: Zeneca, Alderley Park Macclesfield, Cheshire, UK) and an FSH preparation (Gonal-F: Serono, Bari, Italy; or Puregon: Organon, Oss, The Netherlands). Serum oestradiol concentration was measured from every patient on stimulation day 5 or 6, and the range was <0.1–4.93 nmol/l. In addition, serum oestradiol was measured on the ovum retrieval day in poor responders and in women with a high risk for ovarian hyperstimulation syndrome (oestradiol range was 0.56–20.87 nmol/l). Follicular aspiration was carried out 36–38 h after human chorionic gonadotrophin (HCG, Profasi: Serono; or Pregnyl: Organon) administration. The size of the aspirated follicles varied from 10 to 24 mm. The number of oocytes obtained was 0–45 per woman (mean 13.2). Of the oocytes, 85% were mature, 12.5% immature and 2.5% degenerated or otherwise abnormal. For each cell culture experiment, the granulosa cells obtained in the same morning from three to eight patients were pooled, enzymatically dispersed with 0.1% hyaluronidase (Sigma Chemical Co., St Louis, MO, USA), and separated from red blood cells by centrifugation through Ficoll-Paque (Pharmacia Biotech AB, Uppsala, Sweden), as previously described (Erämaa et al., 1995). The cells were plated at a density of 2–5x10⁵ cells/well on 35 mm 6-well dishes (Greiner labortechnik Gmbh, Frickenhausen, Germany) and cultured in 1:1 Dulbecco's modified Eagle's medium (DMEM)-Ham's F-12 medium (Gibco, Grand Island, NY, USA) supplemented with 10% fetal calf serum (FCS; Gibco), 2 mmol/l L-glutamine, and antibiotics (100 IU/ml penicillin and 100 µg/ml streptomycin) at 37°C in a 95% air–5% CO₂ humidified environment. FCS was used because it helps to maintain the differentiation of the granulosa cells and promotes the reappearance of their FSH responsiveness (Schipper et al., 1993). Cell culture media were changed every other day, and the hormonal stimulations were performed on days 5–10 of culture. At this culture stage, human granulosa–luteal cell progesterone production and inhibin/activin and ß_A subunit mRNA expression are optimally responsive to gonadotrophin treatment (Voutilainen et al., 1986; Schipper et al., 1993; Erämaa et al., 1994). The experiments were carried out in triplicate dishes and each experiment was repeated at least three times to make sure that the results were reproducible. The study was approved by the local research ethics committees.

Recombinant human FSH (rhFSH) and luteinizing hormone (rhLH) were gifts from Serono, and recombinant human activin A peptide was generously provided by Dr A.F.Parlow, NIDDK's National Hormone and Pituitary Programme (USA). (Bu)₂cAMP and 12-O-tetradecanoyl phorbol 13-acetate (TPA) were purchased from Sigma, staurosporine from Boehringer Mannheim (Mannheim, Germany), Ro 31-8220 and Gö 6976 from Calbiochem (La Jolla, CA, USA).

RNA analysis
Cytoplasmic RNA was extracted from the cultured cells (Voutilainen et al., 1986). Northern blotting (with 15 µg extracted RNA per lane) and hybridizations were performed as described previously (Liu et al., 1997). The inhibin ß_B mRNA was originally detected with a synthetic oligonucleotide complementary to human inhibin ß_B mRNA (Voutilainen et al., 1991). Since the signal was very weak, an additional oligonucleotide complementary to a different region of the human inhibin ß_B subunit mRNA was designed. The sequence of this oligonucleotide was 5'-GGG CAC GTC CCG CTT GAC GAT GTT GTA CTC-3', corresponding to the nucleotides 1002–1031 of the inhibin ß_B mRNA (GenBank Accession no. M13437; Mason et al., 1986). The two oligonucleotides were labelled separately, and pooled together for hybridization. The oligonucleotides and mouse ribosomal 28S RNA cDNA insert (used as a loading control) (Arnheim, 1979) were labelled with [P³²]deoxycytidine as described previously (Liu et al., 1997). The relative intensities of the autoradiographic signals were quantified by densitometric scanning. All the RNA data shown here were normalized with the respective 28S ribosomal RNA values. The differences in the specific RNA levels were assessed by the Mann-Whitney test. The level of significance was chosen as P < 0.05.

	Results

Top Abstract Introduction Materials and methods Results Discussion Acknowledgements References

 
Our preliminary experiments showed that the expression of inhibin/activin and ß_A subunits was readily detectable, whereas the ß_B subunit mRNA signal was very weak or undetectable by Northern blotting in cultured human granulosa–luteal cells. Due to this low ß_B mRNA expression, we used two oligonucleotides together for hybridization to increase the sensitivity in Northern analysis. As shown in Figure 1, a predominant 4.8 kb transcript of the ß_B subunit was detected in unstimulated granulosa–luteal cells maintained in culture for 8 days. To determine whether different culture conditions could affect the accumulation of the ß_B subunit mRNA, granulosa–luteal cells were first maintained in the culture media supplemented with 10% FCS for 7 days. Thereafter, the culture medium was refreshed with one containing FCS at different concentrations. Figure 1 shows that the expression of the ß_B subunit mRNA was dependent on the FCS concentration in the culture medium, with the ß_B mRNA level in 10% FCS medium almost 3-fold higher than in serum-free medium. It has previously been reported that inhibin/activin and ß_A subunit mRNA expression is increased by cAMP agonists through the protein kinase A pathway (Tuuri et al., 1996). Interestingly, (Bu)₂cAMP (1 mmol/l) reduced the ß_B subunit mRNA accumulation down to 30% of the control (P < 0.05) (Figure 1). The inhibitory effect of (Bu)₂cAMP was detectable after both 24 and 48 h incubation. A representative Northern blot showing the concentration-dependent effect of (Bu)₂cAMP on the ß_B subunit mRNA level is presented in Figure 2.

View larger version (32K): [in this window] [in a new window]   Figure 1. Regulation of inhibin/activin ßB subunit mRNA expression by (Bu)2cAMP (1 mmol/l) in cultured granulosa–luteal cells maintained in media with different concentrations of fetal calf serum (FCS). The dispersed cells were allowed to grow for 7 days in the medium with 10% FCS, and then treated as indicated for 24 h. Cytoplasmic RNA was extracted, and the Northern blot was prepared with 15 µg RNA in each lane. The filter was hybridized with ßB subunit and ribosomal 28S RNA probes. The experiment was repeated three times, and the results were similar.

 

View larger version (31K): [in this window] [in a new window]   Figure 2. A representative Northern blot showing the dose-dependent effect of (Bu)2cAMP (24 h of treatment) on inhibin/activin ßB subunit mRNA accumulation in primary cultures of granulosa–luteal cells. The culture conditions and RNA analyses were similar to those in Figure 1. The experiment was repeated five times, and the results were similar.

 
Since gonadotrophins up-regulate both inhibin/activin and ß_A subunit mRNA expression in granulosa–luteal cells (Erämaa et al., 1994), we tested whether the steady state mRNA levels of the ß_B subunit is also regulated by gonadotrophins. As shown in Figures 3 and 4, both rhLH and rhFSH (at a concentration of 100 IU/l for 24 h) reduced the accumulation of ß_B subunit mRNA to about 40% and 30% of the control respectively (both P < 0.05). The effects of both rhLH and rhFSH were dose-dependent with a half maximal inhibition at about 30 IU/l for both (Figures 3 and 4).

View larger version (34K): [in this window] [in a new window]   Figure 3. A representative Northern blot showing the dose-dependent effect of recombinant human (rh)LH (24 h of incubation) on inhibin/activin ßB subunit mRNA accumulation in primary cultures of granulosa–luteal cells. The culture conditions and RNA analyses were similar to those in Figure 1. The experiment was repeated four times, and the results were similar.

 

View larger version (34K): [in this window] [in a new window]   Figure 4. A representative Northern blot showing the dose-dependent effect of rhFSH (24 h of treatment) on inhibin/activin ßB subunit mRNA accumulation in primary cultures of granulosa–luteal cells. The culture conditions and RNA analyses were similar to those in Figure 1. The experiment was repeated three times, and the results were similar.

 
A protein kinase inhibitor, staurosporine (50 nmol/l), also inhibited the inhibin/activin ß_B subunit mRNA accumulation down to about 20% of the control (P < 0.05); this effect was dose-dependent with a half-maximal inhibition at a concentration between 0.1 and 1.0 nmol/l (Figure 5). However, protein kinase C activator, TPA (160 nmol/l), and inhibitors Ro 31-8220 (5 µmol/l) and Gö 6976 (5 µmol/l) had no significant effect on ß_B subunit mRNA level after 24 h of treatment (data not shown). Higher concentrations of Ro 31-8220 were toxic for the cells.

View larger version (18K): [in this window] [in a new window]   Figure 5. Dose-dependent effects of staurosporine (24 h of treatment) on inhibin/activin ßB subunit mRNA accumulation in primary cultures of granulosa–luteal cells. The culture conditions and RNA analyses were similar to those in Figure 1. Each column represents the mean ± SEM of three experiments (from different patients) with the control levels adjusted to 100.

 
Expression of the inhibin/activin ß_B subunit mRNA was increased (P < 0.05) by activin A addition (10–100 ng/ml) into the culture medium in a dose-dependent manner (Figure 6), in agreement with a previous report (Erämaa et al., 1995). The stimulatory effect of activin A on the expression of the ß_B mRNA was totally blocked by combining the treatment with (Bu)₂cAMP or staurosporine. Co-treatment with rhLH or rhFSH also inhibited the stimulatory effect of activin A on ß_B mRNA accumulation (data not shown).

View larger version (29K): [in this window] [in a new window]   Figure 6. A representative Northern blot showing the dose-dependent effect of activin A (24 h of stimulation) on inhibin/activin ßB subunit mRNA accumulation in primary cultures of granulosa–luteal cells. The cell culture conditions and RNA analyses were similar to those in Figure 1. The experiment was repeated four times, and the results were similar.

 

	Discussion

Top Abstract Introduction Materials and methods Results Discussion Acknowledgements References

 
We studied the regulation of inhibin/activin ß_B subunit mRNA expression by gonadotrophins in cultured human granulosa–luteal cells, and found that both rhLH and rhFSH reduced the accumulation of the ß_B subunit mRNA. Our results fit very well with a previous report documenting lower ß_B subunit mRNA levels in pre-ovulatory follicles and corpus luteum than in antral follicles which have not yet gained full responsiveness to gonadotrophins (Roberts et al., 1993). However, our data seem to be in contrast to a report that HCG, which functions through the LH receptor, was unable to modulate the basal ß_B subunit mRNA levels in cultured human granulosa–luteal cells (Erämaa et al., 1995). This discrepancy may be explained by the assay sensitivity: the basal ß_B subunit mRNA signal in the previous study was hardly detectable and thus additional reduction might not be visualized. Nevertheless, the stimulatory effects of activin A on the ß_B subunit mRNA accumulation was suppressed by HCG in the previous study (Erämaa et al., 1995), indicating that also HCG has an inhibitory effect on the ß_B subunit expression. It is important to note that the cultured cells used in both studies were pre-ovulatory granulosa–luteal cells obtained from IVF patients. Thus the relatively low basal ß_B mRNA expression in cultured granulosa–luteal cells may be caused by the in-vivo gonadotrophin treatment the patients received before follicular aspiration.

There seems to be some discrepancy between ß_B subunit gene expression and inhibin B peptide secretion data. Although gonadotrophins consistently reduce ß_B subunit mRNA (this study), inhibin B secretion is increased by FSH and LH at least during the first 24 h of treatment (Muttukrishna et al., 1997; Vänttinen et al., 2000). This could be explained by the assumption that, after 24 h of gonadotrophin treatment, there are still ß_B subunits which can form inhibin B with increasing amounts of subunits. However, after longer LH or HCG treatment (48 h), inhibin B secretion is no longer increased or may be even decreased (Muttukrishna et al., 1997) when the ß_B subunit production decreases.

The regulation of the inhibin/activin ß_B subunit mRNA expression by FSH may follow a species-specific pattern. In cultured rat granulosa cells, the ß_B mRNA accumulation was increased by FSH treatment, and this stimulation was supposed to be through the cAMP–protein kinase A pathway (Dykema and Mayo, 1994). However, Lanuza et al. (1999) reported that FSH increased more inhibin A than inhibin B production, whereas activin A dramatically stimulated inhibin B secretion in rat granulosa cell cultures, and this is in accordance with a recent human study (Vänttinen et al., 2000).

The physiological significance of the inhibitory effects of gonadotrophins on the inhibin/activin ß_B subunit expression in granulosa–luteal cells remains to be clarified. The cells used in this study were obtained from an unphysiological environment achieved by stimulation with gonadotrophins, and they were already luteinized due to the in-vivo HCG stimulation. However, during the culture the cells become sensitive to FSH and remain responsive to LH, as reported previously (Voutilainen et al., 1986; Schipper et al., 1993). Thus, these granulosa–luteal cells resemble the granulosa cells of pre-ovulatory follicles. It was recently reported that during human menopausal gonadotrophin/HCG treatment protocols, serum activin B (probably of ovarian origin) concentrations decreased gradually, while serum oestradiol concentrations increased (Vihko et al., 1998). This phenomenon might be explained by the reduced ß_B/ or ß_B/ß_A subunit ratios during gonadotrophin treatment. The inhibin dimers may be preferentially generated when the subunit is present in excess of the ß subunits, and likewise, the predominance of the ß subunits tends to favour the production of activin dimers (Mason, 1988). After treatment with gonadotrophins, granulosa–luteal cells express less ß_B subunit (this study), but more subunit (Erämaa et al., 1994), favouring inhibin A, but not inhibin B, formation. Therefore, switching off the ß_B subunit expression by gonadotrophins during follicular maturation may be a key factor in the regulation of the relative secretion of different inhibin and activin forms during the menstrual cycle.

Our results demonstrated that the cAMP-dependent protein kinase A pathway is negatively involved, while staurosporine-sensitive protein kinase may be positively involved in the regulation of the ß_B subunit mRNA expression in cultured human granulosa–luteal cells. Gonadotrophins mediate their effects on human granulosa–luteal cells mainly via increased cAMP production and protein kinase A activation. Inhibition of the ß_B subunit mRNA expression by (Bu)₂cAMP treatment further showed that the regulation pattern of the ß_B subunit expression is different from that of the and ß_A subunits (Tuuri et al., 1996). However, the inhibition of the ß_B subunit mRNA accumulation by cAMP analogues is somewhat surprising, as cAMP has been shown to stimulate ß_B subunit mRNA accumulation in human fetal testicular cells (Erämaa et al., 1992). Besides protein kinase A, staurosporine-sensitive protein kinase seems also to be involved in the regulation of the ß_B subunit mRNA expression. However, the inhibitory effect of staurosporine may be protein kinase C-independent, since the more specific protein kinase C inhibitors Ro 31–8220 and Gö 6976 could not mimic the effect of staurosporine.

Taken together, our results suggest that gonadotrophins are potent inhibitors of the inhibin/activin ß_B subunit mRNA expression in human granulosa–luteal cells, and both cAMP-dependent protein kinase A and staurosporine-sensitive protein kinase pathways are involved in the regulation of the ß_B subunit mRNA accumulation. We suggest that activin-induced and gonadotrophin-inhibited ß_B subunit expression in granulosa cells may be essential in the transition from inhibin B to inhibin A dominance during the menstrual cycle.

	Acknowledgements

Top Abstract Introduction Materials and methods Results Discussion Acknowledgements References

 
Ms Merja Haukka is thanked for her technical assistance. Recombinant human FSH and LH for in-vitro experiments were generously provided by Serono-Nordic (Vantaa, Finland), and activin A peptide by Dr A.F.Parlow, NIDDK's National Hormone and Pituitary Programme, NIH (USA). This study was supported by the Academy of Finland and Kuopio University Hospital Research Contract No. 5107 (to R.V.).

	Notes

 
⁴ To whom correspondence should be addressed at: Department of Pathology, P.O.Box 21, University of Helsinki, FIN-00014 Helsinki, Finland. E-mail: Jiangi.Liu{at}helsinki.fi

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Submitted on September 11, 2000; accepted on January 31, 2001.

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