Dimeric inhibins and activin A in human follicular fluid and oocyte–cumulus culture medium

C.P. Lau1, W.L. Ledger1, N.P. Groome2, D.H. Barlow1 and S. Muttukrishna1,3

1 Nuffield Department of Obstetrics and Gynaecology, University of Oxford, John Radcliffe Hospital and 2 School of Biological and Molecular Sciences, Oxford Brookes University, Oxford, UK


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The concentrations of inhibin A, inhibin B and activin A in follicular fluid and oocyte culture medium were analysed to investigate the production of these peptide hormones by ovarian granulosa cells and oocyte–cumulus complexes, as well as their potential as possible biochemical markers for oocyte quality and fertilizing capacity. Follicular fluids were collected from individual follicles during oocyte retrieval for in-vitro fertilization (IVF). Oocyte–cumulus culture media were collected after in-vitro insemination. The concentrations of dimeric inhibin A, inhibin B and activin A were measured using two-site enzyme-linked immunosorbent assays in the follicular fluid and matched oocyte culture medium. Hormone concentrations were compared with oocyte quality and fertilizing capacity. The concentration of inhibin A in follicular fluid increased while that of inhibin B decreased with increasing follicle size. Follicular fluid concentrations of inhibin A inhibin B and activin A were not significantly different in follicles with differing oocyte quality. Oocyte culture medium concentrations of activin A were significantly higher in morphologically good quality oocytes. There was no relationship between the concentrations of the three hormones and oocyte fertilizing capacity. This study confirms that follicular fluid concentrations of inhibin A may prove to be a marker of follicular growth and maturation. Higher concentrations of activin A produced by good quality oocyte–cumulus complexes suggest that activin A may play a role in oocyte maturation.

Key words: activin A/follicular fluids/inhibin A/inhibin B/oocytes


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Inhibins are dimeric glycoprotein hormones with dissimilar subunits, mainly secreted in women by the ovaries. They consist of {alpha} and ß subunits, linked by disulphide bonds. There are two forms of inhibins (A and B) consisting of a common {alpha} subunit and distinguishable ß subunit. Inhibin A consists of {alpha}A subunits and inhibin B consists of {alpha}B subunits. The ovary also produces the monomeric {alpha} subunit in large quantities. Activins are dimers of the ß subunits linked by disulphide bridges. Activin A is a ßAA dimer, activin AB is a ßAB dimer and activin B is a ßBB dimer. Gonadotrophins and sex steroids regulate granulosa cell secretion of immunoreactive inhibin by rat (Erickson and Hsueh, 1978Go; Suzuki et al., 1987Go; Biscak et al., 1988), bovine (Henderson and Franchimont, 1983Go), human (Hillier et al., 1991aGo) and non-human primate (Hillier et al., 1989Go), as well as bioactive dimeric inhibins and activin A by human (Muttukrishna et al., 1997Go) granulosa–luteal cells in vitro. There is also evidence suggesting that the secretion of both inhibins (A and B) and activin A are regulated by follicle-stimulating hormone (FSH) and human chorionic gonadotrophin (HCG) in vivo (Lockwood et al., 1996Go).

Serum inhibin A concentrations peak prior to the pre-ovulatory luteinizing hormone (LH) surge and in the mid-luteal phase suggesting that inhibin A is a product of the mature dominant follicle and the corpus luteum (Groome et al., 1994; Muttukrishna et al., 1994Go). Inhibin B concentrations peak in early follicular phase (day 4/day 5) and then remain low throughout the rest of the cycle suggesting it is mainly a product of pre-antral follicles (Groome et al., 1996). During the menstrual cycle, mean serum activin A concentrations vary in a biphasic manner, with highest concentrations around mid-cycle and the late luteal/early follicular phases and nadirs in both mid-follicular and mid-luteal phases (Muttukrishna et al., 1996Go).

Classically, inhibin has an endocrine role by inhibiting pituitary production of FSH. Although activin was also initially purified as a substance that increases pituitary FSH secretion, there is increasing evidence for diversified biological activity of activin in various tissues (Vale et al., 1994Go). In-vitro studies show that both inhibin and activin can act directly to modulate androgen synthesis in the theca interna (Hsueh et al., 1987Go). Treatment of cultured human theca cells with recombinant activin A potently inhibits stimulation of androgen production by LH and insulin-like growth factor 1 (IGF-1) (Hillier et al., 1991bGo). Conversely, picomolar amounts of recombinant inhibin markedly augment LH/IGF-stimulated androgen production. Potent augmentation by activin of FSH-inducible aromatase activity has been convincingly demonstrated in granulosa cells in vitro in the rat (Hutchison et al., 1987Go; Miro et al., 1991Go) and in the non-human primate (Miro and Hillier, 1992Go).

Studies in cattle suggest that inhibin A and activin A may play important roles during the final stages of oogenesis and that addition of recombinant inhibins and activins improves the performance of serum-free culture medium for in-vitro maturation of oocytes from cattle and possibly other mammalian species (Stock et al., 1997Go). Recently, follicular fluid concentrations of inhibin A, inhibin B and activin A from individual follicles from regularly cycling women have been measured using specific two-site enzyme-linked immunosorbent assays (Magoffin and Jakimiuk, 1997Go). This study reported a linear relationship between follicular fluid concentrations of inhibin A and follicle size and maturation. A similar study has also been reported using a mouse follicle culture system (Smitz and Cortvrindt, 1998Go). The aim of the present study was to investigate the association between follicular fluid and oocyte–cumulus complex culture medium concentrations of inhibin A, inhibin B and activin A and the quality and the fertilizing capacity of the oocyte. The relationship between the concentrations of these hormones in the follicular fluid and oocyte culture medium, and the quality, fertilizing capacity of the oocyte and follicle size were studied.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Sample collection
Follicular fluid (FF) and matched oocyte cumulus culture medium samples from 20 women undergoing in-vitro fertilization (IVF) treatment (unexplained or tubal factor infertility) were collected. First follicular aspirates before media flushing of the follicle were collected and the volume of FF was also recorded. Any follicle aspirates that were not clear and contaminated with blood were discarded. Oocyte–cumulus complexes were cultured in sterile Petri dishes containing 2 ml culture medium [Tyrode's 6 prepared in house according to Gardner and Lane (1993)] as part of the IVF procedure. Individual follicular fluids and matched oocyte culture media were centrifuged at 600 g for 15 min and the supernatant was stored at –20°C for hormone assays. The mean age of the subjects was 37 ± 4 years. Oocyte culture medium was collected after 16–18 h of insemination. The semen, in all 20 cases, was assessed to be of good quality. Sperm quality was assessed based on the WHO criteria (WHO, 1992) for semen analyses. Briefly, the volume was >=2 ml with a sperm concentration >=20x106/ml. Motility is defined as: (a) rapid (>=25 µm/s), (b) slow (<25 µm/s but still progressive), (c) motile but non-progressive and (d) immotile. The motility was either >=25% type (a) or >=40% progressive [= (a)+(b)] or >=50% motile [= (a)+(b)+(c)]. The morphology was assessed using a strict criteria, >=15% normal forms 5–14% borderline spermatozoa and <5% poor quality spermatozoa.

Hormone assays
Cross-reaction of the culture medium was checked in each assay. The absorbance values of the culture medium alone were similar to the diluent blanks in all three enzyme-linked immunosorbent assays (ELISA).

Inhibin A
All follicular fluid samples were diluted 100 times in Tris HCl + 10% bovine serum albumin (BSA) buffer. Concentrations of inhibin A in the follicular fluid and oocyte culture medium were assayed in duplicate using a specific ELISA reported elsewhere (Muttukrishna et al., 1997Go). The minimum detection limit of the inhibin assay was 2 pg/ml. The inter- and intraplate variations for the inhibin A ELISA were <10%.

Inhibin B
All follicular fluid samples were diluted 100 times in sample buffer. Follicular fluids and oocyte culture media were assayed in duplicate for inhibin B using a specific ELISA (Muttukrishna et al., 1997Go) with some modifications. The sample preparations involved a single oxidation step with no SDS treatment. The sensitivity of the assay was 6 pg/ml, and the mean intra- and interplate variations were <10% respectively.

`Total' activin A
All follicular fluid samples were diluted 10 times in phosphate-buffered saline (PBS) + 1% BSA buffer. Follicular fluids and oocyte culture media were assayed in duplicate for `total' activin A using the recently reported activin A ELISA (Muttukrishna et al., 1997Go). The sensitivity of the assay was 50 pg/ml, and the mean intra- and interplate variations were <12%.

Data obtained from patients' files and classification of oocyte
Information on follicular size, matching oocyte quality and fertilizing capacity (fertilized or unfertilized) was obtained from patients' case records. Follicular diameter assessed by transvaginal ultrasound was recorded in mm (average of three perpendicular planes was taken) and aspirated FF volume was noted in ml. Oocytes were graded by the clinical embryologists according to the amount of cumulus and maturity as reported elsewhere (Saith et al., 1998Go). Briefly, Grade 1 describes oocytes with a fully mature complex, normal amount of cumulus, and slightly dark corona and cumulus. Grade 2 describes oocytes with a fully mature complex, normal amount of cumulus, and signs of post-mature cumulus. Grade 3 describes oocytes with a borderline fully mature complex, normal amount of cumulus, and dark corona or cumulus. Grades 4 and 5 describe immature oocytes. In general, grade 1 is considered to be of good quality, grades 2 and 3 are average qualities, and grades 4 and 5 are unlikely to fertilize. The limitations of this oocyte quality grading system in terms of repeatability and relationship to oocyte maturity were recognized but, as it was the method used in our IVF unit, we had no alternative option.

Statistical analysis
Hormone concentrations in follicular fluid and oocyte culture media were compared between different oocyte qualities and fertilizing capacity. Hormone measurements did not vary significantly from normal distribution (normality tests, Prism statistical software; GraphPad Inc., San Diego, CA, USA) and parametric methods were used for analysis. One-way analysis of variance were carried out with Bonferroni–Dunn post hoc tests to compare the concentrations of hormones in FF and culture medium and different grades of oocytes.

The relationships between the concentrations of inhibin A, inhibin B and activin A in follicular fluids and oocyte culture media and three parameters, follicular size, oocyte grade and fertilizing capacity, were determined by Pearson correlation analysis using the statistics package for social sciences (SPSS) software programme. Mean concentrations of hormones between the fertilized and non fertilized groups were compared by the unpaired Student's t-test using the same programme.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Follicular fluid volume was positively correlated with follicle diameter (r = 0.9, P < 0.001). Larger follicles produced higher concentrations of inhibin A and relatively low concentrations of inhibin B. The inhibin A content of the follicular fluid was positively correlated with the size of the follicle (r = 0.4, P < 0.001), whereas the inhibin B content of the follicular fluid was negatively correlated with the size of the follicle (r = –0.33, P < 0.01). Follicle size was also significantly correlated to oocyte–cumulus culture medium concentrations of inhibin A (r = 0.3, P = 0.03) and inhibin B (r = –0.3, P < 0.01). Oocyte quality was found to be related to oocyte fertilizing capacity (r = 0.3, P < 0.01).

Inhibin A and inhibin B
High concentrations of inhibin A and B (~40–50 ng/ml) were present in the FF. Oocyte–cumulus complex also produced measurable amounts of inhibin A and B in culture. Mean inhibin A concentrations in FF were not different in follicles yielding different grades of oocytes (Figure 1aGo). Inhibin B concentrations in the FF were significantly higher in grade 1 oocyte-containing follicles compared with grade 2 oocyte-containing follicles (Figure 1bGo). Oocyte culture medium concentrations of inhibin A and B did not vary with different grades of oocytes (Figure 2Go). Inhibin B concentrations in the oocyte–cumulus complex media were negatively correlated with fertilizing capacity of the oocytes (r = –0.3, P < 0.01). There were no significant correlations between the concentrations of inhibin A and B in both FF (Figure 3Go) and oocyte culture medium (Figure 4Go) with oocyte fertilizing capacity.



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Figure 1. Individual follicular fluid concentrations of (a) inhibin A, (b) inhibin B, (c) activin A in follicles containing different grades of oocyte. The horizontal line shows the mean value [*P < 0.05; one way analysis of variance (ANOVA)].

 


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Figure 2. Relationship between oocyte quality and oocyte culture medium concentrations of (a) inhibin A, (b) inhibin B, (c) activin A. The horizontal line shows the mean value (*P < 0.05; ANOVA).

 


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Figure 3. Relationship between oocyte fertilizing capacity and follicular fluid concentrations of (a) inhibin A, (b) inhibin B, (c) activin A. The horizontal line shows the mean value.

 


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Figure 4. Relationship between oocyte fertilizing capacity and oocyte culture medium concentrations of (a) inhibin A, (b) inhibin B, (c) activin A. The horizontal line shows the mean value.

 
Total activin A
The concentrations of `total' activin A in the follicular fluid appeared to be lower in follicles containing decreased oocyte quality, although this was not significant (Figure 1cGo). The concentrations of activin A in the oocyte culture medium (Figure 2cGo) was also significantly correlated with the quality of the cumulus–oocyte complex (r = 0.31, P < 0.05). The concentration of activin A in the oocyte culture medium of grade 1 oocytes (875.5 ± 101 pg/ml) was significantly higher (P < 0.05) than that of grade 3 oocytes (539 ± 107.2 pg/ml). However, the concentrations of activin A in both the follicular fluid (Figure 3cGo) and oocyte culture medium (Figure 4cGo) were not significantly correlated to oocyte fertilizing capacity.


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Dimeric inhibins and activin are well known for their actions on pituitary gonadotrophin secretion and intra-ovarian steroidogenesis. Although inhibins and activin are suggested to have an autocrine/paracrine effect on mouse oocyte maturation and embryo development, their actions on human oocyte development and embryogenesis have not been documented. This study is the first attempt to explore the relationships between inhibin A, inhibin B, activin A and the quality and fertilizing capacity of the human oocyte carried out on women undergoing assisted conception. All three hormones are secreted by granulosa cells and hence are all present in the follicular fluid. By measuring the concentrations of these peptide hormones in the follicular fluid using highly sensitive two-site enzyme-linked immunosorbent assays and correlating them with estimates of quality and maturity of the oocyte, their possible significance on the maturation and quality of oocyte was studied. When an oocyte is transferred into culture medium during the in-vitro fertilization procedure, the oocyte–cumulus complex continues to secrete inhibins and activins. Therefore, the concentrations of these hormones in the oocyte culture medium were measured to investigate the relationship between oocyte quality and the potential of cumulus cells to secrete inhibins and activins in culture. Apart from oocyte quality, the concentrations of inhibins and activins were also compared with other parameters such as follicle size and oocyte fertilizing capacity.

In this study, the concentration of inhibin A in the follicular fluid increases with follicle size and FF volume, whereas the concentration of inhibin B in the follicular fluid decreases when follicle size and FF volume increases. This is consistent with a previous report showing that smaller follicles express more ßB subunit mRNA (Hillier, 1991c) and would appear to agree with the changes in concentrations of inhibin A and B in serum seen as the pre-ovular dominant follicle matures (Muttukrishna et al., 1994Go; Groome et al., 1996). Magoffin and Jakimiuk (1997) in their natural cycle study found a significant increase in follicular fluid inhibin B with increasing follicle size (4–14 mm) and a decrease in inhibin B in follicles >14 mm diameter. Our patients all underwent gonadotrophin-releasing hormone (GnRH) agonist and gonadotrophin treatment as part of their IVF cycle, with possible effects on intrafollicular autocrine/paracrine events, so the studies are not directly comparable. However, almost all follicles in the IVF patients in this study were >14 mm diameter and the relationship between inhibin B and follicle size is consistent with the natural follicle data in the above reported study. The relationship between follicle size and the amount of inhibin A (r = 0.28, P = 0.03) and inhibin B (r = –0.3, P < 0.01) in the oocyte–cumulus complex culture medium is consistent with the relationship between FF concentrations of inhibin A (r = 0.4, P < 0.001) and inhibin B (r = –0.33, P = 0.02) with follicle size.

The concentrations of activin A in the oocyte culture medium appeared to be higher in the case of good maturity of cumulus–oocyte complexes, while no relationship was found with inhibin A, inhibin B and activin A in the FF. There was an increase in activin A concentration in oocyte culture medium (60%) in the case of good quality cumulus–oocyte complexes suggesting that activin A may be involved in the maturation of the oocyte. The measurement carried out in this study is `total' activin A (follistatin bound activin A + `free' activin A) and total follistatin concentrations have to be measured in the future in similar studies to evaluate the availability of `free' activin A that is biologically active.

It has been demonstrated (Dyson and Gurdon, 1997Go) that activin signalling is necessary in the early embryonic development of Xenopus. Stock et al. (1997) have demonstrated that both inhibin A and activin A play important roles during the final stages of oogenesis in cattle. Inhibin A and inhibin B were not found to be associated with oocyte quality in this study, possibly identifying a difference between species. Activin receptor mRNA (ActRII) has been shown to be strongly expressed by rat oocytes (Sadatsuki et al., 1993Go; Cameron et al., 1994Go) and human oocytes (Sidis et al., 1998Go), supporting a putative action of activin on this cell type. As activins are structurally related to the transforming growth factor ß peptide family, it is not surprising to find that activins have growth and differentiation promoting effects on oocytes. Furthermore, Alak et al. (1998) have shown that activin A stimulates meiotic maturation of human oocytes in vitro. This study also indirectly supports the hypothesis that increased concentrations of activin A in the oocyte–cumulus culture medium may promote maturation of the oocyte. However, further studies have to be carried out with larger numbers to confirm that activin A promotes human oocyte maturity and/or embryo development.

The lack of correlation between the follicular fluid concentrations of inhibin A, inhibin B and activin A and the chance of fertilization (Figure 3cGo) suggests that these factors do not influence the chance of fertilization. In this study only cases with excellent sperm parameters were included to minimize one variable but it is not possible to control for all extrinsic factors and/or for the intrinsic genetic potential of the oocyte.

The availability of human recombinant inhibins and activins would allow further in-vitro studies to investigate their effect(s) on oocyte development. Activin receptor expression on human oocytes can also be studied to determine the mechanisms controlling the responsiveness of human oocytes to these intrafollicular proteins.

In summary, this study confirms previous reports on inhibin A and inhibin B as markers of follicle maturation. Complete overlap in data between FF and oocyte–cumulus culture medium concentrations of inhibin A, inhibin B and activin A and the quality of oocytes suggests that these hormones could not be biochemical predictors of oocyte quality. Future in-vitro studies are needed to assess the ability of activin A in promoting human oocyte maturation that could be applicable in IVF laboratories.


    Acknowledgments
 
The authors thank the embryologists (Oxford Fertility Unit) for their help with follicular fluid and oocyte culture medium collection and oocyte morphological assessment details. We thank the National Institute for Biological Standards and Controls (Potters Bar, UK) for the human recombinant inhibin A standard and Genentech Inc. (San Francisco, CA, USA) for the human recombinant activin A standard used in the experiments.

S. Muttukrishna is funded by the Wellcome trust. This research was supported by the Oxford Fertility Unit, John Radcliffe Hospital, Oxford, UK.


    Notes
 
3 To whom correspondence should be addressed Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Alak, B.M., Coskun, S., Friedman, C.I. et al. (1998) Activin A stimulates meiotic maturation of human oocytes and modulates granulosa cell steroidogenesis in vitro. Fertil. Steril., 70, 1126–1130.[ISI][Medline]

Bicsak, T., Cajander, S.B., Vale, W. et al. (1988) Inhibin: studies of stored and secreted forms of biosynthetic labelling and immunodetection in cultured rat granulosa cells. Endocrinology, 122, 741–748.[Abstract]

Cameron, V., Nishimura, E., Mathews, L. et al. (1994) Hybridization histochemical localization of activin receptor subtypes in rat brain, pituitary, ovary and testis. Endocrinology, 134, 799–808.[Abstract]

Dyson, S. and Gurdon, J.B. (1997) Activin signalling has a necessary function in Xenopus early development. Curr. Biol., 7, 81–84.[ISI][Medline]

Erickson, G.F. and Hsueh, A.J.W. (1978) Secretion of inhibin by rat granulosa cells in vitro. Endocrinology, 103, 1960–1963.[Abstract]

Gardner, D.K. and Lane, M. (1993) Embryo culture systems. In Handbook of In Vitro Fertilization. CRC Press, Boca Raton.

Groome, N.P., Illingworth, P.J., O'Brien, M. et al. (1994a) Detection of dimeric inhibin throughout the human menstrual cycle by two-site enzyme immunoassay. Clin. Endocrinol., 4, 717–723.

Groome, N.P., Illingworth, P.J., O'Brien, M. et al. (1994b) Measurement of dimeric inhibin-B throughout the human menstrual cycle. J. Clin. Endocrinol. Metabol., 81, 1404–1405.

Henderson, K.M. and Franchimont, P. (1983) Inhibin production by bovine ovarian tissues in vitro and its regulation by androgens. J. Reprod. Fertil., 67, 291–298.[Abstract]

Hillier, S.G. (1991) Regulatory functions for inhibin and activin in human ovaries. J. Endocrinol., 131, 171–175.[ISI][Medline]

Hillier, S.G., Wickings, E.J., Saunders, P.T.K. et al. (1989) Hormonal control of inhibin production by primate granulosa cells. J. Endocrinol., 123, 65–73.[Abstract]

Hillier, S.G., Wickings, E.J., Illingworth, P.I. et al. (1991a) Control of immunoactive inhibin production by human granulosa cells. Clin. Endocrinol., 35, 75–78.

Hillier, S.G., Yong, E.L., Illingworth, P.I. et al. (1991b) Effect of recombinant activin on androgen synthesis in cultured human thecal cells. J. Clin. Endocrinol. Metab., 72, 1206–1211.[Abstract]

Hsueh, A.J.W., Dahl, K.D., Vaughan, J. et al. (1987) Heterodimers and homodimers of inhibin subunits have different paracrine action in modulation of luteinizing hormone-stimulated androgen synthesis. Proc. Natl Acad. Sci. USA, 84, 5082–5086.[Abstract]

Hutchison, L.A., Findlay, J.K., De Vos, F.L. et al. (1987) Effects of bovine inhibin, transforming growth factor-ß and bovine activin A on granulosa cell differentiation. Biochem. Biophys. Res. Commun., 146, 1405–1412.[ISI][Medline]

Lockwood, G.M., Muttukrishna, S., Groome, N.P. et al. (1996) Circulating inhibins and activin A during GnRH-analogue down-regulation and ovarian hyperstimulation with recombinant FSH for in vitro fertilization and embryo transfer. Clin. Endocrinol., 45, 741–748.[ISI][Medline]

Magoffin, D.A. and Jakimiuk, A.J. (1997) Inhibin A, inhibin B and activin A in the follicular fluid of regularly cycling women. Hum. Reprod., 12, 1714–1719.[Abstract]

Miro, F., Smyth, C.D. and Hillier, S.G. (1991) Development-related effects of recombinant activin on steroid synthesis in rat granulosa cells. Endocrinology, 129, 3388–3394.[Abstract]

Miro, F. and Hillier, S.G. (1992) Relative effects of activin and inhibin on steroid hormone synthesis in primate granulosa cells. J. Clin. Endocrinol. Metab., 75, 1556–1561.[Abstract]

Muttukrishna, S., Fowler, P.A., Groome, N.P. et al. (1994) Serum concentrations of dimeric inhibin during the spontaneous human menstrual cycle and after treatment with exogenous gonadotrophin. Hum. Reprod., 9, 1634–1642.[Abstract]

Muttukrishna, S., Fowler, P.A., George, L. et al. (1996) Changes in peripheral serum concentrations of total activin A during the human menstrual cycle and pregnancy. J. Clin. Endocrinol. Metab., 81, 3328–3334.[Abstract]

Muttukrishna, S., Groome, N. and Ledger, W. (1997) Gonadotropic control of secretion of dimeric inhibins and activin A by human granulosa-luteal cells in vitro. J. Assist. Reprod. Genet., 14, 566–574.[ISI][Medline]

Sadatsuki, M., Tsutsumi, O., Yamada, R. et al. (1993) Local regulatory effects of activin A and follistatin on meiotic maturation of rat oocyte. Biochem. Biophys. Res. Commun., 196, 388–395.[ISI][Medline]

Saith, R.R., Srinivasan, A., Michie, D. and Sargent I.L. (1998) Relationships between the developmental potential of human in-vitro fertilization embryos and features describing the embryo, oocyte and follicle. Hum. Reprod. Update, 4, 121–134.[Abstract/Free Full Text]

Sidis, Y., Fujiwara, T., Leykin, L. et al. (1998) Characterization of inhibin/activin subunit, activin receptor, and follistatin messenger ribonucleic acid in human mouse oocytes: evidence for activin's paracrine signalling from granulosa cells to oocytes. Biol. Reprod., 59, 807–812.[Abstract/Free Full Text]

Smitz, J. and Cortvrindt, R. (1998) Inhibin A and B secretion in mouse preantral follicle culture. Hum. Reprod., 13, 927–935.[Abstract]

Stock, A.E., Woodruff, T.K. and Smith, L.C. (1997) Effects of inhibin A and activin A during in vitro maturation of bovine oocytes in hormone- and serum-free medium. Biol. Reprod., 56, 1559–1564.[Abstract]

Suzuki, T., Miyamoto, K., Hasegawa, Y. et al. (1987) Regulation of inhibin production by rat granulosa cells. Mol. Cell. Endocrinol., 54, 185–195.[ISI][Medline]

Vale, W., River, C. and Vaughan, J. (1994) The role of inhibin and activin. In Knobil, E. and Neill, J.D. (eds) The Physiology of Reproduction. Raven Press, New York, pp. 1861.

World Health Organization (1992) WHO Laboratory Manual for the Examination of Human Semen and Sperm–Cervical Mucus Interaction, 3rd edn. WHO, Geneva.

Submitted on January 20, 1999; accepted on June 21, 1999.