Inhibin and activin secretion during murine preantral follicle culture and following HCG stimulation

H. Newton1, Y. Wang1, N.P. Groome2 and P. Illingworth1,3

1 Department of Reproductive Medicine, Westmead Hospital, Westmead, Sydney, NSW 2145, Australia and 2 School of Biological Sciences, Oxford Brookes University, Oxford, UK


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
BACKGROUND: Isolation and culture to antral stage of mouse preantral follicles provides an ideal system for investigating the endocrinology of follicular development to maturity. METHODS: The release of inhibin A, inhibin B, pro-alphaC and activin A was measured at specific time-points throughout an in-vitro culture period of 8 days. At the end of culture, follicles were induced to ovulate in vitro by the addition of HCG and the resulting hormone secretion studied both at 20 h and at 6 h intervals. RESULTS: During the preovulatory culture period, the concentrations of inhibin A, B, pro-alphaC and activin A increased significantly. Compared with control, there was a significant decline at 24 h in the concentrations of inhibin A [P < 0.001; 216 ± 47 versus 823 ± 110 arbitrary mouse units (amu)/ml], inhibin B (P < 0.01; 131 ± 23 versus 361 ± 45 amu/ml) and pro-alphaC (P < 0.001; 14 ± 5 versus 1198 ± 212 pg/ml). In contrast, there was a significant increase in the concentration of activin A (P < 0.001; 1.32 ± 0.04 versus 0.34 ± 0.03 ng/ml). CONCLUSIONS: These data provide clear evidence of a profound change in the relative secretion rates of inhibin and activin relative to ovulation and suggest that the principal role of activin A may be at time of ovulation rather than during follicular development.

Key words: activin/follicular development/HCG/inhibin/mouse


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Inhibins are polypeptide hormones secreted by the granulosa cells of the ovary. The molecule is a dimer composed of an {alpha}-subunit linked by disulphide bridges to one of two ß-chains (A or B). In both women (Groome et al., 1996Go; Lockwood et al., 1996Go) and rodents (Woodruff et al., 1996Go; Woodruff, 1998Go) the ovaries secrete two different forms of inhibin (inhibin A and inhibin B) and at least one form of activin (activin A). Along with sex steroids, inhibins are likely to have an endocrine role in the gonadal–pituitary interactions regulating ovulation in both types of animal. In addition, a number of critical actions within the ovary have been described for inhibins and activins that strongly indicate a paracrine role for these substances in follicle growth and development (Hillier and Miro, 1993Go; Woodruff, 1998Go).

Hitherto, information about the relative forms of inhibin/activin being synthesized and secreted have come from a variety of sources: assay of serum at different stages of the cycle (Groome et al., 1996Go; Woodruff et al., 1996Go); assay of follicular fluids from different sized follicles (Groome et al., 1996Go; Magoffin and Jakimiuk, 1997Go, 1998Go; Schneyer et al., 2000Go); immunohistochemistry and in-situ hybridization on fixed ovaries (Schwall et al., 1990Go; Roberts et al., 1993Go, 1994Go); and in-vitro culture of individual cell types (Bicsak et al., 1986Go, 1987Go; Hillier et al., 1991Go). However, none of these approaches is able to define changes in inhibin/activin secretion by a single developing follicle through the different phases of follicle growth, antrum formation and ultimately ovulation.

In-vitro follicle culture is a new strategy that allows mature oocytes to be harvested from immature follicles isolated from ovarian tissue (Hartshorne, 1997Go; Smitz and Cortvrindt, 1999Go). The technique may ultimately be applicable to the preservation of fertility in patients who have cryopreserved ovarian tissue prior to gonadotoxic treatment for malignant disease. In addition to this clinical perspective, in-vitro follicle growth provides a novel system for investigating the endocrinology of the developing follicle, thereby expanding current knowledge of ovarian physiology.

To date, the murine model is one of the most advanced systems for studying follicle development in vitro. Enzymatic isolation of follicles from the ovarian stroma is an efficient method for the collection of many oocyte–cumulus complexes (Eppig and Schroeder, 1989Go) but this strategy damages the basement membrane and thecal layer, therefore development and endocrine function of the growing follicle may not be a true reflection of the events in vivo (Newton et al., 1999Go). Manual isolation with insulin syringes is a more laborious procedure, which yields fewer numbers, but some thecal cells should remain attached to follicles (Qvist et al., 1990Go; Nayudu and Osborn, 1992Go). Isolated follicles can be grown in culture and induced to ovulate by stimulation with HCG/LH (Cortvrindt et al., 1996Go; Rose et al., 1999Go). Fertilization of the mature in-vitro grown oocytes produces blastocysts with a preimplantational developmental competence comparable to in-vivo grown oocytes (Eppig and O'Brien, 1998Go), and the transfer of embryos to pseudopregnant females has resulted in the birth of healthy pups (Eppig and Schroeder, 1989Go; Spears et al., 1994Go).

The aim of this study was to investigate the endocrine function of isolated follicles during culture, specifically how secretion of inhibin A, inhibin B, proalpha C and activin A varied over time. Furthermore the effect of stimulating ovulation in vitro on the release of these molecules was investigated.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Tissue collection
Ovaries from 3 week old Balb/c mice, killed by cervical dislocation, were collected and transferred into sterile Leibovitz medium supplemented with 10% (v/v) heat inactivated fetal calf serum (FCS), 50 U/ml penicillin and 50 µg/ml streptomycin at 37°C; this medium will subsequently be referred to as antibiotic medium. Unless otherwise stated, all media and culture reagents were obtained from Sigma Chemical Co. Ltd, Sydney, NSW.

Follicle isolation
Preantral follicles measuring 100–135 µm in diameter were isolated under sterile conditions using 27 gauge insulin needles (Terumo, Leuven, Belgium) under a dissecting microscope with a heated stage set at 37°C. Any adhering stroma was removed from the follicles which were then washed in two change of antibiotic medium at 37°C. Follicle morphology was briefly assessed under the dissecting microscope and only follicles with an intact round structure and a spherical centrally located oocyte were chosen for the study.

Follicle culture
Culture medium was prepared from {alpha} minimal essential medium (Life Technologies, Melbourne, Victoria, Australia) supplemented with 10% (v/v) FCS, 50 U/ml penicillin, 50 µg/ml streptomycin, 5.5 µg/ml sodium pyruvate, 3 mmol/l L-glutamine, 10 µg/ml transferrin, 5 µg/ml insulin, 5 ng/ml sodium selenite and 90 mIU/ml recombinant FSH (Gonal-F, Serono). All supplements, including the FSH, were added to the culture medium at all stages of the experimental protocols described below.

Isolated follicles were cultured individually in 96-well sterile flat bottomed plates (Falcon, Becton Dickinson Labware, NJ, USA) containing 100 µl of culture medium at 37°C under an atmosphere of 5% CO2 and 95% air. The in-vitro growth period was 8 days, based on observations of the optimal in-vitro growth period for retrieving mature oocytes (H.Newton, unpublished data). Late on day 2 (cultures were seeded on day 1) medium was removed from each well, taking care to leave a small volume over the follicle, and replenished with 100 µl of fresh medium. Twenty hours later, the spent medium was collected and stored at –20°C for measurements of day 3 inhibin pro-alphaC, inhibin A and B and activin. In a similar manner, fresh medium added to the follicles late on day 5 and collected 20 h later was stored for day 6 measurements. Late on day 8 cultured follicles were randomly divided into two groups. One group received 100 µl of fresh medium (control) whilst the second group received 100 µl of fresh medium supplemented with 2.5 IU/ml Chorulon (Intervet Australia, Sydney, NSW, Australia). Twenty hours later, oocyte–cumulus complexes were retrieved from the wells, in minimal medium, using pulled glass pipettes and oocytes were stripped and assessed for maturation. Spent medium from each well was collected and stored at –20°C for measurements of day 9 inhibin pro-alphaC, inhibin A and B, activin and progesterone measurements, either with or without HCG stimulation. Twenty hours was chosen as the incubation period, since oocytes had to be retrieved at this point on day 9 for assessment of maturation. If follicles deteriorated during the culture period medium from the appropriate well was excluded from the study. The experiment was repeated three times on separate days.

In a further experiment, the follicles were cultured as above for 8 days. After 8 days, the follicles were cultured for a further 30 h with medium changes every 6 h. The follicles were randomly divided into two groups. One group received 100 µl of fresh medium every 6 h (control) whilst the second group received 100 µl of fresh medium supplemented with 2.5 IU/ml Chorulon every 6 h.

Inhibin A and B assays
Inhibin A and B were measured according to previously described protocols (Groome et al., 1994Go; Groome et al., 1996Go). A specific mouse standard for both inhibin A and inhibin B was prepared using spent media from fresh murine follicles cultured individually as described above and used to generate standard curves for murine inhibin A and B. This standard exhibited parallelism with serial dilutions of murine testicular extract and serum from superovulated mice (Y.Wang, unpublished data). The mouse standard preparation was calibrated with human inhibin A and B standard samples. The concentration of mouse standard that gave equivalent immunofluorescence to 1000 pg/ml was defined as 1000 arbitrary mouse units per millilitre (amu/ml) (Y.Wang, unpublished data).

The mouse inhibin B standard curve consisted of spent media at 100% and then dilutions to 50, 37.5, 25, 18.8, 12.5, 6.3 and 4.5% in {alpha} minimal essential medium. This gave a concentration curve with values ranging from 1500 to 35 amu/ml. The limit of detection of the assay was 35 amu/ml. All day 3 test samples were assayed at 100% concentration, whilst day 6 and day 9 samples were diluted to 40% in {alpha} minimal essential medium. All samples were measured in two assays; the intra-assay coefficients of variation were 4.2 and 1.4% and the inter-assay coefficient of variation was 2.8%.

The inhibin A standard curve consisted of spent media at dilutions of 75, 37.5, 25, 18.8, 12.5, 4.5, 2.3 and 1.2% in {alpha} minimal essential medium. This gave a concentration curve with values ranging from 3000 to 47 amu/ml. The limit of detection of the assay was 47 amu/ml. All day 3 test samples were assayed at 100% concentration, whilst day 6 and day 9 samples were diluted to 40% in {alpha} minimal essential medium. All samples were measured in two assays; the intra-assay coefficients of variation were 5.7 and 7.0% and the inter-assay coefficient of variation was 6.3%.

Inhibin pro-alphaC assay
Inhibin pro-alphaC was measured using enzyme-linked immunosorbent assay (ELISA) kit assays (Serotec Limited, Oxford, UK). The standard concentration curve ranged in value from 200 to 1.56 pg/ml. The limit of detection of the assay was 3.125 pg/ml. All the samples were diluted to 5% in culture medium and run in a single assay, the intra-assay coefficient of variance was 1.5%.

Activin assay
Activin was measured using an ELISA kit assay (Serotec). The standard concentration curve ranged in value from 5 to 0.078 ng/ml. The limit of detection of the assay was 0.156 ng/ml. All the test samples were used at 100% concentration and measured in a single assay, the intra-assay coefficient of variance was 3.3%.

Progesterone assay
Progesterone was measured by immunofluorescent assay (Immulite; Diagnostic Products Corporation, CA, USA). All test samples were diluted to 10% in assay diluent.

Statistical analysis
In the initial comparison the data for the paired results (control versus HCG) were analysed with the Student's t-test. For the time-course experiment involving medium changes every 6 h, the HCG treatment data were analysed by two-way analysis of variance taking the type of treatment (control versus HCG) as a between-subject variable and the time since culture started as within-subject variable. All values presented are the mean ± SEM.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Many isolated preantral follicles (71 ± 4.3%) survived the period of in-vitro culture and after 8 days had an expanded granulosa cell population with antral cavity like spaces and a visible, morphologically normal oocyte (Newton and Illingworth, 2001Go). The diameter of follicles increased from 115 ± 2.3 µm on day 1 to 389 ± 19 µm on day 8 (P < 0.0001). Follicles that did not survive the culture period were observed to deteriorate after 72 h in culture. The spherical structure appeared to `collapse' and the oocyte was excluded from the follicular unit (Newton and Illingworth, 2001Go). Follicle loss was likely to be due to manual damage caused during the isolation procedure.

Measurements of inhibin pro-alphaC, inhibin A and B released during 20 h periods on day 3, 6 and 9 indicated that increasing concentrations were produced as the culture proceeded. Between day 3 and day 9, inhibin A and B release increased by 18-fold and 6-fold respectively. The ratio of A:B was 1.30 ± 0.2 on day 3 and increased to 3.07 ± 0.3 on day 9 (P < 0.0001, n = 14–16 follicles per group) (Figure 1a,b,cGo). Activin release also increased during culture and on day 9 the concentration was 5-fold higher than on day 3 (P < 0.0001, n = 23) (Figure 1dGo).



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Figure 1. (a) Inhibin A, (b) inhibin B, (c) pro-alphaC and (d) activin release during in-vitro culture of murine preantral follicles grown in serum supplemented medium for 8 days. Each time point represents production during a 20 h period; n = 14–23 follicles per group. The release of each increased significantly during cultureP < 0.0001.

 
On day 8 of culture, medium was supplemented with HCG to stimulate ovulation. Twenty hours later, the medium was collected and inhibin and activin concentrations were compared to the control unstimulated medium. Following HCG stimulation, inhibin A and B concentrations were 1220 ± 121 and 348 ± 37 amu/ml respectively, which was significantly lower than in the control group (2521 ± 307 amu/ml and 703 ± 67 amu/ml respectively) (P < 0.0001, n = 24–28 follicles per group, Figure 2a,bGo). There was no difference between the ratio of A:B in the stimulated or the control group (3.05 ± 0.17 versus 3.07 ± 0.32 respectively, P = 0.96). In a similar manner, pro-alphaC release was significantly higher in the spent medium from the control (2469 ± 204 pg/ml) than the stimulated group (632 ± 59 pg/ml) (P < 0.0001, n = 20 follicles per group, Figure 2cGo). In contrast, activin concentrations in the control group were 640 ± 44 pg/ml and after 20 h of HCG stimulation the level rose to 1141 ± 46 pg/ml (P < 0.0001, n = 37 follicles per group, Figure 2dGo).



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Figure 2. (a) Inhibin A, (b) inhibin B, (c) pro-alphaC and (d) activin release on day 9 from preantral follicles grown in vitro for 8 days and either stimulated (+HCG) or not stimulated (–HCG) for 20 h with HCG; n = 20–37 follicles per group, *P < 0.001.

 
The results of the 6 h experiment are shown in Figure 3Go. During the initial 6 h period of culture, there was significant secretion of inhibin A, inhibin B, pro-alphaC and activin A. Following addition of HCG, the inhibin A concentration fell from 822.7 ± 110 amu/ml at baseline to 216 ± 46.6 amu/ml at 24 h (effect of HCG, P < 0.0001; effect of time P < 0.05; interaction, P < 0.001: n = 16) and the inhibin B concentration fell from 360.6 ± 45 amu/ml at baseline to 131 ± 23 amu/ml at 24 h (effect of HCG, P < 0.0001; effect of time, P < 0.05; interaction, P < 0.01; n = 16). The most marked fall was noted in the concentration of pro-alphaC, which declined dramatically from 1198 ± 212 pg/ml at baseline to 14.1 ± 5.0 pg/ml at 24 h (effect of HCG, P < 0.0001; effect of time, P < 0.01; interaction, P < 0.001; n = 16). In stark contrast to the results for inhibins and pro-alphaC, the secretion of activin A rose during the 24 h culture period from a baseline concentration of 0.34 ± 0.03 ng/ml to a peak of 1.32 ± 0.037 ng/ml at 24 h (effect of HCG, P < 0.0001; effect of time, P < 0.0001; interaction, P < 0.0001; n = 16).



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Figure 3. (a) Inhibin A, (b) inhibin B, (c) pro-alphaC and (d) activin release on day 9 from preantral follicles grown in vitro for 8 days and either stimulated (+HCG) or not stimulated (–HCG) for 30 h with HCG. n = 16 follicles per group. Following F-test for interaction between HCG and time the probability of the null hypothesis being true is as shown: *P < 0.01; **P < 0.001;***P < 0.0001.

 
The maturation rate of oocytes released from the follicles that received HCG stimulation was 73 ± 13%. Follicles that were not stimulated with HCG did not ovulate. The functional capacity of the HCG was further supported by progesterone concentrations measured in medium collected from follicles on day 9 following HCG stimulation (151 ± 16 ng/ml) or after no HCG stimulation (9 ± 2 ng/ml) (P < 0.0001, n = 11 follicles per group).


    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
This study has examined the endocrine function of isolated preantral follicles during in-vitro growth in serum-supplemented culture. The effect of inducing ovulation by the addition of HCG to the medium was also investigated. In agreement with previous observations of preantral mouse follicle culture, the results suggest that inhibin A and B are secreted in similar quantities throughout development (Smitz and Cortvrindt, 1998Go). These data are therefore consistent with in-situ hybridization studies on human ovaries that demonstrate a low level of expression of ßA-subunit even in small follicles, although the expression is maximal in large dominant follicles (Schwall et al., 1990Go; Roberts et al., 1993Go). Despite the pattern of mRNA expression, previous studies of human follicular fluid had found a predominance of the B form of dimeric inhibin protein in small follicles (Groome et al., 1996Go; Magoffin and Jakimiuk, 1997Go; Schneyer et al., 2000Go). Whether the finding in this paper represents a species difference will not be clear until human preantral follicle culture can be achieved to the same extent.

As murine follicles developed in culture, inhibin A production began to predominate over inhibin B. This is consistent with previous observations in a variety of experimental systems. Cortvrindt and Smitz found that culturing intact murine follicles resulted in an increase in inhibin A secretion at the same stage (Smitz and Cortvrindt, 1998Go), and in human studies circulating concentrations of the A form of inhibin have been found to increase in the late follicular phase (Groome et al., 1994Go). Furthermore, studies of follicular fluid have revealed that inhibin A concentrations increase with increasing follicular size (Magoffin and Jakimiuk, 1997Go; Schneyer et al., 2000Go) and the expression of ßA-subunit is greater in larger follicles (Roberts et al., 1993Go). The cellular mechanism for the differential control of inhibin A and inhibin B secretion is unknown as is the physiological significance of this effect.

The most striking observations in this study are the dramatic changes in the relative concentrations of inhibin and activin following administration of HCG. A dramatic fall in the concentration of inhibin A, inhibin B and pro-alphaC was observed after the addition of HCG. In contrast the concentration of activin A increased significantly. No specific inhibin receptor has yet been identified and a consensus is emerging that inhibin primarily acts by antagonising the actions of activin (Lewis et al., 2000Go). When one thus considers the changes in a ratio of the relative concentrations of inhibin and activin during follicular development (Figure 4Go), a clear pattern emerges. During follicular development, the actions of activin are likely to be limited by the relative excess of inhibin forms present. However, following ovulation, the relative ratio is reversed, leading to an excess of activin A being available for biological actions related to the processes of ovulation such as oocyte maturation. The marked fall in pro-alphaC secretion is particularly noteworthy. This fall, almost to nothing, raises the possibility that it is a fall in {alpha}-subunit synthesis rather than an increase in ßA synthesis that is changing the balance of secretion from primarily the {alpha}/ß heterodimer inhibin to primarily the ß/ß homodimer activin. In order preferentially to secrete the heterodimer inhibin rather than the homodimer activin, the inhibin {alpha} subunit is required to be expressed in the cell in considerable excess to the inhibin ß-subunit. (Mason et al., 1987Go). These data thus suggest that the principal function of the excess (relative to ß) {alpha}-subunit secretion in the ovarian follicle may be to promote secretion of the biologically suppressive inhibin to inhibit the actions of the potentially much more active substance, activin.



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Figure 4. The ratio between activin A and total dimeric (inhibin A + inhibin B) concentration in medium of cultured follicles according to the stage of the follicle (based on the data presented in this publication).

 
To what extent do these data represent a purely mouse phenomenon, particularly as the mouse corpus luteum (unlike the human corpus luteum) does not secrete inhibin? There are some indirect data to suggest that a similar process is possible in human ovulation. Most of the available data on human follicular fluid relate to the follicle prior to ovulation. In contrast, there are few data on human follicular fluid inhibin/activin concentrations in the periovulatory period. One study examined samples of follicular fluid collected at different time intervals relative to the onset of the LH peak (Lenton et al., 1988Go). The ({alpha}-subunit) inhibin concentration, as measured by the {alpha}-subunit-based Monash 1968 assay, was found to decline quite markedly following the mid-cycle LH peak. A more recent study reported higher follicular fluid activin concentrations in older women than younger women and speculated that this may be because the follicles from older women were collected closer to ovulation (Klein et al., 2000Go). The activin concentration in post-HCG follicular fluid from women undergoing IVF (Fujiwara et al., 2000Go) was found to be 4-fold higher than pre-ovulatory follicular fluid measurements of the same group (Schneyer et al., 2000Go) while the inhibin B concentrations were significantly lower in the post-HCG follicular fluid (Fujiwara et al., 2000Go) than in the pre-ovulatory follicle (Schneyer et al., 2000Go). Clearly, however, these latter data may simply be a result of the excessive FSH stimulation of the IVF treatment regime. However, the limited available data on human follicular fluid are not inconsistent with the possibility of changes in the inhibin/activin ratio relative to ovulation, and further research is warranted.

In this context, there is one action of activin A that is critical: the action on the oocyte. The oocyte contains abundant activin receptors (Manova et al., 1995Go), and in both human and mouse, activin A has consistently been observed to promote oocyte maturation (Alak et al., 1998Go; Sidis et al., 1998Go; Smitz et al., 1998Go). Enhanced local synthesis of activin is thus likely to be a critical mediator of the effect of HCG/LH on oocyte maturation. We postulate that the action of HCG in the follicle is to reduce synthesis of {alpha}-subunit, thereby favouring secretion of ß-subunit dimers that act on the oocyte to promote oocyte maturation. This hypothesis awaits further testing. In particular, little is known about the concentration of the activin-binding proteins follistatins in follicular fluid around the time of the physiological LH surge.

In conclusion, these findings suggest that, in the mouse follicle at least, the principal paracrine role of activin A in the follicle may be in association with ovulation rather than with follicular selection and development. Similar studies of human follicular function are necessary to investigate the relevance of these findings to human ovulation.


    Notes
 
3 To whom correspondence should be addressed at: Department of Reproductive Medicine, Westmead Hospital, Westmead, NSW 2119, Australia. E-mail: peteri{at}westgate.wh.usyd.edu.au Back


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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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Submitted on October 13, 2000; accepted on September 15, 2001.