1 Department of Reproductive Medicine, Westmead Hospital, Westmead, Sydney, NSW 2145, Australia and 2 School of Biological Sciences, Oxford Brookes University, Oxford, UK
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Abstract |
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Key words: activin/follicular development/HCG/inhibin/mouse
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Introduction |
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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., 1996; Woodruff et al., 1996
); assay of follicular fluids from different sized follicles (Groome et al., 1996
; Magoffin and Jakimiuk, 1997
, 1998
; Schneyer et al., 2000
); immunohistochemistry and in-situ hybridization on fixed ovaries (Schwall et al., 1990
; Roberts et al., 1993
, 1994
); and in-vitro culture of individual cell types (Bicsak et al., 1986
, 1987
; Hillier et al., 1991
). 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, 1997; Smitz and Cortvrindt, 1999
). 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 oocytecumulus complexes (Eppig and Schroeder, 1989) 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., 1999
). 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., 1990
; Nayudu and Osborn, 1992
). Isolated follicles can be grown in culture and induced to ovulate by stimulation with HCG/LH (Cortvrindt et al., 1996
; Rose et al., 1999
). 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, 1998
), and the transfer of embryos to pseudopregnant females has resulted in the birth of healthy pups (Eppig and Schroeder, 1989
; Spears et al., 1994
).
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.
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Materials and methods |
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Follicle isolation
Preantral follicles measuring 100135 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 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, oocytecumulus 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., 1994; Groome et al., 1996
). 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 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
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 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
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.
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Results |
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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 = 1416 follicles per group) (Figure 1a,b,c). 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 1d
).
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Discussion |
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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, 1998), 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., 1994
). Furthermore, studies of follicular fluid have revealed that inhibin A concentrations increase with increasing follicular size (Magoffin and Jakimiuk, 1997
; Schneyer et al., 2000
) and the expression of ßA-subunit is greater in larger follicles (Roberts et al., 1993
). 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., 2000). When one thus considers the changes in a ratio of the relative concentrations of inhibin and activin during follicular development (Figure 4
), 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
-subunit synthesis rather than an increase in ßA synthesis that is changing the balance of secretion from primarily the
/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
subunit is required to be expressed in the cell in considerable excess to the inhibin ß-subunit. (Mason et al., 1987
). These data thus suggest that the principal function of the excess (relative to ß)
-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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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., 1995), and in both human and mouse, activin A has consistently been observed to promote oocyte maturation (Alak et al., 1998
; Sidis et al., 1998
; Smitz et al., 1998
). 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
-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.
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Notes |
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References |
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Submitted on October 13, 2000; accepted on September 15, 2001.