Molecular Human Reproduction, Vol. 5, No. 8, 726-731,
August 1999
© 1999 European Society of Human Reproduction and Embryology
FSH-induced resumption of meiosis in mouse oocytes: effect of different isoforms
1 Laboratory of Reproductive Biology, Section 5712, Juliane Marie Center for Children, Women and Reproduction, University Hospital of Copenhagen, Blegdamsvej 9, DK-2100 Copenhagen, Denmark, 2 Department of Reproductive Biology, Instituto Nacional de la Nutricion Salvador Zubiran, Mexico City, Mexico, and 3 Wallaceville Animal Research Centre, Upper Hutt, New Zealand
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Abstract |
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The ability of different isoforms of follicle stimulating hormone (FSH) to induce the resumption of meiosis in cultured mouse oocytes was evaluated. Oocytes were cultured in the presence of hypoxanthine to prevent spontaneous resumption of meiosis. Using serial dilutions of the isoform fractions representing less acidic isoforms (pI 6.43–5.69), mid-acidic (pI 5.62–4.96) and acidic (pI 4.69–3.75), the concentration which caused 50% of the oocytes to resume meiosis and undergo germinal vesicle breakdown (GVBD) after a culture period of 24 h (i.e. ED50% GVBD) was determined. The FSH concentration of the isoform fractions was determined by radioimmunoassay, radio-receptor assay or through cAMP release in a Chinese hamster ovary-cell line expressing the human FSH-receptor. Determined by radioimmunoassay, the (ED50% GVBD) values were: less acidic 6.4 ± 0.3 IU/l (mean ± SD), mid-acidic 6.1 ± 0.7 IU/l and acidic 12.2 ± 0.7 IU/l. The less and mid-acidic isoforms were significant lower than the acidic (P < 0.0005). Similar relationships between the isoform fractions were obtained by the two other FSH assays. The results demonstrate that FSH isoforms with a pI of >5.0 induced resumption of meiosis significantly more efficiently than acidic isoforms. Less and mid-acidic isoforms may exert an important physiological function by inducing the resumption of meiosis in oocytes from pre-ovulatory follicles during the mid-cycle gonadotrophin surge.
FSH isoforms/meiosis/mouse oocytes
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Introduction |
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Follicle stimulating hormone (FSH) is composed of an
- and a ß-subunit with two possible N-linked glycosylation sites located on each of the two subunits. As a consequence, FSH exists as a family of isohormones, which differ in their electric charge due to variance in their oligosaccharide structures including variations in the total number of charged terminal sialic acid and, to a lesser extent, the sulphate residues (Dahl and Stone, 1992
). Separation based on charge shows that FSH derived from the pituitary, serum, or urine all contain isoforms within a broad spectrum of isoelectric points (pI) of between 4 and 8 (Wide, 1985; Ulloa-Aguirre et al., 1988b, 1992a; Stanton et al., 1992). Furthermore, studies in experimental animals and in humans have demonstrated that FSH isoforms, in addition to charge heterogeneity, also show differences in the complexity of the carbohydrate branching pattern (Anobile et al., 1998; Ulloa-Aguirre et al., 1988a). Acidic FSH isoforms more often show a complex branching pattern with bi-, tri- and even tetra-antennary carbohydrate side-chains, than less acidic FSH isoforms, which more often lack branching of the carbohydrate moieties.
In females, the prime target organ of FSH is the ovary, where FSH stimulates follicular development, oocyte maturation and ovulation (Chappel, 1995; Ulloa-Aguirre et al., 1995). The composition of FSH isoforms in the circulation varies throughout the menstrual cycle, being more acidic in the early follicular phase and becoming less acidic as ovulation approaches (Wide and Bakos, 1993; Anobile et al., 1998). It appears that the concentration of circulating oestradiol is a major regulator of the isoform mixture released by the pituitary and that rising oestradiol values during the pre-ovulatory phase of the cycle promote the release of less acidic isoforms (Padmanabhan et al., 1988; Wide and Bakos, 1993; Zambrano et al., 1995). In contrast, progesterone has been shown to possess the opposite effect, at least in the short term (Ulloa-Aguirre et al., 1992b; Wide et al., 1995, 1996). Acidic isoforms stay longer in circulation than the less acidic ones (de Leeuw et al., 1996), and acidic isoforms show, independent of assay, constantly lower bioactivity in vitro than less acidic isoforms (Chappel, 1995, Ulloa-Aguirre et al., 1995; Lambert et al., 1998). Taken together, the functional significance of the FSH isoform variations throughout the follicular phase remains unresolved. This has prompted a number of studies to address the question of the in-vivo biological activity of FSH and its different isoforms. In the majority of studies, various in-vitro assays with diverse end-points such as oestradiol production by granulosa or Sertoli cell cultures, cAMP production by Chinese hamster ovary (CHO) cells transfected with the human FSH-receptor, or inhibin production by granulosa cells have been used (Lambert et al., 1998). Recently, the ability of intact mouse follicles to increase in size during culture has also been used (Vitt et al., 1998). However, no clear-cut data on the relative in-vivo potencies of various FSH isoforms have yet emerged. It has, however, been suggested that less acidic isoforms actually possess specific functions at the receptor level (Anobile et al., 1998; Lambert et al., 1998; Timossi et al., 1998a).
One of the obstacles in defining a specific role for the less acidic isoforms predominating just around ovulation, may relate to a lack of information on the precise physiological function of FSH during the mid-cycle gonadotrophin surge. This fact makes it difficult to define suitable end-points for the effect of FSH isoforms. The mid-cycle gonadotrophin surge induces two distinct biological events, namely resumption of meiosis in oocytes contained in large pre-ovulatory follicles and follicular rupture, i.e. the ovulatory process itself. The physiological regulation of resumption of meiosis is probably regulated by luteinizing hormone (LH) and FSH acting together, but recent studies suggest that FSH has an important specific function in this process by actively promoting resumption of meiosis (Downs et al., 1988; Byskov et al., 1997; Yding Andersen et al., 1999). This has been highlighted by the fact that isolated cumulus enclosed oocytes (CEO) from large pre-ovulatory follicles respond to FSH in physiological concentrations by resuming meiosis, whereas even very high concentrations of recombinant LH or human chorionic gonadotrophin (HCG) (i.e. 1500 IU/l) have no effect on meiosis, probably reflecting the fact that receptors for LH/HCG are absent or expressed at very low levels on the cumulus cells immediately surrounding the oocyte (Byskov et al., 1997; Eppig et al., 1997). There is now good evidence to suggest that the cumulus cells of pre-ovulatory follicles represent a specific specialized form of granulosa cells (Eppig et al., 1997). Therefore, CEO may represent a physiological intact model exclusively expressing FSH receptors, which could serve as a suitable way to monitor the effect of various FSH isoforms. In an attempt to monitor the activity of various FSH isoforms in an assay mimicking the actions of FSH around the time of ovulation, the aim of the present study was to evaluate the ability of various FSH isoforms to induce resumption of meiosis in mouse oocytes in vitro.
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Animals
Immature female mice (B6D2-F1 strain C57B1/2J) were kept under controlled conditions of light and temperature with free access to food and water. Ovarian stimulation was performed when the mice weighed 11–16 g and consisted of an i.p. injection of Gonadoplex (Leo, Copenhagen, Denmark) containing 7.5 IU per mouse (pregnant mare's serum gonadotrophin 5 IU and human serum gonadotrophin 2.5 IU). The animals were killed by cervical dislocation 44–48 h later. The experiments were performed according to the rules of the Danish Authorities for Animal Care, Ministry of Justice.
Media
The media used for the culture of oocytes consisted of -minimum essential medium (-MEM), with Earle's balanced salt solution (EBSS), 4 mmol/l hypoxanthine (HX), 3 mg/ml bovine serum albumin (BSA), 0,23 mmol/l pyruvate, 2 mmol/l glutamine, 100 IU/ml penicillin and 100 µg/ml streptomycin as described earlier (Byskov et al., 1997). This medium served as control medium. Test media consisted of control media supplemented with FSH isoform fractions.
Oocyte assay
The ovaries were recovered and placed in HX medium where an initial cleaning and removal of connective tissue was carried out. Oocytes were isolated from the ovaries by puncturing individual follicles using 25 gauge needles. Isolation of oocytes was performed in HX medium to prevent germinal vehicle breakdown (GVBD) until the tests were carried out. The oocytes were then washed three times in control medium before starting the experiments.
CEO were cultured separately in 4-well dishes (Nunclon, Roskilde, Denmark), 0.4 ml medium in each well containing control medium or medium supplemented with FSH isoform fractions in a 100% humidified atmosphere of 5% CO2 with 95% air at 37°C. The culture period was 22–24 h.
In each 4-well dish, one well always served as a control and contained oocytes cultured in the control medium. Each well contained 30–40 CEO. Determination of the ED50% GVBD (see below for definition) was repeated three to five times for each FSH isoform fraction.
At the end of the culture period, GVBD was scored by examining the oocytes in an inverted microscope. The percentage of oocytes with GVBD per total number of oocytes (% GVBD) was calculated.
Isolation of FSH isoforms
FSH isoforms were isolated from human pituitary extracts as previously described (Zambrano et al., 1996). Total glycoprotein extracts from anterior pituitary glands were obtained using a previously-described method (Jones et al., 1970). A chromatofocusing column (50x1 cm) was used to isolate the FSH isoform fractions (Ulloa-Aguirre et al., 1992a; Timossi et al., 1998b). Each isoform fraction was then transferred to a dialysis membrane (molecular weight cut-off 12 kDa) and dialysed against deionized water and 10 mmol/l ammonium carbonate (pH 7.5). After freeze-drying, the isoforms were dissolved in 100 mmol/l ammonium bicarbonate (pH 7.4) and applied to an affinity column with monoclonal anti-LH antibodies in order to remove any LH that had co-eluted with the FSH isoform fractions. This procedure removed >90% of the immunoreactive LH present in the original concentrate (Ulloa-Aguirre et al., 1992a; Timossi et al., 1998b). Before testing, the FSH isoform fractions in the oocyte assay were thoroughly dialysed against the control medium, divided into aliquots and kept at –20°C until use.
Measurement of FSH concentration
Radioimmunoassay
The reagents used for the measurement of human FSH were provided by the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK). Iodination of the purified human FSH (human FSH-I-4) was performed using the solid-phase oxidizing agent 1,3,4,6-tetrachloro-3 , 6 -diphenyl glycoluril (Iodogen) method (Salacinski et al., 1981; Yding Andersen et al., 1992). In brief, 50 µl of iodogen solution (40 µg/ml in chloroform) was dried under a flow of nitrogen in a glass tube. Ten µl of Na125I (0.33 mCi) and 10 µg FSH in 40 µl was allowed to react for 10 min in a 0.1 mol/l sodium phosphate buffer (pH 7.4). The reaction was stopped by the addition of 500 µl of 0.05 mol/l sodium phosphate buffer (pH 7.4) and separation of protein-bound and free [125I] was done on a PD-10 column containing Sephadex G-25 (Pharmacia-Upjohn, Uppsala, Sweden). The labelled FSH was further purified by QAE-Sepharose ion exchange chromatography as previously described (Moore et al., 1997). A 2 ml column equilibrated with 20 mmol/l Tris–HCl pH 8.6 was eluted with successive 2 ml fractions of 20 mmol/l Tris–HCl, pH 8.6 containing 100, 120, 140, 160, 180 mmol/l KCl. Finally the column was eluted with 6 ml of 200 mmol/l KCl. Each of the fractions was tested for the number of counts and the ability to specifically bind the antibody.
The FSH isoform fractions were diluted in a buffer consisting of 0.05 mol/l sodium phosphate buffer (pH 7.4) and 5 mg/ml BSA. As a standard, the reference preparation LER-907 (1 mg LER-907 contains 53 IU FSH, 2nd International Reference Preparation) was used. This standard, and crude pituitary extracts, show a similar degree of charge heterogeneity (Chappel et al., 1986). The rabbit polyclonal anti-human FSH antiserum (human FSH-6) was used in a final dilution of 1:150.000. This antiserum is characterized by having <0.1% cross-reactivity with highly-purified human LH and undetectable reactivity with the free -subunit. Bound and free [125I]-FSH was separated by adding 100 µl Sac-Cel (Wellcome Reagents Ltd., Beckenham, UK). The supernatant was discarded and the pellet was counted. Inter-and intra-assay coefficients of variation of a sample containing 25 IU/l were 7 and 5% respectively.
Radio-receptor assay
The radio-receptor assay used membrane preparations prepared from bovine testes, [125I]-ovine FSH as the label and the S-20 standard from National Institutes of Health (Bethesda, MD, USA) as a standard, and was performed as previously described (Cheng, 1975; Moore et al., 1997). The intra-assay variation for a 5.7 ng/ml sample was 6.6%. All samples were measured in the same assay.
CHO-cell assay
The CHO-cell assay for FSH bioactivity (Albanese et al., 1994) was developed by constructing a CHO-cell line, which stably expresses the recombinant human FSH receptor and which, upon stimulation with FSH, releases cAMP. In brief, a sufficient number of cells were propagated in -MEM supplemented with 10% (v/v) fetal calf serum (FCS), penicillin/streptomycin and Geneticin 418 (all Gibco, Life Technologies, Paisley, Scotland) to allow for a transfer of 250 000 cells to each well in the actual assay. After overnight incubation, the cells were washed twice with PBS containing 10 mg/ml BSA and incubated with 250 µl -MEM supplemented with 0.25 mmol/l IBMX and 1 mg/ml BSA. Standards (S-20, NIH, Bethesda, USA) and unknowns were diluted in PBS containing 10 mg/ml BSA added in 20 µl and tested in triplicate for each dilution. After an incubation period of exactly 4 h at 37°C, the medium from each well was pipetted into tubes and stored at –20°C until assayed for cAMP. Measurement of cAMP was performed according to a previously described radioimmunoassay (Moore et al., 1997), and the amount of cAMP generated by the unknowns was related to a standard. The intra- and inter-assay coefficients of variation were 15 and 18% respectively.
Statistical analysis
Results are presented as mean ± SD. The ED50% GVBD value for the FSH isoforms were compared using Student's t-test.
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Separation of anterior pituitary FSH extracts by chromatofocusing disclosed the presence of multiple distinct peaks of FSH immunoreactivity within a pH window of 7.1–3.75 (Figure 1
). The FSH isoforms were pooled into eight different fractions ensuring that the highest concentration of FSH within a single peak and the closely related neighbouring fractions were pooled into one fraction (as indicated in Figure 1). In the present study, the following fractions were used: fraction 2 (pI 6.43–5.69) representing a less acidic FSH isoform; fraction 4 (pI 5.62–4.96) representing a mid-acidic FSH isoform; and fraction 7 (pI 4.69–3.75) representing an acidic FSH isoform.
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Each of the three different FSH isoform fractions was tested in the oocyte assay using a total of 2663 CEO, including controls. Each isoform fraction was tested in serial dilutions and the dilution at which 50% of the oocytes resumed meiosis was determined (equivalent dose of 50% GVBD, i.e. ED50% GVBD) as indicated in Figure 2
, which shows a representative assay for the determination of the ED50% GVBD for the less acidic FSH isoform.
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The mean ED50% GVBD value for each of the isoform fractions was related to the FSH concentration measured by radioimmunoassay, radio-receptor assay and by the CHO-cell assay (Table I
). A significantly higher concentration of the acidic FSH isoform fraction was required to induce 50% of the oocytes to resume meiosis independent of the assay used to determine the FSH concentration, compared with the less and mid-acidic FSH isoform fractions (P < 0.0005). The less and mid-acidic isoform fractions showed similar potencies for inducing resumption of meiosis. Based on the results from the radioimmunoassay, and the radio-receptor assay, double the amount of the acidic isoform fraction was needed to stimulate resumption of meiosis to a similar degree as the less and mid-acidic isoforms, whereas according to the CHO-cell assay a less pronounced effect was observed although the difference remained highly significant (P < 0.001).
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Discussion |
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To our knowledge this study is the first to use CEO for evaluation of the biological activity of the naturally occurring FSH isoforms in vitro. The major finding is that less and mid-acidic FSH isoforms are significantly more potent in inducing mouse CEOs to resume meiosis in vitro. Based on the FSH measurements with the radioimmunoassay and the radio-receptor assay, almost double the concentration of acidic FSH isoforms was required to induce 50% of the oocytes to resume meiosis, compared with that of less and mid-acidic isoforms. Based on the FSH measurements with the CHO-cell assay, the acidic FSH isoform concentration needed to be increased by ~50% in order to induce a similar response to the less and mid-acidic isoforms. Collectively, our data confirm and extend results showing a higher biological activity of less acidic isoforms in vitro, compared with acidic ones (Chappel, 1995
; Ulloa-Aguirre et al., 1995). The FSH-induced resumption of meiosis (as used in this study) mimicks a physiological process occurring during the mid-cycle gonadotrophin surge and the strong activity of the less and mid-acidic isoforms compared with the acidic ones may, for the first time, indicate a specific function for the increased release of less acidic FSH isoforms during the mid-cycle surge of gonadotrophins. Our study thereby supports the notion that the secretion of different FSH isoforms occurring during the follicular phase of the menstrual cycle may represent an important mechanism through which the pituitary gland regulates gonadal function (Zambrano et al., 1995).
There are inherent methodological difficulties in measuring the concentration of different FSH isoforms (Chappel, 1995; Ulloa-Aguirre et al., 1995). It is possible that the different oligosaccharide residues themselves represent antigenic determinants which are recognized by certain antibodies, as has been shown for HCG (Chen and Bahl, 1992), or the carbohydrate side-chains may alter the conformation of the FSH molecule in such a way that certain antigenic determinants may be exposed or masked and thereby change the binding due to steric or charge effects. There is an ongoing debate on these problems, which has not yet been resolved (Jeffcoate, 1997). In order to circumvent these problems, most studies try to measure FSH isoform concentrations by employing different assays. In the present study, we used three different assays, which essentially all showed similar results, although they were not directly comparable due to the use of different standards. However, in a recent study (Zambrano et al., 1996) which resembled our study by the radioimmunoassay method used and the way the FSH isoforms were isolated, demonstrated that different isoform fractions showed dilution curves parallel to the standard curve in assays employing different polyclonal and/or monoclonal antibodies, indicating a similar efficacy of some immunoassay methods in measuring the different FSH isoforms. Using this radioimmunoassay assay we measured the FSH concentration at which 50% of the oocytes resumed meiosis to be ~6 IU/l for the less and mid-acidic isoforms and ~12 IU/l for the acidic isoforms. These values correlate well with that obtained by us using unfractionated FSH, where 50% of the oocytes resumed meiosis with a concentration of ~8 IU/l (Byskov et al., 1997). This supports a correct measurement of the concentration of FSH in different isoform fractions and indicates that the sensitivity of the bioassay is sufficient.
A number of studies have measured the intrafollicular concentrations of FSH, which most likely represent the concentration to which the CEO are exposed in vivo. In fluid from pre-ovulatory follicles obtained from women in their natural menstrual cycle, almost constant values were found in the follicular phase (McNatty, 1978), with a minor increase in concentrations around ovulation to ~4–6 IU/l. In women undergoing ovarian stimulation with exogenous gonadotrophins similar concentrations of 4–6 IU/l were found close to follicular rupture (Filicori et al., 1996). Although there are several confounding factors in comparing these results [e.g. different assay systems, FSH isoform profile of follicular fluid (FF)] with those of the present study, it is tempting to speculate that the similar levels of FSH in human FF and the ED50% GVBD concentration of less and mid-acidic FSH isoforms actually indicates that intrafollicular FSH exerts a physiological action as inducing meiotic resumption in connection with the mid-cycle surge of gonadotrophins. Furthermore, in the early follicular phase where circulating levels of FSH reach similar heights as during the mid-cycle surge, the predominant acidic FSH isohormone profile is insufficient for stimulating oocytes to resume meiosis untimely.
The same standards were used for the radio-receptor assay and the CHO-cell assay. Although the relative potencies of the isoforms were identical with the two assays, the actual levels differed around four times, being highest in radio-receptor assay. This may be due to differences in both origin (heterologous versus homologous) and end-points (binding versus signal transduction) between the two assay systems (Zambrano et al., 1999).
In the present study, less and mid-acidic isoforms showed similar results. This is in contrast with most other studies in which the mid-acidic isoform fraction has shown results intermediate to the less acidic and more acidic isoforms. However, in other studies, the less acidic FSH isoform fraction often has a pI range similar to our mid-acidic (pI 5.62–4.96) fraction. In fact, one study (Vitt et al., 1998), showed a significantly higher growth rate of mouse follicles cultured in a FSH isoform fraction with a pI of 5.0–5.6 compared with mid- (4.5–5.0) and acidic (3.6–4.6) fractions. In another study (Harris et al., 1998), which examined differences in oestradiol and inhibin production by granulosa–lutein cells, the less acidic isoform fraction had a pI of 5.3–5.5. Therefore, our study is actually in agreement with other studies showing that FSH isoforms with a pI of >5.0 exert higher responses in in-vitro bioassays. Furthermore, the present study shows that no additional activity is gained by using isoform fractions with a pI above 5.0–5.7.
In conclusion, the present study demonstrates that FSH isoforms with a pI of >5.0 have a significant higher ability to induce resumption of meiosis in cultured mouse CEO compared to that of more acidic isoforms. Around twice as high a concentration of acidic isoforms are required to provoke a response similar to that of isoforms with a pI of >5.0. Around 6 IU/l of FSH isoforms with a pI of >5.0 which, in vivo, increase just around the time of ovulation, is required to induce 50% of the mouse oocyte to resume meiosis. Almost similar concentrations of FSH are found in follicular fluid close to follicular rupture making it conceivable that less acidic isoforms actually may exert an important physiological function in inducing resumption of meiosis in oocytes from pre-ovulatory follicles in connection with the mid-cycle surge of gonadotrophins.
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Acknowledgments |
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The technical assistance by Winny Ng Chie, Wayne Young, Tiny Roed and Anette Winkel is gratefully acknowledged. Dr A.F.Parlow and the National Hormone and Pituitary Program, USA is thanked for providing the reagents for RIA measurements. Dr K.P.McNatty is thanked for his interest in this work. This study was supported by the Danish Medical Research Council (Grant Nos. 9400824, 9502022 and 9602272), and from the Consejo Nacional de Ciencia y Tecnologia (CONACyT), Mexico (grants 0004P-N9505 and G016M).
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4 To whom correspondence should be addressed
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Submitted on February 22, 1999; accepted on May 4, 1999.
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