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Endocrinology Vol. 143, No. 1 107-116
Copyright © 2002 by The Endocrine Society

REPRODUCTION-DEVELOPMENT

Acidic Mix of FSH Isoforms Are Better Facilitators of Ovarian Follicular Maturation and E2 Production than the Less Acidic

Christine R. West1, Nichole E. Carlson, James S. Lee, Alan S. McNeilly, Tejinder Pal Sharma, Wen Ye and Vasantha Padmanabhan

Department of Pediatrics (V.P., J.S.L.), Biostatistics (N.E.C., W.Y.), and the Reproductive Sciences Program (C.R.W., T.P.S., V.P.), University of Michigan, Ann Arbor, Michigan 48109; and Medical Research Council Human Reproductive Sciences Unit (A.S.M.), Edinburgh, Scotland EH3 9ET, United Kingdom

Address all correspondence and requests for reprints to: Vasantha Padmanabhan, Reproductive Sciences Program, 300 North Ingalls Building, Room 1109 SW, Ann Arbor, Michigan 48109-0404. E-mail: vasantha{at}umich.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
FSH is secreted as a mix of isoforms with varying biologic attributes. To determine the functional significance of FSH heterogeneity, an acidic (ovine pituitary FSH; C-FSH) and less acidic mix (C-FSH exposed to neuraminidase; N-FSH) were administered to prepubertal lambs. Production of GnRH- induced less acidic FSH was blocked with a competitive GnRH receptor antagonist, Nal-Glu. Beginning 24 h after Nal-Glu, lambs were injected with C-FSH or N-FSH and LH. Controls included untreated, GnRH-treated, and Nal-Glu-treated groups. Blood samples were obtained at 2-h intervals. Plasma FSH levels were similar before treatment and increased over time in the C-FSH but not the N-FSH group (P < 0.001). Three of the six GnRH-treated ewes exhibited an LH surge. Peak E2 concentrations in the GnRH-treated animals were achieved 30–36 h after initiation of treatment. Peak circulating E2 levels tended to be higher in the C-FSH than in the GnRH-treated group. Only two of six N-FSH-treated ewes had a serum E2 rise. The C-FSH ewes had more estrogenic follicles than the GnRH and N-FSH groups (P < 0.05). Our findings show that C-FSH clears more slowly than N-FSH, and C-FSH is a better facilitator of follicular development and maturation than N-FSH.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
FSH CONSISTS OF A FAMILY of related isoforms differing in their charge, metabolic clearance rates, and in vitro biological activities (1, 2, 3, 4). A mixture of circulating gonadotropin isoforms reaches target tissues to influence a variety of biologic end points. Although the factors affecting final gonadotropin isoform distribution within the circulation are multifaceted and complex, it is evident that endocrine changes regulate the proportion of FSH isoforms both within the pituitary and in the peripheral circulation (1, 2, 3, 4). For instance, studies characterizing changes in FSH heterogeneity during onset of puberty (5, 6) and the late follicular phase (7, 8, 9, 10) have suggested that changes in FSH heterogeneity may be important in the pubertal process and reproductive cyclicity.

Considering the multitude of functions FSH mediates such as maturation of follicles, prevention of follicular atresia, aromatase induction, granulosa cell proliferation, LH receptor induction, and induction of its own receptors (11, 12, 13), changes in FSH heterogeneity that occur during various physiologic states may have important biologic consequences in the mediation of the many FSH-induced functions and thus are of physiologic importance. A large body of evidence has been generated using chemically and enzymatically produced isoform mixes to show that the FSH isoforms vary in their receptor-binding ability, immunologic detection, in vitro biologic activity, and metabolic clearance (1, 2, 3, 4). More recent studies with gonadotropins suggest that the different FSH isoforms vary not only in their biologic potency but also in their ability to mediate the different FSH functions (3, 4, 14, 15, 16). Missing, however, are studies that establish the relevance of FSH heterogeneity in vivo during the maturational process and ovulatory cyclicity, especially using natural pituitary FSH isoforms. Such studies have been hampered by the difficulty in obtaining naturally occurring FSH isoforms in large quantities.

To overcome this limitation, in this study we blocked the endogenous production of less acidic FSH isoforms in prepubertal ewes with Nal-Glu, a competitive GnRH receptor antagonist, and provided them with two different mixes of ovine pituitary FSH, a more acidic native mix (purified ovine pituitary FSH, C-FSH) and a less acidic mix generated by exposing the C-FSH to neuraminidase (N-FSH). Previous studies have shown that FSH released in a GnRH-associated pulse is less acidic, fast clearing, and more biopotent than what is secreted in the tonic mode (1, 3, 17). The C-FSH and N-FSH mixes varied in their profile of FSH isoform distribution and to a certain degree mimicked the distribution profile of circulating FSH that elutes during the prepubertal and pubertal period (5). Specifically, we tested the hypothesis that C-FSH is a better facilitator of ovarian follicular development/maturation than N-FSH. Prepubertal female lambs were used as the in vivo model to test this hypothesis.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
General materials
The GnRH antagonist Nal-Glu [Ac-D2Nal1,D4ClPhe2,D3Pal3,Arg5,4-(methoxybenzoyl)-D-2-aminobutyric acid6, D-Ala10] GnRH used in these studies was synthesized at the Salk Institute under contract NO1-HD-02906 with the NIH and made available by the Contraceptive Development Branch, Center for Population Research, NICHD. Highly purified ovine pituitary FSH (code G4–227B; potency 50 times NIH-S1) was provided by Dr. Harold Papkoff (University of California, Davis, CA).

Generation of more acidic and less acidic mixes of FSH isoforms
To address the physiological relevance of FSH heterogeneity in the facilitation of follicular development during the pubertal process, we compared the efficacy of an acidic and a less acidic mix of FSH isoforms. The ability of neuraminidase to cleave terminal sialic acid residues from purified ovine pituitary FSH was exploited to generate a less acidic mix of asialo FSH isoforms (N-FSH). Untreated ovine pituitary FSH (C-FSH) was used as the acidic mix of FSH isoforms.

To maintain molar equivalency between the two mixes during treatment, we began with 50 ml ovine pituitary FSH stock solution [85.0 ± 1.7 µg/ml as measured by a validated RIA (18), mean ± SE, n = 4] in phosphate buffer (0.1 M, pH 5.1) containing 0.1% BSA. Following thorough mixing, the solution was split into two equal 25-ml aliquots. One unit of neuraminidase (type VI: chromatographically purified from Clostridium perfringens; N3001; Sigma, St. Louis, MO) in 0.5 ml of the above-described buffer was added to one aliquot, and 0.5 ml buffer alone was added to the other. Both aliquots were then incubated for 2.5 h at 37 C with mixing (100 rpm in a Dubnoff shaking water bath). At the end of this time, 17 mg sodium azide was added to each aliquot to stop the reaction (the sodium azide concentration in each delivered pulse of FSH was 64 µg/animal several magnitude lower than the reported LD 50 of sodium azide, which is 27 mg/kg (orally in the rat) (MSDS 52906, Mallinckrodt Baker Inc., Phillipsburg, NJ) (19). This treatment was shown to give limit digests of this material in analytical pilot studies and essentially paralleled what was reported earlier (20). After processing, final FSH concentrations averaged 89.2 ± 2.6 and 88.1 ± 4.3 µg/ml in the C-FSH and N-FSH, respectively, suggesting no loss of immunoreactivity because of neuraminidase digestion.

Chromatofocusing of FSH
Distribution patterns of FSH isoforms in the two mixes of FSH were determined following analytical chromatofocusing of C-FSH and N-FSH. Details of the chromatofocusing procedure were described previously (7). Equal volumes of the two FSH stocks containing equimolar concentrations of FSH were dialyzed against 0.025 mol/liter imidazole-HCL, pH 7.4, overnight and then applied to a preequilibrated 15 x 0.9 cm column PBE 94 resin (Pharmacia, Piscataway, NJ). After entry of the sample, polybuffer (Pharmacia, pH 4.0) was used to elute the sample, and 2-ml fractions were collected. When the pH of the eluent reached 4.0 (i.e. after collection of 80 fractions), 1 M NaCl was used to elute any remaining FSH, and 10 additional 2-ml fractions were collected. The pH and immunoreactive FSH content of the fractions were determined. Recovery of FSH following analytical chromatofocusing separation, assessed by summing up FSH recovered in all fractions and relating to starting amount chromatofocused, was 94% and 89% for the C-FSH and N-FSH, respectively.

Animal experimentation
Pilot studies: validation of a method to block GnRH action long term. To test the efficacy of the C-FSH and N-FSH in facilitating follicular development and E2 production, it was essential to block the action of GnRH and limit the endogenous production of less acidic FSH isoforms. Previous studies have shown GnRH facilitates the release of fast-clearing, less acidic FSH isoforms in ovary-intact female animals (1, 3, 17). We conducted two pilot studies. In the first pilot study, six prepubertal lambs (~25 wk of age) were bled for 33 h at 30-min intervals. Beginning 8 h into the collection, all animals were administered bolus injections of Nal-Glu (10 µg/kg body weight) iv at 8-h intervals. In previous studies we had shown that iv injections of Nal-Glu suppresses LH release with the duration of suppression being dependent on the dose of Nal-Glu used. A 10-µg/kg dose suppressed LH for up to 10 h (21). In the second pilot study, after collecting samples every 30 min for 3 h, lambs were injected sc with Nal-Glu (50 µg/kg body weight) at 12-h (n = 3) or 24-h (n = 3) intervals and blood collection continued every hour for 72 h. Previous studies have shown administration of GnRH-antagonist sc provides long-term suppression of LH (22). We compared the suppression of LH observed with administration of Nal-Glu at the doses of 50 µg/kg sc every 12 or 24 h and 10 µg/kg iv every 8 h. We concluded that administration of 50 µg/kg Nal-Glu sc at 12-h intervals achieved complete suppression of LH during the 72-h study (Fig. 1Go) as opposed to the 24-h interval sc delivery and the 8-h interval iv delivery in which there was incomplete LH suppression. Hence, this mode of Nal-Glu delivery was chosen as the approach for blocking endogenous GnRH action in the main study.



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  		Figure 1. The efficacy of Nal-Glu, a competitive GnRH antagonist in suppressing circulating LH concentrations (as an index of GnRH action). Left panel shows response to Nal-Glu administered iv at 8-h intervals. Top and bottom right panels show responses to sc administration of 50 µg/kg body weight of Nal-Glu given every 12 and 24 h, respectively. Shaded area represents the Nal-Glu treatment period.

 
Main study. The objective of the experiment was to determine the differential effects of C-FSH and N-FSH on follicular maturation. The experimental design is summarized in Fig. 2Go. Treatment groups consisted of: 1) an untreated control group (n = 6; mean body weight = 44.7 ± 2.2 kg); 2) a positive control group that received GnRH (iv 2 ng/kg; n = 6; mean body weight = 47.0 ± 1.6 kg); 3) a negative control group that received Nal-Glu (sc 50 µg/kg; n = 6; mean body weight = 45.3 ± 1.5 kg); 4) a group that received Nal-Glu sc plus C-FSH (iv 8 µg/pulse; n = 6; mean body weight = 46.3 ± 1.5 kg); and 5) a group that received Nal-Glu sc plus N-FSH (8 µg/pulse; n = 6; mean body weight = 46.5 ± 1.7 kg). LH (15 µg/pulse) was coadministered with both mixes of FSH because earlier studies have shown that LH is required along with FSH for achieving normal steroidogenesis (23); this paralleled the physiologic drive; and outcomes from C-FSH and N-FSH drive can be compared with that achieved following GnRH drive, a known stimulator of both LH and FSH. The experiment was conducted using prepubertal Suffolk lambs (25–30 wk of age) maintained under normal husbandry conditions at the Sheep Research Facility in Ann Arbor, Michigan. All experimental procedures were approved by the Committee for the Use and Care of Animals at the University of Michigan.



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  		Figure 2. Schematic showing the design of the study. Arrows indicate the time of administration of Nal-Glu. C-FSH and N-FSH were injected in conjunction with purified ovine pituitary LH every 2 h from 0 to 24 h and hourly from 25–48 h. Starting from 6 h before Nal-Glu administration, blood samples were collected at 2-h intervals throughout the course of the study. Closed boxes represent the 2-h pulse bleed conducted at the end of study to assess delivery patterns of C-FSH and N-FSH.

 
Administration of Nal-Glu (diluted in 5% glucose-sterile H2O) occurred every 12 h in the indicated groups beginning 24 h before administration of GnRH or FSH plus LH. Lambs were fitted with indwelling catheters in the jugular vein and blood samples were collected every 2 h, starting 6 h before administration of Nal-Glu and 30 h before the administration of GnRH or FSH plus LH. A second indwelling catheter was placed in the other jugular vein of lambs that received pulsatile GnRH or FSH plus LH to prevent contamination of the sampling catheter. GnRH or gonadotropins were diluted in 0.1% BSA-saline and were administered every 2 h for the first 24 h (0–24 h) and then hourly for the next 24 h (25–48 h). From 46–48 h, blood samples were collected every 6 min to characterize the circulatory pattern of LH and FSH following pulsatile administration of GnRH or FSH plus LH. When sampling overlapped with administration of GnRH or gonadotropins, the blood sample was collected first, followed by administration of GnRH or gonadotropins. Patency of the catheters was maintained with heparinized saline.

Lambs were euthanized with Beuthanasia (Schering-Plough Corp. Animal Health Corps, Kenilworth, NJ) 48 h after the initiation of GnRH or gonadotropin treatment. The ovaries were collected immediately after euthanasia and the diameters of follicles were measured. Follicular fluid (5–50 µl) was aspirated from all follicles greater than or equal to 3 mm in diameter and frozen at -80 C for subsequent measurement of intrafollicular E2 levels. Follicular responses of animals in the various treatment groups were assessed by counting the number of follicles reaching sizes greater than or equal to 3 mm and determining their estrogenic status by classifying them as estrogenic (potential preovulatory, >50 ng/ml E2), estrogen active (10–50 ng/ml E2), and nonestrogenic (<10 ng/ml) based on criteria established by McNeilly et al. (24).

Radioimmunoassays. Peripheral LH and FSH were assayed in duplicate using well-validated RIAs (18, 25). An ovine serum-based standard, B1371, calibrated against NIH-LH-S1 and NIDDK-ovine FSH-1 were used as the reference standards in the LH and FSH assays, respectively. All samples from a given animal were measured in a single assay. Sensitivity (two standard deviations from the buffer control) of the LH (n = 10) and FSH (n = 9) assays averaged 0.16 ± 0.01 and 0.05 ± 0.01 ng, respectively. Samples were assayed in duplicate 200-µl (25 µl for high values) volumes for the LH and 100–200 µl in the FSH assay. The half-maximal displacement points of the LH and FSH assays were 0.68 ± 0.02 and 0.48 ± 0.02 ng, respectively. Intraassay coefficients of variation averaged 7.2 ± 0.9 and 8.3 ± 0.5, respectively, for the LH and FSH assays. Interassay coefficients of variation of the LH averaged 10.3%, 7.1%, 6.8%, and 9.21% for the four quality pools measuring 0.8, 22.2, 74.1, and 195.9 ng/ml, respectively. Interassay coefficients of variation of the FSH averaged 9.3%, 6.8%, 5.0%, and 4.4% for the four quality pools 3.4, 8.3, 22.5, and 25.9 ng/ml, respectively.

Plasma E2 concentrations were determined in duplicate diethyl ether extracts of 200 µl plasma using a modification (26) of the E2 MAIA assay (Serono-Baker Diagnostics Inc., Allentown, PA). The sensitivity and 50% displacement point of this E2 assay averaged 0.06 ± 0.01 and 1.19 ± 0.13 pg (n = 15 assays), respectively. All samples were assayed in duplicate 200-µl aliquots. Intra- and interassay coefficient of variation of this E2 assay averaged 13.9% ± 0.8% and 13.5% ± 3.5% (n = 5 quality control pools), respectively. The median variance ratio for LH, FSH, and E2 assays averaged 0.06 ± 0.01 and 0.03 ± 0.00, and 0.05 ± 0.01, respectively. Follicular fluid E2 concentrations were determined using a well-validated in-house RIA (27). The sensitivity, half-maximal displacement, intra- and interassay coefficient of variation, and median variance ratio of this E2 assay (n = 2) averaged 1.38 pg, 27.5 pg, 12.02%, and 11.45% (based on four quality control pools), respectively.

Statistical analyses
To compare the patterns of LH, FSH, and E2 concentrations over time between treatments, the 12 time points of 2-h observation for each of the 3 d of study were divided into four 6-h intervals. Mean hormone concentration was calculated for each 6-h interval. Repeated-measures ANOVA was performed to test for day, time, and treatment effect for each hormone. All data were analyzed by repeated-measures ANOVA; general linear models or mixed models procedure of SAS, Cary, NC) to evaluate the effects of treatment, and log (base-e) transformed before analysis. A Tukey adjustment was made for all pairwise comparisons.

To assess effects of Nal-Glu, the ratios of mean hormone concentration at each 6-hr time period after Nal-Glu were compared with the mean hormone concentration for the 6 h before Nal-Glu administration (baseline). Repeated-measures ANOVA was used to test for time differences between the control and Nal-Glu treatments and to test for Nal-Glu suppression of LH, FSH, and E2 (i.e. if the mean ratio was less than 1). All ratios were log (base-e) transformed before analysis, and a Tukey adjustment was used for all pairwise comparisons. One ewe was missing her baseline E2 concentration (loss of sample), and that concentration was imputed. For analysis of E2 data, the maximum value of E2 achieved during the 3 d of study in the GnRH, C-FSH, and N-FSH groups were analyzed by ANOVA following log (base-e) transformation. Two different analyses were performed, one including all animals and the other including only those ewes that showed an estrogen response (in the C-FSH group only five of the six showed an E2 response, and in the N-FSH group only 2 of the six ewes showed an E2 increase).

Follicular data were analyzed using absolute number of follicles as well as the percentage distribution within size classes as the variables. For the first, data were transformed using square root transformation. Tukey adjustment was used when comparing the means of different groups for all the variables. Two different analyses were performed, one using all ewes in each group and the other including only those that showed follicles greater than or equal to 3 mm.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Distribution profile of C-FSH and N-FSH
Comparison of the distribution pattern of FSH isoforms in the C-FSH and N-FSH revealed a relatively less acidic mix of FSH isoforms in the N-FSH than C-FSH (Fig. 3Go). One hundred percent of FSH isoforms in C-FSH eluted in fractions with pH less than 5.4 and the salt peak. In contrast, 50% of isoforms in N-FSH eluted in pH range more than 5.4. Comparison of the distribution profiles of C-FSH and N-FSH with previously published distribution of FSH in the prepubertal and pubertal animals during onset of puberty (5) (Fig. 3Go, bottom left) revealed similar directionality of changes although they did not exactly match. In terms of similarities, 50% of FSH isoforms in the N-FSH and pubertal lambs existed in pH range more than 5.4. On the contrary, although none of the C-FSH eluted at pH more than 5.4, approximately 18% of FSH in prepubertal lambs eluted in this pH range. The proportion of C-FSH eluting in the salt peak was also much greater in C-FSH, compared with the proportion eluting in prepubertal lambs. The present approach did facilitate the generation of two different FSH isoform mixes, one more acidic and one less acidic that were similar to the directionality of changes observed in the prepubertal and pubertal lambs but failed to completely reproduce what was present in the prepubertal and pubertal lambs. Although C-FSH and N-FSH differed in the FSH isoform distribution profiles, they had similar immunopotencies (Fig. 3Go, inset).



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  		Figure 3. Top panels show the elution patterns of FSH isoforms as measured by the FSH RIA (closed circles) following chromatofocusing separation of C-FSH and N-FSH. Inset in top shows the displacement curves of the original ovine pituitary FSH (C-FSH-pre), ovine pituitary FSH subjected to neuraminidase digestion (N-FSH), and ovine pituitary FSH processed in parallel with N-FSH but in the absence of neuraminidase (C-FSH-post), all plotted against starting FSH equivalents. Bottom panels show the percent C-FSH and N-FSH isoforms eluting in pH ranges less than 5.4, more than 5.4 and the salt peak. For comparison, the distribution patterns of circulating FSH in prepubertal and pubertal lambs from a previously published study (5 ) are shown on the bottom left.

 
Patterns of gonadotropin release before and after treatment
Figure 4Go summarizes the circulating patterns of LH achieved following the various treatments. As predicted by the pilot study (Fig. 1Go), administration of 50 µg/kg Nal-Glu sc at 12-h intervals effectively suppressed LH below the detection limit of the assay throughout the study in the negative control animals (Nal-Glu group) (P < 0.01). Reflective of Nal-Glu suppression, LH concentrations in the C-FSH and N-FSH treatment groups were significantly lower on d 1 than that of the GnRH nonsurge group. On d 2 (d 1 of gonadotropin treatment) levels of LH in the C-FSH and N-FSH group were comparable with that of the GnRH nonsurge group. On d 3 (d 2 of gonadotropin treatment) LH concentration in the C-FSH and N-FSH were higher than the GnRH nonsurge group. Pulsatile administration of GnRH resulted in the induction of LH surges in three of the six animals studied. As expected and consistent with the efficacy of Nal-Glu in blocking GnRH action, none of the animals in the C-FSH or N-FSH groups showed an LH surge. Although the replacement levels of LH achieved in the C-FSH and N-FSH groups were similar, the final levels of LH achieved in these two groups were higher than those seen at 48 h in the GnRH nonsurge group (P < 0.001 for C-FSH/nonsurge GnRH comparison and P < 0.01 for N-FSH/nonsurge GnRH comparison).



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  		Figure 4. Mean circulating concentrations of LH achieved in the different treatment groups. Because three of the six GnRH-treated animals had an LH surge during this period, mean circulating levels of LH in the nonsurge (NS) and surge (S) subsets are shown separately. Note the efficacy of Nal-Glu in suppressing LH (top right) and the similarity in replacement patterns LH achieved in the C-FSH and N-FSH groups (bottom panels). Shaded area represents the Nal-Glu treatment period.

 
Patterns of circulating FSH in the five treatment groups are shown in Fig. 5Go. Nal-Glu, although effective in suppressing LH levels, had minimal effect on circulating FSH levels. FSH levels in the GnRH surge group, as one would expect, showed a decline before the onset of the surge. Although C-FSH and N-FSH did not show loss of immunoreactivity following neuraminidase treatment and similar molar concentrations of C-FSH as N-FSH were administered, circulating levels of FSH in the C-FSH group increased progressively from the time of administration. In contrast, circulating levels of FSH did not change during the course of N-FSH treatment. At the 42- to 48-h interval, FSH levels in the C-FSH group were 3 times higher than that of the N-FSH-treated group and 5 times higher than that of the GnRH nonsurge group (P < 0.001, Fig. 5Go). FSH levels achieved in the N-FSH treated animals were not different from GnRH nonsurge group. The amplitude of N-FSH pulses as determined from 6-min samples during the last 2 h of study (46–48 h) was slightly lower than that of C-FSH (Fig. 5Go, insert). The amplitudes of FSH and LH pulses following GnRH administration were not evaluated because of the potential confounding effects of endogenous pulses of GnRH and the expected loss of pituitary sensitivity that would follow the generation of LH surges in three of the six animals.



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  		Figure 5. Mean circulating concentrations of FSH achieved in the different treatment groups. Note the inability of Nal-Glu to suppress FSH as it did LH. Mean circulating levels of FSH in the nonsurge (NS) and surge (S) groups are shown separately. The indent shows FSH pulse amplitude achieved following pulsatile administration of C-FSH and N-FSH. Shaded area represents the Nal-Glu treatment period. Note the increased circulating levels of C-FSH but not of N-FSH achieved following administration of similar molar concentrations of C-FSH as N-FSH.

 
Follicular responses
Concentrations of E2 in follicular fluid were used to determine estrogenic status and as an index of follicular maturation. Estrogenic status was defined using previously established criteria (24). Follicles with less than 10 ng/ml follicular fluid were classed as nonestrogenic, follicles with 10–50 ng/ml E2 were classed as estrogen active, and follicles with more than 50 ng/ml E2 were classed as estrogenic (potential preovulatory) follicles. The total number of follicles greater than or equal to 3 mm in size and their relative estrogenic status are summarized in Table 1Go, and the percentage of distribution in Fig. 6Go. The total number of follicles in the 2.0- to less than 3-mm size range as well as follicles in the greater than or equal to 3-mm range was not significantly different among groups. No differences in absolute number of estrogen-active follicles were evident among the various treatment groups. However, the largest number of estrogenic follicles were found in the C-FSH-treated group (P < 0.05 when all ewes were included in the analysis and P < 0.01 when only those animals that had follicles greater than or equal to 3 mm were included in the analysis). Pairwise comparisons of the means with Tukey adjustment found that the mean number of estrogenic (potential preovulatory) follicles in the C-FSH group were significantly larger than the mean number of estrogenic follicles in the Nal-Glu treated (negative control) group. No estrogenic follicles were found in the Nal-Glu-treated animals. All the greater than or equal to 3-mm follicles (100%) in the GnRH surge group were nonestrogenic.


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  		Table 1. Total number of follicles greater than or equal to 3 mm in size and their relative estrogenic status

 


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  		Figure 6. Shown are the percent distribution of estrogenic (>50 ng/ml E2), estrogen active (10–50 ng/ml E2), and nonestrogenic (<10 ng/ml) follicles in the various treatment groups. For percent calculation animals that did not show follicle greater than or equal to 3 mm are excluded (see Table 1Go for the number of animals responding). For the GnRH-treatment group the nonsurge (NS) and surge (S) subsets are shown separately.

 
ANOVA revealed significant differences in percentage distribution of estrogenic (P < 0.05) and nonestrogenic (P < 0.001) follicles among groups (those animals not having follicles greater than or equal to 3 mm were not included in this analysis). Among all groups, the largest percentage of estrogenic follicles and the smallest number of nonestrogenic follicles were found in the C-FSH group. Pairwise comparison of means with Tukey adjustment found that the percentage of estrogenic follicles of C-FSH group was significantly greater than the Nal-Glu group (P < 0.05). On the contrary, the percentage of nonestrogenic follicles of C-FSH group were significantly smaller (19.8% ± 12.3%) than the GnRH surge group (100%). The majority of the greater than or equal to 3-mm follicles in the Nal-Glu-treated animals (93.7% ± 6.2%) were nonestrogenic. This is consistent with the ability of Nal-Glu to arrest follicular growth. Thirty percent of the greater than or equal to 3-mm follicles in the GnRH-treated animals that did not show an LH surge during the study period were estrogenic. Only three of the N-FSH-treated animals had follicles greater than or equal to 3 mm, and only two of these had estrogenic follicles.

Circulating E2 responses
Mean circulating levels of E2 achieved in the various groups are summarized in Fig. 7Go. E2 levels remained low and constant throughout the Nal-Glu treatment period. Pulsatile GnRH administration increased circulating E2 concentrations in all animals (P < 0.01). In the three animals that showed an LH surge during the study period, peak circulating E2 levels achieved during the presurge period averaged 2.63 ± 0.19 pg/ml. In the three animals that did not surge during the study period, circulating E2 concentrations at hour 48 averaged 1.74 ± 0.34 pg/ml. Corresponding means for the Nal-Glu group was 0.32 ± 0.05 pg/ml. Mean levels of E2 in the five C-FSH-treated animals with follicles greater than or equal to 3.0 mm tended to be higher than the GnRH-treated group and averaged 3.67 ± 0.73 pg/ml. One animal did not show follicles greater than or equal to 3 mm and its E2 concentration throughout collection stayed at 0.22 pg/ml. Two of the N-FSH-treated animals that had an estrogenic follicle achieved peak E2 levels of 2.76 and 3.02 pg/ml, respectively. Overall, although E2 tended to increase over days in all three groups, the daily incremental increases in E2 were higher in the C-FSH group than the GnRH group. The mean E2 concentrations in the other four averaged 0.40 ± 0.09 pg/ml. Circulating levels of E2 in each group correlated well with the estrogenic status of the follicular population and the total E2 produced by the greater than or equal to 3 mm follicles (not shown).



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  		Figure 7. Mean circulating concentrations of E2 achieved in the different treatment groups. Each E2 value represents a measure from three pooled samples (e.g., -24 is a measure of a sample obtained by pooling equal volumes of plasma from -28, -26, and -24 h; -12 of -16, -14 and -12 h; 0 of -4, -2, and 0 h and so on) and hence likely to be an underestimate. For the GnRH-treatment group, the nonsurge (NS) and surge (S) subsets are shown separately. Shaded area represents the Nal-Glu treatment period. Note the ability of Nal-Glu to suppress E2, ability of GnRH and C-FSH in inducing E2 production, and the relatively reduced potency of N-FSH to elicit E2 response of magnitude such as that seen in the GnRH and C-FSH treatment groups. The increase at 48 h in the N-FSH group is the outcome of two of the six animals responding to N-FSH administration.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Previously, we had outlined four criteria for establishing the biologic significance of FSH heterogeneity (3, 4). Criteria 1 and 2, namely the existence of FSH heterogeneity and its neuroendocrine regulation, have been adequately met and remain uncontested. A mix of FSH isoforms resides in the pituitary and circulation and is under neuroendocrine control (1, 2, 3, 4). In general, increases in E2 and GnRH in gonad-intact models lead to increased release of less acidic FSH isoforms and progesterone counteracts this increase (3, 4). Evidence supporting criterion 3, namely that it should change in a biologically meaningful manner, also exists. For example when sustained gonadal stimulation is essential, such as during the early follicular phases of the estrous/menstrual cycles in the female when follicular recruitment and growth of the follicle is occurring, a more acidic mix of FSH isoform prevails in the circulation (3, 4, 7, 8, 9, 10). On the contrary, at times when an acute and a potent signal is essential to facilitate selection and accelerated growth of the preovulatory follicle, such as that seen during pubertal onset and the preovulatory period, increased proportion of less acidic FSH isoforms are present in the circulation (3, 4, 5, 6, 7, 8, 9, 10).

Missing, however, is the evidence for fulfilling criterion 4 that links manifestation of physiologic changes in FSH heterogeneity with biologic consequences in vivo. Results of this study, which show C-FSH to be a better facilitator of ovarian follicular maturation/differentiation and E2 production than the N-FSH, provide evidence that the type of FSH signal delivered does indeed dictate biologic consequences. These results differ from the in vitro studies of Vitt et al. (28), which show less acidic isoforms to be better facilitators of follicular maturation and E2 production than the acidic isoforms. Such opposing outcomes bring to the forefront the dissociation between in vitro and in vivo studies and the need for assessment of functional significance at the in vivo level.

The ability of C-FSH to promote follicular maturation appears to relate in part to the differences in clearances of C-FSH and N-FSH. In spite of administering equimolar concentrations of FSH in a pulsatile manner, circulating levels of FSH in the N-FSH-treated animals showed no FSH increase. Previous studies have shown that asialo ovine FSH clears faster than intact FSH in mouse in vivo bioassays (32 vs. 565 min for asialo and intact, respectively) (29). Similarly, studies with rat and human FSH have also shown a high degree of correlation between the metabolic clearance rate, charge, and sialic acid content of the FSH isoforms (1, 2, 3, 4). That pituitary FSH from ovariectomized ewes are more acidic and clear more slowly than FSH obtained from intact ewes (30, 31) are consistent with these findings. Although ovine FSH contains high amounts of terminal sulfate residues (32), our results with N-FSH agree with earlier studies (1, 2, 3, 4, 29, 30, 31) in showing a major role for sialic acid in determining the circulatory half-life of FSH in sheep.

Considering the expected differences in clearance kinetics of C-FSH and N-FSH, our rationale for selecting C-FSH and N-FSH for testing the physiologic relevance of FSH heterogeneity needs to be reviewed. First, the proportion of less acidic and acidic FSH isoforms in C-FSH and N-FSH, broadly speaking, reflect the relative proportion of less acidic and acidic FSH isoforms prevailing during the prepubertal period and the onset of puberty, respectively. It should, however, be recognized that C-FSH and N-FSH are not true mimics of circulating FSH because purification of FSH from the pituitary leads to elimination of less acidic FSH isoforms, and the processing of FSH in circulation involves more than removal of sialic acid. As such, structural differences are likely to exist between our test preparations and the circulating prepubertal and pubertal FSH. Second, N-FSH, similar to circulating FSH at the time of onset of puberty in female lambs (5), is more bioactive in eliciting E2 increases in in vitro FSH bioassays (33). Third, in spite of its fast clearance, N-FSH may facilitate different types of FSH receptor(s) interactions, couple to different signaling cascades, and result in different outcomes than what would be achieved by the slow clearing C-FSH. For example, FSH can lead to calcium increases within minutes of its addition to target cells (34, 35). Similarly, effective activation of adenyl cyclase by N-FSH is also possible because G proteins can have half-lives in the order of seconds (36). More recent studies have also shown that the ovine FSH receptor gene undergoes alternate splicing and generates multiple transcripts and FSH function is predominantly mediated through an alternatively spliced novel growth factor type 1 receptor that exists in the ovary (37, 38). Considering all the above, N-FSH administered in a pulsatile manner has the ability to provide an acute yet potent stimulus and exert qualitatively different effects on target tissues than C-FSH.

In this regard, the mode of FSH presented is also worthy of some comment. Because of the long circulating half-life of available FSH, FSH is conventionally administered once or twice daily during gonadotropin stimulation protocols (39, 40, 41). In this study, FSH was administered in a pulsatile manner at 2-h intervals for the first 24 h and then hourly for the next 24 h for the following reasons: 1) The mode of delivery paralleled the pattern of GnRH/LH secretion heralding the onset of puberty (5, 42); 2) our recent study showed that, although not clearly discernible at the peripheral level, FSH secretion is comprised of both basal and episodic components and each pulse of GnRH was associated with an FSH pulse (18); and 3) Nal-Glu administration blocked GnRH action and ablated the GnRH-associated component of pulsatile FSH secretion (Padmanabhan, V., N. P. Evans, G. E. Dahl, F. J. Karsch, and J. Van Cleeff, unpublished data). GnRH administration in intact animals is shown to increase the release of less acidic FSH isoforms (5). The levels of circulating FSH we achieved following pulsatile N-FSH also paralleled that of the GnRH-treated group (although it failed to mimic the fall that occurred during the late follicular phase of the GnRH-driven animals) but much lower than that achieved with C-FSH. Frequent sampling did confirm that N-FSH was delivered in the intended pulsatile mode (Fig. 5Go, inset).

In terms of outcome measures (follicular development and E2 production), C-FSH facilitated maturation of a greater number of follicles than GnRH, thus possibly accounting for the higher level of circulating E2 achieved by this group of lambs. Considering that the breed we were working with was selected for twinning, an optimal outcome would be achievement of dominance by two follicles per animal. GnRH-treated animals provided the optimal drive in this context because they showed development of one or two estrogenic follicles. In contrast, two of the C-FSH-treated animals showed four to six estrogenic follicles. From the standpoint of overcoming human infertility with exogenous FSH, the outcome achieved with C-FSH is optimal for in vitro fertilization protocols but not for gonadotropin stimulation protocols in which the end goal is to facilitate monoovular condition. It has been long debated whether a short-acting FSH would help facilitate monoovular condition.

Our findings with N-FSH show that provision of less acidic FSH isoforms throughout follicular development (as done in this study) is not an optimal alternative. Although N-FSH administration limited the development of estrogenic follicles to one or two in the two animals that responded, they were not optimal in terms of consistency of response among animals. Only two of the six animals driven with N-FSH developed estrogenic follicles. Inconsistent responses we achieved with N-FSH may relate to when N-FSH was administered during follicular development. In this study, prepubertal lambs were stimulated throughout with one or the other form of FSH, namely C-FSH or N-FSH (Fig. 8Go, left and middle panels). This drive contrasts with the type of FSH the ovary sees during natural pubertal progression or during ovulatory cycles (Fig. 8Go, right panel). Previous studies have shown that during the prepubertal period (5) and the early follicular phase of the estrus/menstrual cycle (5, 6, 7, 8, 9), the ovary is provided with an acidic mix of FSH. In contrast, during pubertal onset and late follicular phase of the estrus/menstrual cycle ovary receives a different type of signaling via the less acidic mix of FSH (5, 6, 7, 8, 9).



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  		Figure 8. Schematic showing the pattern of FSH/GnRH delivery, the nature and levels of circulating FSH expected in the circulation, maturational and estrogenic status of the follicles, and circulating levels of E2 achieved. Note the nature of FSH in the three groups: more acidic in C-FSH, less acidic in N-FSH, more acidic during the first part, and less acidic during the latter part of GnRH administration as predicted based on the nature of C-FSH and N-FSH delivered and the nature of circulating FSH seen from a previous study in the GnRH-drive group (5 ). Larger number of estrogenic follicles and the higher level of circulating E2 is the outcome in C-FSH group, inconsistent response in N-FSH group (small number of animals responding optimally and others did not), and optimal response in the GnRH-driven animals (optimum response will be one to two follicles achieving dominance).

 
Provision of C-FSH and N-FSH in the sequence mimicking what naturally occurs during pubertal transition and early to late follicular phase transition, namely C-FSH during the first 24 h and N-FSH for the subsequent 24 h, may help achieve optimal outcomes both in terms of number of follicles becoming estrogenic and consistency of animal responses. As such, the switch from more acidic to a less acidic mix of FSH signal during pubertal onset and reproductive cyclicity may be a means to limit the amount of circulating FSH and thus restrict the number of follicles being selected. Although this premise remains to be tested, it is in agreement with the better success rate achieved with the physiologic step-down approach for avoiding multifollicular cycles (43, 44).


   Acknowledgments
 
We are grateful to Douglas D. Doop and Gary McCalla for the quality care and maintenance of the ewes used in this study; Drs. Gordon Niswender and Leo E. Reichert for supplying LH assay reagents; Dr. Morton B. Brown for statistical advice; Dr. Harold Papkoff for the provision of purified pituitary FSH; and the National Pituitary Hormone Program for their generous gift of the FSH standard and FSH antisera.


   Footnotes
 
Preliminary report, 11th International Congress of Endocrinology, Sydney, Australia, October 2000, p. 120 (Abstract P102).

This work was supported by USPHS Grant HD 23812.

1 Present address: Pfizer, Inc., Global Research and Development, Ann Arbor, Michigan 48105. Back

Abbreviations: C-FSH, Acidic native mix (purified ovine pituitary FSH); N-FSH, less acidic mix generated by exposing the C-FSH to neuraminidase.

Received June 19, 2001.

Accepted for publication September 25, 2001.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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