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BOR - Papers in Press, published online ahead of print May 25, 2005.
Biol Reprod 2005, 10.1095/biolreprod.105.039602
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BIOLOGY OF REPRODUCTION 73, 610–618 (2005)
DOI: 10.1095/biolreprod.105.039602
© 2005 by the Society for the Study of Reproduction, Inc.

Gonadotropin Control of Inhibin Secretion and the Relationship to Follicle Type and Number in the hpg Mouse1

Yuan Wang 2, 3, Helen Newton 3, Jenny A. Spaliviero 4, Charles M. Allan 4, Benjamin Marshan 3, David J. Handelsman 4, and Peter J. Illingworth 3

Department of Reproductive Medicine,3 Westmead Hospital, Westmead, New South Wales 2145, Australia ANZAC Research Institute,4 University of Sydney, Sydney, New South Wales 2139, Australia


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Inhibin is secreted in two distinct heterodimeric forms, A and B, but the mechanism for the differential control of these two forms is unclear. To evaluate the relationship between secretion of inhibin forms and folliculogenesis, the effects of gonadotropins on inhibin concentrations were studied in parallel with stereological enumeration of ovarian follicle types in gonadotropin-deficient hypogonadal (hpg) female mice treated with recombinant human FSH (10 IU/day), hCG (1 IU/day), or both for 20 days. Treatment with FSH alone significantly increased blood concentrations of both inhibin A and inhibin B, whereas hCG alone had no effect on either inhibin. The combination of FSH and hCG further increased the concentration of inhibin A but had no effect on the concentration of inhibin B beyond that of FSH. The number of primordial follicles per ovary was significantly reduced in FSH-treated hpg mice, but was not affected by hCG treatment. Antral follicles were absent in the untreated hpg mice, present following treatment with FSH, and were present in only limited numbers following hCG treatment alone. Preovulatory follicles were observed only in the wild-type and combined FSH and hCG treatment groups. These results demonstrate that secretion of both inhibins is associated with the presence of antral follicles. Inhibin A secretion is increased by the presence of preovulatory follicles, whereas the concentration of inhibin B is not affected. The observed effects of gonadotropins on inhibin A and B secretion may be explained by corresponding gonadotropin effects on follicle development.

follicle, follicle number, follicle-stimulating hormone, gonadotropins, hpg, human chorionic gonadotropin, inhibin, ovary, stereology


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Inhibin A and inhibin B are heterodimeric glycoproteins consisting of an alpha subunit and either a ßA (inhibin A) or a ßB (inhibin B) subunit, and are defined on the basis of their inhibitory effect on pituitary FSH secretion [1]. Extensive studies in the 1980s and early 1990s demonstrated that inhibin is secreted by both granulosa and Sertoli cells in response to FSH by a cyclic AMP-dependent pathway [2–4]. However, these observations were derived from the use of immunoassay formats based entirely on alpha subunit epitopes and were thus unable to distinguish between inhibin A, inhibin B, and biologically inactive forms of the free alpha subunit. Studies examining the level of inhibin subunit mRNA expressed in response to FSH have suggested that the synthesis of both alpha and beta subunits was similarly regulated by FSH [4–6], but these studies could not define the resulting secreted forms of inhibin.

Sensitive and specific assay formats have since been developed for human inhibin A and human inhibin B [7]. These assays have been applied with only limited methodological adaptations to the study of inhibin secretion in a variety of species, including rhesus monkey [4], stump tailed macaque [8], hamster [9], rat [10], and mouse [11]. Use of these assays has demonstrated that the two hormones, inhibin A and inhibin B, have different cyclic patterns in the serum of both women [7] and rats [10]. In addition, there are differences in the concentrations of the two inhibins in follicular fluid [12, 13] as well as in the patterns of mRNA expression of the inhibin subunits at various stages of follicle development [14–16].

These observations strongly suggest that inhibin A and inhibin B are regulated in different ways by the two gonadotropins. Although studies in women have demonstrated that inhibin B secretion is closely controlled by FSH [17– 19], data regarding the role of LH in regulating inhibin secretion are more limited.

The ovary is a heterogeneous organ consisting of follicles in various stages of development and maturation, which are likely to have distinctive sensitivity to both LH and FSH in governing follicular ripening and inhibin secretion, yet these relationships are not yet fully understood. Previous in vivo work to investigate the relationship between inhibin secretion and follicular development has been limited. A recent description of a number of mouse gonadotropin-knockout models [16] has demonstrated that as individual follicles mature, a change occurs in the pattern of inhibin secretion. However, this study did not quantify the changes in follicle dynamics observed. Indeed, most quantitative studies of ovarian follicle number have used a two-dimensional approach [20, 21] to estimate the number of ovarian follicles at various follicle stages. This assumes particles are homogeneously distributed within three-dimensional structures, and when applied to the morphologically heterogeneous ovary, this creates considerable potential for quantitative errors and bias. Systematic three-dimensional stereology has proved to be a fundamental advance and has now been developed and applied in a wide range of fields [22, 23]. In particular, the combination of optical sections to define a three-dimensional counting box within a single thick tissue section (the optical dissector) with use of the Cavalieri principle [24], offers a particularly powerful tool for obtaining a total count of particles, such as follicular types within a whole structure such as the ovary.

The congenitally hypogonadotropic (hpg) mouse [25, 26] provides a unique null reproductive hormone background on which it is possible to evaluate the individual effects of FSH and LH [27–29]. In the present study, we used this genetic model to study the effects of FSH and LH on the circulating concentrations of inhibins A and B. Previous investigations of the effects of gonadotropins on inhibin concentration have been based on studies of the short-term effects of gonadotropins [4]. To investigate the effects of development of different follicle types from initiation of antral follicle growth in the hypogonadal mouse, we studied the animals for a duration of 20 days. This approach was combined with a systematic stereological assessment of the number and type of follicles developing within the ovary to better understand the physiological basis for differences in the respective rates of secretion of inhibin A and inhibin B.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals and Animal Care

As described previously [30], the hpg mice were from the University of Sydney colony that was established from original stock provided by Dr. Harry Charlton, Department of Human Anatomy, University of Oxford, Oxford, U.K. [25]. Mice were genotyped by a duplex polymerase chain reaction (PCR)-based assay [31]. The genotype is deduced from PCR products that distinguish three genotypes (N/N, N/hpg, and hpg/hpg). Homozygous normal (N/N) and heterozygous normal (N/hpg) were phenotypically indistinguishable with normal reproduction, and no significant difference was found in the follicle counts between these two types; the similar numbers of each were recruited as normal controls (mice of the wild type).

Mice were housed in groups of three to four in standard mouse cages and maintained under controlled conditions (lights-on, 0700–1900 h; temperature, 20–24°C), with free access to mouse cubes and water.

All experiments were approved by the Animal Ethics Committee of the University of Sydney in accordance with the National Health and Medical Research Council guidelines for animal experimentation and care of laboratory animals.

Treatment

Five groups of mice (n = 4–17 per group) were used in the principal study: untreated hpg mice and litter-mate (wild-type) controls, hpg mice treated with daily i.p. injection of FSH (10 IU, recombinant human FSH, Gonal-F; Serono, French's Forest, NSW, Australia), hCG (1 IU, recombinant human hCG; Ovidrel; Serono), or both FSH and hCG together. All treatments were started immediately after weaning (Day 21) and continued for 20 days. All mice were killed at 41 days of age, 24 h after the last injection.

The dosage of FSH and hCG was identified in preliminary experiments as being the minimum required to elicit an ovarian response in the treated animals. Treatment with FSH alone at 1.5 IU/day for 20 days, or 5 IU/ day for 7 or 16 days, was found to have no stimulatory effect on either ovarian weight or inhibin B secretion. Changes in these parameters were observed only when FSH was given at 10 IU/day for 7 and 16 days.

A time-course study was carried out to investigate the chronology of the short-term changes in serum inhibin A and inhibin B following gonadotropin treatment. After weaning (21–24 days after birth), six groups of hpg mice with six mice in each group received treatment with FSH (10 IU/day) alone, or with FSH (10 IU/day) and hCG (1 IU/day) together for 1, 4, and 7 days, respectively.

All mice were killed 24 h after the last injection. Mice were killed by exsanguination under ketamine (Parke-Davis, Caringbah, NSW, Australia)/ rompun (Bayer Australia Ltd., Botany, NSW, Australia) anesthesia. Cardiac serum was stored at –20°C. Mouse ovaries were quickly removed, dissected free of surrounding fat, weighed, fixed in 4% formaldehyde in PBS (pH 7.2–7.4) for 24 h, and stored in 70% ethanol. Only one ovary from each mouse was used for stereology study.

The ovaries were dehydrated and embedded in glycomethacrylate (GMA) according to the manufacturer's instructions (Technovit 7100; Kulzer and Co., Friedrichsdorf, Germany). Each ovary was cut into serial 25-µm sections using a Polycut S microtome (Reichert Jung, Nossloch, Germany), and stained with toluidine blue for light micrographs and periodic acid-Schiff (PAS) for stereology.

Serum was collected and stored at –20°C until later assay. Progesterone and estradiol were measured by standard immunofluorescent immunometric assay (Immulite; DPC). The lower limit of detection was 73.4 pmol/L for estradiol and 600 pmol/L for progesterone. Coefficients of variation were 9.1% for estradiol and 12.9% for progesterone.

Inhibin assays were performed according to previously published procedures [7]. The human standards used were the World Health Organization-National Institute for Biological Standards and Control standard code 91/624 containing recombinant DNA-derived human inhibin, an immunopurified concentrate of inhibin that forms from human follicular fluid [7] for inhibins A and B, respectively.

Validation of the Use of the Human Inhibin Assay Format in Murine Samples

The human inhibin assays were validated for use in mouse serum as follows. Human inhibin A and B standards were serially diluted (1:2) with inhibin-free castrated male mouse serum (CMMS) from 1000 pg/ml to 7.8 pg/ml. Equine chorionic gonadotropin-stimulated female mouse serum, mouse ovary, and testis homogenates were similarly serially diluted from original medium to 1/128 in CMMS. Mouse follicle culture medium was diluted serially with both CMMS and blank culture medium. The resulting dilution curves for mouse inhibin A and inhibin B were compared with each other and with human standard curves for parallelism and linearity. Parallelism of the dilution curves was evaluated by analysis of covariance (SPSS version 8; F test). Assay curves were judged as being consistent with parallelism if the P value for the test of the sum of squares associated with parallelism was ≥ 0.05, and were judged as nonparallel otherwise. Linearity was analyzed by linear regression, with a P value of < 0.05 being considered as consistent with linearity.

The resulting dilution curves are shown in Figure 1. In both inhibin A and B assays, mouse samples and tissue extracts, and conditioned follicle culture medium gave linear dilution curves but were consistently nonparallel with the human inhibin standards. As a result, for all subsequent mouse inhibin assays, inhibin concentrations were interpolated from mouse standards using an arbitrary unitage. The concentration of mouse standard that gave equivalent immunofluorescence to 1000 pg/ml was defined as 1000 arbitrary mouse units per milliliter (amu/ml).



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  		FIG. 1. Comparison of serial dilutions of human inhibin standard with serial dilutions of mouse preparations: (a) serial dilution of the mouse inhibin A in mouse serum, mouse ovary extract, mouse follicle culture medium, and human inhibin A standard; (b) serial dilution of the mouse inhibin B in mouse serum, mouse ovary and testis extract, mouse follicle culture medium, and human inhibin B standard; (c) serial dilution of the human inhibin A standard and the mouse reference preparation aligned to cross at 1000 pmol/L arbitrarily equivalent to 1000 amu/mL; (d) serial dilution of the human inhibin B standard and the mouse reference preparation aligned to cross at 1000 pmol/L arbitrarily equivalent to 1000 amu/mL

The limit of detection of the resulting assay formats was 7.8 amu/ml for inhibin A and 8.0 amu/ml for inhibin B. The intraassay coefficient of variation was 5.3% for inhibin A and 5.4% for inhibin B. The interassay coefficient of variation (CV) was 5.2% for inhibin A and 5.8% for inhibin B.

Stereology Principles

The optical dissector principle was applied to estimate the numerical density of the follicles in the ovary using a light microscope modified to employ a microcator to monitor scanned depth and CAST software (Olympus, Albertslund, Denmark). The nucleolus of each oocyte was used as the index particle, and follicle development stages were characterized as described here. The largest cross-section of the primordial follicle contains a single small oocyte with a visible nucleolus and one layer of two to four flat progranulosa cells, and a basement membrane without a theca layer. Follicles containing a single granulosa layer with both fusiform and cuboidal cells were counted as primordial; the primary follicle contains an enlarged oocyte with a visible nucleolus surrounded by a single layer of cuboidal follicle cells; the secondary follicle contains more than one layer of follicle cells around the enlarging oocyte with no antrum present; the antral follicle has antral spaces scattered or distinct, apart from the changes of granulosa cells and oocyte; and the preovulatory follicle has a single antral cavity with a well-developed stalk of granulosa cells. Follicles in advanced atresia could not be counted because the degenerating oocytes lacked nucleoli.

Sections for follicle counting were selected using uniform sampling from a randomized start through a random number table. The number of counting frames and frame step size required was based on the average size of the ovary, the frequency of section selection (1/5), and the requirement for a minimum limit number of follicles to be counted (100–120 follicles in this study) per ovary for optimal estimation. On this basis, an optical dissector frame size of 26 446 µm2, and a frame step size of 250 µm x 250 µm were derived using an iterative approach and used to estimate the number of follicles at different developing stages.

The area of the ovary in each selected section was obtained after delineation of the ovary using a 10x objective. The follicle numbers were estimated by using unbiased sample frames created by CAST grid software (Olympus) on images scanned directly to a real-time screen at 40x oil-immersion. When counting in a frame, the stage was moved down in the direction of the z-axis. A follicle was counted (Fig. 2) when the nucleolus of its oocyte within the counting box came into focus through the central 18 µm of the 25-µm sections and did not touch the forbidden solid lines. Nucleoli in the top 2 µm and bottom 5 µm (to ensure complete penetration of the dye) of the sections were not counted to safeguard against surface artifacts such as irregularities in the section surface and missing nucleoli.



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  		FIG. 2. Diagrammatic representation of the process for counting follicles in a thick section. As shown in (A), to avoid edge artifacts, the top 2 µm and bottom 5 µm surfaces of the section were not counted, leaving a dissector thickness of 18 µm for counting. B, C) The resulting images at two different depths within the section, with the solid line indicating the left-hand and lower borders (nucleoli crossing these lines excluded), whereas the interrupted line indicates the right-hand and upper borders (nucleoli crossing these borders included). Original magnification x40

Calculation of Follicle Number

The follicle density (number per unit volume) was calculated as the total sum of the follicles counted divided by the total dissector volume according to the optical dissector principle. The volume of the ovary was derived from the sum of the area of all selected sections multiplied by the section thickness (25 µm) and inverse of sampling frequency (f 1/5), according to the Cavalieri principle. The total number of primordial and primary follicles in the ovary was thus obtained by multiplying follicle density by the volume of the ovary. Secondary, antral, preovulatory follicles and corpora lutea were counted in every frame in every section.

The reproducibility of follicle number estimates was evaluated by the CV between identical recounts for each type of follicle. The between-count CV for the total number of follicles per ovary was as follows: primordial follicles, 2.7%; primary follicles, 3.6%; secondary follicles, 4.0%; and antral follicles, 8.6%.

Statistical Analysis

The data relating to serum inhibin A, inhibin B, progesterone, estradiol, mouse ovary weight, and follicle number per ovary were expressed as the mean ± SEM.

The presence of statistically significant differences among various treatment groups in ovarian weight and hormone assays were determined using one-way analysis of variance (ANOVA) followed by two-tailed t-test for the ANOVA secondary analysis. P < 0.05 was taken as indicating statistical significance.

The data in the short time-course were analyzed by two-way ANOVA to investigate the interaction between treatment and duration of treatment followed by post hoc two-tailed t-testing to analyze individual differences between treatment groups.

The number of follicles per ovary in wild-type and untreated hpg mice were compared by a two-tailed unpaired t-test. The follicle number data were evaluated by two-way ANOVA to examine the effects of FSH treatment and hCG treatment, as well as the interaction between FSH and hCG. A P value of < 0.05 was considered as indicating a significant difference.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Ovarian Weight

One-way ANOVA demonstrated significant variation between the ovarian weights in the different groups (P < 0.001). On post hoc analysis, treatment of hpg mice with FSH alone (1.01 ± 0.10 mg; P < 0.0001) and hCG alone (0.89 ± 0.09 mg; P < 0.0001) significantly increased ovary weight compared with that of untreated hpg animals (0.23 ± 0.19 mg). However, these weights were lower than those in wild-type mice (2.90 ± 0.15 mg), whereas combined FSH and hCG treatment caused an increase above that of wild-type controls (3.73 ± 0.54 mg; P < 0.01).

Inhibin Concentrations

As shown in Figure 3, there was significant variation in the concentrations of both inhibin A and inhibin B between the different groups (inhibin A, P < 0.001: inhibin B, P < 0.001). Post hoc testing demonstrated that compared with untreated animals, FSH alone stimulated inhibin A secretion significantly (23.46 ± 3.87 amu/ml; P < 0.05), while combined FSH and hCG treatment caused a further increase in inhibin A concentration (67.85 ± 12.1 amu/ml; P < 0.01), which was similar to the concentration in wild-type controls (53.61 ± 7.49 amu/ml; P = NS). In contrast, hCG alone had no effect on inhibin A secretion. Treatment with FSH alone stimulated inhibin B secretion (101.58 ± 21.99 amu/ml; P < 0.05) compared with the untreated state (9.11 ± 0.97 amu/ml). Combined FSH and hCG treatment stimulated inhibin B concentration (97.62 ± 25.32 amu/ml; P < 0.05), but there was no significant increase compared with that of the FSH-alone group. Human chorionic gonadotropin alone had no effect on inhibin B secretion.



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  		FIG. 3. Serum concentrations of inhibin A (amu/mL), inhibin B (amu/ mL), progesterone (nmol/L), and estradiol (pmol/L) in age-matched normal controls (n = 9), hpg controls (n = 4), hpg mouse treated with FSH alone (n = 9), FSH and hCG (n = 6), and hCG alone (n = 5). Values are mean ± SEM. *P < 0.05, **P < 0.01

Short Time-Course Study

As shown in Figure 4, there was a steady increase in both ovarian weight and inhibin concentrations in the first 7 days of treatment in both the FSH and combined treatment groups. Following 7 days of FSH treatment alone, the ovarian weight was similar to that observed in the group that had received FSH for 20 days. The inhibin A concentration rose slowly in the animals treated with FSH alone, and it was below the limit of detection until treatment had been administered for 7 days. In the animals treated with combined gonadotropins, the inhibin A concentration rose more rapidly, and following 7 days of treatment it was 20.9 ± 7.7 amu/ml. The inhibin B concentration rose steadily in the FSH-treated group, and after 4 days of treatment it was clearly elevated above baseline (54.8 ± 26.9 amu/ml). The inhibin B concentration in the combined treatment group rose rapidly and was clearly elevated in the group that had received treatment for only a single day (77.0 ± 33.1 amu/ ml). However, no difference was observed in inhibin B concentrations between the FSH and the combined FSH and hCG group at 4 or 7 days.



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  		FIG. 4. Ovarian weights and serum concentrations of inhibin A (amu/ mL) and inhibin B (amu/mL) in hpg mice treated with either FSH alone or FSH and hCG combined, and killed at 1, 4, or 7 days of treatment (n = 6 at each time point). Values are mean ± SEM

Steroid Concentrations

Combined FSH and hCG treatment produced a significant increase in progesterone secretion (P < 0.01). Neither gonadotropin alone stimulated progesterone secretion. No significant difference in estradiol concentration was noticed between the different study groups.

Stereology

Representative sections are shown in Figure 5, and details of follicle numbers are shown in Table 1.



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  		FIG. 5. Representative figures illustrating the effects on ovarian histology of wild-type ovaries (original magnification x100), ovaries from untreated hpg animals (original magnification x400), ovaries from hpg animals treated with FSH alone (original magnification x100), hCG alone (original magnification x100), and combined FSH and hCG (original magnification x100). CL, corpus luteum; M, primordial; P, primary; S, secondary; A, antral


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  		TABLE 1. The number of follicles per ovary (mean ± SEM)

Primordial follicles The number of primordial follicles observed in the untreated hpg animals was significantly higher than that in the wild type (P < 0.05). FSH treatment alone or in combination with hCG significantly reduced the number of primordial follicles (P < 0.005), whereas hCG treatment alone had no effect (P = 0.377).

Primary follicles There was no significant difference in the number of primary follicles between untreated hpg mice, wild-type mice, and mice treated with FSH alone. In contrast, hCG, either alone or in combination with FSH, caused a significant increase in the number of primary follicles (P < 0.001).

Secondary follicles A significantly higher number of secondary follicles was found in wild-type mice (P < 0.001) and in mice treated with FSH only (P < 0.001) compared with follicles in untreated hpg mice. Treatment with hCG (either alone or in combination with FSH) had no significant effect (P = 0.057).

Antral and preovulatory follicles No antral and preovulatory follicles were found in untreated hpg mice, whereas in wild-type mice, both antral and preovulatory follicles were clearly observed. FSH treatment significantly increased the number of antral follicles (P < 0.001) compared with untreated hpg mice, as did hCG treatment (P < 0.05). Preovulatory follicles were observed in those hpg mice that received combined FSH and hCG treatment, as well as in wild-type mice. No preovulatory follicles were found in the ovaries of untreated hpg mice and hpg mice treated with either FSH or hCG alone.

Corpora lutea were found only in wild-type (5.7 ± 0.66 per ovary) and combined FSH + hCG treatment (4.3 ± 0.56 per ovary) groups.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
We have investigated the relative roles of FSH and LH in regulating secretion of the different forms of inhibin through a study of inhibin concentrations in parallel with a quantitative study of changes in the type and number of follicles observed in the ovaries following gonadotropin stimulation in congenitally gonadotropin-deficient mice.

Administration of pure recombinant human FSH led to an increase in ovarian weight and progesterone concentration (only when administered along with hCG), and significantly increased circulating inhibin B concentrations. The lack of effect on estradiol concentration was unexpected, and the explanation is not clear. In contrast to the effects on progesterone, hCG either alone or in addition to FSH, had no effect on the concentrations of inhibin B. The circulating inhibin A concentration was also increased in response to FSH without any further change produced by treatment with hCG alone. However, unlike inhibin B, the inhibin A concentration was significantly increased by the addition of hCG along with FSH. These results are consistent with a recent observational study of a number of mouse single-gonadotropin-knockout models [16] and provide strong experimental evidence that, in mice, inhibin B secretion is primarily regulated by FSH but not by LH (hCG), whereas inhibin A is regulated by both gonadotropins. This finding is similar to the results of previous physiologic studies in women [17, 18, 32].

The effects of gonadotropin on inhibin concentration were accompanied by changes in the number and type of follicles observed following gonadotropin treatment. FSH alone stimulated follicular development as far as the antral stage, whereas treatment with both FSH and hCG was required for further follicle development to the preovulatory stage. This is likely to be closely related to the observed changes in inhibin concentration.

Studies based on in situ hybridization have demonstrated that both the inhibin ßA and ßB genes are expressed in the granulosa cells of antral follicles, with the ßB form being the dominant subtype [33]. On further development to a preovulatory follicle, the expression of ßB diminishes, while ßA expression rises [33]. Previous work from our laboratory using incubated follicles [34] has demonstrated that follicles cultured in the presence of FSH produce significantly more inhibin B than inhibin A, with inhibin B secretion occurring at an earlier stage of follicle culture.

A time-course study was performed to clarify the chronology of the changes observed in inhibin A and inhibin B concentrations. The findings were consistent with the follicle development data, in that following treatment with FSH, there was an early rise in inhibin B concentration, which approached the maximum value within 7 days, whereas the inhibin A concentration showed only a limited rise despite 7 days of FSH treatment. This is consistent with the relatively rapid increase in inhibin B concentration originating from early antral follicles.

The chronic administration of hCG to induce development of preovulatory follicles did not produce any further increase in inhibin B concentration. In contrast, the concentration of inhibin A was further increased. This is unlikely to be a result of ovulation, as the rodent corpus luteum does not secrete inhibin [35, 36]. Instead, it is likely that the increased inhibin A concentration is a result of the additional (i.e., FSH) effect of LH in enhancing the differentiation and maturation of FSH-stimulated follicles to a more advanced preovulatory stage [11]. The findings in this study are similar to those of previous observations in women of a strong correlation between inhibin A concentration and LH action during recovery after administration of a GnRH antagonist in the late-follicular phase [17].

This is the first study to apply contemporary methodology using the combination of optical dissector and the Cavalieri principle to study the effect of gonadotropins on ovarian follicle development in the mouse ovary. Before the development of three-dimensional stereology, ovarian morphometric studies were based on two-dimensional probes. The use of single histology sections generates volume-weighted, not number-weighted probes, and correction factors are required [20, 37], thus producing a significant variance in absolute values for numbers of follicles per ovary [38]. In contrast, application of the stereology methodology has a number of inherent strengths. With the application of the dissector and unbiased counting rules associated with the dissector, the follicles of different size are counted with equal probability independent of the their size, shape, and distribution. A similar stereological method with the fractionator/dissector combination was recently used to estimate the follicular numbers in the mouse ovary [39], giving further evidence that new stereology could give an unbiased and assumption-free estimation of follicle numbers in mouse ovary. One limitation in this methodology for the study of ovarian follicle dynamics is that, in using the nucleolus as the defining particle, it is not possible to count atretic follicles (in which the nucleolus is absent), with the result that follicle loss cannot be quantified.

In the present study, using the combination of optical dissector and the Cavalieri principle, the estimates of tissue volume and numerical density are derived from the same set of tissue sections, and no correction factor for volume changes is required. In addition, the use of GMA as an embedding medium in light microscopy provides a significant advantage in quantitative analysis, because tissue shrinkage is less pronounced than with paraffin, and dimensional changes during stretching and mounting are highly reproducible [40, 41].

The findings in the present study showed that FSH treatment resulted in a significant decrease in the number of primordial follicles. We found that the groups with FSH present (wild-type, FSH alone, and FSH with hCG) all had significantly lower numbers of primordial follicles than the two groups in which FSH was absent (hpg and hCG alone). This was surprising, as the early stages of primordial follicle recruitment are generally considered to be independent of gonadotropin [42, 43], particularly as the duration of treatment was relatively short. However, our finding is in agreement with some previous studies that suggest a role for gonadotropins in primordial follicle recruitment [44– 49]. The use of an unbiased methodology is important in avoiding the systematic bias induced by the effects of FSH on the size and volume of the remainder of the ovary. We were unable to locate any other stereological studies using the relatively less-biased three-dimensional approach with optical dissector and Cavalieri combination to study primordial follicle number following FSH treatment.

The mechanism for the observed effect of FSH on primordial follicle number is not clear. It is generally accepted that FSH and LH are unlikely to exert direct actions on primordial follicles because functional gonadotropin receptors have not yet developed in primordial follicles [50–52]. However, FSH receptor mRNA transcripts have been detected in granulosa cells from as early as the primordial stage [53], raising the possibility of a mechanism for FSH direct action. In addition, recent work has suggested that the FSH interaction with its receptor can influence early transition of follicles from the resting pool into the growing pool by modulating production of GDF-9 by oocytes, and stem cell factor by somatic cells [54]. On the other hand, the observed reduction in primordial follicle numbers may simply be an indirect effect of increased Mullerian hormone secretion from the larger number of growing follicles [55].

One unexpected finding was the significantly increased number of primary follicles found in the hCG-treated groups, both alone and combined with FSH-treated groups, when compared with untreated wild-type and hpg mice with or without FSH treatment. This finding was in agreement with a previous study [47], whereas no change in the number of primary follicles was found after ovarian exposure of LH in other studies [56, 57]. The fact that the number of primary follicles increased without any change in the number of primordial follicles suggests that this effect reflects a prevention of atresia in the early stages of development rather than an increased rate of recruitment from the primordial pool. Changes in the rate of atresia may also explain differences in the total number of follicles observed between different treatment groups. However, while some follicles suggestive of atresia were observed during study of the sections, it was not possible to apply stereological methodology to this aspect due to the lack of a defining particle in an atretic follicle.

High doses of human FSH had to be administered before any effect on either ovarian morphology or inhibin B concentration was observed. The reason for this is unclear. Mouse antibody formation to the human protein is one possibility. FSH antibody was previously detected when recombinant human FSH was given to mice at 0.5 IU, 1 IU, or 10 IU/day for 6 wk [58]. Male mice when given hCG via i.p. injection at 1 IU/day three times per week for 6 wk did not produce antibody; whereas given at 10 IU/day or 100 IU/day three times weekly, an hCG antibody was detected [59]. The other possibility is the potential variations in the interaction of human gonadotropins with rodent gonadotropin receptors [60]. Another possibility is that the relative potency of human FSH on mouse FSH receptors may differ from that of murine FSH, but their relative biopotencies are not known, although human and rat have been compared [60]. Regardless of the reason, the critical issue is whether the high doses are producing a supraphysiological response. This appears unlikely as the observed changes in the two principal end points of ovarian weight and inhibin B secretion are similar to those recently reported by our group in a transgenic FSH-secreting construct with much lower circulating concentrations of FSH [27].

We found significant nonparallelism between serial dilutions of mouse inhibin preparations and the human standards. This was consistent in that all mouse preparations exhibited linear dilutions in parallel to each other but with nonparallelism with the human standards for both inhibin A and inhibin B. This finding differs from previous studies in mice [61, 62], although it is notable that the data presented in one study suggested nonparallelism without reaching statistical significance [61]. As a result of our findings we developed a mouse standard from conditioned mouse medium and used this for the inhibin assays. In the light of our findings, future studies requiring mouse inhibin assays should use a mouse rather than a human standard for accurate quantification.

In summary, we have combined immunoassay with three-dimensional stereology to explore the relationship between the gonadotropin regulation of inhibin A and inhibin B secretion and concurrent ovarian follicle development. This work provides experimental data to support previous suggestions that FSH exerts an influence on the number of primordial follicles in the ovary, contrary to the hypothesis that the earliest stages of follicle development are gonadotropin-independent. Antral follicles stimulated by FSH alone were the main source of inhibin B secretion, whereas hCG-induced follicle maturation resulted in a significant increase in inhibin A secretion. These data demonstrate differential control of the two forms of inhibin by LH and FSH.


   ACKNOWLEDGMENTS
 
We sincerely thank Ms. Lija Arthur and Ms. Elaine Philips for their technical guidance in assay technique. We also thank Ms. Julie Simpson, Mr. Adam Koch, and Ms. Kirsten Mctavish for genotyping the mice. We are grateful for the provision of rhFSH and rhCG from Serono Australia.


   FOOTNOTES
 
1 Supported by NHMRC grant 211244. Back

2 Correspondence: Yuan Wang, Department of Reproductive Endocrinology, Westmead Hospital, Westmead, NSW 2145 Australia. FAX: 02 9845 7793; yuanw{at}westgate.wh.usyd.edu.au Back

Received: 5 January 2005.

First decision: 25 January 2005.

Accepted: 18 May 2005.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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