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Endocrinology, doi:10.1210/en.2006-1265
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Endocrinology Vol. 148, No. 5 2301-2308
Copyright © 2007 by The Endocrine Society

Increased Oocyte Degeneration and Follicular Atresia during the Estrous Cycle in Anti-Müllerian Hormone Null Mice

Jenny A. Visser, Alexandra L. L. Durlinger, Isolde J. J. Peters, Edwin R. van den Heuvel, Ursula M. Rose, Piet Kramer, Frank H. de Jong and Axel P. N. Themmen

Department of Internal Medicine (J.A.V., A.L.L.D., P.K., F.H.d.J., A.P.N.T.), Erasmus MC, 3000 CA Rotterdam, The Netherlands; and Department of Pharmacology (I.J.J.P., E.R.v.d.H., U.M.R.), NV Organon, 5342 CC Oss, The Netherlands

Address all correspondence and requests for reprints to: Jenny A. Visser, Ph.D., Department of Internal Medicine, Room Ee532, Erasmus MC, P.O. Box 2040, 3000 CA Rotterdam, The Netherlands. E-mail: j.visser{at}erasmusmc.nl.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Anti-Müllerian hormone (AMH) plays an important role in folliculogenesis. AMH null mice display an increased recruitment of primordial follicles. Nevertheless, these mice do not have proportionally more preovulatory follicles. Therefore, AMH null mice provide an interesting genetic model to study the regulation of species-specific number of preovulatory follicles. We studied the follicle pool throughout the estrous cycle at 4 months of age. Analysis of the follicle pool revealed that AMH null mice have an increased and earlier cyclic recruitment of growing follicles despite a blunted FSH surge at estrus. However, FSH levels at estrus were apparently too low to support growth to the preovulatory stage because an increased level of atresia was observed, which neutralized the increased cyclic recruitment. When AMH null mice were subjected to a superovulation scheme, the rise in FSH levels resulted in the rescue of the recruited cohort of growing follicles. Analysis of the follicle pool also revealed that the increased recruitment of primordial follicles in AMH null mice was neutralized by an increased loss of follicles during the transition from small preantral to large preantral follicle. This major loss of follicles was not completely reflected by a corresponding augmentation of atresia but did correspond with an increased number of oocyte remnants observed in AMH null mice. We conclude that a combination of increased oocyte degeneration and increased follicular atresia neutralizes the increased initial and cyclic recruitment in AMH null mice to a normal number of preovulatory follicles.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
FOLLICLE DEVELOPMENT in the ovary occurs along several sequential stages, which are controlled by endocrine and paracrine factors. During the fetal period, a limited number of oocytes is produced, giving rise to the so-called primordial follicle pool, a pool that cannot be replenished during life. The size of the primordial follicle pool decreases significantly with age, resulting in the menopausal transition in women, and limited fertility in female mice older than 12 months (1). Throughout life, follicles are recruited from this dormant primordial follicle pool for further growth and development. After puberty, a second recruitment step takes place, i.e. at every new cycle a limited number of follicles is recruited from the cohort of small growing follicles (cyclic recruitment), followed by a final follicle selection of a subset for dominance and ovulation (2). Thus, the majority of follicles that start to grow will not reach this preovulatory stage and are destined to be removed through atresia, a process that involves apoptosis (3). In fact, the default developmental pathway for follicles, especially follicles of the preantral and early antral stage, is to undergo atresia (4). To rescue these follicles to the preovulatory stage the increase in circulating FSH during each cycle is crucial. Indeed, in FSH null mice follicles do not develop beyond the large preantral stage (5). However, FSH acts only as a survival factor for a limited number of follicles. According to the current understanding, FSH levels need to rise to a critical threshold concentration, which is achieved within the microenvironment of the follicle. This microenvironment is established by intraovarian growth factors, such as TGFß family members, epidermal growth factor, IGF, and fibroblast growth factor, which can either augment or diminish FSH action (6, 7). The precise interplay among intraovarian growth factors, ovarian feedback signals, and gonadotropins results in the development and ovulation of a fixed, species-specific number of follicles. These growth factors are also important for the early, hormone-independent, initial recruitment and growth of follicles. In particular, members of the TGFß family have recently been studied in more detail and were shown to regulate follicle recruitment, granulosa and theca cell proliferation, steroidogenesis, oocyte maturation, and ovulation (8, 9).

Previously we have shown that anti-Müllerian hormone (AMH), an ovary-specific member of the TGFß family, regulates both primordial follicle recruitment and FSH responsiveness of growing follicles (10, 11, 12, 13). Studies of AMH null mice revealed that AMH regulates the rate of entry of primordial follicles into the growing pool. In AMH null mice, more primordial follicles are recruited, and as a result, 4-month-old AMH null mice contain fewer primordial follicles but almost 3-fold smaller-growing follicles, compared with wild-type mice (10). This increase in number of growing follicles was found despite a lower serum FSH level, suggesting that in the absence of AMH, follicles have become more sensitive to FSH. This finding was confirmed in an in vitro culture system of mouse preantral follicles in which AMH inhibited FSH-induced follicle growth (12). Furthermore, an in vivo study, in which FSH levels were modulated, showed that in the presence of both low and high serum FSH concentrations more growing follicles were found in AMH null mice, compared with wild-type mice (11). However, despite the increased recruitment of primordial follicles and the increased FSH responsiveness of growing follicles in the absence of AMH, AMH null ovaries do not have a proportionally increased number of preovulatory follicles (10). This suggests that, in AMH null mice, the increased number of growing follicles is corrected to a normal amount of preovulatory follicles during folliculogenesis. Therefore, AMH null mice provide an interesting genetic model to characterize the mechanisms that control follicle development and ensure the species-specific number of preovulatory follicles. More insight into the mechanisms that control folliculogenesis will lead to a better understanding of reproductive disorders in women.

Here we determined the number of nonatretic and atretic follicles throughout the estrous cycle in detail. We observed that AMH null mice display an increased and earlier recruitment of growing follicles despite lower FSH levels. This increase in cyclic recruitment is counteracted by an increase in atresia on all days of the estrous cycle. More strikingly, we found that in AMH null mice a significant number of follicles is removed during the transition from small preantral to large preantral follicle, which is reflected by an increased number of oocyte remnants in AMH null mice.

The combination of both mechanisms of follicle removal, oocyte degeneration and follicular atresia, results in the final establishment of a normal number of preovulatory follicles in AMH null mice, despite the initial increase in number of growing follicles.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Wild-type and AMH null female mice on a C57BL/6J background were generated as described previously (10). The animals were obtained from the Animal Facility of the Erasmus Medical Center in Rotterdam (The Netherlands) and were kept under standard animal housing conditions in accordance with the National Institutes of Health guidelines for the Care and Use of Experimental Animals. The animals were killed at 4 months of age on each day of the ovarian cycle, i.e. proestrus, estrus, metestrus, diestrus-1, and diestrus-2. For each day six wild-type and six AMH null females were collected. To determine the day of the cycle daily vaginal smears were taken for a period of at least 2 wk and examined as described previously (10). In agreement with previous results, no difference in either the length or regularity of the estrous cycle was found between AMH knockout (AMHKO) or wild-type mice. At the selected day of the estrous cycle, animals were killed at 1400 h by decapitation. Blood samples were collected and serum was isolated as described previously (10). Serum samples were stored at –20 C until assayed for FSH or AMH. Ovaries and uteri were removed and weighed. The ovaries were fixed overnight in Bouin’s fluid at room temperature. For histological examination of the follicle population, fixed ovaries were embedded in paraffin, and after routine histological procedures, 8-µm sections were mounted on slides and stained with hematoxylin and eosin.

Superovulation and in vitro fertilization assay
Female mice, aged 2–13 months, were injected sc with 7.5 IU urinary FSH (Humegon) in saline at random stages of the cycle to initiate follicular development (n = 5–13 per age group). After 46 h, 7.5 IU urinary human chorionic gonadotropin (Pregnyl) was administered to induce ovulation, and 18 h later animals were killed by cervical dislocation. Oviducts and ovaries were removed to isolate the ovulated oocytes from the oviducts. The in vivo ovulated oocytes were collected in T6 medium [98 mM NaCl, 1.4 mM KCl, 0.5 mM MgCl2.6H2O, 1.8 mM CaCl2.2H2O, 0.4 mM Na2HPO4.2H2O, 25 mM NaHCO3, 25 mM Na-lactate, 0.5 mM Na-pyruvate, 5.6 mM glucose, 0.2% (vol/vol) phenol red, 15 g/liter BSA] and incubated with capacitated mouse sperm (1 x 106 cells/ml) at 37 C and 5% CO2 in air for 4 h. Thereafter fertilized oocytes were washed once in and subsequently transferred to medium of 95 mM NaCl, 2.5 mM KCl, 0.35 KH2PO4, 0.2 mM D (+) glucose, 0.2 mM Na-pyruvate, 1 mM L-glutamine, 0.01 mM EDTA, 0.2 mM MgSO4.7H2O, 1.7 mM CaCl2.2H2O, 11.8 mM Na-lactate, 25 mM NaHCO3, 1% (vol/vol) essential amino acids (50 times), 0.5% (vol/vol) nonessential amino acids (100 times), 0.2% (vol/vol) phenol red, 1 g/liter BSA. After incubation for 20 h at 37 C and 5% CO2 in air, two-cell embryos were transferred to fresh medium of 95 mM NaCl, 2.5 mM Kcl, 0.35 KH2PO4, 0.2 mM D (+) glucose, 0.2 mM Na-pyruvate, 1 mM L-glutamine, 0.01 mM EDTA, 0.2 mM MgSO4.7H2O, 1.7 mM CaCl2.2H2O, 11.8 mM Na-lactate, 25 mM NaHCO3, 1% (vol/vol) essential amino acids (50 times), 0.5% (vol/vol) nonessential amino acids (100 times), 0.2% (vol/vol) phenol red, 1 g/liter BSA to grow to the (hatched) blastocyst stage in 3 d.

Ovarian histology and follicle counting
Follicle count was performed as described previously (10), using serial sections of both ovaries. With the exception of primordial follicles (diameter < 20 µm), both nonatretic and early atretic follicles were included in the study. Follicles were divided into four groups based on their mean diameter, which was determined by measuring two perpendicular diameters in the section in which the nucleolus of the oocyte was present: small preantral follicles (20–170 µm), large preantral follicles (171–220 µm), small antral follicles (221–310 µm), and large antral follicles (>311 µm) (10). Within the ovary, two forms of follicle loss can be distinguished (14). Follicles from the preantral stage onward are removed through follicular atresia, which is driven by granulosa cell apoptosis. Atretic follicles were identified by the presence of pyknotic nuclei in granulosa cells (15, 16). Follicles in stage 1a and stage 1b of atresia are defined as early atretic follicles. In addition, follicles at the primordial, primary, and small preantral stage are removed through oocyte degeneration, which is driven by apoptosis of the oocyte (17, 18, 19). These oocyte remnants (convoluted and condensed, or fragmented) were counted in every sixth section to prevent recounting. In addition, the number of fresh corpora lutea, i.e. corpora lutea formed during the last ovarian cycle, was counted in serial sections of ovaries collected on the day of estrus. Newly formed corpora lutea can be distinguished from older ones by their irregular size and the morphology of the granulosa-luteal cells (10).

Hormone analyses
Serum FSH was determined by RIA using rat FSH as ligand and antibodies against ovine FSH (20). All results are expressed in terms of NIDDK-rat FSH-RP-2 National Institute of Diabetes and Digestive, and Kidney Diseases reference preparation rat FSH-RP-2. The intraassay coefficient of variation was 11.8% and all samples were measured in one assay. AMH serum levels were measured with an in-house AMH ELISA(1). In brief, mouse serum samples diluted in high-performance ELISA buffer (reference M1940; Mast Group Ltd., Merseyside, UK) were added to duplicate wells on AMH antibody-coated microplates. After incubation at room temperature and washing with PBS/Tween 20 (0.005% Tween 20), plates were incubated with biotinylated capture antibody diluted in 1% (wt/vol) casein buffer. Next, plates were washed with PBS/Tween 20 and incubated with polyhorseradish peroxidase conjugate (Mast Group, reference M2051) diluted in 1% (wt/vol) casein buffer. After washing with PBS/Tween 20 and deionized water, tetramethylbenzidine substrate (Insight Biotechnology International, Wembley, UK) was added. After incubation at room temperature in the dark, the chromogenic reaction was stopped with 6% (vol/vol) phosphoric acid and absorbance was read at 450 nm with a reference wavelength set at 655 nm using a microplate reader (Bio-Rad Laboratories, Hemel Hempstead, UK). A human AMH standard line was included for each plate (1). The intraassay coefficient of variation was 4.2% and all samples were measured in one assay.

Statistical analysis
Results are presented as the mean ± SEM. The data were evaluated for statistical differences either by one-way or two-way ANOVA, followed by Duncan’s new multiple range test or an independent samples t test in case of the presence of significant difference. SPSS, Inc. (version 11.0.1; SPSS, Inc., Chicago, IL) computer software was used for the analysis. Differences were considered significant at P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Ovarian and uterine weight
Initially, ovarian weights were determined as an indication of AMH effects on the ovary. During the estrous cycle, ovarian weight of both AMH null and wild-type females did not vary (Table 1Go). However, the ovarian weight of AMH null females was significantly increased (1.6-fold, P < 0.0001, by two-way ANOVA), compared with ovaries of wild-type females. This increase in ovarian weight was present at all days of the estrous cycle.


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  		TABLE 1. Ovarian and uterine weights in wild-type and AMHKO female mice during the estrous cycle

 
Uterine weight is an estrogen sensitive parameter and therefore was determined to reflect changes in ovarian estrogen production. As expected, in wild-type females uterine weight changed during the estrous cycle being lowest at metestrus and highest at proestrus. Uteri of AMH null females showed a similar weight change during the cycle and their weight did not differ significantly from wild-type uteri (Table 1Go).

Number of corpora lutea
In our previous study (10), we found that ovaries of AMH null mice contain a similar number of fresh corpora lutea (CL), compared with ovaries of wild-type littermates. To confirm this result, the number of fresh CL was counted in both ovaries on the day of estrus as a reflection of the number of ovulations during the last estrous cycle. The average number of fresh CL present did not differ between AMH null (8.8 ± 0.3) and wild-type ovaries (8.2 ± 0.6).

Number of nonatretic follicles
The number of nonatretic follicles was determined in 4-month-old mice throughout the estrous cycle to determine possible differences in follicle dynamics between wild-type and AMH null mice. At the large antral stage, the number of nonatretic follicles did not differ between wild-type and AMH null ovaries (Fig. 1Go). Furthermore, the change in follicle numbers throughout the estrous cycle was similar in both genotypes, showing an absence of large antral follicles at estrus due to the ovulatory surge of LH.


Figure 1
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  		FIG. 1. Number of nonatretic follicles in 4-month-old mice during the estrous cycle. The numbers of nonatretic small preantral, large preantral, small antral, and large antral follicles were determined throughout the estrous cycle in wild-type mice (closed symbols) and AMH null mice (open symbols). Data represent means ± SEM (n = 6 pairs of ovaries). *, Significantly different from wild-type, P < 0.05; #, significantly different from other cycle days, analyzed per genotype, P < 0.01. P, Proestrus; E, estrus; M, metestrus; D1, diestrus 1; D2, diestrus 2.

 
With respect to the smaller follicle classes, AMH null ovaries contained significantly more follicles, compared with wild-type ovaries (P < 0.0001 for preantral follicles, P < 0.001 for small antral follicles, by two-way ANOVA) (Fig. 1Go). The number of small preantral follicles in ovaries of AMH null mice was 2- to 3-fold higher, compared with ovaries of wild-type mice, at all days of the cycle (Fig. 1Go). In both mouse strains, the number of small preantral follicles remained constant throughout the estrous cycle. Also at the large preantral stage, the number of follicles in ovaries of AMH null mice was increased 2- to 3-fold, compared with ovaries of wild-type mice, with an exception of data on diestrus 2 (Fig. 1Go). Whereas in wild-type ovaries the number of large preantral follicles remained constant during the estrous cycle, in ovaries of AMH null mice the number of this class of follicles was significantly increased at estrus (P < 0.05, by post hoc test) (Fig. 1Go). At the subsequent small antral stage, this increase in follicle number at estrus was observed in both wild-type and AMH null ovaries (P < 0.05, by post hoc test), albeit the increase was more pronounced in AMH null mice (Fig. 1Go). As a result, the number of small antral follicles was significantly different (P < 0.05, by post hoc test) in ovaries of AMH null mice, compared with ovaries of wild-type mice at estrus, whereas at other days of the cycle no significant difference was observed (Fig. 1Go).

Number of atretic follicles
Because the increase in number of preantral follicles in AMH null mice was not reflected by more large antral follicles, we studied whether ovaries of AMH null mice showed increased atresia. Analysis of the number of early atretic follicles during the estrous cycle revealed that the number of early atretic large antral follicles did not differ between both strains (Fig. 2Go). At the smaller follicular stages, ovaries of AMH null mice contained significantly more early atretic follicles (P < 0.0001, by two-way ANOVA) (Fig. 2Go). In both genotypes, most atretic follicles were observed during the large preantral and small antral stage, varying between 10 and 20 atretic follicles in ovaries of wild-type mice and between 30 and 50 follicles in ovaries of AMH null mice (Fig. 2Go). Despite the low number of early atretic follicles in the small preantral class, still more atretic follicles were found in AMH null ovaries (Fig. 2Go). In ovaries of both wild-type and AMH null mice, the number of atretic follicles remained at a constant level during the estrous cycle.


Figure 2
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  		FIG. 2. Number of early atretic follicles in 4-month-old mice during the estrous cycle. The numbers of early atretic small preantral, large preantral, small antral, and large antral follicles were determined throughout the estrous cycle in wild-type (closed symbols) and AMH null mice (open symbols). Data represent means ± SEM (n = 6 pairs of ovaries). *, Significantly different from wild-type, P < 0.05. P, Proestrus; E, estrus; M, metestrus; D1, diestrus 1; D2, diestrus 2.

 
Oocyte remnants
The major difference in the number of atretic follicles was found in follicles of the large preantral and small antral stage. In addition, our calculations indicate that the major drop in follicle numbers occurred during the transition from small preantral to large preantral follicle as shown in Fig. 3AGo at estrus. This pattern was also observed at all other days of the cycle (results not shown). The reduction in follicle number was present in ovaries of both wild-type and AMH null mice but was much more pronounced in AMH null mice. This drop in follicle numbers was, however, not reflected by the number of early atretic follicles (Fig. 2Go), which were identified based on the presence of pyknotic nuclei in granulosa cells (Fig. 3CGo) (15, 16). Therefore, we determined the number of oocyte remnants in ovaries isolated at estrus, as a reflection of oocyte apoptosis occurring during the earliest stages of follicle development (Fig. 3CGo) (14). Ovaries of AMH null mice contained almost 2-fold more oocyte remnants than ovaries of wild-type littermates (P < 0.001, by post hoc test) (Fig. 3BGo).


Figure 3
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  		FIG. 3. Number of nonatretic follicles and oocyte remnants in 4-month-old mice at estrus. A, The nonatretic follicle population was determined in wild-type mice (open bars) and AMH null mice (black bars) at estrus. The major loss of follicles occurs during the small preantral to large preantral follicle transition. B, Comparison of the number of oocyte remnants in wild-type and AMH null mice at estrus. Numbers were determined in every sixth section to prevent double counting. Data represent the mean ± SEM (n = 6 pairs of ovaries). *, Significantly different from wild-type, P < 0.05. C, Photomicrograph depicting degenerating oocytes (convoluted and condensed, or fragmented, arrows) and early atretic follicle (pyknotic bodies in granulosa cells, small arrows).

 
Hormone levels
FSH and AMH measurements were performed to study changes in levels during the estrous cycle. Wild-type mice displayed a normal pattern of serum FSH levels during the estrous cycle with elevated levels at estrus, reflecting the secondary FSH peak, which declined at metestrus to the lowest levels observed at diestrus 1 and 2, and proestrus. In contrast, in AMH null mice, the secondary FSH peak at estrus was blunted and, when compared with wild-type mice, the lowest levels were reached a day later (diestrus 2) of the cycle (Fig. 4Go). In wild-type mice, serum AMH levels were increased at estrus and gradually declined to basal levels during the following days of the cycle (Fig. 4Go). Obviously, in AMH null mice, AMH levels were undetectable (results not shown).


Figure 4
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  		FIG. 4. FSH and AMH levels during the estrous cycle in 4-month-old mice. FSH levels were determined in wild-type (closed symbol, solid line) and AMHKO (open symbol, solid line) female mice. AMH levels (closed symbol, dashed line) were determined in wild-type female mice. Data represent the mean ± SEM (n = 6 mice). *, Significantly different from wild-type, P < 0.05; #, significantly different from other cycle days, analyzed per genotype, P < 0.05. P, Proestrus; E, estrus; M, metestrus; D1, diestrus 1; D2, diestrus 2.

 
Superovulation and in vitro fertilization
Previously (10) and in the present study, we observed that ovaries of 4-month-old AMH null mice show an enhanced recruitment of small antral follicles despite lower FSH levels. Nevertheless, FSH levels were not sufficient to select more of these follicles, compared with wild-type mice, for development to the preovulatory stage because we also observed an increase in atresia. To determine whether a rise in FSH levels would rescue this increased cohort of growing follicles, AMH null mice of various ages were subjected to a superovulation protocol. Indeed, at 4 and 5 months of age, when the cohort of growing follicles is increased in ovaries of AMH null mice, AMH null mice yielded significantly more oocytes compared with wild-type mice (P < 0.05, by post hoc test) (Table 2Go). In wild-type mice, the number of retrieved oocytes remained relatively constant with increasing age. However, in AMH null mice, the number of retrieved oocytes declined rapidly from 5 months of age onward, and at 10 and 13 months of age, significantly fewer oocytes were retrieved from AMH null mice (P < 0.05, by post hoc test) (Table 2Go). A similar percentage of retrieved oocytes from wild-type and AMH null mice developed to the two-cell and (hatched) blastocyst stage after in vitro fertilization and in vitro culture. Furthermore, in both strains the percentage decreased in mice of 10 months and older (Table 2Go).


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  		TABLE 2. Number of retrieved oocytes and percentage two-cell embryos and (hatched) blastocysts after superovulation of wild-type and AMHKO female mice

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study, we characterized the follicle numbers, hormone levels, and oocyte quality in wild-type and AMH null mice in detail. We observed that throughout the estrous cycle uterine weight of AMH null mice did not differ from that of wild-type mice, indicating that estradiol levels in AMH null mice are similar to those of wild-type mice. Because estradiol is mainly produced by large antral and preovulatory follicles (21), this implies that the number of these follicles would be the same in wild-type and AMH null mice. Indeed, the number of corpora lutea, as a reflection of the number of preovulatory follicles, did not differ. Analysis of the follicle pool of both strains revealed that also the number of large antral follicles did not differ. Before the large antral follicle stage, however, AMH null mice have significantly more follicles, which explains the increase in weight of AMH null ovaries, compared with wild type, observed in this and our previous study (10). Analyses of follicle numbers throughout the estrous cycle showed that in particular the number of preantral follicles was increased, confirming a role for AMH in initial follicle recruitment (10, 12).

In cycling wild-type animals the secondary FSH surge at estrus results in the recruitment of small antral follicles (2, 21). Interestingly, in AMH null mice, this FSH-dependent recruitment of small antral follicles was more pronounced than in wild-type mice as seen in Fig. 1Go. The presence of more follicles at the appropriate stage in AMH null mice could explain this enhanced recruitment. However, it is also possible that in the absence of AMH follicles display an increased sensitivity toward FSH. Indeed, in our previous in vivo study, we observed more growing follicles in AMH null mice in presence of both low and high serum FSH levels. In our in vitro study, we observed that FSH-dependent follicle growth of cultured mouse preantral follicles was inhibited by AMH (11). In addition, in the present study, we also observed an increased recruitment at an earlier follicular stage in the absence of AMH. This enhanced and premature recruitment in the presence of lower estrus FSH levels suggests an increased sensitivity toward FSH in AMH null mice. Despite this enhanced sensitivity, most of these follicles do not reach the preovulatory stage, suggesting that cyclic recruitment and selection for dominance may require different threshold levels of FSH. Such a difference in FSH threshold levels may be partially explained by the expression pattern of AMH and the AMH type II receptor in the ovary. AMH and its type II receptor display a similar expression pattern being expressed in small growing follicles with highest expression in large preantral and small antral follicles (13, 22, 23, 24). This window of AMH expression is also reflected by the AMH serum levels, which showed a small but significant increase at estrus, correlating with the increased number of the small antral follicles at estrus. Thus, in the AMH null mice, the lower FSH levels are sufficient to recruit the small growing follicles because the absence of AMH renders the follicles more sensitive to FSH. FSH sensitivity of large growing follicles is similar in wild-type and AMH null mice because the AMH type II receptor is not expressed in follicles of this size class. As a result, FSH levels in AMH null mice are now too low to support the follicles for dominance and a normal number of ovulations ensues. Indeed, a rise in FSH levels in a superovulation scheme demonstrated that AMH null follicles can be rescued and supported to the preovulatory stage, resulting in a large increase in number of retrieved oocytes in AMH null mice, compared with wild-type mice. It has been shown that the increased number of follicles capable of ovulating upon gonadotropin administration reflects a reduction in the number of follicles undergoing atresia (25, 26). Indeed, the number of oocytes retrieved in both wild-type and AMH null mice corresponds with the total number (nonatretic and atretic) of small antral follicles observed at estrus.

With increasing age, the number of retrieved oocytes from AMH null mice declined to numbers similar to those obtained in wild-type mice. This age-related decline in number of retrieved oocytes could reflect the depletion of the primordial follicle pool of AMH null mice at an earlier age, compared with wild-type mice (10). On the other hand, it cannot be ruled out that the enhanced cyclic recruitment of follicles weakens in aging AMH null mice due to age-related changes in the ovarian feedback system. Nevertheless, the absence of AMH did not seem to affect the capacity of oocytes to be fertilized and develop further because the percentage of two-cell embryos and (hatched) blastocysts obtained from the retrieved oocytes did not differ between AMH null and wild-type mice. These findings show that the correction of follicle numbers in AMH null mice is not dependent of oocyte/follicle quality but is an intrinsic mechanism of the ovary to obtain the species-specific number of preovulatory follicles.

Our results suggest that the correction of follicle number occurs at two levels. In both AMH null and wild-type mice, atretic follicles were observed during the large preantral and small antral stage. These findings are in agreement with previous studies in rodents showing that early antral follicles are most susceptible to atresia (15, 27). However, throughout the estrous cycle, ovaries of AMH null mice contained significantly more atretic follicles, compared with wild-type mice, thereby neutralizing the enhanced cyclic recruitment observed in AMH null mice. Because FSH is the major survival factor for early antral follicles, the blunted FSH surge in AMH null mice most likely is the predominant cause for this increase in atresia at this follicular stage, as discussed above, although a direct effect of AMH on follicle survival cannot be ruled out. Raised inhibin levels could explain the blunted FSH surge in AMH null mice because inhibin suppresses FSH synthesis and FSH release from the pituitary gland (28). In our previous study (10), we observed increased serum inhibin levels at estrus in AMH null mice. Unfortunately, we were unable to measure serum inhibin levels in this study.

Despite the increase in number of atretic follicles, atresia may not be the sole explanation for the normalization of follicle numbers in AMH null mice. We observed a major decline in follicle numbers during the transition from small preantral to large preantral follicles. This drop in preantral follicle number was not reflected by a corresponding level of atretic follicles. However, the duration of full follicular development (primordial to preovulatory follicle) should also be taken into account. Estimates suggest that complete follicular development lasts 19 d or even more than 5 wk (reviewed in Ref. 29). Pedersen (30) showed that, in particular, the transition from small preantral to large preantral follicle requires 14 d. Therefore, conclusions based on follicle numbers obtained during one cycle have to be made with caution. Nevertheless, the decline in follicle number during the small preantral to large preantral stage was more pronounced in AMH null mice, suggesting that, in addition to follicular atresia, other mechanisms may play a role in establishing the number of follicles that will continue to grow to the preovulatory stage after initial recruitment. The limited data available suggest that postnatal oocyte apoptosis also contributes to the fine-tuning of follicle growth and atresia (14, 17, 31, 32). Loss of follicles through oocyte degeneration occurs predominantly during the early follicular stages, from primordial up to small antral follicles. However, little is known about the mechanisms responsible for apoptosis in oocytes of immature follicles. It has been suggested that local factors, either antiapoptotic or proapoptotic stimuli, regulate oocyte apoptosis (14, 32), in contrast to follicular atresia in which FSH plays a dominant role.

Loss of proapoptotic Bax expression in Bax null mice and targeted expression of antiapoptotic Bcl2 in oocytes resulted in fewer atretic small preantral follicles, as determined by the number of oocyte remnants, with more nonatretic follicles (18, 19). Interestingly, similar to AMH null mice, also in these mouse models no difference in the ovulation rate was observed between transgenic and wild-type mice despite the increase in number of growing follicles. These findings support the presence of two regulatory mechanisms for number of follicles: a gonadotropin-independent mechanism resulting in oocyte atresia and a gonadotropin-dependent mechanism resulting in follicular atresia. In AMH null mice, both mechanisms appear to be enhanced because we also observed, besides the increase in follicular atresia, an increased number of oocyte remnants. It has been suggested that the degeneration of oocytes is a fast process (17, 33). However, it is unclear how long their remnants remain present in the ovary.

Our results suggest that AMH plays a role in fine-tuning the balance of follicle growth and death by serving as a survival factor for small growing follicles, either directly or indirectly through regulation of other factors. Furthermore, our results show that in particular initial recruitment is a wasteful process because the major loss of follicles occurs before the FSH-dependent recruitment of follicles. A better understanding of the process of follicle recruitment might provide tools to control early folliculogenesis, which in the long run might help to preserve ovarian function in women at risk for premature ovarian failure, such as young women treated for childhood cancer. Furthermore, our results suggest that AMH contributes in determining the FSH threshold levels required for cyclic follicle recruitment. Interestingly, serum AMH levels are increased in patients with polycystic ovarian syndrome (34, 35), in which the number of small antral follicles is generally increased. It is possible that AMH contributes to the aberrant selection of follicles. Increased knowledge of the mechanisms underlying cyclic recruitment may prove to be useful in understanding the pathophysiology of polycystic ovarian syndrome.


   Acknowledgments
 
The authors thank Bas Karels for histological support.


   Footnotes
 
Disclosure Statement: J.A.V., A.L.L.D., I.J.J.P., E.R.v.d.H., U.M.R., P.K., and F.H.d.J. have nothing to disclose. A.P.N.T. consults for Diagnostic Systems Laboratories.

First Published Online January 25, 2007

Abbreviations: AMH, Anti-Müllerian hormone; AMHKO, AMH knockout; CL, corpora lutea.

Received September 14, 2006.

Accepted for publication January 17, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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