help button home button Endocrine Society Endocrinology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Endocrinology, doi:10.1210/en.2007-0674
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
148/11/5451    most recent
Author Manuscript (PDF)
Right arrow Purchase Article
Right arrow View Shopping Cart
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Clelland, E. S.
Right arrow Articles by Peng, C.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Clelland, E. S.
Right arrow Articles by Peng, C.
Endocrinology Vol. 148, No. 11 5451-5458
Copyright © 2007 by The Endocrine Society

Inhibition of Premature Oocyte Maturation: A Role for Bone Morphogenetic Protein 15 in Zebrafish Ovarian Follicles

Eric S. Clelland, Qian Tan, Ari Balofsky, Rosie Lacivita and Chun Peng

Department of Biology, York University, Toronto, Ontario, Canada M3J 1P3

Address all correspondence and requests for reprints to: Dr. Chun Peng, Department of Biology, York University, 4700 Keel Street, Toronto, Ontario, Canada M3J 1P3. E-mail: cpeng{at}yorku.ca.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Bone morphogenetic protein-15 (BMP-15) is a member of the TGF-β superfamily known to regulate ovarian functions in mammals. Recently, we cloned zebrafish BMP-15 (zfBMP-15) cDNA and demonstrated that it may play a role in oocyte maturation. In this study, we further investigated the role of BMP-15 in zebrafish follicular development and oocyte maturation using an antiserum developed for zfBMP-15 and by microinjection of follicles with antisense zfBMP-15 N-morpholino oligonucleotides or an expression construct containing zfBMP-15 cDNA. Injection with antiserum caused a significant decrease in maturation-incompetent [insensitive to maturation-inducing hormone (MIH)] early growth phase follicles and a concomitant increase in mature follicles in vivo. In vitro maturation assays showed that incubation with antiserum resulted in a significant increase in oocyte maturation as compared with follicles incubated in preimmune serum or media control. Next, early growth phase follicles were collected and preincubated with either antiserum, preimmune serum, or medium control before treatment with MIH or human chorionic gonadotropin (hCG). Antiserum significantly increased oocyte maturation in response to MIH, but not to hCG, and enhanced basal maturation rate in longer-term incubations. Knockdown of BMP-15 in early growth stage follicles with a BMP-15 antisense oligonucleotide resulted in increased oocyte maturation, whereas microinjection of BMP-15 cDNA into oocytes significantly reduced MIH- and hCG-induced oocyte maturation in normally competent, mid-growth-phase follicles. Collectively, these findings suggest that BMP-15 modulates follicular growth and prevents premature oocyte maturation in zebrafish, in part, by suppressing the sensitivity of follicles to MIH.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE TGF-β SUPERFAMILY of growth and differentiation factors has been shown to play important roles in regulating mammalian ovarian functions such as follicular development and maturation, production of steroids, and regulation of gonadotropin receptors (1, 2, 3, 4, 5, 6). Less is known about the role of TGF-β superfamily in the ovary of lower vertebrates such as fish. Activin and inhibin have been reported to stimulate oocyte maturation in zebrafish (7, 8, 9), whereas TGF-β has an inhibitory effect on oocyte maturation in zebrafish (10) and steroid production in goldfish (11). Bone morphogenetic protein-15 (BMP-15) mRNA is highly expressed in the zebrafish ovary, testis, and brain. Neutralization of endogenous BMP-15 enhances basal and human chorionic gonadotropin (hCG)-induced zebrafish oocyte maturation, whereas addition of recombinant human BMP-15 (rhBMP-15) has the opposite effect (12). Anti-Müllerian hormone (13) and growth and differentiation factor-9 (GDF-9) (14) are also expressed in zebrafish ovary and may modulate ovarian function.

The discovery of GDF-9 and BMP-15 (also known as GDF-9B) has significantly advanced our understanding of early follicular development, particularly in mammalian systems. GDF-9 is the product of a somatic gene and is expressed in a variety of tissues including ovary, testis, brain, bone, etc. (15, 16). BMP-15 is the product of an X-linked gene in mammals (17, 18) and was previously reported on chromosome 6 in zebrafish, based on comparison of the cDNA sequence with the zebrafish genomic sequence (12). However, in the latest version of the zebrafish genome ensemble (http://www.ensembl.org/Danio_rerio/blastview/), the gene is now assigned to chromosome 7. In mammals, BMP-15 is highly expressed in oocytes (17, 18, 19) and pituitary (20) and to a lesser extent in other tissues (21). It is widely expressed in zebrafish with the highest expression in gonads (12).

GDF-9 and BMP-15 are intraovarian regulatory factors. In early, pituitary hormone-independent follicles, they act as mitogens to stimulate the growth and differentiation of granulosa and theca cells and regulate follicular response to FSH (22), exerting control over the expression of other regulatory factors such as kit ligand (23, 24) and gremlin (25). Knockout of GDF-9 arrests follicular development at the primary stage in the mouse (26), whereas naturally occurring mutations in sheep BMP-15 genes such as FecXG,B,I,H,L (27, 28, 29) or the GDF-9 gene FecGH (27) cause primary follicle arrest in homozygous ewes, although interestingly, heterozygous ewes are hyperfertile (30). Mutations in BMP-15 and GDF-9 genes have also been implicated in human premature ovarian failure (31, 32, 33, 34). GDF-9 may also play a role in differentiation of primordial follicles because it was recently reported that knockdown of GDF-9 using small interfering RNA blocked the FSH-induced differentiation of somatic cells to primordial follicles in hamsters (35). In antral follicles, GDF-9 controls antrum expansion and steroid synthesis by regulating the expression of cyclooxygenase 2 (Cox2), hyaluronan synthase 2 (Has2), steroidogenic acute regulator protein (StAR), LH receptor, and urokinase plasminogen activator (uPA) (36). Recent studies suggest BMP-15 acts synergistically with GDF-9 to regulate these factors (37, 38). They also work cooperatively to stimulate inhibin production and down-regulate FSH-induced progesterone secretion (39, 40) and to prevent premature follicular atresia (41).

Although the important role of BMP-15 in mammalian oocytes has become increasingly clear, there remains scarce information regarding its role in fish ovaries. To better understand the role of BMP-15 in the zebrafish ovary, a series of experiments was conducted. Here we describe the effect of immunoneutralizing endogenous BMP-15 on oocyte maturation in vitro and on follicle numbers in vivo. The effects of BMP-15 knockdown (by N-morpholino oligonucleotide microinjection) or overexpression of BMP-15 [via microinjection of zebrafish BMP-15 (zfBMP-15) expression construct] on oocyte maturation in vitro are also described. Our observations are discussed with regard to the functional role of BMP-15 in zebrafish ovary particularly as pertains to follicular development and oocyte maturation.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Zebrafish were obtained from a local supplier (Fish and Bird Emporium, Churchill, Ontario, Canada). Fish were maintained under a 14-h light, 10-h dark cycle at 28 C in 10-liter tanks in a circulating freshwater AHAB system (Aquatics Habitats, Apopka, FL). Fish were fed twice daily, ad libitum, with Basic Food Flakes (Tropic Aquaria, Brampton, Ontario, Canada) supplemented daily with newly hatched brine shrimp (San Francisco Bay Brand, Newark, CA). Fish were killed in accordance with the regulations of the Canada Council for Animal Care. They were anesthetized in tricaine (3-aminobenzoic acid ethyl ester; Sigma-Aldrich Canada, Oakville, Ontario, Canada) and decapitated.

Antiserum
A peptide (MQTFISELGVADIPL) epitope located at the C terminus of zfBMP-15 was selected as the antigen for production of polyclonal antiserum as previously described (12). Sodium azide (0.02%) was added to the serum as preservative upon receipt from the supplier (Sigma Genosys, Oakville, Ontario, Canada). To remove the sodium azide before in vitro or in vivo application, antiserum or preimmune serum was centrifuged at 4 C for 5 min at 13,000 x g through 10-kDa cutoff Microcon columns (Millipore Corp., Bedford, MA) as per the manufacturer’s instructions. The sera were stored at –20 C until use.

Oocyte maturation assays
Fish were killed as described above. Ovaries from eight to 10 females were excised, rinsed, and placed into sterile Cortland’s medium at room temperature (8). The ovaries were teased into separate follicles using transfer pipettes (Samco Scientific Corp., San Fernando, CA) without trypsinization. Fish follicular development has been classified into five stages (42): stages I and II, previtellogenic follicles with sizes smaller than 0.34 mm; stage III (0.35–0.69 mm), the growth phase when vitellogenesis takes place; stage IV (greater than 0.69 mm), follicles undergoing maturation; and stage V, mature eggs (Fig. 1Go). Previous studies have shown that small stage III follicles (stage III-1, 0.35–0.51 mm) are incompetent to undergo maturation in response to hormones, whereas larger follicles (stage III-2, 0.52–0.69 mm) are sensitive to maturation-inducing hormones (MIH) (8, 42, 43). Therefore, follicles were staged according to their size and oocyte maturation assays conducted as previously described (12). Briefly, follicles were incubated in 1 ml medium (~20 per well) at 28 C in 24-well culture plates. Cortland’s medium (control) was supplemented with BMP-15 antiserum or preimmune serum (1:100 dilution), recombinant hCG (kindly provided by Dr. A. F. Parlow, National Hormone and Peptide Program, Torrance, CA; 100 ng/ml), or the MIH 17{alpha},20β-dihydroxyprogesterone (Sigma-Aldrich; 10 ng/ml), as indicated. In some experiments, antiserum was neutralized using 10 µg/ml of the synthetic target epitope before incubation. Preincubation experiments were conducted using MIH-insensitive stage III-1 follicles. These follicles were incubated for 18 h with antiserum, preimmune serum, or control media, rinsed, and incubated for either an additional 6 h with control or MIH or for an additional 24 h with control or hCG.


Figure 1
View larger version (72K):
[in this window]
[in a new window]

  		FIG. 1. Schematic drawing (A) and micrograph (B) of zebrafish folliculogenesis. In the primary growth stage (stage I), follicles appear translucent and are ranged from 7–140 µm in size. Stage II follicles (0.15–0.34 mm) have cortical alveoli, typically surrounding the germinal vesicle. Vitellogenesis takes place in stage III follicles, whereas oocyte maturation occurs in stage IV. During oocyte maturation, germinal vesicle (GV) migrates from center to the periphery and the GV membrane breaks down. Stage V is the mature egg.

 
Intraperitoneal injection of gravid zebrafish
Female zebrafish were briefly anesthetized, blotted dry, and weighed. Fish were injected ip (1 µl/100 mg body weight) with antiserum, preimmune serum, or saline (Cortland’s medium without added BSA), allowed to recover for about 5 min in still water, and then transferred to 3-liter tanks in the AHAB system. A maximum of five similarly injected fish were placed in each tank. Fish were fed normally and killed 72 h after injection. The ovaries from each fish were removed and rinsed in saline. One ovary was placed in a 5-cm culture dish containing clean saline and teased into separate follicles. The follicles were collected and stored in 4% paraformaldehyde in saline at 4 C until counted. The contralateral ovary was rinsed in saline, blotted dry, and flash frozen in liquid nitrogen for future analyses. Fixed follicles were later rinsed in saline to remove the formaldehyde, staged, and counted. The proportion of follicles at various developmental stages was determined for each individual fish.

Expression construct of zfBMP-15
The open reading frame of zfBMP-15 cDNA (GenBank accession number AY954923) (12) was inserted into the pCMV-4B expression vector (Stratagene, La Jolla, CA) upstream of a flag tag and transformed into DH5{alpha} Escherichia coli cells. Empty vector and zfBMP-15 plasmid DNA was isolated using a high-speed maxi-prep kit (QIAGEN Canada, Mississauga, Ontario, Canada) and quantified by UV spectrophotometry. Human embryonic kidney 293T cells (American Type Culture Collection, Manassas, VA) were transfected with 15 µg plasmid DNA using 25-kDa polyethylenimine for 5 h (Sigma) as previously described (44). The cells were washed and cultured for 48 h in DMEM (Invitrogen) containing 10% fetal bovine serum.

N-morpholino oligonucleotides
The antisense morpholino oligonucleotide spanning the start codon of zfBMP-15 gene (5'-CCGCTGGTAGCCTTCATGTTCAGCC-3') and a missense N-morpholino control oligonucleotide (5'-CCcCTcGTAGgCTTCATcTTCAcCC-3') were purchased from GeneTools LLC (Philomath, OR).

Microinjection
Ovarian follicles were microinjected with N-morpholino oligonucleotides or rzfBMP-15 plasmid DNA to assess the effects of BMP-15 knockdown or overexpression on oocyte maturation. Glass micropipettes were pulled from 1-mm glass tubes on a WPI model PUL-1 horizontal pipette puller (World Precision Instruments Inc., Sarasota, FL). Microinjection was completed using a WPI model PV830 Pneumatic Pico Pump supplied by nitrogen gas. The pico pump was adjusted until the volume of the microinjected solution was approximately 1 nl, based on the diameter of the solution droplets measured under the dissecting microscope.

For knockdown studies, the N-morpholinos were diluted to 0.5 mM. Antisense or missense oligonucleotides (approximately 0.5 ng/follicle) or water control was injected into stage III-1 follicles. The follicles were allowed to sit at room temperature for 30–60 min, and any that showed signs of leakage were discarded. The remaining follicles were incubated for 24 h in Cortland’s medium and scored for oocyte maturation, and 80–100 follicles for each injection group were scored in an experiment. For overexpression studies, mid-growth-phase follicles with diameters ranging from 0.50–0.60 mm were microinjected with zfBMP-15 plasmid DNA or its control vector (1 ng/follicle). The injected follicles were incubated for 6 h, treated with MIH for 6 h or with hCG for 24 h, and scored for oocyte maturation. Each experiment was repeated three to five times.

Protein extraction and Western blotting
The 293T cells were lysed in RIPA buffer and centrifuged at 13,000 x g at 4 C for 30 min to collect the supernatant. Zebrafish follicle lysates were prepared by homogenizing approximately 80 follicles in lysis buffer, followed by 30 sec of sonication. Protein concentration was determined using a Bradford assay (45), and 10 µg total protein from BMP-15 overexpression experiments or 20 µg total protein from BMP-15 knockdown experiments were separated by SDS-PAGE and transferred to a polyvinylidene difluoride membrane (Millipore). The membrane was washed in Tris-buffered saline/Tween 20 and blocked with 5% skim-milk plus 2% fetal bovine serum in Tris-buffered saline/Tween 20 and probed with zfBMP-15 antiserum (1:2000), anti-flag (Sigma A2220, 1:1000), and anti-acetylated tubulin (Sigma T6793, 1:1000) antibodies. The primary antibodies were removed, the blot was washed and probed with horseradish peroxidase-conjugated secondary antibody [1:5000, antimouse (NA931V) or antirabbit (NAV934V); Amersham Biosciences Inc., Baie d’Urfe, Quebec, Canada] and signal detected by ECL (ECL Plus; Amersham).

Statistical analyses
Where a single stage of follicle was used in an experiment, differences in oocyte maturation were analyzed by one-way ANOVA and Student-Newman-Keuls testing for normally distributed data or Kruskal-Wallis testing when normality testing failed, using InStat (Graphpad Software, San Diego, CA). Results are expressed as mean ± SEM for the number of experiments (n) given in each figure. P ≤ 0.05 was considered significant, although the P value presented on each figure is the least significant value obtained for that analysis. For the in vitro experiment testing the effect of antiserum on various growth stage follicles (see Fig. 3Go) and the in vivo injection experiment (see Fig. 4Go), the effect of treatment was determined for each stage independently using ANOVA as above.


Figure 3
View larger version (13K):
[in this window]
[in a new window]

  		FIG. 3. Effects of BMP-15 antiserum on maturation of oocytes in different sizes of follicles. Follicles at stages III and IV were separated into different groups according to their sizes and incubated with Cortland’s medium, preimmune serum (PIS), or BMP-15 antiserum (AS, 1:100 dilution) for 24 h. Bars represent mean ± SEM of three experiments. Different letters denote statistical significance occurring within each size group (P ≤ 0.01).

 

Figure 4
View larger version (16K):
[in this window]
[in a new window]

  		FIG. 4. BMP-15 antiserum causes precocious follicular development and oocyte maturation in zebrafish in vivo. A, Female zebrafish (n = 5–8 fish per group) were ip injected with saline or anti-BMP-15 antiserum (AS) at different dilution (undiluted or diluted 1:5 or 1:50 with saline) at the volume of 1 µl/100 mg body weight. The fish were killed 72 h after injection, and the follicles from each fish were staged and counted. B, Undiluted AS, preimmune serum (PIS), or saline was injected into female zebrafish (n = 15–16 fish per group), and follicles were staged at 72 h after injection. Bars represent mean ± SEM. Letters above bars denote statistical significance (P < 0.05).

 

   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effects of BMP-15 antiserum on oocyte maturation in vitro
We have previously shown that incubation with zfBMP-15 antiserum leads to enhanced maturation of zebrafish oocytes (12). To confirm the specificity of the BMP-15 antiserum, large growth phase follicles were incubated for 24 h in medium containing Cortland’s medium (control), antiserum, or preimmune serum with or without neutralizing target epitope (Fig. 2AGo). Maturation was clearly enhanced by the antiserum (P ≤ 0.001) but not significantly affected by the preimmune serum. The effect of antiserum on oocyte maturation was neutralized by the addition of the target epitope. Representative micrographs of stage III-2 follicles incubated for 24 h (Fig. 2BGo) are provided to illustrate the maturation assay. These results confirm the specificity of the effect of BMP-15 antiserum on zebrafish oocyte maturation.


Figure 2
View larger version (46K):
[in this window]
[in a new window]

  		FIG. 2. Target epitope neutralizes the effect of BMP-15 antiserum on oocyte maturation. A, Stage III-2 zebrafish follicles were incubated in Cortland’s medium (control), BMP-15 antiserum, or preimmune serum (1:100 dilution), with or without the target epitope that was used to generate the BMP-15 antibody (10 µg). BMP-15 antiserum significantly enhanced oocyte maturation, and this effect was inhibited by the target epitope. Bars represent mean ± SEM of three experiments. Different letters denote statistical significance (P ≤ 0.01). B, Representative micrographs of 24-h in vitro maturation experiments showing ooplasmic clearing: Cortland’s medium (1 ), BMP-15 antiserum (2 ), and MIH incubation (3 ). Scale bars, ~0.65 mm.

 
To determine whether the effect of BMP-15 on oocyte maturation is dependent on the developmental stage of the oocytes, we tested the effect of BMP-15 antiserum on oocyte maturation using different stages of follicles. When we selected stage III and IV follicles and divided them into groups based on their sizes, we found that small follicles at early growth phase (stage III-1, 0.35–0.51 mm in diameter), which do not mature in response to MIH, hCG, or activin (8), showed greatest response to the BMP-15 antiserum when compared with the controls. Although the BMP-15 antiserum led to higher mean percentages of oocytes undergoing maturation, no significant differences between the antiserum treatment and the controls were found in later developmental stages (Fig. 3Go).

Effects of BMP-15 antiserum on follicular development in vivo
To determine the function of BMP-15 on follicular development, female zebrafish were injected with saline control or antiserum either undiluted, 1:5 diluted, or 1:50 diluted in saline, and 3 d later, the fish were killed and follicles staged and counted as described (Fig. 4AGo). Undiluted antiserum produced the most pronounced changes in follicle number, particularly in stage III-1 and stages IV and V (P ≤ 0.05), and this concentration was used in subsequent experiments. Gravid females were injected with saline control, antiserum, or preimmune serum and killed 72 h after injection (Fig. 4BGo). The follicles from a single ovary of each fish were staged, quantified, and expressed as the percentage of the total number. Neither antiserum nor preimmune serum injection had a significant effect on the number of primary and cortical alveolar follicles. However, antiserum injection caused a very significant decrease (P ≤ 0.001) in the number of early growth phase (stage III-1) follicles, compared with control and preimmune serum-injected fish. A concomitant increase in the number of maturing follicles and mature eggs (stages IV and V) was also observed. The BMP-15 antiserum also had a moderate effect on the number of stage III-2 follicles compared with control but was not significantly different from preimmune serum injection. There was no significant difference in follicle numbers between saline and preimmune serum-injected fish.

Effects of BMP-15 antiserum on sensitivity of oocytes to gonadotropin and MIH
The results from both in vitro and in vivo studies suggest that BMP-15 acts on the small growth phase follicles. Because oocytes in this stage are incompetent to undergo maturation in response to LH analog or MIH, we examined whether BMP-15 may play a role in regulating the sensitivity of oocytes to hCG and MIH. Stage III-1 follicles were pretreated with antiserum, preimmune serum, or Cortland’s medium (control) for 18 h, followed by incubation with MIH for 6 h (Fig. 5AGo) or hCG for 24 h (Fig. 5BGo) because MIH and hCG usually induce oocyte maturation after 2–6 and 18–24 h of incubation, respectively (10). Similar to previous reports (8, 43), neither MIH nor hCG induced oocyte maturation in these small early stage follicles. However, in follicles pretreated with BMP-15 antiserum, MIH increased oocyte maturation by 4-fold when compared with the control (Fig. 5AGo). Antiserum pretreatment induced 2- to 3-fold higher oocyte maturation rate compared with those preincubated in preimmune serum or Cortland’s medium (Fig. 5AGo). However, this increase is statistically significant only in follicles that were further incubated for 24 h (Fig. 5BGo). Interestingly, hCG did not enhance oocyte maturation in any of the pretreatment groups (Fig. 5BGo).


Figure 5
View larger version (15K):
[in this window]
[in a new window]

  		FIG. 5. BMP-15 antiserum induces responsiveness of small growth follicles to MIH. Stage III-1 follicles were incubated with Cortland’s medium, antiserum, or preimmune serum for 18 h, washed, and further incubated with MIH (10 ng/ml) for 6 h (A) or hCG (100 ng/ml) for 24 h (B). Bars represent mean ± SEM of three to four experiments. Statistical differences are denoted by different letters (P ≤ 0.01).

 
Effects of zfBMP-15 overexpression and knockdown on oocyte maturation
To further confirm the physiological role of BMP-15 in oocyte maturation, we used microinjection to overexpress BMP-15 or to silence expression of BMP-15 in the zebrafish oocytes. First, an expression plasmid carrying the coding cDNA sequence of zfBMP-15 was made, and the expression of recombinant zfBMP-15 was confirmed in 293 cells. The 293 cells were transfected with the zfBMP-15 cDNA or the empty vector (as negative control); cell lysates were collected at 48 h after transfection and probed with both anti-flag and anti-BMP-15 antisera. As shown in Fig. 6AGo, both antibodies detected three bands with approximate molecular mass of 45, 28, and 14 kDa in zfBMP-15 cDNA-transfected cells. Similarly, three protein bands were detected in zebrafish oocytes microinjected with zfBMP-15 cDNA but not in its negative control. In mid-growth-phase follicles, microinjection with zfBMP-15 cDNA significantly inhibited (P ≤ 0.001) both MIH-induced (Fig. 6BGo) and hCG-induced oocyte maturation (Fig. 6CGo) relative to empty vector injections. On the other hand, microinjection of an antisense N-morpholino oligonucleotide to knock down endogenous BMP-15 produced a significant increase (P ≤ 0.001) in oocyte maturation of small growth phase follicles, compared with missense morpholino injection or water control (Fig. 7AGo). The efficiency of the antisense oligonucleotide knockdown was confirmed by Western blots (Fig. 7BGo).


Figure 6
View larger version (14K):
[in this window]
[in a new window]

  		FIG. 6. Overexpression of BMP-15 suppresses MIH- and hCG-induced oocyte maturation. Mid-growth-phase follicles (0.50–0.60 mm) were microinjected with plasmid DNA carrying the coding sequence of zfBMP-15 or its control vector. A, Western blot analysis of BMP-15 in oocyte lysates or 293T cell lysates using anti-flag and anti-BMP-15 antibodies. Equal loading was confirmed by Western blot probed with antitubulin antibody. BMP15, pCMV4B-zfBMP-15; EV, empty vector (pCMV4B). B, Oocytes were injected with zfBMP-15 plasmid DNA and at 6 h after injection incubated with or without MIH (10 ng/ml) for 6 h. C, Oocytes were injected with zfBMP-15 plasmid DNA and at 6 h after injection incubated with or without hCG (100 ng/ml) for 24 h. Bars represent mean ± SEM of four (B) or three (C) experiments. Different letters denote statistical significance (P ≤ 0.001).

 

Figure 7
View larger version (19K):
[in this window]
[in a new window]

  		FIG. 7. Knockdown of BMP-15 by N-morpholino oligonucleotide increases maturation in small growth phase oocytes. A, Stage III-1 follicles (0.35–0.51 mm) were injected with antisense morpholino or missense morpholino oligonucleotides (0.5 ng/follicle) or water, and 24 h after injection, oocyte maturation was scored. Bars represent mean ± SEM of five experiments (oligos) or three experiments (water). Different letters denote statistical significance (P ≤ 0.001). B, The efficiency of the knockdown was confirmed by Western blot, using anti-BMP-15 antibody. Equal loading was confirmed by Western blot using antitubulin antibody.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In the present study, we have continued our investigation into the role of BMP-15 in zebrafish ovaries. We confirmed our previous findings that neutralization of BMP-15 using its antiserum enhances oocyte maturation. We provided novel in vivo data that suggest that BMP-15 mainly acts on early growth phase follicles to inhibit their growth and maturation. Finally, using overexpression and gene-silencing approaches, we showed that knockdown of BMP-15 induced precocious oocyte maturation in early growth phase follicles, whereas overexpression of BMP-15 reduced gonadotropin- and MIH-induced oocyte maturation in hormone-sensitive growth phase follicles. Taken together, our findings demonstrate that BMP-15 plays a physiological role in the zebrafish ovary to prevent precocious oocyte maturation.

In the present study, anti-zfBMP-15 antiserum was used in both in vivo and in vitro experiments to determine the role of BMP-15 in the zebrafish ovary. Several lines of evidence suggest that the effect of the antiserum is specific. First, the peptide we used to produce the antiserum was carefully selected to reduce the possibility of cross-reactivity with other related proteins. For example, only four of 15 amino acids are identical to its closest homolog, zebrafish GDF9. Second, the effect of antiserum on oocyte maturation could be reversed by preincubation with the antigen that was used to develop the antiserum. Third, Western blot analyses showed that in zfBMP-15 cDNA-transfected 293 cells or injected zebrafish oocytes, the antiserum detected three protein species with the molecular sizes predicted to be the BMP-15 monomer, dimer, and precursor. These bands were also detected by an anti-flag antibody that reacts to the C-terminal flag tag of the zfBMP-15 expression construct. Finally, knockdown of BMP-15 using an antisense oligonucleotide produced similar effects as BMP-15 antiserum on oocyte maturation. Thus, the effects of BMP-15 antiserum on follicle growth in vivo and oocyte maturation in vitro are most likely due to its ability to neutralize endogenous BMP-15.

One of the most interesting findings of this study is that ip injection of BMP-15 antiserum affects follicular development in vivo. Because it takes about 10 d for follicles to progress through development from stage I to stage V (9, 46), we reasoned that 3 d would be sufficient to determine whether inhibition of endogenous BMP-15 by antiserum injection would have an effect on follicular development. When fish were examined 72 h after injection, BMP-15 antiserum injection caused a significant reduction in the number of stage III-1 follicles and a concomitant rise in the number of maturing and mature (stage IV and V) follicles. The results suggest inhibition of BMP-15 is insufficient to promote precocious development in previtellogenic follicles but can accelerate development in vitellogenic follicles. Immunoneutralization of BMP-15 and GDF-9 has been performed in vivo in sheep, where long-term inoculation leads to sterility in normal ewes (47, 48), whereas short-term inoculation before the breeding leads to increased fecundity (49), the effects of antiserum injection mimicking the effects of naturally occurring BMP-15 or GDF-9 gene mutations. Antisera has also been used to neutralize the effect of BMP-15 and GDF-9 in vitro, nullifying the effects of GDF-9 in mouse mural granulosa cells (50) and both GDF-9 and BMP-15 in ovine granulosa cells (40).

Fish ovarian follicular development is divided into the growth phase and maturation phase. In general, during the growth phase, oocytes accumulate vitellogenin and increase in size under the stimulation of FSH and estrogen (51). During oocyte maturation, LH induces MIH production (52) and enhances the expression of membrane progestin receptors (mPRs) (53). MIH binds to the mPRs, leading to activation of maturation-promoting factor and, subsequently, oocyte maturation (54). It has been reported that stage III-1 follicles do not respond to hCG or MIH (8, 43). Although the cause of such hormone insensitivity is not clear, several factors, such as activin, have been shown to increase the sensitivity of these follicles to MIH (43, 55, 56). Interestingly, in the present study, we found that suppression of endogenous BMP-15 expression using a morpholino antisense oligonucleotide or immunoneutralization of BMP-15 using its antiserum induced the precocious maturation of these small follicles. When we tested the effect of BMP-15 antiserum on oocyte maturation using follicles at different stages of development, we found that the BMP-15 antiserum has the most potent effect on oocyte maturation in stage III-1 follicles. Preincubation of stage III-1 follicles with BMP-15 antiserum rendered these follicles sensitive to MIH. However, BMP-15 antiserum did not significantly affect the sensitivity of follicles to hCG. Previously, we have shown that incubation of stage III-2 follicles with human recombinant BMP-15 reduced hCG-induced oocyte maturation to control levels (12). Here we report that microinjection of a zfBMP-15 expression construct into mid-growth-phase follicles also inhibits hCG- and MIH-induced oocyte maturation. These findings are consistent with data from the in vivo experiments that suggest that BMP-15 acts on small growth phase follicles to suppress the sensitivity of follicles to hormones and prevent them from undergoing maturation.

Although we found that BMP-15 can inhibit premature development and maturation of oocytes, the mechanisms underlying its action are not clear. Suppression of gonadotropin receptors may be a probable mechanism because BMP-15 has been shown to inhibit FSH receptors in mammals (57). However, this cannot explain the finding that suppression of endogenous BMP-15 by antisense oligonucleotide or antiserum is sufficient to induce oocyte maturation in vitro in the absence of gonadotropin or why hCG does not augment basal oocyte maturation in smaller follicles pretreated with the BMP-15 antiserum. Thus, the most likely sites of action for BMP-15 are the production of MIH and/or MIH receptors. Previously, we have shown that TGF-β1 down-regulates expression of 20β-hydroxysteroid dehydrogenase, a key enzyme involved in MIH production, and one of the isoforms of MIH receptors, mPRβ (58). These may also be potential targets of BMP-15 in the zebrafish ovary.

In mammals, BMP-15 and GDF-9 play critical roles in early folliculogenesis, but their relative importance appears to be species specific. BMP-15 is necessary for early folliculogenesis in monoovulatory species, such as human and sheep, but not in polyovulatory mice (22, 59). Although we did not observe significant effects of BMP-15 antiserum on stage I and stage II follicles in this study, more experimentation, especially with longer-term treatment of BMP-15 antiserum, is necessary to determine whether BMP-15 exerts an effect on early folliculogenesis. GDF-9 may play a role in this process because it has been shown that GDF-9 mRNA is highly expressed in previtellogenic follicles but falls significantly upon uptake of vitellogenin (14). Whether BMP-15 and GDF-9 protein levels are regulated throughout follicular development and maturation and whether these two factors cooperate to control folliculogenesis in fish remain to be determined.

In summary, using in vivo and in vitro studies along with gain-of-function and loss-of-function approaches, we have demonstrated that BMP-15 exerts regulatory effects of small early growth phase follicles to prevent precocious development and maturation of oocytes in zebrafish, mostly by suppressing their sensitivity to MIH.


   Acknowledgments
 
We are grateful to Dr. Shunichi Shimasaki of the Department of Reproductive Medicine, University of California, San Diego, for advice and discussion on the project and for critically reviewing the manuscript. We thank Dr. A. F. Parlow and National Hormone and Peptide Program for providing recombinant hCG.


   Footnotes
 
This study was supported by a grant from the Natural Science and Engineering Research Council of Canada and an Ontario Premier’s Research Excellent Award to C.P. C.P. is a recipient of a Mid-Career Award from Ontario Women’s Health Council and Canadian Institute of Health Research.

Disclosure Statement: The authors have nothing to declare.

First Published Online July 26, 2007

Abbreviations: BMP-15, Bone morphogenetic protein-15; GDF-9, growth and differentiation factor-9; hCG, human chorionic gonadotropin; MIH, maturation-inducing hormone; mPR, membrane progestin receptor; zfBMP-15, zebrafish BMP-15.

Received May 18, 2007.

Accepted for publication July 16, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Peng C 2003 The TGF-β superfamily and its roles in the human ovary and placenta. J Obstet Gynaecol Can 25:834–844[Medline]
  2. Peng C, Mukai ST 2000 Activins and their receptors in female reproduction. Biochem Cell Biol 78:261–279[CrossRef][Medline]
  3. Shimasaki S, Moore RK, Erickson GF, Otsuka F 2003 The role of bone morphogenetic proteins in ovarian function. Reprod Suppl 61:323–337[Medline]
  4. Knight PG, Glister C 2006 TGF-β superfamily members and ovarian follicle development. Reproduction 132:191–206[Abstract/Free Full Text]
  5. Knight PG, Glister C 2003 Local roles of TGF-β superfamily members in the control of ovarian follicle development. Anim Reprod Sci 78:165–183[CrossRef][Medline]
  6. Juengel JL, McNatty KP 2005 The role of proteins of the transforming growth factor-β superfamily in the intraovarian regulation of follicular development. Hum Reprod Update 11:143–160[Medline]
  7. Pang Y, Ge W 1999 Activin stimulation of zebrafish oocyte maturation in vitro and its potential role in mediating gonadotropin-induced oocyte maturation. Biol Reprod 61:987–992[Abstract/Free Full Text]
  8. Wu T, Patel H, Mukai S, Melino C, Garg R, Ni X, Chang J, Peng C 2000 Activin, inhibin, and follistatin in zebrafish ovary: expression and role in oocyte maturation. Biol Reprod 62:1585–1592[Abstract/Free Full Text]
  9. Ge W 2005 Intrafollicular paracrine communication in the zebrafish ovary: the state of the art of an emerging model for the study of vertebrate folliculogenesis. Mol Cell Endocrinol 237:1–10[CrossRef][Medline]
  10. Kohli G, Hu S, Clelland E, Di Muccio T, Rothenstein J, Peng C 2003 Cloning of transforming growth factor-β1 (TGF-β1) and its type II receptor from zebrafish ovary and role of TGF-β1 in oocyte maturation. Endocrinology 144:1931–1941[Abstract/Free Full Text]
  11. Calp MK, Matsumoto JA, Van Der Kraak G 2003 Activin and transforming growth factor-β as local regulators of ovarian steroidogenesis in the goldfish. Gen Comp Endocrinol 132:142–150[CrossRef][Medline]
  12. Clelland E, Kohli G, Campbell RK, Sharma S, Shimasaki S, Peng C 2006 Bone morphogenetic protein-15 in the zebrafish ovary: complementary deoxyribonucleic acid cloning, genomic organization, tissue distribution, and role in oocyte maturation. Endocrinology 147:201–209[Abstract/Free Full Text]
  13. Rodriguez-Mari A, Yan YL, Bremiller RA, Wilson C, Canestro C, Postlethwait JH 2005 Characterization and expression pattern of zebrafish Anti-Mullerian hormone (Amh) relative to sox9a, sox9b, and cyp19a1a, during gonad development. Gene Expr Patterns 5:655–667[CrossRef][Medline]
  14. Liu L, Ge W 2007 Growth differentiation factor 9 and its spatiotemporal expression and regulation in the zebrafish ovary. Biol Reprod 76:294–302[Abstract/Free Full Text]
  15. Wu X, Matzuk MM 2002 GDF-9 and BMP-15: oocyte organizers. Rev Endocr Metab Disord 3:27–32[CrossRef][Medline]
  16. Fitzpatrick SL, Sindoni DM, Shughrue PJ, Lane MV, Merchenthaler IJ, Frail DE 1998 Expression of growth differentiation factor-9 messenger ribonucleic acid in ovarian and nonovarian rodent and human tissues. Endocrinology 139:2571–2578[Abstract/Free Full Text]
  17. Dube JL, Wang P, Elvin J, Lyons KM, Celeste AJ, Matzuk MM 1998 The bone morphogenetic protein 15 gene is X-linked and expressed in oocytes. Mol Endocrinol 12:1809–1817[Abstract/Free Full Text]
  18. Laitinen M, Vuojolainen K, Jaatinen R, Ketola I, Aaltonen J, Lehtonen E, Heikinheimo M, Ritvos O 1998 A novel growth differentiation factor-9 (GDF-9) related factor is co-expressed with GDF-9 in mouse oocytes during folliculogenesis. Mech Dev 78:135–140[CrossRef][Medline]
  19. Otsuka F, Yao Z, Lee T, Yamamoto S, Erickson GF, Shimasaki S 2000 Bone morphogenetic protein-15. Identification of target cells and biological functions. J Biol Chem 275:39523–39528[Abstract/Free Full Text]
  20. Otsuka F, Shimasaki S 2002 A novel function of bone morphogenetic protein-15 in the pituitary: selective synthesis and secretion of FSH by gonadotropes. Endocrinology 143:4938–4941[Abstract]
  21. Galloway SM, McNatty KP, Cambridge LM, Laitinen MP, Juengel JL, Jokiranta TS, McLaren RJ, Luiro K, Dodds KG, Montgomery GW, Beattie AE, Davis GH, Ritvos O 2000 Mutations in an oocyte-derived growth factor gene (BMP15) cause increased ovulation rate and infertility in a dosage-sensitive manner. Nat Genet 25:279–283[CrossRef][Medline]
  22. Shimasaki S, Moore RK, Otsuka F, Erickson GF 2004 The bone morphogenetic protein system in mammalian reproduction. Endocr Rev 25:72–101[Abstract/Free Full Text]
  23. Otsuka F, Shimasaki S 2002 A negative feedback system between oocyte bone morphogenetic protein 15 and granulosa cell kit ligand: its role in regulating granulosa cell mitosis. Proc Natl Acad Sci USA 99:8060–8065[Abstract/Free Full Text]
  24. Thomas FH, Vanderhyden BC 2006 Oocyte-granulosa cell interactions during mouse follicular development: regulation of kit ligand expression and its role in oocyte growth. Reprod Biol Endocrinol 4:19[CrossRef][Medline]
  25. Pangas SA, Jorgez CJ, Matzuk MM 2004 Growth differentiation factor 9 regulates expression of the bone morphogenetic protein antagonist gremlin. J Biol Chem 279:32281–32286[Abstract/Free Full Text]
  26. Yan C, Wang P, DeMayo J, DeMayo FJ, Elvin JA, Carino C, Prasad SV, Skinner SS, Dunbar BS, Dube JL, Celeste AJ, Matzuk MM 2001 Synergistic roles of bone morphogenetic protein 15 and growth differentiation factor 9 in ovarian function. Mol Endocrinol 15:854–866[Abstract/Free Full Text]
  27. McNatty KP, Smith P, Moore LG, Reader K, Lun S, Hanrahan JP, Groome NP, Laitinen M, Ritvos O, Juengel JL 2005 Oocyte-expressed genes affecting ovulation rate. Mol Cell Endocrinol 234:57–66[CrossRef][Medline]
  28. Bodin L, Di Pasquale E, Fabre S, Bontoux M, Monget P, Persani L, Mulsant P 2007 A novel mutation in the bone morphogenetic protein 15 gene causing defective protein secretion is associated with both increased ovulation rate and sterility in Lacaune sheep. Endocrinology 148:393–400[Abstract/Free Full Text]
  29. Chu MX, Sang LH, Wang JY, Fang L, Ye SC 2005 [Study on BMP15 and GDF9 as candidate genes for prolificacy of Small Tail Han sheep]. Yi Chuan Xue Bao 32:38–45 (Chinese)[Medline]
  30. Hanrahan JP, Gregan SM, Mulsant P, Mullen M, Davis GH, Powell R, Galloway SM 2004 Mutations in the genes for oocyte-derived growth factors GDF9 and BMP15 are associated with both increased ovulation rate and sterility in Cambridge and Belclare sheep (Ovis aries). Biol Reprod 70:900–909[Abstract/Free Full Text]
  31. Di Pasquale E, Beck-Peccoz P, Persani L 2004 Hypergonadotropic ovarian failure associated with an inherited mutation of human bone morphogenetic protein-15 (BMP15) gene. Am J Hum Genet 75:106–111[CrossRef][Medline]
  32. Di Pasquale E, Rossetti R, Marozzi A, Bodega B, Borgato S, Cavallo L, Einaudi S, Radetti G, Russo G, Sacco M, Wasniewska M, Cole T, Beck-Peccoz P, Nelson LM, Persani L 2006 Identification of new variants of human BMP15 gene in a large cohort of women with premature ovarian failure. J Clin Endocrinol Metab 91:1976–1979[Abstract/Free Full Text]
  33. Dixit H, Rao LK, Padmalatha V, Kanakavalli M, Deenadayal M, Gupta N, Chakravarty B, Singh L 2005 Mutational screening of the coding region of growth differentiation factor 9 gene in Indian women with ovarian failure. Menopause 12:749–754[CrossRef][Medline]
  34. Dixit H, Rao LK, Padmalatha VV, Kanakavalli M, Deenadayal M, Gupta N, Chakrabarty B, Singh L 2006 Missense mutations in the BMP15 gene are associated with ovarian failure. Hum Genet 119:408–415[CrossRef][Medline]
  35. Wang C, Roy SK 2006 Expression of growth differentiation factor 9 in the oocytes is essential for the development of primordial follicles in the hamster ovary. Endocrinology 147:1725–1734[Abstract/Free Full Text]
  36. Elvin JA, Clark AT, Wang P, Wolfman NM, Matzuk MM 1999 Paracrine actions of growth differentiation factor-9 in the mammalian ovary. Mol Endocrinol 13:1035–1048[Abstract/Free Full Text]
  37. Hussein TS, Thompson JG, Gilchrist RB 2006 Oocyte-secreted factors enhance oocyte developmental competence. Dev Biol 296:514–521[CrossRef][Medline]
  38. Su YQ, Wu X, O’Brien MJ, Pendola FL, Denegre JN, Matzuk MM, Eppig JJ 2004 Synergistic roles of BMP15 and GDF9 in the development and function of the oocyte-cumulus cell complex in mice: genetic evidence for an oocyte-granulosa cell regulatory loop. Dev Biol 276:64–73[CrossRef][Medline]
  39. McNatty KP, Juengel JL, Reader KL, Lun S, Myllymaa S, Lawrence SB, Western A, Meerasahib MF, Mottershead DG, Groome NP, Ritvos O, Laitinen MP 2005 Bone morphogenetic protein 15 and growth differentiation factor 9 co-operate to regulate granulosa cell function in ruminants. Reproduction 129:481–487[Abstract/Free Full Text]
  40. McNatty KP, Juengel JL, Reader KL, Lun S, Myllymaa S, Lawrence SB, Western A, Meerasahib MF, Mottershead DG, Groome NP, Ritvos O, Laitinen MP 2005 Bone morphogenetic protein 15 and growth differentiation factor 9 co-operate to regulate granulosa cell function. Reproduction 129:473–480[Abstract/Free Full Text]
  41. Hussein TS, Froiland DA, Amato F, Thompson JG, Gilchrist RB 2005 Oocytes prevent cumulus cell apoptosis by maintaining a morphogenic paracrine gradient of bone morphogenetic proteins. J Cell Sci 118:5257–5268[Abstract/Free Full Text]
  42. Selman K, Wallace RA, Sarka A, Qi X 1993 Stages of oocyte development in the zebrafish, Brachydanio rerio. J Morphol 218:203–224[CrossRef]
  43. Pang Y, Ge W 2002 Gonadotropin and activin enhance maturational competence of oocytes in the zebrafish (Danio rerio). Biol Reprod 66:259–265[Abstract/Free Full Text]
  44. Xu G, Zhong Y, Munir S, Yang BB, Tsang BK, Peng C 2004 Nodal induces apoptosis and inhibits proliferation in human epithelial ovarian cancer cells via activin receptor-like kinase 7. J Clin Endocrinol Metab 89:5523–5534[Abstract/Free Full Text]
  45. Bradford M 1976 A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254[CrossRef][Medline]
  46. Wang Y, Ge W 2004 Developmental profiles of activin βA, βB, and follistatin expression in the zebrafish ovary: evidence for their differential roles during sexual maturation and ovulatory cycle. Biol Reprod 71:2056–2064[Abstract/Free Full Text]
  47. Juengel JL, Hudson NL, Heath DA, Smith P, Reader KL, Lawrence SB, O’Connell AR, Laitinen MP, Cranfield M, Groome NP, Ritvos O, McNatty KP 2002 Growth differentiation factor 9 and bone morphogenetic protein 15 are essential for ovarian follicular development in sheep. Biol Reprod 67:1777–1789[Abstract/Free Full Text]
  48. McNatty KP, Hudson NL, Whiting L, Reader KL, Lun S, Western A, Heath DA, Smith P, Moore LG, Juengel JL 2006 The effects of immunizing sheep with different BMP15 or GDF9 peptide sequences on ovarian follicular activity and ovulation rate. Biol Reprod 76:552–560[CrossRef][Medline]
  49. Juengel JL, Hudson NL, Whiting L, McNatty KP 2004 Effects of immunization against bone morphogenetic protein 15 and growth differentiation factor 9 on ovulation rate, fertilization, and pregnancy in ewes. Biol Reprod 70:557–561[Abstract/Free Full Text]
  50. Gilchrist RB, Ritter LJ, Cranfield M, Jeffery LA, Amato F, Scott SJ, Myllymaa S, Kaivo-Oja N, Lankinen H, Mottershead DG, Groome NP, Ritvos O 2004 Immunoneutralization of growth differentiation factor 9 reveals it partially accounts for mouse oocyte mitogenic activity. Biol Reprod 71:732–739[Abstract/Free Full Text]
  51. Nagahama Y, Yoshikuni M, Yamashita M, Tokumoto T, Katsu Y 1995 Regulation of oocyte growth and maturation in fish. Curr Top Dev Biol 30:103–145[Medline]
  52. Patino R, Yoshizaki G, Thomas P, Kagawa H 2001 Gonadotropic control of ovarian follicle maturation: the two-stage concept and its mechanisms. Comp Biochem Physiol B Biochem Mol Biol 129:427–439[CrossRef][Medline]
  53. Tokumoto M, Nagahama Y, Thomas P, Tokumoto T 2006 Cloning and identification of a membrane progestin receptor in goldfish ovaries and evidence it is an intermediary in oocyte meiotic maturation. Gen Comp Endocrinol 145:101–108[CrossRef][Medline]
  54. Thomas P, Pang Y, Zhu Y, Detweiler C, Doughty K 2004 Multiple rapid progestin actions and progestin membrane receptor subtypes in fish. Steroids 69:567–573[CrossRef][Medline]
  55. Wang Y, Ge W 2003 Spatial expression patterns of activin and its signaling system in the zebrafish ovarian follicle: evidence for paracrine action of activin on the oocytes. Biol Reprod 69:1998–2006[Abstract/Free Full Text]
  56. Pang Y, Ge W 2002 Epidermal growth factor and TGF{alpha} promote zebrafish oocyte maturation in vitro: potential role of the ovarian activin regulatory system. Endocrinology 143:47–54[Abstract/Free Full Text]
  57. Otsuka F, Yamamoto S, Erickson GF, Shimasaki S 2001 Bone morphogenetic protein-15 inhibits follicle-stimulating hormone (FSH) action by suppressing FSH receptor expression. J Biol Chem 276:11387–11392[Abstract/Free Full Text]
  58. Kohli G, Clelland E, Peng C 2005 Potential targets of transforming growth factor-β1 during inhibition of oocyte maturation in zebrafish. Reprod Biol Endocrinol 3:53[CrossRef][Medline]
  59. Pangas SA, Matzuk MM 2004 Genetic models for transforming growth factor β superfamily signaling in ovarian follicle development. Mol Cell Endocrinol 225:83–91[CrossRef][Medline]




This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
148/11/5451    most recent
Author Manuscript (PDF)
Right arrow Purchase Article
Right arrow View Shopping Cart
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Clelland, E. S.
Right arrow Articles by Peng, C.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Clelland, E. S.
Right arrow Articles by Peng, C.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Endocrinology Endocrine Reviews J. Clin. End. & Metab.
Molecular Endocrinology Recent Prog. Horm. Res. All Endocrine Journals