| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Regular Article |
b Departments of Epidemiology/Preventive Medicine,
c Anatomy/Neurobiology, and
d Medicine, University of Maryland School of Medicine, Baltimore, Maryland 21201
e National Institute of Allergy and Infectious Diseases, Frederick, Maryland 21702
ABSTRACT
Little is known about the embryonic factors that regulate the size of the primordial follicle endowment at birth. A few studies suggest that members of the B-cell lymphoma/leukemia-2 (bcl-2) family of protooncogenes may be important determinants. Thus, the purpose of this study was to test whether bcl-2 regulates the size of the primordial follicle pool at birth. To test this hypothesis, three lines of transgenic mice (c-kit/bcl-2 mice) were generated that overexpress human bcl-2 in an effort to reduce prenatal oocyte loss. The overexpression was targeted to the ovary and appropriate embryonic time period with the use of a 4.8-kilobase c-kit promoter. This promoter provided two to three times more expression of bcl-2 in the ovaries with minimal or no overexpression in most nongonadal tissues. On Postnatal Days 8–60, ovaries were collected from homozygous c-kit/bcl-2 and nontransgenic littermates (controls) and processed for histological evaluation of follicle numbers. All lines of c-kit/bcl-2 mice were born with significantly more primordial follicles than control mice (P
0.05). By Postnatal Days 30–60, however, there were no significant differences in follicle numbers between c-kit/bcl-2 and control mice. These results indicate that bcl-2 overexpression increases the number of primordial follicles at birth, but that the surfeit of primordial follicles is not maintained in postnatal life. These data suggest that it is possible that the ovary may contain a census mechanism by which excess numbers of primordial follicles at birth are detected and removed from the ovary by adulthood.
apoptosis, follicle, gene regulation, oocyte development, ovary
INTRODUCTION
Each female mammal is born with a finite reserve of primordial follicles in her ovaries [1]. This reserve is deposited in embryonic life. In mammals, germ cells migrate from outside the embryo proper, through the dorsal mesentery of the hind-gut, to the indifferent gonad [1–4]. The germ cells then lose their motile characteristics and begin to proliferate rapidly [3–5]. As a result, the number of germ cells increases dramatically. For example, the number of germ cells increases from approximately 25 000 on Embryonic Day (ED) 14.5 to about 85 000 by ED 18.5 in the mouse [3]. Following this extensive proliferation, the number of germ cells is reduced by almost half via mechanisms that involve apoptosis [6–8]. The remaining germ cells eventually enter meiosis and become quiescent [9, 10]. At this time, the germ cells are termed oocytes [1]. Many of these oocytes, but not all, will be surrounded by somatic cells to form the primordial follicles [1].
Although studies suggest that proliferation and apoptosis play critical roles in the establishment of the primordial follicle pool, little is known about the factors that regulate these processes. Until recently, most work has focused on the role of the c-kit receptor and its ligand (steel factor) in ovarian development [11–19]. In female mice, c-kit is abundant in proliferating germ cells, absent in germ cells once they undergo the transition to early meiosis, and detectable again once the oocytes enter the diplotene stage of meiosis after birth and through adulthood [11, 14, 15, 20]. During embryonic life, c-kit and its ligand play a central role in directing migration and survival of germ cells [15, 21–25]. During adulthood, it is believed that the c-kit receptor and steel factor continue to regulate follicular function by maintaining meiotic arrest and promoting growth and differentiation of thecal cells [20, 22].
The B-cell lymphoma/leukemia-2 (bcl-2) family of protooncogenes may also be involved in the establishment of the primordial follicle pool. Bcl-2 family members can be divided into two distinct groups, those that promote apoptosis (e.g., bax, bcl-xshort, bid, and bad) and those that promote cell survival (e.g., bcl-2, bcl-xlong, and bcl-w) [26–29]. Several investigators have shown that these factors play a central role in regulating apoptosis in nonreproductive and reproductive tissues [6, 26, 29–37]. Thus, the purpose of this study was to test the hypothesis that the cell survival factor, bcl-2, helps regulate the size of the primordial follicle pool at birth. Specifically, the goals of this study were to 1) generate transgenic mice that overexpress bcl-2 in the developing ovary, 2) test whether simple overexpression of bcl-2 during embryogenesis is sufficient to increase the size of the primordial follicle pool at birth, and 3) determine whether an increase in the size of the primordial follicle pool at birth affects the number of follicles in postnatal life.
MATERIALS AND METHODS
Construction of the Transgene
The c-kit promoter (4.8 kilobase [kb]) was subcloned into NotI-HindIII sites in the human bcl-2 cDNA open reading frame (1.9 kb) in pBlueScript II KS. The c-kit promoter was provided by Drs. Kitamura and Tsujimura (Department of Pathology, Osaka University Medical School, Osaka, Japan). The human bcl-2 cDNA was provided by Dr. Stanley Korsmeyer (Program in Molecular Oncology, Dana Farber Cancer Institute, Boston, MA). The c-kit/bcl-2 transgene construct (6.7 kb) then was used to generate three lines of homozygous transgenic mice (c-kit/bcl-2 mice) as described below.
Generation of Transgenic Animals
The c-kit/bcl-2 construct was injected into the pronucleus of fertilized oocytes from FVB/N donors and the fertilized eggs were transferred to pseudopregnant dams. Pregnant dams were allowed to deliver pups naturally. At 3 wk, the pups were screened for the presence of the c-kit/bcl-2 transgene via Southern blot analysis. First, genomic DNA was extracted from tail snips using proteinase K digestion and phenol-chloroform extraction. DNA (40 µg) then was digested with BamHI, resolved through 1.0% agarose gels, and blotted to nylon membranes. The nylon membranes were prehybridized in QuikHyb solution (Stratagene, Inc., La Jolla, CA) in sealed plastic bags at 65°C. After 1 h, 32P-labeled probe (EcoRI fragment of human bcl-2) was added to the bag. This probe was selected because it hybridizes to two main sequences of DNA, the transgenic human bcl-2 and the endogenous mouse bcl-2 (Fig. 1). The blot then was incubated for 2 h at 60°C, washed twice in 1x saline-sodium citrate buffer (SSC) containing 0.1% SDS at room temperature, and washed twice in 0.1x SSC containing 0.1% SDS at 65°C. The washed blot then was wrapped in plastic wrap and exposed to autoradiographic film overnight at -70°C.
|
Mice were considered negative for the c-kit/bcl-2 transgene (-) if there was a single band for the endogenous mouse bcl-2 (Fig. 1). These negative mice were bred together to establish a line of nontransgenic mice (controls, -/-). Founder (F1) mice were considered heterozygous for the c-kit/bcl-2 transgene (+) if there was a band for the transgene and the endogenous mouse bcl-2 (Fig. 1). Heterozygous mice from the same founder line were bred together to generate mice that were homozygous (+/+) for the c-kit/bcl-2 transgene.
To ensure that potential homozygotes were true homozygotes, they were bred with control mice (-/-). The offspring then were screened for the presence of the c-kit/bcl-2 gene using the polymerase chain reaction (PCR), forward primers to the c-kit promoter (5'-CCT GTC TTA GAG GCA CAA GC-3'), and reverse primers to the human bcl-2 gene (5'-CTT CTC CCA GCG TCG CCA T-3'). The conditions for the PCR were as follows: 30 cycles of 94°C for 45 sec, 55°C for 1 min, and 72°C for 3 min. PCR products then were subjected to agarose gel electrophoresis. The presence of a 320-base pair (bp) band indicated that a mouse was positive for the c-kit/bcl-2 transgene and the absence of a 320-bp band indicated that a mouse was negative for the transgene. Potential homozygotes that gave birth to a litter of offspring that all were positive for the transgene were considered to be true homozygotes. These mice then were mated with other true homozygotes from the same founder line to establish three colonies (lines) of c-kit/bcl-2 homozygotes.
All transgenic and control mice were housed in the University of Maryland School of Medicine Animal Facility, maintained under a 12D:12L cycle, provided food and tap water ad libitum. At selected time points (Postnatal Days [PDs] 8, 12, 27, and 60), some animals were killed by halothane inhalation, their ovaries were removed and processed for histological evaluation, immunohistochemistry, or Western blot analysis. PN 8 was selected because it is a time point when the primordial follicle pool is fully formed in the mouse [1], PN 12 was selected because it is a time point when follicular growth is fully in process [1], PN 27 was selected because it is a time point shortly after puberty is completed, and PN 60 was selected because it is a time point when the mice should be cycling adults [1]. All procedures involving animal care, killing, and tissue collection were approved by the University of Maryland School of Medicine Institutional Animal Use and Care Committee and conducted in accordance with the Guide for Care and Use of Laboratory Animals.
Immunohistochemistry for Bcl-2
Ovaries were fixed in 4% paraformaldehyde for at least 24 h. After fixation, the tissues were dehydrated, embedded in Paraplast (VWR Scientific, Baltimore, MD), serially sectioned (5 µm), and mounted on glass slides. The sections then were processed for immunolocalization of human Bcl-2 using a commercially available monoclonal antibody against human Bcl-2 (1:50 dilution; Dako Corp., Carpinteria, CA) as the primary antibody. The secondary antibody and visualization reagents were used according to the manufacturer's instructions from the HistoMouse-SP Kit (Zymed Laboratories, San Francisco, CA). As a control for background staining, some tissues were incubated with secondary antibody, but no primary antibody.
Western Blot Analysis
At PNs 8–60, ovaries were collected from transgenic and control mice and immediately snap-frozen in a dry ice and ethanol bath. In some experiments, nongonadal tissues (i.e., liver, kidney, skin, brain, and bladder) were also collected and snap-frozen in a dry ice and ethanol bath. Each frozen sample was gently homogenized in lysis buffer (40 mM Tris-HCL pH 8.0, 1% NP40, 150 mM NaCl, 2.5 mM EDTA, 20 mM NaF, 20% glycerol) containing a protease inhibitor cocktail tablet (Roche Diagnostics GmbH, Mannheim, Germany). After homogenization, the amount of protein in each sample was evaluated using a bicinchoninic acid (BCA) protein assay kit (Pierce, Rockford, IL). Protein lysates (40 µg/lane) then were subjected to Western blot analysis using a 1:1000 dilution of monoclonal antibody specific for Bcl-2 (Dako) as the primary antibody and a 1:2000 dilution of horseradish peroxidase (HRP)-conjugated anti-mouse polyclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA) as the secondary antibody. The Bcl-2 antibody cross-reacts with the human and mouse proteins, allowing the detection of both proteins in the Western blot. Immune complexes were visualized using an enhanced chemiluminescence (ECL) detection kit (Cell Signaling Technologies, Beverly, MA). To ensure that proteins were loaded equally in each lane, the blots were stripped using ImmunoPure elution buffer (Pierce) and incubated with ß-actin (1:1000 dilution, Santa Cruz Biotechnology) and HRP-conjugated anti-mouse polyclonal antibody. Scanning densitometry then was used to estimate the relative increase in bcl-2 expression in transgenic mice compared with control mice.
Histological Evaluation of Follicle Numbers
Ovaries were fixed in Kahle's solution (4% formalin, 28% ethanol, and 0.34 N glacial acetic acid) for at least 24 h. After fixation, the tissues were dehydrated, embedded in Paraplast (VWR Scientific), serially sectioned (8 µm), mounted on glass slides, and stained with Weigert's hematoxylin-picric acid methylene blue. A stratified sample consisting of every tenth section was used to estimate total number of primordial, primary, preantral, and antral follicles per ovary. The selected sections from one ovary (approximately 20–30 sections per ovary) were randomized and the number of primordial, primary, preantral, and antral follicles was counted in each of the entire sections. Only follicles with a visible nucleus were counted to avoid double counting. In addition, all follicles were counted without knowledge of the genotype, transgenic line, and age of the animal.
Follicles were scored as "primordial" if they contained an intact oocyte with a visible nucleolus surrounded by a single layer of fusiform-shaped granulosa cells. Follicles were scored as "primary" if they consisted of an intact, enlarged oocyte with a visible nucleolus and a single layer of cuboidal granulosa cells. Follicles were scored as "preantral/antral" if they contained an oocyte with a visible nucleolus and more than one layer of granulosa or thecal cells. In several cases, follicles of this type also contained antral spaces. Often, follicles contained a single granulosa layer that consisted of both fusiform and cuboidal cells. Follicles of this type were scored as primordial even though they were likely to be primordial follicles undergoing transition to the primary stage. Transitional follicles were scored in this way because there were no significant differences in the number of transitional follicles between control and transgenic animals. In addition, only a small percentage of follicles per ovary (less than 10%) were in transition from the primordial to the primary stage at the selected time points. In some ovaries, follicles contained more than one layer of cuboidal granulosa cells, but less than two complete layers. Follicles of this type were counted as preantral/antral follicles. To obtain an estimate of the total number of follicles per ovary, the number of primordial, primary, or preantral/antral follicles present in the marked sections was multiplied by 10 to account for the fact that every tenth section was used in the analysis and by 8 to account for section thickness.
Statistical Analysis
All data were analyzed using SPSS statistical software (SPSS, Inc., Chicago, IL). The mean number of follicles was calculated using ovaries from at least five different animals per genotype at each time point. Differences between the mean number of follicles in transgenic and control ovaries were evaluated by both the Mann-Whitney rank test and ANOVA, with statistical significance assigned at P 0.05. When a significant P value was obtained with ANOVA, the Scheffé test was used in the post hoc analysis.
The mean fold increase in bcl-2 expression in transgenics compared with controls was calculated using protein lysates from at least four separate pools of ovaries (approximately 4–6 ovaries per pool). Differences between the fold increase in bcl-2 expression over time were evaluated by ANOVA, with statistical significance assigned at P 0.05. When a significant P value was obtained with ANOVA, the Scheffé test was used in the post hoc analysis. All results are presented as mean ± SEM.
RESULTS
Transgenic Animals
Three founder mice (two females and one male) were identified by Southern blot analysis (Fig. 1). When these founder mice were used to generate three lines of homozygous c-kit/bcl-2 mice, the transmission of the transgene appeared to follow Mendelian inheritance patterns. In addition, there did not appear to be any effects of the transgene on the gross appearance of the mice, pregnancy rates, litter size, birth weight, or the age at vaginal opening.
Bcl-2 Expression Pattern
The c-kit/bcl-2 mice expressed human bcl-2 in approximately 20%–30% of the oocytes of primordial follicles, whereas the control mice did not express human bcl-2 in the oocytes of primordial follicles (Fig. 2). This expression was present in ovaries from all three transgenic lines and did not appear to change over time (Fig. 3A). It also did not appear to differ consistently between the three transgenic lines of mice (P 
0.45). All three lines expressed approximately 2.9 ± 0.5-fold more bcl-2 than controls at PN 7 and still expressed about 2.3 ± 0.6-fold more bcl-2 than controls at PN 27. In contrast, there was minimal or no overexpression of the transgene in nongonadal tissues such as the liver, kidney, skin, brain, and bladder (Fig. 3B).
|
|
Effect of Bcl-2 on the Size of the Primordial Follicle at Birth
All three lines of transgenic mice were born with significantly more primordial follicles than controls (Fig. 4). Line 1 had approximately 23% more primordial follicles than controls (n = 16 for line 1, n = 11 for controls; P 0.01). Line 2 had 32% more primordial follicles than controls (n = 10 for line 2, n = 11 for controls; P 0.01) and line 3 had 73% more primordial follicles than controls (n = 5 for line 3, n = 11 for controls; P 0.01). In contrast, there were no significant differences in the numbers of primary and preantral/antral follicles between transgenic and control mice at PN 8 (Fig. 4).
|
Follicle Numbers in Postnatal Life
On PN 8, all three lines of transgenic mice had significantly more primordial follicles than controls (Figs. 4 and 5A; P 0.01). On PN 12, the transgenic mice still had more primordial follicles than controls (Fig. 5A; P 0.05). Line 1 had approximately 20% more primordial follicles than controls (n = 9 for line 1, n = 6 for controls; P 0.05). Line 2 had 34% more primordial follicles than controls (n = 8 for line 2, n = 6 for controls; P 0.05) and line 3 had 24% more primordial follicles than controls (n = 10 for line 3, n = 6 for controls; P 0.05). By PN 27, the mice from lines 1 and 2 had similar numbers of primordial follicles as controls, while the mice from line 3 still had approximately 33% more primordial follicles than controls (Fig. 5A, P 0.05). By PN 60, all three lines of transgenic mice had similar numbers of primordial follicles as controls (Fig. 5A).
|
There were no significant differences between the numbers of primary follicles in line 1, line 2, and controls at any time point (Fig. 5B). On PN 27, line 3 had 44% more primary follicles than controls (Fig. 5B, P 0.05), but this difference disappeared by PN 60. In addition, there were no significant differences between the numbers of preantral/antral follicles in the three transgenic lines compared with controls at any time point (Fig. 5C).
DISCUSSION
Every female mammal is born with a finite, nonrenewable supply of primordial follicles in her ovaries [1, 7]. Little is known about the embryonic factors that regulate the development of this endowment or how its size at birth affects the rates of follicular growth in postnatal life. Thus, one purpose of the current study was to test the hypothesis that simple overexpression of bcl-2 during embryonic life affects the size of the primordial follicle pool at birth. To test this hypothesis, we first generated transgenic mice that overexpress bcl-2 in the developing ovary. These mice are unique because the c-kit promoter drives overexpression of bcl-2 in the oocytes of developing primordial follicles. Other investigators have developed transgenic mice that overexpress bcl-2 in the ovary [35, 37]. These investigators, however, used either the inhibin 
subunit promoter or a fragment of the zona pellucida promoter to drive overexpression of bcl-2 in the postnatal ovary [35, 37]. The inhibin promoter targets the expression to the granulosa cells of large follicles, whereas the zona pellucida promoter targets the oocytes contained in primary and antral follicles [35, 37]. The phenotype in these animals differs from the phenotype in the c-kit/bcl-2 mice. Targeted overexpression of bcl-2 by the inhibin promoter inhibits apoptosis in large follicles and promotes germ cell tumorigenesis [35]. Targeted overexpression of bcl-2 by the zona pellucida promoter reduces the number of atretic small preantral follicles and prevents spontaneous and chemotherapy-induced apoptosis of the oocyte [37].
The current study is the first to show that bcl-2 can increase the size of the primordial follicle pool at birth. It is likely that bcl-2 increases the size of the primordial follicle pool by decreasing the number of germ cells that undergo apoptosis in embryonic life. This hypothesis is supported by studies that suggest that 1) the number of primordial follicles at birth is a function of the number of germ cells present in embryonic life, 2) massive numbers of germ cells are normally depleted from the developing ovary via apoptosis, and 3) bcl-2 acts as an antiapoptotic factor in the ovary and several nongonadal tissues [1, 6–8, 28, 29, 33–37].
Our study also examined the effect of the size of the primordial follicle pool at birth on follicle numbers in postnatal life. Many investigators propose that the number of follicles in the primordial endowment is the major factor that determines the timing and rate at which follicles begin to grow [1, 38–40]. It is believed that an excess number of primordial follicles may alter the number of follicles that grow to the primary, preantral, and antral stages. Our data do not support this hypothesis. In transgenic lines 1 and 2, an excess number of primordial follicles at birth did not change the number of follicles that grew to the primary, preantral, and antral follicles during postnatal life. In line 3, there was a brief increase in the number of primary follicles, but this increase was not maintained nor did it result in an increase in the number of preantral or antral follicles.
Our data differ from those derived from animals that were treated with busulfan, a chemical that destroys primordial germ cells of embryos [41]. When rats were exposed to various doses of busulfan in utero, there was an inverse correlation between the number of primordial follicles in the ovary at birth and the rate at which they began to grow. In rats given the highest dose of busulfan, the surviving follicles began to grow very early in life, exhausting the follicular reserve during the prepubertal period [41]. It is possible that a reduced size of the primordial follicle pool (as observed with busulfan treated animals), but not an increased size of the primordial follicle pool (as observed with c-kit/bcl-2 mice) alters the rate at which follicles grow. Perhaps there are compensatory mechanisms in the ovary to regulate follicular growth when the primordial follicle pool is depleted, but not when the primordial follicle pool is excessive.
The size of the primordial follicle pool has been proposed to affect the timing of estrous cyclicity and the menopausal transition [42–45]. Thus, we initially expected that mice born with an excess number of primordial follicles would maintain this excess, experience an early onset of cyclicity, experience a late age at reproductive senescence, or a combination of these. Instead, we found that mice born with a surfeit of primordial follicles did not maintain the excess in postnatal life. This finding suggests that the c-kit/bcl-2 mice may have a normal onset of cyclicity and age at reproductive senescence. In fact, our observations support this hypothesis because the transgenic mice experience vaginal opening (one marker for the onset of cyclicity) at the same time as nontransgenic controls.
It is likely that the excess primordial follicles in postnatal life are removed from the ovary via programmed cell death or other unknown mechanisms. This contention is supported by our data, which indicate that the excess number of primordial follicles simply "disappear" from the ovary and do not grow into larger follicles. However, we cannot rule out the possibility that the excess primordial follicles grow into antral follicles and that the antral follicles rapidly undergo atresia before we can detect them. We also cannot rule out the possibility that the hormonal changes that occur during puberty play a role in the elimination of the surfeit of primordial follicles. In two of the three transgenic lines, the excess number of primordial follicles was completely removed shortly after puberty (PN 27). In one transgenic line, the number of excess primordial follicles was significantly reduced after puberty (PN27) compared with before puberty (PN12).
Regardless of mechanism underlying the removal of the excess primordial follicles, it is likely that the follicles are not lost due to changes in the total amount of bcl-2 expression over time because our data indicate that bcl-2 overexpression is maintained at the times the follicle numbers return to normal. In lines 1 and 2, primordial follicles returned to normal by PN 27, a time when bcl-2 was still overexpressed in these two lines. In line 3, primordial follicle numbers returned to normal by PN 60 even though bcl-2 was still overexpressed at this time.
In conclusion, we have developed a transgenic mouse model that overexpresses bcl-2 in the oocytes of the developing ovary. We used this mouse model to show that simple overexpression of bcl-2 increases the size of the primordial follicle pool at birth and that an excess number of primordial follicles at birth is not maintained in postnatal life. Future studies should examine the mechanisms by which bcl-2 increases the size of the primordial follicle pool at birth because such studies will lead to an improved understanding of the factors that regulate the size of the primordial follicle pool at birth. Future studies should also examine the mechanisms by which excess primordial follicles at birth are removed from the ovary during postnatal life. Such studies will lead to an increased understanding of the factors that regulate follicular growth and atresia, the timing of estrous cyclicity, and the onset of reproductive senescence.
ACKNOWLEDGMENTS
We thank Ms. Lynn Van Ruiten for her help with the figures and Ms. Despina Almiroudis for her help with the Western blots. We also thank Dr. Jonathan Tilly who first conceived of the idea to generate transgenic mice using the c-kit and the bcl-2 open reading frame.
FOOTNOTES
First decision: 3 November 2000.
1 Supported by National Institutes of Health grant HD38955 to J.A.F., grant CA68033 to P.A.F., and grant AG13844 to A.N.H. 
2 Correspondence: Jodi A. Flaws, University of Maryland School of Medicine, Department of Epidemiology and Preventive Medicine, 660 W. Redwood Street, Baltimore, MD 21201. FAX: 410 706 1503; jflaws{at}epi.umaryland.edu
Accepted: November 15, 2000.
Received: October 10, 2000.
REFERENCES
This article has been cited by other articles:
|
|
 |
|
  C. M. Tingen, S. K. Bristol-Gould, S. E. Kiesewetter, J. T. Wellington, L. Shea, and T. K. Woodruff Prepubertal Primordial Follicle Loss in Mice Is Not Due to Classical Apoptotic Pathways Biol Reprod, July 1, 2009; 81(1): 16 - 25. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Rashidi and D. Shanley Evolution of the menopause: life histories and mechanisms Menopause Int, March 1, 2009; 15(1): 26 - 30. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J C Sadeu, T Adriaenssens, and J Smitz Expression of growth differentiation factor 9, bone morphogenetic protein 15, and anti-Mullerian hormone in cultured mouse primary follicles Reproduction, August 1, 2008; 136(2): 195 - 203. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. S. Albamonte, M. A. Willis, M. I. Albamonte, F. Jensen, M. B. Espinosa, and A. D. Vitullo The developing human ovary: immunohistochemical analysis of germ-cell-specific VASA protein, BCL-2/BAX expression balance and apoptosis Hum. Reprod., August 1, 2008; 23(8): 1895 - 1901. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. R Greenfeld, M. E Pepling, J. K Babus, P. A Furth, and J. A Flaws BAX regulates follicular endowment in mice Reproduction, May 1, 2007; 133(5): 865 - 876. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K.R. Barnett, C. Schilling, C.R. Greenfeld, D. Tomic, and J.A. Flaws Ovarian follicle development and transgenic mouse models Hum. Reprod. Update, September 1, 2006; 12(5): 537 - 555. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. Tomic, M. S. Frech, J. K. Babus, R. K. Gupta, P. A. Furth, R. D. Koos, and J. A. Flaws Methoxychlor Induces Atresia of Antral Follicles in ER{alpha}-Overexpressing Mice Toxicol. Sci., September 1, 2006; 93(1): 196 - 204. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
F. Jensen, M. A Willis, M. S Albamonte, M. B Espinosa, and A. D Vitullo Naturally suppressed apoptosis prevents follicular atresia and oocyte reserve decline in the adult ovary of Lagostomus maximus (Rodentia, Caviomorpha). Reproduction, August 1, 2006; 132(2): 301 - 308. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
W. Jefferson, R. Newbold, E. Padilla-Banks, and M. Pepling Neonatal Genistein Treatment Alters Ovarian Differentiation in the Mouse: Inhibition of Oocyte Nest Breakdown and Increased Oocyte Survival Biol Reprod, January 1, 2006; 74(1): 161 - 168. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. P. Miller, R. K. Gupta, C. R. Greenfeld, J. K. Babus, and J. A. Flaws Methoxychlor Directly Affects Ovarian Antral Follicle Growth and Atresia through Bcl-2- and Bax-Mediated Pathways Toxicol. Sci., November 1, 2005; 88(1): 213 - 221. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. Tomic, K. P. Miller, H. A. Kenny, T. K. Woodruff, P. Hoyer, and J. A. Flaws Ovarian Follicle Development Requires Smad3 Mol. Endocrinol., September 1, 2004; 18(9): 2224 - 2240. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Borgeest, K. P. Miller, R. Gupta, C. Greenfeld, K. S. Hruska, P. Hoyer, and J. A. Flaws Methoxychlor-Induced Atresia in the Mouse Involves Bcl-2 Family Members, but Not Gonadotropins or Estradiol Biol Reprod, June 1, 2004; 70(6): 1828 - 1835. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M Myers, K L Britt, N G M Wreford, F J P Ebling, and J B Kerr Methods for quantifying follicular numbers within the mouse ovary Reproduction, May 1, 2004; 127(5): 569 - 580. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Balla, N. Danilovich, Y. Yang, and M. R. Sairam Dynamics of Ovarian Development in the FORKO Immature Mouse: Structural and Functional Implications for Ovarian Reserve Biol Reprod, October 1, 2003; 69(4): 1281 - 1293. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Mazaud, C. J. Guigon, A. Lozach, N. Coudouel, M. G. Forest, H. Coffigny, and S. Magre Establishment of the Reproductive Function and Transient Fertility of Female Rats Lacking Primordial Follicle Stock after Fetal {gamma}-Irradiation Endocrinology, December 1, 2002; 143(12): 4775 - 4787. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Borgeest, D. Symonds, L. P. Mayer, P. B. Hoyer, and J. A. Flaws Methoxychlor May Cause Ovarian Follicular Atresia and Proliferation of the Ovarian Epithelium in the Mouse Toxicol. Sci., August 1, 2002; 68(2): 473 - 478. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Embree-Ku and K. Boekelheide Absence of p53 and FasL Has Sexually Dimorphic Effects on Both Development and Reproduction Experimental Biology and Medicine, July 1, 2002; 227(7): 545 - 553. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. Tomic, S.G. Brodie, C. Deng, R.J. Hickey, J.K. Babus, L.H. Malkas, and J.A. Flaws Smad 3 May Regulate Follicular Growth in the Mouse Ovary Biol Reprod, April 1, 2002; 66(4): 917 - 923. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. K. Pru and J. L. Tilly Programmed Cell Death in the Ovary: Insights and Future Prospects Using Genetic Technologies Mol. Endocrinol., June 1, 2001; 15(6): 845 - 853. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
