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Journal of Clinical Endocrinology & Metabolism , doi:10.1210/jc.2005-0219
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The Journal of Clinical Endocrinology & Metabolism Vol. 90, No. 8 4664-4670
Copyright © 2005 by The Endocrine Society

Germ Cell Proliferation and Apoptosis in the Developing Human Ovary

N. Fulton, S. J. Martins da Silva, R. A. L. Bayne and R. A. Anderson

Medical Research Council Human Reproductive Sciences Unit, Centre for Reproductive Biology, University of Edinburgh, Edinburgh EH16 4SB, United Kingdom

Address all correspondence and requests for reprints to: Dr. R. A. Anderson, Medical Research Council Human Reproductive Sciences Unit, University of Edinburgh Chancellors’ Building, 49 Little France Crescent, Edinburgh EH16 4SB, United Kingdom. E-mail: r.a.anderson{at}hrsu.mrc.ac.uk.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Context: The regulation of germ cell proliferation and loss during human ovarian development is poorly understood. This is of particular interest at the time leading up to the formation of primordial follicles, at 18 wk gestation onward.

Objective: The objective of the study was to identify and quantify germ cell proliferation and apoptosis and expression of caspases in the human fetal ovary.

Design: This study was a laboratory investigation.

Setting: The study was conducted at a research institute.

Methods: Cell proliferation and apoptosis were detected using immunohistochemical localization of phosphorylated histone H3 and cleaved caspase-3, respectively. Caspases were also detected by immunoblotting.

Results: The overall proportion of germ cells in mitosis remained constant between 14 and 19 wk but showed increasing clustering. Caspase-2, -3, -7, -8, and -9 were detected by immunoblotting. There was a significant increase in germ cell apoptosis. A specimen of 20 wk gestation showed similar phosphorylated histone H3 but markedly lower cleaved caspase-3 expression than earlier gestations. Cleaved caspase-3 was not expressed in oocytes that had formed primordial follicles.

Conclusions: These results indicate that as primordial follicle formation is initiated and progresses, there is an increase in both mitotic activity and apoptosis of those germ cells that have not reached the apparently protective environment of the primordial follicle.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
FEMALE REPRODUCTIVE POTENTIAL is characterized by the presence of a finite number of oocytes within primordial follicles in the ovary, whose depletion spells the menopause in the human. This number is primarily determined during fetal life when primordial follicles are formed after entry of germ cells into meiosis (whereupon they are known as oocytes) and their association with somatic cells. Primordial follicles are first observed in the human ovary from approximately 18 wk gestation. Although the number of primordial follicles then increases rapidly, the number of oocytes in the ovary shows a marked decline in all species (1, 2) to approximately 30% at the time of birth in the human.

Before this loss, there is considerable expansion of the germ cell number such that the total number of germ cells in the human ovary rises approximately 10-fold from some 300,000 at 2 months post coitum (pc, equivalent to 10 wk gestation) to a peak of 3.4 million at 5 months pc/22 wk gestation (2). In parallel to this, there is also ongoing loss of germ cells. The number of degenerating germ cells in the human ovary rises to a peak also at 5 months pc at which time some 20% of germ cells were identified as being atretic (2). Germ cell loss has been described as occurring in three waves (1, 2), with numerically the most important wave coincident with the appearance of primordial follicles. Whereas the human data are limited, they are in general in keeping with data from animal species. In the mouse the peak number of germ cells is found at embryonic d 13 (3), the time of transition from mitosis to meiosis, with approximately two thirds of germ cells lost by the time of birth. Recent detailed analysis of the germ cell population in the fetal mouse ovary has demonstrated a steady loss from embryonic d 13.5 to birth (4), i.e. over the duration of meiotic prophase, indicating the possible involvement of several mechanisms in this loss rather than a single one. This finding is in contrast to others who have reported that the decrease in germ cell number was more restricted in time to the early neonatal period, which was associated with the breakdown of clusters of oocytes and the formation of primordial follicles (5). In the sheep, a model of a large mammalian monoovulatory species, 75% of germ cells in the ovary are lost between gestation d 75 and 90, when primordial follicle formation is occurring (6, 7).

There is clear evidence that germ cell loss in the developing ovary occurs through apoptosis (8, 9, 10, 11), although other mechanisms associated with mitotic arrest may also be involved (12). Apoptotic cells have been identified by in situ labeling in both mouse (5) and human (13, 14, 15). Using two separate techniques [terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling and immunolocalization of cleaved poly(ADPribose) polymerase (PARP)], germ cell apoptosis appeared restricted to the period between 18 and 22 d pc, coincident with the breakdown of germ cell cysts (5). Comparable quantitative studies have not been performed using human ovaries. The Bcl-2 family of evolutionarily conserved, pro- and antiapoptotic proteins is implicated in the survival or demise of numerous cell types and has been identified as regulators of apoptosis within the mammalian ovary (16, 17). Involvement of these pathways in the determination of germ cell number in the human fetal ovary is indicated by the immunolocalization of some members, including Bcl-2, Mcl-1, and Bax (14, 15, 18). Caspases are the mammalian homologs of the cysteine protease CED3 in Caenorhabditis elegans, responsible for the execution of cell death. Three main families of caspases have been identified: initiators and effectors of apoptosis and those involved with cytokine activation (19, 20). Gene knockout experiments of Bcl2 and caspase family members have demonstrated their importance in oocyte and follicle apoptosis (21); however, there are no data regarding caspase expression in the developing human ovary.

In the present study, we investigated the expression of markers of cell proliferation [proliferating cell nuclear antigen (PCNA) and phosphorylated histone H3 (phospho-H3)] and key caspases in the developing human ovary. We quantified germ cell proliferation and apoptosis during the key period of ovarian development leading up to primordial follicle formation. These data allow comparison with other species in which such quantification has been performed and provide a basis for analysis of the causes and mechanisms of the regulation of germ cell number in the human ovary.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Tissue samples

Human fetal ovaries were obtained after medical termination of pregnancy from six to eight fetuses at each gestation. Written consent was obtained according to the national guidelines (22). The study was approved by the Lothian Pediatrics/Reproductive Medicine research ethics subcommittee. Termination of pregnancy was induced by treatment with mifepristone (200 mg, orally), followed by misoprostol (Pharmacia, Surrey UK; 200 mg every 3 h, per vaginam). None of the terminations was for reasons of fetal abnormality, and all fetuses used in this study appeared morphologically normal. Gestational age was determined by ultrasound examination before termination and was confirmed by subsequent direct measurement of foot length.

Ovaries were dissected and either stored at –70 C for subsequent protein extraction or fixed in Bouins fixative for 2 h, followed by transfer to 70% ethanol before processing into paraffin using standard histology techniques.

Immunohistochemistry

Paraffin-embedded ovaries were cut into 5-µm sections and mounted onto electrostatically charged microscope slides (VWR, Poole, UK), dried overnight, and then dewaxed and rehydrated using conventional methods. Endogenous peroxidases were quenched in 3% hydrogen peroxide in methanol for 30 min at room temperature. After a wash in water, slides were transferred into Tris-buffered saline [TBS; 0.05 M Tris, 0.85% NaCl (pH 7.6)] for 5 min and blocked for 30 min in normal serum (Diagnostics Scotland, Carluke, UK) diluted 1:4 in TBS containing 5% BSA. Sections were blocked with avidin (0.01 M; 15 min) and then biotin (0.001 M; 15 min; both from Vector Laboratories, Peterborough, UK) with washes in TBS in between.

Primary antibodies used were rabbit polyclonal IgG anti-phospho-H3 directed against serine 10 (Upstate Biotechnology, Milton Keynes, UK), rabbit polyclonal anticleaved caspase-3 (New England Biolabs, Hitchin, UK), rabbit polyclonal anti-c-kit (Dako Cytomation, Ely, Cambridgeshire, UK) and mouse monoclonal anti-PCNA (Dako). Antibodies were diluted 1:200, 1:50, 1:20, and 1:100, respectively. Antigen retrieval was performed in boiling citrate (0.01 M) for 2 min followed by immediate cooling in cold water for phospho-H3 and cleaved caspase-3. Antigen retrieval was not necessary for detection of c-kit and PCNA. Sections were incubated in the respective antibodies overnight at 4 C. Bound antibody was detected using a swine antirabbit biotinylated secondary antibody or a rabbit antimouse biotinylated secondary antibody diluted at 1:500 (Dako) for 30 min at room temperature followed by incubation in avidin/biotin horseradish peroxidase-linked complex (Dako) for 30 min at room temperature according to the manufacturer’s instructions. Visualization of the bound antibody was realized using 3, 3'-diaminobenzidine tetrahydrochloride (Dako). Sections were counterstained using hematoxylin.

Quantification of phospho-H3 and cleaved caspase 3 expression

Histological analyses were performed using a BH-2 microscope (Olympus, Tokyo, Japan) fitted with a Hitachi color camera and an automatic stage (Prior Scientific Instruments Ltd., Cambridge, UK). Image Pro Plus software 4.5.1 with Stereologer Pro 5 software (Media Cybernetics UK, Workingham, Berkshire, UK) was used for cell counts and diameter measurements. Analyses were performed to determine the number of immunostained cells both per unit area and as a proportion of the total number of germ cells present. Three representative sections from each ovary were analyzed. The total area of each section was measured, and the total number of positively stained cells (phospho-H3 or cleaved caspase-3) in the whole section was counted using the manual tag mode in the Image Pro Plus program, giving the number of immunostained cells per 10,000 µm2. To calculate the total number of germ cells in a tissue section to correct for changes in germ cell size, 20 random areas each of 12,877 µm2 were chosen by the Stereologer. The numbers of germ cells in each area were then counted, summed, and calculated as number of germ cells per unit area. Germ cells were identified by characteristic morphology, confirmed by c-kit immunostaining, particularly at earlier gestations and at the periphery of the sections. For each measure, the mean of values from the three sections was taken.

Measurement of germ cell diameter was performed on sections across the gestational range. Data were calculated as mean of two diameters at right angles. A total of 77–117 cells were counted for each gestational age.

Immunoblotting

Fetal ovaries were homogenized in lysis buffer containing 80 mM Tris (pH 6.8), 1% (vol/vol) glycerol, 1% (wt/vol) sodium dodecyl sulfate and a Complete Mini protease inhibitor cocktail tablet (Roche Diagnostics, Mannheim, Germany). Total protein quantification was performed using the BCA protein assay kit (Pierce, Rockford, IL). Protein extracts of human lymphoblastoid U937 cells were used as controls. Cells were ether untreated or UV irradiated for 5 min to induce apoptosis and then incubated at 37 C in 5% CO2 for 2 h.

Proteins were boiled in a 1:3 volume of 4x reduced sample buffer [20% 250 mM Tris (pH 6.8), 4% sodium dodecyl sulfate, 10% ß-mercaptoethanol, and 0.1% bromophenol blue]. Ten micrograms total protein was loaded onto either 5 or 12% SDS-PAGE gels with high-molecular-weight Rainbow protein markers run in parallel (Amersham Biosciences, Little Chalfont, UK). Gels were blotted onto polyvinyl difluoride membrane (Amersham Biosciences) and then blocked in TBS and Tween 20 [10 mM Tris (pH 7.5), 150 mM NaCl, 0.1% Tween 20] containing 5% powdered milk before overnight incubation at 4 C with primary antibody. Rabbit polyclonal antibodies to human caspase-2 (Abcam Ltd., Cambridge, UK) and caspase-3 (cleaved and uncleaved), -7, and -9, and PARP (New England Biolabs), and mouse monoclonal antibody to caspase-8 (New England Biolabs) were diluted 1:1000 in TBS and Tween 20 containing 5% powdered milk. Primary antibodies were omitted as a negative control. Bound antibody was detected using horseradish peroxidase-linked secondary antibodies diluted 1:10,000 (Cell Signaling Technology, New England Biolabs) and detected using the ECL Plus Western blotting detection system and Hyperfilm enhanced chemiluminescence (both from Amersham Biosciences).

Statistics

Data are expressed as mean ± SEM over 2-wk gestation intervals (14–15, 16–17, and 18–19 wk). Changes in expression of phospho-H3 and cleaved caspase 3 with gestation were analyzed by nonparametric ANOVA.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Germ cell proliferation

Initial experiments investigated the expression of PCNA as a widely used marker of cell proliferation. PCNA was observed in the majority of both somatic and germ cells of the fetal ovary at all gestations examined including endothelial cells and cells of the surface epithelium (Fig. 1Go, A and B). Although intense expression was observed in many oogonia, weaker PCNA staining persisted in the largest oocytes at later gestations, which were in the process of forming primordial follicles (Fig. 1BGo). The widespread distribution of PCNA did not render it a useful quantitative marker of proliferation; however, it does indicate active cell proliferation in the fetal ovary.



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  		FIG. 1. Immunohistochemical localization of PCNA and phospho-H3 in the human fetal ovary. PCNA expression, 16-wk (A) and 20-wk (B) ovaries. Phospho-H3 expression, 17-wk (C and D), 19-wk (E), and 20-wk (F) ovaries. Positive staining is brown, and sections are counterstained with hematoxylin. se, Surface epithelium; gc, germ cell; s, somatic cell; pf, primordial follicle; a, germ cell in anaphase. Scale bar (A), 20 µm; refers to all except D, 50 µm.

 
Phospho-H3 expression was explored as a potentially more quantifiable marker of mitosis. Specific immunostaining for phospho-H3 was detected in both germ cells and somatic cells at all gestations, but, in contrast to PCNA, it was expressed in only a few cells (Fig. 1Go, C–F). The majority of cells expressing phospho-H3 were germ cells, but immunopositive somatic cells were also detected. Although the low frequency of immunopositive germ cells within the ovary at all gestations with phospho-H3 expression did not reveal a striking gradient between the cortex and medulla, the presence of such a gradient was suggested by the finding that no oocytes within the more centrally located primordial follicles expressed phospho-H3 (Fig. 1FGo). Phospho-H3 expression in germ cells was quantified from 14 to 20 wk gestation (Fig. 2AGo). The number of immunopositive germ cells per unit area decline from 0.2 ± 0.04 per 10,000 µm2 at 14–15 wk to 0.11 ± 0.02 per 10,000 µm2 at 18–19 wk (P = 0.05). A single specimen at 20 wk gestation showed similar expression to those at 18–19 wk (0.12 per 10,000 µm2).



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  		FIG. 2. Quantification of phospho-H3 expression in the human fetal ovary. A, Number of phospho-H3-immunopositive germ cells per 10,000 µm2. B, Germ cell diameter. C, Number of germ cells per 10,000 µm2. D, Percentage of germ cells expressing phospho-H3. E, Clustering of phospho-H3-immunopositive germ cells. Data expressed as the percentage of phospho-H3-immunopositive germ cells in groups of two or more. Mean ± SEM, n = 6–8 ovaries from different fetuses in each gestational age group except 20 wk, n = 1. Data derive from 77–112 germ cells per gestation (B). *, P = 0.05; **, P ≤ 0.0001 by ANOVA.

 
The above results need to be taken in context. As germ cells increase in size with maturity, there will be fewer germs cells per unit area at later gestations. The above data may not therefore accurately reflect the proportion of germ cells that are mitotically active. Direct measurement of cell diameter confirmed an increase in germ cell size with increasing gestation from 12.3 ± 0.2 µm at 14–15 wk to 16.5 ± 0.6 µm at 20 wk (P < 0.0001, Fig. 2BGo). This was accompanied by a corresponding decrease in the total number of counted germ cells per unit area from 16.9 per 10,000 µm2 at 14–15 wk to 10.8 ± 0.5 per 10,000 µm2 (P = 0.0001, Fig. 2CGo). Recalculation of the number of germ cells expressing phospho-H3 as a percentage of the total number of germ cells present per unit area using these data showed no change with increasing gestation, 1.02 ± 0.20% at 14–15 wk vs. 0.99 ± 0.18% at 18–19 wk (Fig. 2DGo).

Whereas most germ cells expressing phospho-H3 were surrounded by immunonegative cells, it was apparent that in all specimens some were arranged in clusters of two cells or more. The number of phospho-H3 expressing germ cells in clusters as a proportion of the total number of immuno-positive cells was calculated in each specimen. This showed that the proportion of phospho-H3 expressing germ cells in clusters increased with gestation from 27 ± 5% at 14–15 wk to 42 ± 4% at 18–19 wk (P = 0.05, Fig. 2EGo).

Germ cell apoptosis

Caspase expression in human fetal ovary was investigated by immunoblotting. Expression of uncleaved caspase-2, -3, -7, -8, and -9 was detected (Fig. 3AGo), as was expression of cleaved caspase-8. Cleaved caspase-3 (Fig. 3AGo), -7, and -9 were not detected in fetal ovary although cleaved caspase-3 and -9 were detected in the positive control of UV-treated U937 cells. The caspase target PARP and its cleaved form were detected in the U937 control cells before and after induction of apoptosis, respectively. Both PARP and its cleaved product were detected in human fetal ovary at all gestations examined (Fig. 3AGo).



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  		FIG. 3. Caspase expression in the human fetal ovary. A, Western blots of caspase-2, -3, -7, -8, -9, cleaved caspase-3 and -8, PARP, and cleaved PARP in ovaries of 13, 15, and 17 wk gestation. Controls consisting of lymphoblastoid U937 cells untreated (C–) or after UV irradiation to induce apoptosis (C+). Molecular sizes (kilodaltons) are indicated. B and C, Immunohistochemical localization of cleaved caspase-3 in the human fetal ovary at 16 and 20 wk, respectively. One of a cluster of germ cells is expressing cleaved caspase-3 (B), and two germ cells are immunopositive (C), but the oocyte within a primordial follicle is immunonegative. Positive staining is brown, and sections are counterstained with hematoxylin. gc, Germ cell; s, somatic cell; pf, primordial follicle. Scale bar, 50 µm and refers to both B and C.

 
Although cleaved (activated) caspase-3 could not be detected by immunoblotting, it was readily detected by immunohistochemistry. Specific expression was detected in a few cells in each section of fetal ovary examined (Fig. 3BGo): the low prevalence of these cells is likely to explain the immunoblotting result. These cells could mostly be identified as germ cells and showed typical pyknotic morphology, although in some cases the apoptotic process appeared to have progressed too far for reliable identification. Pregranulosa cells intermingled with germ cells, however, did not express cleaved caspase-3, nor did the oocytes or granulosa cells of primordial follicles (Fig. 3CGo).

Quantification of expression of cleaved caspase-3 was performed as for phospho-H3. This demonstrated that cleaved caspase-3 expression was increased at 18–19 wk, compared with earlier gestations both when expressed per unit area (P = 0.03, Fig. 4AGo) and after correction for the fewer number of germ cells per area at later gestations (Fig. 4BGo). The proportion of germ cells expressing cleaved caspase-3 increased from 0.95 ± 0.11% at 14–15 wk to 2.36 ± 0.45% (P = 0.02) at 18–19 wk. The single specimen of 20 wk gestation showed the lowest prevalence of cleaved caspase-3 expression of all ovaries examined at 0.11 per 10,000 µm2, compared with 0.28 ± 0.05 per 10,000 µm2 at 18–19 wk.



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  		FIG. 4. Quantification of cleaved caspase-3 expression in the human fetal ovary. A, Number of cleaved caspase-3-immunopositive cells per 10,000 µm2. B, Percentage of germ cells immunopositive for cleaved caspase-3. Mean ± SEM, n = 6–8 ovaries from different fetuses in each gestational age group except 20 wk, n = 1. *, P = 0.03; **, P = 0.02 by ANOVA.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
It is during the weeks investigated in the present study that germ cells in the human ovary progressively enter meiosis and associate with stromal cells to form primordial follicles, the essential structure of the female gonad and determinant of reproductive life span. This is therefore a crucial period in organogenesis. Whereas the concept that the ovary is endowed with a finite number of female germ cells has recently been questioned in the rodent (23), the classic studies of Baker (2) showing how the number of germ cells within the human ovary increases to a maximum at midgestation and then falls dramatically by the time of birth, remain undisputed. This decline continues throughout life, accelerating in the later reproductive years as the menopause approaches (24, 25). The present study was undertaken to examine germ cell proliferation and loss by apoptosis in the developing human ovary using quantitative techniques and investigate the expression of caspases as mediators of the apoptotic process. Crucial to this has been the ability to maximize the quality of histological preservation of the tissues with minimization of postmortem changes.

Cell proliferation was detected using two immunomarkers. PCNA, a marker of DNA replication, is widely used for the detection of cell proliferation (26, 27). Histone H3 becomes phosphorylated on serine 10 during chromosomal condensation at prophase and is dephosphorylated during telophase (28, 29). Phospho-H3 can be detected by immunohistochemistry, and quantification in mammalian tissues shows a close relationship with mitotic index (30). PCNA staining demonstrated proliferation of both germ cells and somatic cells in the human fetal ovary during the second trimester, with widespread immunostaining. The intensity and thus detection of PCNA expression shows variability according to antibody concentration (31). Additionally, PCNA is also present at times of DNA repair and may be detected in quiescent cell populations, and can also be increased by growth factors independently of mitosis (32), all of which limit its value as a reliable quantitative marker of cell proliferation (27). By contrast, phospho-H3 gave very high contrast staining as previously reported (30), suitable for quantification. The majority of phospho-H3-positive cells were germ cells, with only occasional somatic cells labeled. Similar data have been obtained in the sheep, in which cell proliferation in the fetal ovary was detected by in vivo incorporation of bromodeoxyuridine (7). Those data demonstrated that stromal cells and pregranulosa cells that were closely associated with oogonia before primordial follicle formation (i.e. at a developmental stage analogous to that studied here) showed only very limited mitotic activity. In the sheep, the surface epithelium was also identified as highly mitotically active. Whereas the surface epithelium in the human was found here to show intense expression of PCNA, expression of phospho-H3 was not detected. Comparable activity to that seen in germ cells would, however, be more difficult to detect using this technique because of the small number of epithelial cells per section, compared with germ cells, and the small number of germ cells stained in each section. Oocytes within primordial follicles were not immunostained, indicating that histone H3 is not phosphorylated in prophase of meiosis. In maize histone H3 is phosphorylated in late meiotic prophase/early metaphase associated with chromosome cohesion (33), in contrast to earlier phosphorylation in mitosis associated with chromosome condensation (28).

The number of phospho-H3 germ cells per unit area of the ovary showed a significant decrease with increasing gestation. However, germ cell size increases over this time period and the number of germ cells per unit area decreases correspondingly. Correction for the changing number of germ cells per unit area indicated that the proportion of germ cells in mitosis was stable. Additionally, germ cells are increasingly entering meiosis as development progresses, thus leaving the pool of potentially mitotically active (and thus phospho-H3-positive) cells. Data from Baker (2) indicate that the percentage of germ cells not in meiosis (i.e. oogonia) falls by more than 50% across the gestational range studied here. This suggests that those germ cells yet to enter meiosis are increasing their mitotic activity by approximately a factor of 2 across the second trimester. Supportive evidence for this is provided by analysis of the distribution of phospho-H3-positive germ cells. In the mouse, the presence of synchronously dividing germ cell cysts has been clearly demonstrated (34). Intercellular bridges are present between the germ cells within these cysts (34) and have also been demonstrated in the human (35), linking developmentally similar germ cells. At all gestations, phospho-H3-positive germ cells were frequently observed in clusters as well as singly, which may represent a cyst-like pattern of organization as described in the mouse. The proportion of phospho-H3-positive germ cells in clusters increased with gestation, consistent with increasing mitotic activity in germ cell cysts. These data thus demonstrate that germ cell mitosis in the human ovary continues, whereas other oocytes are forming primordial follicles, and indeed those germ cells not yet in meiosis are increasingly mitotically active.

The expression of representative caspases of both the initiator and effector functional groups was demonstrated by immunoblotting. Initiator caspase-8 and -9, and effector caspase-2, -3, and -7, were detected in ovary specimens across the gestational range examined. Caspase-2 was of particular interest because inactivation of this enzyme causes a significant increase in the pool of primordial follicles in the mouse postnatal ovary (36); thus, it appears to be an important determinant of germ cell death before primordial follicle formation. Different caspases may be important in mediating separate apoptotic pathways in fetal germ cells: whereas caspase-2 deficiency conferred resistance to cytokine deprivation, it did not abrogate the effect of a meiotic recombination defect (37). Caspase-2 deficiency was also associated with resistance to chemotherapy-induced oocyte death in oocytes from adult animals (36). In contrast, knockout of the proapoptotic Bcl-2 member Bax prolonged reproductive life span by reducing loss of ovarian follicles in the postnatal ovary (38). Germ cell number during development was, however, unaffected despite its expression in the fetal ovary in both rodent and human (14, 15).

Caspase-3 was also readily detected by immunoblotting. Although the activated cleaved form was not detectable in the fetal ovary by this method, it was localized by immunohistochemistry in all specimens examined. The major cell type expressing cleaved caspase-3 appeared to be germ cells, although in some cases the morphological events accompanying apoptosis precluded clear identification. In keeping with our previous data demonstrating apoptosis by in situ labeling (15), cleaved caspase-3 was not expressed in germ cells in the later stages of primordial follicle formation. Quantification of the number of cleaved caspase-3-positive cells demonstrated an increase between 14 and 19 wk gestation, although the single specimen at 20 wk gestation, which showed significantly greater maturity in terms of the number of primordial follicles present, showed a low level of expression. Analysis of caspase-3 knockout mice suggests that this enzyme is primarily of importance in granulosa cell apoptosis but not germ cell development (39). Activated caspase-3 has, however, been localized to both the oocyte and granulosa cells of atretic follicles in the mouse ovary (40) but was not detected in primordial follicles. The present data suggest that caspase-3 is involved in at least one of the pathways of apoptosis regulating germ cell number in the developing human ovary and that activity of this pathway may peak as primordial follicles start to be assembled.

The prevalence of apoptosis increases during the second trimester but may fall as primordial follicle formation becomes widespread. In the mouse, germ cell apoptosis has been reported to show a dramatic increase immediately before primordial follicle formation as germ cell cysts break down (5). The present data suggest a more gradual increase in the human, but the underlying processes appear to be essentially similar. Interaction with somatic pregranulosa cells is believed to be essential for germ cell survival at this time. It has been proposed that each oocyte needs to associate with a sufficient number of pregranulosa cells (7), although the factors involved have not been established. This process may be analogous to the determination of the density of innervation during the development of the nervous system (41), and intriguingly, neurotrophins have been implicated in the process of primordial follicle formation in both rodent and human (42, 43, 44).

The timing of changes in germ cell proliferation and apo-ptosis investigated here are crucial for defining the mechanisms and pathways involved. Several trophic factors have been identified in the developing ovary and have been shown to reduce germ cell loss in vitro in the human (45, 46) as well as rodents (9, 47, 48). Differences between these factors in how they change during development may reflect varying requirements at different stages of the transition from mitotic proliferation to meiosis and primordial follicle formation. For example, expression of activin increases transiently in human germ cells shortly before primordial follicle formation (46), whereas germ cells and somatic cells continue to express c-kit and neurotrophin 4, respectively, throughout this process (43, 45). It is likely that other mechanisms are also involved. The separation of clusters of germ cells into single cells over a short time period at primordial follicle formation has been suggested to be the key event in the mouse (5), although a recent detailed analysis demonstrated a more continuous loss through later gestation and into the neonatal period (4). Whereas we have not performed a directly comparable analysis in the human, the present data show germ cell proliferation and loss across a range of gestations. Whereas there was an increase in apoptosis at the time primordial follicles are increasingly formed, this was not dramatic, consistent with a more continuous process with multiple mechanisms rather than the relatively sudden wave reported by Pepling and Spradling. However, the variable stages of germ cell development present in the human at any one gestation may have blunted this. Previous reports of apoptosis in the human fetal ovary have included only limited quantification (13, 14, 15), with none attempting a systematic analysis.

In conclusion, these data demonstrate germ cell proliferation and apoptosis during the period of development leading up to primordial follicle formation. Germ cell proliferation continues, but there appear to be changes in its organization with increasingly synchronous mitosis in adjacent germ cells. Apoptosis also increases, resulting in increased germ cell loss immediately preceding primordial follicle formation, which appears to offer a protected environment. These data provide further insight into the regulation of a fundamental aspect of reproductive lifespan in the human female.


   Acknowledgments
 
We are greatly indebted to Joan Creiger and the staff of the Bruntsfield Suite (Royal Infirmary of Edinburgh) for their assistance in the supply of samples for this study.


   Footnotes
 
This work was supported by United Kingdom Medical Research Council.

First Published Online May 24, 2005

Abbreviations: PARP, Poly(ADPribose) polymerase; pc, post coitum; PCNA, proliferating cell nuclear antigen; phospho-H3, phosphorylated histone H3; TBS, Tris-buffered saline.

Received February 2, 2005.

Accepted May 17, 2005.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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