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Department of Obstetrics and Gynecology (T.E.V., J.S.T.) and Department of Pathology (T.E.V., F.S., R.H., J.S.T.), University of Oulu, FIN-90220 Oulu, Finland; Children’s Hospital and Program for Developmental and reproductive Physiology, Biomedicum (M.A., M.H.), University of Helsinki, FIN-00290 Helsinki, Finland; Department of Physiology (H.B.), S-41345 Gothenburg University, Gothenburg, Sweden; Department of Endocrinology and Fertility, Division of Obstetrics and Gynecology (M.D., E.R.t.V.), University Hospital 3015 Utrecht, The Netherlands; Department of Pediatrics (M.H.), Washington University in St. Louis, St. Louis, Missouri 63110
Address all correspondence and requests for reprints to: Juha S. Tapanainen, M.D., Ph.D., Clinic of Obstetrics and Gynecology, University of Oulu, 90220 Oulu, Finland. E-mail: juha.tapanainen{at}oulu.fi
Abstract
The majority of oocytes present in fetal ovaries are depleted before birth, and only about 400 will ovulate during the normal fertile life span. Studies on animals have shown that apoptosis is the mechanism behind oocyte depletion and follicular atresia. In the present study, we investigated the extent and localization of apoptosis in human fetal (aged 13–40 weeks) and adult ovaries. Furthermore, the expression of apoptosis-regulating proteins, bcl-2 and bax, and the relationship of transcription factor GATA-4 were studied. Apoptosis was found in ovarian follicles throughout fetal and adult life. During fetal development, apoptosis was localized mainly to primary oocytes and was highest between weeks 14–28, decreasing thereafter toward term. Expression of bcl-2 was observed only in the youngest fetal ovaries (weeks 13–14), and bax was present in the ovaries throughout the entire fetal period. In adult ovaries, apoptosis was detected in granulosa cells of secondary and antral follicles, and Bcl-2 and bax were expressed from primary follicles onwards. During fetal ovarian development, GATA-4 messenger RNA and protein were localized to the granulosa cells, with expression being highest in the youngest ovaries and decreasing somewhat toward term. The expression pattern of GATA-4 suggests that it may be involved in the mechanisms protecting granulosa cells from apoptosis from fetal to adult life. The results indicate that depletion of ovarian follicles in the human fetus occurs through intrinsic mechanisms of apoptosis in oocytes, and later in adult life the survival of growing follicles may be primarily determined by granulosa cell apoptosis.
AT AN EARLY stage of human embryo development, germ cells separate from somatic cells and migrate to the gonadal ridge. The majority of oogonia continue to increase their number by mitosis and by the 20th week of gestation about 7 million are present (1). Thereafter, oogonia enter meiosis and become enveloped by a layer of epithelial cells, so that around the 24th week of gestation almost all oogonia, which have now entered the primary oocyte stage, are individually surrounded. Once meiosis is initiated, the destiny of the oocytes is determined, and they can either degenerate or complete meiosis but cannot return to mitotic proliferation. At this time the number of oocytes has started to reduce dramatically and only about two million are present at birth, and 400,000 at puberty and during adult life only about four hundred oocytes will eventually reach full maturation and ovulate (1). The fate of the nonovulatory follicles is atresia, which occurs in the ovary from fetal life to menopause (2, 3), and the results of studies carried out in both animals and humans have shown that apoptosis is the mechanism behind this process (4, 5, 6, 7).
Apoptosis is an active energy-consuming process controlled by a number of intracellular proteins (8). Cells destined to undergo apoptosis show membrane blebbing, condensation of chromatin and cytosolic contents, and finally fragmentation of DNA by endonucleases into multiples of 185–200 bp (9). Apoptosis appears abundantly in several tissues during human embryonic development (10), and it also occurs in adult tissues. Cells with a high regeneration and division rate, and cells under endocrine control are particularly susceptible to apoptosis (11).
The hormonal changes leading to human ovarian follicular atresia are still poorly understood. Gonadotropins, together with estrogens, operate as survival factors for follicles (12, 13), whereas androgens have been shown to induce atresia and apoptosis in follicles (12). However, regulation of the intracellular mechanisms operating in atretic cells is still mostly unclear. Among several other factors, members of the bcl-2 protein family, bcl-2 and bax, have been shown to regulate apoptosis in the ovary (14, 15, 16). Bax causes apoptosis by inducing permeabilization of mitochondrial membranes and opening of mitochondrial porin channels (17). Bcl-2 is capable of blocking the induction of apoptosis, first at the mitochondrial level by forming heterodimers with bax (17, 18), and secondly after bax-induced pore formation (19). In bcl-2-deficient mice, decreased numbers of follicles are present after birth (20), and excessive expression of bcl-2 leads to decreased follicular apoptosis and atresia (21, 22). Supporting the crucial role of this protein family in the regulation of apoptosis in the ovary, bax-deficient mice have abnormal follicles with an excessive number of granulosa cells (23, 24).
GATA-4 belongs to a family of zinc finger transcription factors termed the GATA-binding proteins, which regulate gene expression, differentiation, and cell proliferation in a variety of tissues (25, 26). In the ovary, GATA-4 expression is localized to granulosa cells of primary and antral follicles (27, 28). In addition, the expression of GATA-4 is stimulated by exogenous gonadotropins in cultured gonadal cell lines (27, 29), suggesting that the expression of GATA-4 within these tissues is associated with hormonal signaling or cell proliferation. Of interest, down-regulation of GATA-4 expression accompanies follicular atresia in adult mouse ovaries (27). Of the other GATA-binding proteins, down-regulation of GATA-1 and GATA-2 have been associated with increased apoptosis in hematopoietic and other nonovarian cells (30, 31). Similarly, GATA-4 might function as a cell survival factor within the gonad. The expression of GATA-4 is absent or very low in fetal rodent ovaries as studied by immunohistochemistry (32), but there are no reports on its expression in human fetal ovaries.
Although apoptosis and its regulation in the fetal gonads of experimental animals have been studied extensively, very little is known about the mechanisms of follicular demise in human fetal ovaries (33). In the adult ovary, apoptosis has been found to occur incrementally with follicular size, and it seems to be dependent on the estrogen-androgen balance within the follicles (7). The role of other factors known to regulate apoptosis in animal models is not well defined in the human ovary. To gain further insight into the character of oocyte fate during human fetal development and adult follicular atresia, we have now investigated apoptosis, apoptosis-regulating factors (bcl-2 and bax) and the transcription factor GATA-4 in fetal (13–40 weeks) and adult ovaries.
Materials and Methods
Human ovaries
Adult normal ovarian tissue was obtained from patients (aged 22–45) undergoing ovariectomy as a result of uterine myomas (n = 4) or focal endometrial cancer (n = 4, stages I and II). Ovarian tissue from 8 fetuses (fetal age 13–22 and 33 weeks) with normal karyotype, were obtained after spontaneous and therapeutic abortions because of maternal disease. In addition, 6 neonates (fetal age 23–39 weeks at birth) who died because of perinatal asphyxia or infection in 48 h after birth. Fetal samples with detectable autolysis were excluded from the study. All of the ovarian samples were fixed in 4% buffered formaldehyde for 24 h and embedded in paraffin. Ovaries from three fetuses were snap-frozen in liquid nitrogen and stored at -70 C for RNA extraction and Northern blot analysis. The study was approved by the Ethics Committee of Oulu University.
In situ DNA 3'-end labeling
Apoptosis was qualitatively identified in the ovaries by using an in situ DNA 3'-end labeling kit (Oncor, Gaithersburg, MD). Paraffin sections of ovaries were rehydrated through an alcohol series. Permeability of the cell membranes was increased by incubating the sections in 400 µg of proteinase K (Roche Molecular Biochemicals, Mannheim, Germany) in 200 mL of PBS for 15 min. Endogenous peroxidase activity was inhibited by quenching the samples for 5 min in 5% hydrogen peroxide. DNA fragmentation was identified by applying terminal transferase enzyme with digoxigenin-labeled nucleotides to the samples and incubating for 1 h under coverslips. Antidigoxigen antibody was used to recognize the digoxigen-labeled nucleotide chains attached to the 3'-ends of sample DNA. A color reaction was produced with diaminobenzen in the presence of 0.03% hydrogen peroxide and the sections were lightly counterstained with hematoxylin.
Bcl-2, bax, and GATA-4 immunohistochemistry
Paraffin sections were deparaffinized in xylene and hydrated gradually through graded alcohols. Endogenous peroxidase activity was blocked with 3% hydrogen peroxide in methanol. Nonspecific binding was prevented by using 1 mL of FCS in 4 mL PBS. Primary antibody was applied to the sample and it was incubated overnight. Bcl-2 was detected by using monoclonal mouse antihuman bcl-2 (DAKO Corp., Glostrup, Denmark) and bax by using polyclonal rabbit antihuman bax (PharMingen, San Diego, CA). Biotinylated rabbit antimouse immunoglobulin was used as a secondary antibody to the monoclonal antibody and biotinylated goat antirabbit immunoglobulin was used for the polyclonal antibody. A color reaction was produced with diaminobenzen in the presence of 0.03% hydrogen peroxide.
GATA-4 immunohistochemistry was carried out using a goat polyclonal antimouse GATA-4 IgG directed to an epitope which has 100% identity with the corresponding human sequence (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). A commercially available avidin-biotin immunoperoxidase system was used to visualize bound antibody (Vectastain Elite ABC Kit, Vector Laboratories, Inc., Burlingame, CA). 3-Amino-9-ethylcarbazole (Sigma, St. Louis, MO) was used as the chromogen and the development reaction occurred in the presence of 0.03% hydrogen peroxide.
RNA extraction and Northern blotting
Total RNA from human fetal ovaries was extracted by using the guanidine isothiocyanate-cesium chloride method (34), and RNA was quantified by absorbance at 260 nm. For Northern blots, 11 µg RNA was size-fractionated in 1.5% agarose gels and transferred to Biodyne Transfer membrane (Pall Europe Ltd., Portsmouth, UK). The filter was UV-cross-linked (Stratalinker 1800, Stratagene, La Jolla, CA) before hybridization.
Labeling of complementary DNA (cDNA) probes, and filter hybridization
As probes for filter hybridization, we used human GATA-4
(35) cDNA. Human cyclophilin cDNA was used as control for
even loading in filter hybridization. All the cDNA inserts were labeled
with [32P]-
-deoxy-CTP (3000 Ci/mmol;
Amersham Pharmacia Biotech, Arlington Heights, IL) and a
Prime-a-gene kit (Promega Corp., Madison, WI) was used.
The probes were purified with Nuck Trap columns
(Stratagene) and used at 1–3 x
106 dpm/mL in hybridization solution containing
50% formamide, 6x SSC, 0.1% Ficoll, 0.1% polyvinylpyrrolidone,
0.1% BSA, 100 µg salmon sperm DNA/mL 100 µg yeast RNA/mL and 0.5%
SDS. Northern blots were hybridized for 16 h at 42 C and washed
once for 20 min at RT and once at 50 C with 1x SSC-0.1% SDS. Filters
were exposed to Agfa Curix Ortho x-ray film with Trimax T4/T8
intensifying screens (3 M, Ferrania, Italy) at
-70 C.
In situ hybridization
Sections of human fetal ovaries fixed in 4% paraformaldehyde and embedded in paraffin were subjected to in situ hybridization using human GATA-4 riboprobes transcribed from a 575-bp GATA-4 cDNA (27). Tissue sections were incubated with 1 x 106 cpm of [32P]-labeled (1000–3000 Ci/mmol, Amersham Pharmacia Biotech, Arlington Heights, IL) antisense or sense riboprobe in a total volume of 80 µL, following the protocol described in detail elsewhere (27).
Light microscopy
All the tissue samples were evaluated using light microscopy. In early fetal ovaries the follicle structures and different cell types, except oocytes, could not be reliably detected. Therefore, up to the 15th week the cells were classified as oocytes and pregranulosa cells. Thereafter, granulosa cells could be identified in the developing follicle structures, which became more evident. At least 50 oocytes were counted in 3–5 fields of vision, and the proportion of apoptotic oocytes was calculated. The results of in situ detection of apoptosis in granulosa and stromal cells, and immunohistochemistry, were evaluated by means of a histograde system: -, negative; ± , moderately positive; +, positive; ++, strongly positive; +++, very strongly positive. In adult ovaries the follicle was considered apoptotic if any of the granulosa cells showed staining in in situ 3'-end labeling.
Results
Apoptosis during fetal development
In the youngest fetal ovaries, the follicle structures were
not evident and most of the apoptotic cells were recognized as oocytes
(Fig. 1
). Although occasional
nonidentified cells were apoptotic, the oocytes still represented the
overwhelming majority of the labeled cells, and this was also the case
after the 14th week, when some follicular structures and granulosa
cells could be identified (Table 1).
After a slight increase in the number of apoptotic oocytes between
weeks 13 and 14, the extent of apoptosis remained stable, and it
started to decrease during the last quarter of fetal life. At term,
only occasional apoptotic oocytes were present in the ovaries.
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Immunostaining of bcl-2 was observed only in the pregranulosa
cells of the two youngest fetuses (Table 1
, Fig. 2A). Bax immunostaining was persistent in
the ovaries from the 13th week of development until term (Fig. 2, D–F). Before the 14th week, pregranulosa cells were stained, but no
significant staining was detected in the oocytes. Thereafter bax
immunostaining was localized to the oocytes and granulosa cells. During
the second half of fetal life, bax immunostaining remained relatively
stable in oocytes and granulosa cells, but was negligible in the stroma
(Table 1, Fig. 2, E and F).
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The expression of GATA-4 protein during fetal ovarian development
was studied by immunohistochemistry, in situ hybridization,
and Northern blot analysis. High GATA-4 expression was found in the
ovaries of the youngest fetuses (weeks 13–14). As soon as some
follicle structures became evident, GATA-4 protein was localized mainly
in the granulosa cells and to a lesser extent in stromal/pregranulosa
cells (Table 1, Fig. 2, G–H). No GATA-4-positive oogonia or oocytes
were detected at any stage of development. From week 24–25 onwards,
the number of GATA-4 positive granulosa cells as well as the intensity
of the immunostaining were lower than in the ovaries obtained from
earlier fetal ages. A few antral follicles observed in the ovaries of
fetuses older than 27 weeks showed intense staining. To confirm the
results of immunohistochemistry, RNA analysis was performed. In
situ hybridization demonstrated abundant GATA-4 messenger RNA
(mRNA) in fetal ovaries (Fig. 3).
Northern blot analysis of GATA-4 in fetal ovaries showed a single mRNA
band of approximately 4.4 kb at both ages studied (Fig. 3). This
corresponds to the size of GATA-4 transcripts described earlier in the
human ovary (28).
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To study the specific ovarian cell types involved in apoptotic DNA
degradation in adult life, in situ 3'-end labeling of DNA
was performed on histological sections from human ovaries. Apoptotic
cells were primarily located in the innermost layer of granulosa cells
of antral (Fig. 4, A and B) and atretic
follicles. Only occasional apoptotic cells were detected in the
secondary follicles and no sign of apoptosis was found in primordial or
primary follicles (Table 2). No apoptotic
oocytes were observed in adult ovaries. This may be due to the fact
that only a few oocytes were present in the follicles of analyzed
sections. Furthermore, the number of follicles was markedly lower than
in fetal ovaries. Hence, the chance of finding positive cells was
relatively low.
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In adult ovaries, bcl-2 immunostaining was observed mainly in the
granulosa cells of secondary and antral follicles (Fig. 4, C and D). No
significant staining was observed at earlier stages of
folliculogenesis. Bax immunostaining was found in the granulosa and
theca cells of primary, secondary, and antral follicles, and the number
of positive follicles increased with follicular maturation (Table 2 and
Fig. 4, E and F).
GATA-4 expression was negligible in primordial follicles but clearly
detectable in primary, secondary, and antral follicles, confirming our
earlier results (28). GATA-4 protein was detected in
granulosa and theca cells, and to a lesser degree also in stromal cells
(Fig. 4, G and H).
Discussion
Our results demonstrate that a significant proportion of oocytes in the human ovary degenerate during fetal life by the mechanism of apoptosis. This is already evident at 13 weeks of gestation, with a high number of apoptotic oocytes and some occasional pregranulosa cells. Later in fetal life pregranulosa cells remained positive and some labeled follicular granulosa cells were also detected. A high number of apoptotic oocytes was observed until 28–32 weeks, whereafter they decreased in number. Although the number of oocytes reaches a maximum of 7 million by the 20th week of gestation (1, 4), our results show that apoptosis is already present in the ovary at the 13th week, and the process covers the whole fetal period.
In adult ovaries, apoptosis was detected primarily in the antral follicles and mainly in the innermost layer of granulosa cells. Occasional apoptotic cells were also observed in the secondary follicles, but not at earlier stages of folliculogenesis. These results support previous findings showing that apoptosis occurs incrementally with growing follicular size (7, 36) and that apoptosis only slightly affects the early growing follicles (37).
Similarly to adult ovaries, where apoptosis was detected in granulosa cells of growing follicles, apoptosis in fetal ovaries was highest when marked granulosa cell proliferation occurred. In the adult ovary, apoptosis is localized mainly to granulosa cells and is the mechanism of deleting recruited follicles that do not reach the dominant follicle stage. In the fetus, apoptosis mostly deletes primary oocytes. This indicates that oocytes may have an intrinsic mechanism for activation of apoptosis during ovarian development, a fact that has also been suggested by Perez et al. (38). Apoptotic oocytes were not detected in adult tissues, which could be due to the fact that only very few oocytes were present in the analyzed sections. Hence, it is not possible to determine whether the same intrinsic mechanism also occurs in adults. In every ovarian cycle, hundreds of primordial follicles enter the growing stage (37), and most of these never reach the antral follicle stage, where most of the granulosa cell apoptosis was observed. This suggests that the fate of early growing follicles is dependent on the quality of the oocyte, while in the later stages the destiny of the oocyte is more dependent on the status of the surrounding granulosa cells and the follicle as a whole.
In the fetal ovary, high GATA-4 expression was observed at 13 weeks, and it decreased somewhat with fetal age. This expression pattern of GATA-4 in human fetal ovary partially contrasts the earlier findings on mice showing the lack of GATA-4 expression in late gestation (32). As soon as follicular structures became evident, GATA-4 staining could be localized to pregranulosa/stromal cells, but not in the oocytes. In adult ovaries GATA-4 staining was present mainly during the period of active proliferation of the granulosa cells. We have shown earlier that in cultured mouse granulosa cells down-regulation of GATA-4 is associated with increased apoptosis and in mouse ovaries GATA-4 expression in granulosa cells is associated with a low apoptosis rate of growing follicles (27). Similarly, although we were not able to perform functional studies, the high expression of GATA-4 in human fetal ovaries suggests that this protein may be involved in the mechanisms that protect granulosa cells from apoptosis.
The expression of bcl-2 was observed in fetal ovaries only, until week 14. A previous report indicates that bcl-2 is present in the human fetal ovary from the 6th to the 12th week of development (39). Down-regulation of bcl-2 was associated with an increased rate of apoptosis at the 14th week of fetal life, suggesting that bcl-2 may have a protective role in the survival of the oocytes in the very early stages of fetal development. Immunostaining of a proapoptotic member of the bcl-2 protein family, bax, was observed in all fetal ovaries from the 13th to the 40th week. Its expression in granulosa and stromal cells decreased slightly toward term, while relatively stable expression was observed in the oocytes throughout fetal life. However, this did not correlate with oocyte apoptosis, which decreased in late gestation. The expression of bax may not necessarily reflect its actual proapoptotic potential, because its activity is dependent on its intracellular localization (40, 41, 42), and the inability of bax to promote apoptosis in the later part of fetal life could be due to the cytosolic localization of the protein.
In adult ovaries, bcl-2 and bax were expressed mostly in the secondary and antral follicles. Bax protein was also present in the theca cells, where bcl-2 was negligible. However, no apoptosis was detected in the theca cells, although a low bcl-2 to bax ratio predisposes cells to apoptosis (43). However, other members of the bcl-2 family can interact with bcl-2 and bax and affect their anti- or proapoptotic function, and may play a role in counterbalancing the proapoptotic effect of bax both in fetal and adult ovaries.
The present results demonstrate that depletion of ovarian follicles during human fetal development occurs through the mechanism of apoptosis, which is particularly extensive in the oocytes during the first three quarters of gestation. During adult life, apoptosis is mainly located in the granulosa cells of growing follicles. Thus, in fetal as well as in adult ovaries, the destiny of early follicles may be determined by intrinsic mechanisms of apoptosis in the oocyte, whereas later, during FSH-dependent stages of follicular development, granulosa cell apoptosis plays a major role in follicular demise. Several agents, such as the bcl-2 family, are likely to participate in the regulation of apoptosis in the human ovary. In the light of the present results and earlier observations, we also propose that GATA-4 may have a role in mediating anti-apoptotic effects in granulosa cells.
Acknowledgments
We thank Mirja Ahvensalmi for skilled technical assistance and Dr. J. P. de Bruin for valuable comments.
Footnotes
1 This work was supported by grants from the Sigrid Jusélius
Foundation (to T.V., M.H., and J.T.); and Research Funds from the
Academy of Finland (to T.V., M.H., and J.T.); Oulu University Hospital
(to T.V. and J.T.); Helsinki University Central Hospital (to M.A. and
M.H.); and Helsinki University (M.A. and M.H.). 
Received November 9, 2000.
Revised March 20, 2001.
Accepted March 25, 2001.
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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]
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G. Meduri, P. Touraine, I. Beau, O. Lahuna, A. Desroches, M. C. Vacher-Lavenu, F. Kuttenn, and M. Misrahi Delayed Puberty and Primary Amenorrhea Associated with a Novel Mutation of the Human Follicle-Stimulating Hormone Receptor: Clinical, Histological, and Molecular Studies J. Clin. Endocrinol. Metab., August 1, 2003; 88(8): 3491 - 3498. [Abstract] [Full Text] [PDF]
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I. Ketola, J. Toppari, T. Vaskivuo, R. Herva, J. S. Tapanainen, and M. Heikinheimo Transcription Factor GATA-6, Cell Proliferation, Apoptosis, and Apoptosis-Related Proteins Bcl-2 and Bax in Human Fetal Testis J. Clin. Endocrinol. Metab., April 1, 2003; 88(4): 1858 - 1865. [Abstract] [Full Text] [PDF]
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 D.N. Modi, S. Sane, and D. Bhartiya Accelerated germ cell apoptosis in sex chromosome aneuploid fetal human gonads Mol. Hum. Reprod., April 1, 2003; 9(4): 219 - 225. [Abstract] [Full Text] [PDF]
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M. Anttonen, I. Ketola, H. Parviainen, A.-K. Pusa, and M. Heikinheimo FOG-2 and GATA-4 Are Coexpressed in the Mouse Ovary and Can Modulate Mullerian-Inhibiting Substance Expression Biol Reprod, April 1, 2003; 68(4): 1333 - 1340. [Abstract] [Full Text] [PDF]
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S. Kiiveri, J. Liu, M. Westerholm-Ormio, N. Narita, D. B. Wilson, R. Voutilainen, and M. Heikinheimo Differential Expression of GATA-4 and GATA-6 in Fetal and Adult Mouse and Human Adrenal Tissue Endocrinology, August 1, 2002; 143(8): 3136 - 3143. [Abstract] [Full Text] [PDF]
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P. S. Hartley, R. A. L. Bayne, L. L. L. Robinson, N. Fulton, and R. A. Anderson Developmental Changes in Expression of Myeloid Cell Leukemia-1 in Human Germ Cells during Oogenesis and Early Folliculogenesis J. Clin. Endocrinol. Metab., July 1, 2002; 87(7): 3417 - 3427. [Abstract] [Full Text] [PDF]
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