TISSUE-SPECIFIC STEM CELLS
No Evidence for Neo-Oogenesis May Link to Ovarian Senescence
in Adult Monkey
JIHONG YUAN,a,b DONGDONG ZHANG,a LEI WANG,a MENGYUAN LIU,a JIAN MAO,a YU YIN,a XIAOYING YE,a
NA LIU,a JIHONG HAN,a YINGDAI GAO,c TAO CHENG,c DAVID L. KEEFE,d LIN LIUa*
a
State Key Laboratory of Medicinal Chemical Biology, The 2011 Collaborative Innovation Center of Tianjin for
Medical Epigenetics and College of Life Sciences, Nankai University, Tianjin, China; bKey Laboratory of Ministry
of Health on Hormones and Development, Metabolic Diseases Hospital, Tianjin Medical University, Tianjin, China;
c
The State Key Laboratory of Experimental Hematology, Institute of Hematology, Center for Stem Cell Medicine,
Chinese Academy of Medical Sciences and Peking Union Medical College, Tianjin, China; dDepartment of
Obstetrics and Gynecology, New York University Langone Medical Center, New York, USA
Key Words. Germline stem cells • Meiosis • Oogenesis • Monkey • Ovary • Aging
ABSTRACT
Female germline or oogonial stem cells transiently residing
in fetal ovaries are analogous to the spermatogonial stem
cells or germline stem cells (GSCs) in adult testes where
GSCs and meiosis continuously renew. Oocytes can be generated in vitro from embryonic stem cells and induced pluripotent stem cells, but the existence of GSCs and neo-oogenesis
in adult mammalian ovaries is less clear. Preliminary findings of GSCs and neo-oogenesis in mice and humans have
not been consistently reproducible. Monkeys provide the
most relevant model of human ovarian biology. We searched
for GSCs and neo-meiosis in ovaries of adult monkeys at various ages, and compared them with GSCs from adult monkey
testis, which are characterized by cytoplasmic staining for
the germ cell marker DAZL and nuclear expression of the
proliferative markers PCNA and KI67, and pluripotency-
associated genes LIN28 and SOX2, and lack of nuclear
LAMIN A, a marker for cell differentiation. Early meiocytes
undergo homologous pairing at prophase I distinguished by
synaptonemal complex lateral filaments with telomere perinuclear distribution. By exhaustive searching using comprehensive experimental approaches, we show that proliferative
GSCs and neo-meiocytes by these specific criteria were undetectable in adult mouse and monkey ovaries. However, we
found proliferative nongermline somatic stem cells that do
not express LAMIN A and germ cell markers in the adult
ovaries, notably in the cortex and granulosa cells of growing
follicles. These data support the paradigm that adult ovaries
do not undergo germ cell renewal, which may contribute
significantly to ovarian senescence that occurs with age.
STEM CELLS 2013;31:2538–2550
Disclosure of potential conflicts of interest is found at the end of this article.
INTRODUCTION
A remarkable decline in the ovarian follicle pool accompanies
ovarian aging in women, leading to reproductive aging and
menopause [1–3]. Ovarian aging reduces fertility in women
who delay attempts at childbearing, and contributes to
menopause-related health problems, psychological stress, and
increased risk of genetic diseases in offspring. Oocyte renewal
or germ cell regeneration, if it did take place in adult human
ovaries, would have important implications for the millions of
women worldwide who attempt childbearing at midlife, and
others who have lost ovarian function following chemo or
radiation therapy [4, 5].
Extensive evidence accumulated since the 1950s supports
the paradigm that in females of most mammalian species,
oogonia, the germline stem cells (GSCs) that produce oocytes,
exist only during a brief window of oogenesis in the fetal
period of development [6] and that females are born with a
nonrenewable pool of follicles that declines progressively
with age [4, 5, 7–11]. In mice, oogonia and oocytes in early
stages of meiosis, with their chromosomes undergoing recombination, are abundant during fetal development, but disappear
shortly after birth [12, 13]. This well-substantiated paradigm
was challenged by observations suggesting that ovarian surface epithelium (OSE) of adult mice may contain cells that
can sustain postnatal follicular renewal [14]. Recently, putative oogonial stem cells with proliferative capacity were identified in adult mouse and human ovaries [15], but these
findings and the functional tests were not confirmed by other
laboratories [16–19]. Oocytes can be generated in vitro from
embryonic stem cells, induced pluripotent stem cells, and
stem cells from skin [20–23]. Yet in vitro conversion of the
OSE cells [10, 24, 25] and reprogramming cells to germ-like
cells does not prove the existence of GSCs and neo-oogenesis
in vivo [13, 18].
Author contributions: J.Y., D.Z., L.W., M.L., Y.Y., J.M., X.Y., and N.L.: collection/assembly of data and data analysis/interpretation;
J.H., Y.G., and T.C.: provision of study materials and data analysis/interpretation; D.L.K. and L.L.: conception and design, manuscript
writing, and revision. J.Y., D.Z., and L.W. contributed equally to this article.
Correspondence: Lin Liu, Ph.D., College of Life Sciences, Nankai University, Tianjin 300071, China. Telephone: 86-22-23500752; Fax: 8622-23500752; e-mail: [email protected] Received January 30, 2013; accepted for publication July 5, 2013; first published online in
C AlphaMed Press 1066-5099/2013/$30.00/0 doi: 10.1002/stem.1480
STEM CELLS EXPRESS July 29, 2013. V
STEM CELLS 2013;31:2538–2550 www.StemCells.com
Yuan, Zhang, Wang et al.
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GSCs are present as spermatogonial stem cells (SSCs)
and maintain their self-renewal and germ cell renewal continuously in the adult testis [26–28]. A similarity in self-renewal
and survival mechanisms between human and mouse SSCs
exists [29], and mouse and human SSCs share conserved gene
expression of GSC regulatory molecules [30]. These data
potentially allow for the extrapolation of the knowledge about
mouse SSCs to the human germline, which is difficult to
study [30]. DAZL is remarkably highly expressed in enriched
SSCs of mice and humans, in contrast to testis somatic cells
[30]. Mice and rhesus spermatogonia also express similar
markers of germ cells (VASA, DAZL) and stem/progenitor
spermatogonia (PLZF and GFRa1) [31]. Although spermatogonia express their own specific genes, germline markers for
SSCs were validated and are conserved from mouse systems
to monkey and human testis, and might be used also for putative female GSCs in monkey and human ovaries. In addition,
absent or minimal expression of Lamin A/C also can be used
to identify somatic stem cells or progenitor stem cells in adult
mammalian tissues [32, 33]. Lamin A/C expression marks
both mouse and human embryonic differentiation [34, 35].
Meiotic prophase I encompasses many unique features,
including homologous chromosomal searching and pairing,
and formation of synaptonemal complexes (SCs) [36, 37].
SCs composed of three main SC proteins (Scp 1–3) form a
tripartite structure in early meiotic germ cells, begin to assemble at leptotene, and complete SC formation at pachytene
[38]. Both the expression and distribution of SCP3 are frequently used as meiotic markers [39]. Also, early meiocytes
form telomere bouquets, and their perinuclear distribution at
the termini of SC lateral or axial filaments, indicative of
homologous pairing at leptotene to pachytene of prophase I,
represents an essential step for subsequent meiotic recombination [40]. Homologous pairing and perinuclear distribution of
telomeres can serve as decisive indicators of neo-oogenesis in
postnatal mammalian ovaries.
The rhesus monkey provides a suitable model for the
ovarian aging that is ubiquitous in women. Macaca mulatta
(rhesus monkey) females reach sexual maturity at 3–5 years
of age, remain reproductively active over 20 years, and
undergo pathological and hormonal changes characteristic of
the human female climacteric [41, 42]. As in women, ovaries
in rhesus monkeys suffer depletion of follicular reservoir [43].
Whether the adult rhesus monkey ovary exhibits GSCs and
neo-oogenesis has not been systematically examined. We rigorously searched for evidence of GSCs and neo-meiosis in
ovaries from adult monkeys of various ages, using various
experimental approaches.
MATERIALS
AND
METHODS
Experimental Design
1. We attempted to isolate and characterize GSCs from testis
using mice and compared with those of female mouse ovaries. Proliferative GSCs were identified by conventional
bromodeoxyuridine (BrdU) incorporation, and by strong
nuclear PCNA and KI67 expression and absence of nuclear
membrane protein LAMIN A, costaining with DAZL or
VASA using immunofluorescence microscopy.
2. We searched in adult monkey ovaries from different age
group for proliferative GSCs marked by nuclear PCNA or
KI67 colocalized with cytoplasmic DAZL or VASA and
additional pluripotency-associated genes LIN28 or SOX2,
in comparison with those of adult monkey testis served as
positive controls.
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3. We looked for neo-oogenesis in adult monkey ovaries
using defined markers specific for early meiocytes, SCP3
lateral element structures ending with perinulcear distribution of telomeres by immunofluorescence microscopy, and
compared with the early spermatocytes served as positive
controls. SCP3 element structures also were examined for
mouse fetal and adult ovaries and testis, to validate the
monkey data.
4. We counted follicles of various stages and measured serum
hormone levels from ovaries of monkeys at different age.
5. We performed experiments to analyze expression of genes
specific for primordial germ cells (PGCs), cell proliferation, early meiotic pairing and recombination, and folliculogenesis by conventional PCR, and expression of selected
proteins by immunoblot in monkey ovaries compared with
testis.
Collection of Tissues
The general care and housing of rhesus monkeys were provided
by Guangxi Xiong Sen Primate Experimental Animal Development Corporation. The use of monkeys and mice for this study
and the protocol for collecting monkey and mouse tissues were
approved by the Institutional Animal Care and Use Committee
at Nankai University. A minimal number of monkeys were used
in this study to minimize ethical concerns. Female monkeys
were randomly chosen from three groups according to their
reproductive ages at 3–4 years (young), 7–8 years (middle-age),
and 18–19 years (old), with three monkeys in each age group.
Male monkeys were approximately 5 years old. Female and
male rhesus monkeys were euthanized, and ovaries, kidney,
liver, serum, and testis were collected. Half of each ovary and
testis were fixed in 4% paraformaldehyde (PFA) for 24 hours at
room temperature and then embedded in paraffin. Another half
of each ovary and testis, liver and kidney were cut into small
pieces, snap-frozen in liquid nitrogen, and stored at 280 C for
RNA, DNA and protein extraction, and serum for hormone
assays was stored at 280 C.
Isolation and Purification of Mouse Testicular Cells
Isolation of testicular cells was performed based on methods previously published [44, 45], with slight modifications. Testis were
isolated from 4 and 32-week-old mice and washed in phosphatebuffered saline (PBS) containing 3% penicillin and streptomycin.
Testis tissues were dissected and minced by sterile surgical
instrument, rinsed with PBS, and centrifuged (Eppendorf, Hamburg, Germany) at 1,000 rpm for three times to remove the blood
cells. The tissue was placed in a Petri dish containing 1 mg/ml
Collagenase IV (GIBCO, Grand Island, NY) and 10 lg/ml DNase
I (QIAGEN, Chatsworth, CA), digested at 37 C in 5% CO2 incubator for 15 minutes, and then centrifuged at 1,000 rpm for 3
minutes. The tissue was digested in 1 mg/ml Dispase II (Roche,
Berlin, Germany) and 10 lg/ml DNase I for another 15 minutes
and filtered with a 40-lm diameter filter. After centrifugation at
1,200 rpm for 3 minutes, a small mass of red blood cells at the
bottom of the pellet was carefully removed and the pellet was
resuspended in SSCs medium without growth factor, the cells
counted, and prepared in suspension at the concentration of 1 3
107 cells per milliliter.
The isolated testicular cells were cultured in 100 mm dish
and purified by a differential plating method for three times
[46]. After culture in incubator for 4–8 hours, Sertoli cells and
fibroblast cells adhere to the bottom of the dish while SSCs
could not. SSCs were resuspended carefully by pipeting and
transferred to a new gelatin-coated dish. The procedure was
repeated twice such that SSCs were enriched, and the attached
cells were cultured further. Both SSCs and attached cells were
collected and washed with Hanks’ balanced saline solution
(HBSS).
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Isolation of Ovarian Cells
Isolation of ovarian cells was performed based on the method
described [15]. Briefly, ovaries from six mice of 7–8-week were
pooled and dissociated by mincing, followed by a two-step enzymatic digestion involving a 15-minute incubation with 800 U/ml
collagenase (type IV; prepared in HBSS) and a 10-minute incubation with 0.05% trypsin-EDTA. Digestions were carried out in
the presence of 1 lg/ml DNase I (Sigma-Aldrich, St. Louis, MO)
to minimize stickiness within the cell preparations, and trypsin
was neutralized by addition of 10% fetal bovine serum (Hyclone,
Logan, UT). Ovarian dispersants were filtered through a 70-lm
nylon mesh, and the filtrate was centrifuged at 1,200 rpm/minute
and washed with HBSS.
Flow Cytometry Analysis
For each experiment, ovarian cells, SSCs, and attached cells were
designed for both DDX4 staining and negative control (staining
without the first antibody). After washing, cells were blocked in a
solution composed of 1% fatty-acid-free bovine serum albumin
with either 1% normal goat serum in HBSS for 20 minutes on
ice. Cells were then reacted for 20 minutes on ice with a 1:10
dilution of primary antibody (DDX4, ab13840). After washing
with HBSS, cells were incubated with a 1:500 dilution of goat
anti-rabbit IgG conjugated to Alexa Fluor 488 (Invitrogen, Carlsbad, CA) for 20 minutes on ice, and washed with HBSS. Labeled
cells were then filtered again (35-lm pore diameter) and subjected to flow cytometry analysis using a BD FACSCalibur flow
cytometer (BD Biosciences, San Jose, CA), gated against negative controls. Ovarian cells and SSCs that were DDX4 positive
were sorted according to the method used by White et al. [15],
and cell cycles were analyzed using a BD FACSCalibur flow
cytometer (BD Biosciences).
In Vivo BrdU Incorporation and BrdU Staining
BrdU incorporation assay was performed according to a method
described by Staszkiewicz et al. [47]. Briefly, 8–10-week-old
female and male C57BL/6J mice (n 5 3) were injected intraperitoneally with BrdU at a concentration of 50 lg/g of b.wt. (Sigma
Co., St. Louis, MO) twice daily, at 7:00 a.m. and 6:00 p.m. for 3
consecutive days. Control animals (littermates; n 5 2) were
injected with saline. The ovaries and testis were collected and
fixed in 3.7% PFA 1 day after BrdU injection and further preparation method for section was the same as the method for follicle
counts.
Coimmunostaining of BrdU and PCNA, VASA, or LAMIN
A was performed based on the methods described [48], with
slight modifications. Briefly, sections were deparaffinized,
hydrated, treated with hydrogen peroxide (0.3% in PBS) for 10
minutes), subjected to high-pressure antigen recovery sequentially
in 0.01% citrate buffer (pH 6.0) for 2 minutes, and denatured in
2 N HCl for 20 minutes at room temperature. The sections were
then incubated in 5% normal goat serum, followed by incubation
in anti-BrdU (1:200; A21304; Invitrogen) and PCNA (1:400)
overnight at 4 C, and incubated with appropriate secondary antibody diluted by 1:200 for 2 hours at room temperature. For costaining with LAMIN A and VASA, the section was incubated in
anti-BrdU overnight at 4 C, and then with LAMIN A or VASA
overnight at 4 C. After washing in PBS three times, the sections
were incubated with the secondary antibodies. The sections were
rinsed thoroughly three times and stained with 1 lg/ml Hoechst
33342 for 20 minutes to stain nuclei, washed in PBS three times,
mounted in Vectashield (H-1000, Vector Laboratories, Burlingame, CA), and photographed with Zeiss Axio Imager Z1 Carl
Zeiss, Oberkochen, Germany.
Immunocytochemistry and Fluorescence Microscopy
Briefly, after deparaffinizing, rehydrating, and washing in 0.01 M
PBS (pH 7.2–7.4), sections were incubated with 3% H2O2 for 20
minutes at room temperature to block endogenous peroxidase,
Germline Stem Cells and Neo-Oogenesis in Monkey
subjected to high-pressure antigen recovery sequentially in 0.01%
citrate buffer (pH 6.0) for 2 minutes, incubated with blocking
solution (5% goat serum in PBS) for 20 minutes at room temperature, and then incubated with the diluted primary antibodies
overnight at 4 C. Blocking solution without the primary antibody
served as negative control. After washing with PBS, sections
were incubated with appropriate secondary antibodies (Alexa
Fluor 568, 488, or 594, Invitrogen). The sections were then
stained with 1 lg/ml Hoechst 33342 for 20 minutes for reveal of
nuclei, washed, mounted in Vectashield (H-1000, Vector Laboratories), and photographed with a Zeiss Axio Imager Z1 (Carl
Zeiss). The following primary antibodies were used for immunocytochemistry: VASA (ab13840, Abcam, Cambridge, MA)
(1:500), SCP3 (NB 300-232, Novus Biologicals, Littleton, CO)
(1:400), KI-67 (AB9260, Millipore, Billerica, MA; and 14–569982 eBioscience, San Diego, CA) (1:500), PCNA (SC25280, Santa
Cruz, CA) (1:400), TRF2 (05–521, Millipore) (1:400), LAMIN A
(ab26300, Abcam), SOX2 (AB5603, Millipore) (1:100), LIN28
(ab46020, Abcam) (1:100), and DAZL (ab134139, Abcam)
(1:500).
Testis and ovaries were continuously sectioned as described
below, and sections at every 30 section intervals were subject
to immunocytochemistry, and carefully examined under 340
objective to search for putative GSCs as shown by colocalized
immunostaining of strong nuclear PCNA, cytoplasmic DAZL,
nuclear LIN28, or SOX2, and absence of LAMIN A, and early
meiocytes marked by SCP3 filament structure with bouquet
clustering and perinuclear distribution of telomere indicated by
TRF2. These sections showed consistent results by examination
under the fluorescence microscopy. About 14–22 section images
from adult ovaries depending on the age group were examined
to estimate the number of GSCs and early meiocytes, in comparison with similar number of testis sections served as positive
control.
Follicle Counts
Fixed specimens from monkey testis and ovaries were subsequently dehydrated with graded alcohols, cleared in xylene, and
embedded in paraffin wax. It was impractical to section the entire
ovary of each animal, considering that the aim of the study was
to demonstrate ovarian senescence over time, so serial 5-lm sections of half of each ovary were cut and placed on silanized
slides. One out of every 20 serial sections was stained with
hematoxylin and eosin Y (H&E), and analyzed for the number of
follicles in four different developmental stages, and numbers of
primordial, primary, secondary, and antral follicles were classified
and counted using slightly modified standard methods [43, 49].
Primordial, primary, and intermediate-stage follicles were identified by the presence of an oocyte surrounded by a single layer of
flat, squamous, or cuboidal cells. Growing follicles were characterized as having more than one layer of granulosa cells with no
visible antrum. Antral follicles possessed small areas of follicular
fluid (antrum) or a single large antral space. Only those follicles
containing an oocyte with a clearly visible nucleus were scored,
so we could not exclude the possibility of losing de novo forming
follicles if any.
Immunohistochemistry for Detection of SCs
Briefly, slides were deparaffinized and rehydrated, incubated in
3% H2O2 for 20 minutes at room temperature to block endogenous peroxidase, and incubated sequentially with 5% goat serum
for 20 minutes after high-pressure antigen recovery, SCP3 rabbit
polyclonal antibody (NB 300-232, Novus) diluted 1:400 in
blocking solution at 4 C overnight, and then HRP polymer-goat
anti-mouse IgG (Maixin_Bio, Beijing, China) for 15 minutes.
Signals were detected by 3,3-diaminobenzidine substrate (Maixin_Bio). Slides were slightly counterstained with hematoxylin
(Sigma-Aldrich) and examined using light microscopy. Negative
controls were incubated with blocking solution containing no
antibodies.
Yuan, Zhang, Wang et al.
RT-PCR
Cryopreserved ovarian tissues were fully ground in liquid nitrogen and total RNA was isolated using the RNeasy Mini Kit
(74104, Qiagen, Valencia, CA). Total RNA was initially treated
with RNase-free DNase I (79254, Takara Bio Inc., Shiga, Japan)
to remove contaminating genomic DNA, and 1 lg of RNA was
used to synthesize the first strand cDNA using the M-MLV
Reverse Transcriptase (28025-013, Invitrogen) with oligod(T)18
(D511, Takara) primers. Amplification via 33 cycles of PCR was
then performed using Takara Ex Taq Hot Start Version
(DRR006, Takara) with primer sets specific for each gene
designed with dnaman5.2.2 (supporting information Table S1).
Parallel amplification of GAPDH was used to verify cDNA synthesis from total RNA isolated from each tissue. For each cycle,
the template was denatured at 94 C for 30 seconds, annealed at
58 C for 30 seconds, and extended at 72 C for 1 minute. Products of RT-PCR were run on 1% agarose gels and visualized by
staining with ethidium bromide.
Western Blot
Ovarian tissues and testis or liver were fully ground with glass
homogenizer and lysed in SDS sample buffer at 99 C for 10
minutes, and 25 lg of whole-tissue extract proteins was separated
on 10% SDS-polyacrylamide gels and transferred to polyvinylidine difluoride membranes (Millipore). Nonspecific binding was
blocked by incubation in 5% skim milk in TBST for 2 hours at
room temperature. Blots were incubated for 12–16 hours at 4 C
with anti-PCNA (Mouse mono-IgG2a; Santa Cruz, sc-25280),
(Tris-Buffered Saline and Tween 20), anti-LAMIN A (Rabbit polyclonal; Abcam, ab26300-100), anti-VASA (Rabbit polyclonal;
Abcam, ab13840), anti-TRF2 (Mouse monoclonal; Upstate, 05–
521), anti-DAZL (Rabbit polyclonal; Abcam, ab34139), or antib-ACTIN (Rabbit polyclonal; Santa Cruz, sc1616R), followed by
wash for 10 minutes three times. Blots were incubated at room
temperature for 2 hours with horseradish peroxidase conjugated
monkey-anti-rabbit IgG (GE Healthcare 371624) or goat antimouse IgG (H1L) (ZB2305) and washed three times for 30
minutes. Bound antibody was detected using Enhanced ECL
Amersham prime western blotting detection reagent (GE Healthcare RPN2232).
Hormone Assays
Serum samples from nine female monkeys were assayed twice
for progesterone (P4), testosterone (T), and estradiol (E2). P4, T,
and E2 levels were determined by direct radioimmunoassay using
commercial kits (R0205PR-B, R0204PR-B, and R0206PR-B,
China Diagnostics Medical Corporation, Beijing, China). Quality
control serum, sterilized distilled water, and five series diluted
standard samples for a standard curve were tested for each serum
sample. The intra- and inter-assay coefficients of variability for
P4, T, and E2 were below 10% and 15%.
Statistical Analysis
Statistical analyses were performed by ANOVA and means compared by Fisher’s protected least-significant difference using StatView software from SAS Institute Inc. (Cary, NC). p-Value <.05
was considered statistically significant.
RESULTS
Identification of GSCs Using Mouse Testis by Live
Cell Fluorescence-Activated Cell Sorting and
Immunofluorescence
Spermatogonia stem cells (SSCs), GSCs in testes, renew continuously in the male [26–28]. The putative GSCs isolated
from adult mouse and human ovaries were selected by
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fluorescence-activated cell sorting of cells expressing DDX4
(DEAD (Asp-Glu-Ala-Asp) box polypeptide 4, also called
VASA or MVH) [15], so we used this same method [15] to
isolate DDX4 expressing cells from mouse testes and ovaries.
SSCs exhibited very strong DDX4 fluorescence intensity, in
the range of 100–1,000. Some cells from testes also showed
weaker fluorescence, in the range of 10–100 (supporting
information Fig. S1), while other cells expressed only background fluorescence, below 10. These cells appeared to be
spermatocytes and somatic cells, respectively. In contrast,
cells isolated from adult ovaries exhibited only low levels of
DDX4 fluorescence (10–100). Cells with fluorescence in the
intense range (100–1,000), like that seen in SSCs, were not
detectable in cells extracted from the ovary (supporting information Fig. S1).
Next, we examined mouse testis for markers known to
label actively dividing cells and germ cells using immunofluorescence microscopy. Actively dividing stem cells take up
BrdU [48]. Labeling for proliferating cell nuclear antigen
(PCNA) also indicates the presence of dividing cells, including stem cells [50–52], and serves as an alternative to BrdU
incorporation. In testis, SSCs with small nuclei (white arrowheads, around 6 lm) located close to the basal membrane and
showed high BrdU incorporation, but they did not label for
VASA (supporting information Fig. S2A). SSCs had small
nuclei and stained both for BrdU and PCNA (supporting
information Fig. S2B). Testes cells showing cytoplasmic
VASA staining had relatively large nuclei (white arrows,
9–10 lm) (supporting information Figs. S2C, S3A), lacked
BrdU incorporation, and their location and appearance suggest
that they are primary spermatocytes. Some spermatocytes also
stained for diffuse, weaker PCNA. It appears that VASA
marked germ cells, but not necessarily GSCs. Indeed, VASA
protein is not expressed in oogonia or gonocytes during the
first trimester human fetal ovary [53]. These data suggest that
VASA may not reliably identify proliferative GSCs in monkeys or humans.
Nuclear LAMIN A is expressed in differentiated cells, but
not in somatic stem cells [33, 54, 55]. Thus, absence of
LAMIN A could provide an additional marker to identify
stem cells. We verified that BrdU-positive nuclei lacked
LAMIN A, while basal membrane cells showed LAMIN A
staining in mouse testis (supporting information Fig. S2D).
Notably, DAZL (deleted in azoospermia-like), a germ cell
marker, labeled cells with both small (with BrdU incorporation and PCNA staining) and large nuclei (without BrdU
incorporation, and with only diffuse PCNA staining) (supporting information Fig. S2E, S2F). Together, male mouse GSCs
have relatively small nuclei, about 6 lm in diameter, incorporate nuclear BrdU, lack nuclear LAMIN A, and stain for
nuclear PCNA and cytoplasmic DAZL. These criteria could
be used to identify GSCs (supporting information Fig. S3B).
GSCs Are Not Detectable in Monkey and Mouse
Ovaries
KI67 also labels proliferative cells, including stem cells [56–
59]. Monkey spermatogonia (GSCs) with round or oval
shaped nuclei stained strongly for both KI67 and PCNA (Fig.
1A), and also exhibited cytoplasmic DAZL staining (arrowheads) (Fig. 1B). Meiocytes or spermatocytes showed punctuate staining for KI67 and strong cytoplasmic staining of
DAZL. Strong nuclear staining for KI67 in proliferative cells
also was found in the cortex of ovaries from monkeys of various ages, but these cells showed no cytoplasmic staining for
DAZL (arrowheads) (Fig. 1B), although punctuate staining of
KI67 also was found in oocytes with weak peripheral staining
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Germline Stem Cells and Neo-Oogenesis in Monkey
Figure 1. Germline stem cells (GSCs) found in adult monkey testis but not in adult monkey ovaries by coimmunostaining of DAZL and KI67.
(A): Colocalized immunostaining of PCNA with KI67 in spermatogonial stem cells (SSCs) of monkey testis. (B): Identification of SSCs (GSCs)
by colocalized strong positive staining of KI67 and cytoplasmic DAZL staining in small nuclei from monkey testis, in contrast to the absent cytoplasmic DAZL staining and strong KI67-positive small nuclei (white arrowheads) of adult monkey ovaries. Note, spotted KI67 pattern in the
oocyte appears to be similar to that of spermatocytes. Scale bar 5 20 lm.
for DAZL in primordial and primary follicles. By careful
examination of sections processed by immunocytochemistry,
we estimated that an average of 24.3 6 3 (mean 6 SE) cells/
section (refer to Materials and Methods) were positive for
both PCNA and DAZL which may represent actively dividing
GSCs in adult monkey testis. In contrast, putative GSCs by
these criteria were not detectable in all immunostained sections from adult monkey ovaries for each age group. In addition, some oocytes showed weak staining for DAZL in young
and middle-aged adult monkey ovaries, but not in old monkey
ovaries (Fig. 1B). Spermatocytes at different stages also
showed punctuate nuclear staining for KI67 and strong cytoplasmic staining for VASA, and oocytes with these properties
were found in monkey ovaries (supporting information Fig.
S4). Some granulosa cells of growing follicles showed strong
KI67 staining, indicative of active proliferation, but they were
negative for VASA or DAZL. Together, these observations
demonstrate that GSCs homologous to SSCs in testis by these
specific criteria were undetectable in adult monkey ovaries.
Likewise, staining for strong nuclear PCNA and cytoplasmic DAZL also was found in monkey SSCs localized to
the peripheral seminiferous tubules (Fig. 2). Comparatively,
early spermatocytes in monkey testes showed diffuse, weak
staining for PCNA and strong cytoplasmic DAZL staining.
Small nuclei (<10 lm) with strongly positive PCNA
staining also were found in ovaries of young, middle-age,
and old monkeys, but these cells had no cytoplasmic DAZL
staining colocalized with the nuclear PCNA (Fig. 2), suggesting that these cells represent proliferative cells but not
necessarily GSCs.
In adult mouse ovaries, cytoplasmic VASA staining
appeared only in cells with large nuclei (white arrow) not incorporating BrdU. This antibody more likely labels primary
oocytes (supporting information Fig. S5A). Notably, nuclei positive for both BrdU and PCNA appeared in the cortex of mouse
ovaries (indicated by arrowhead), with some nuclei showing diffuse PCNA but negative for BrdU (arrows) (supporting information Fig. S5B). Strongly PCNA-positive nuclei lacked
cytoplasmic VASA (arrowhead), but oocytes stained for cytoplasmic VASA and diffuse PCNA (arrow) (supporting information Fig. S5C). Some cells incorporated BrdU and expressed
LAMIN A (arrow) (supporting information Fig. S5D), consistent with their differentiated cell types. BrdU-positive cells lacking nuclear LAMIN A (white arrowheads) were found in adult
mouse ovaries, but these BrdU-positive cells lacked cytoplasmic
DAZL staining (yellow arrows, supporting information Fig.
S5E), as did the strongly PCNA-positive cells in the epithelia
(yellow arrows) (supporting information Fig. S5F), providing
evidence against their germ cell origin and consistent with these
cells being somatic stem cells.
Yuan, Zhang, Wang et al.
2543
Figure 2. Immunocytochemistry of markers for germ cells and meiocytes by coimmunostaining of DAZL and PCNA in adult monkey ovaries
compared with testis. While meiocytes and germ cells in the testis are positive for DAZL in the cytoplasm and diffusely and lightly stained by
PCNA in the nucleus, spermatogonia stem cells are strongly stained by PCNA and weakly stained by DAZL in the cytoplasm. Nuclei strongly
stained for PCNA in monkey ovarian cortex do not exhibit typical cytoplasmic DAZL staining, in contrast to spermatogonial stem cells in the
adult testis. Young monkey at the age of 3–4 years; middle-aged monkey at 7–8 years; old, reproductive aging monkeys at 18–19 years. Scale
bar 5 20 lm.
Pluripotency-associated gene LIN28 orchestrates PGC
specification [60], and is strongly expressed in spermatogonia
in mice, non-human primates, and human, maintains the GSC
state in the developing human ovary, and might be a marker
for rare GSCs [61–63]. Likewise, SOX2 may also mark GSCs
in the testis [64]. LIN28 was found to express in the nuclei,
colocalized with PCNA and in the cytoplasm of putative
GSCs in the monkey testis, whereas PCNA-positive cells
were found in adult monkey ovaries, but did not show specific nuclei and cytoplasmic staining pattern for LIN28, unlike
GSCs in the testis (supporting information Fig. S6A). Also,
nuclear staining of SOX2 colocalized with strong nuclear
staining of PCNA was found in the monkey testis, but no specific nuclear SOX2 coimmunostaining with strong PCNA in
ovaries (supporting information Fig. S6B).
Neo-Oogenesis Is Absent in Adult Mouse and
Monkey Ovaries
During early meiosis in both males and females, telomere
bouquet clustering and perinuclear distribution are associated
with homologous bivalents identified by SC protein 3 (SCP3)
elements. These structures are essential for homologous chromosome pairing and synapsis and provide definitive markers
for prophase I of meiosis [36, 38–40, 65], and their presence
in adult ovaries would provide evidence of neo-meiosis. We
validated the presence of neo-meiosis in adult monkey testis
by coimmunostaining of DAZL with the telomere-associated
protein TRF2 (supporting information Fig. S7A), or coimmunostaining for SCP3 and TRF2 (supporting information Fig.
S7B). DAZL was strongly expressed in the cytoplasm of spermatocytes, which had perinuclear, bouquet clustering of telomeres, as shown by TRF2 immunostaining (supporting
www.StemCells.com
information Fig. S7A). DAZL also stained the cytoplasm of
premeiotic cells, with telomeres centered in the nuclei. Early
spermatocytes exhibited typical perinuclear distribution of
telomeres and distinct SCP3 lateral filaments connecting telomeres, as shown by TRF2 at termini of SCP3 filaments during
pachytene stage, and bouquets at the leptotene stage (supporting information Fig. S7B), and thus are distinguishable from
GSCs.
DAZL staining was found in mouse early spermatocytes,
meiocytes, and to a lesser extent in oocytes at different stages
(supporting information Fig. S8). Short, punctuate foci of
SCP3 immunostaining were found in fetal testis, with
increased staining in day 10 testis. SCP3 immunostaining
formed lateral filament structures in adult mouse testis (supporting information Fig. S9), like those of adult monkey testis. Aggregates of SCP3 presumably represent degraded or
fragmented SCP3 proteins, appearing after prophase I of
meiosis. Alternatively, insufficient levels of SCP3 proteins
may have failed to assemble distinct lateral filament structures, forming punctuated and fragmented SCP3 staining.
Likewise, no distinct SCP3 lateral filaments but punctuate
SCP3 staining were found in mouse newborn day 10 and
adult ovaries, while distinct staining of SCP3 lateral filaments
found in early meiocytes of mouse fetal ovaries (supporting
information Fig. S9), consistent with the notion that neomeiosis arises in fetal ovaries and adult testis, but not in postnatal ovaries.
Adult monkey testes undergo continuous early meiosis, as
shown by perinuclear telomere TRF2 distribution and homologous pairing indicated by SCP3 filaments, but these characteristic early meiocytes were absent in adult monkey ovaries,
regardless of age (Fig. 3). We estimated an average of
46.4 6 0.7 early meiocytes/section (refer to Materials and
2544
Germline Stem Cells and Neo-Oogenesis in Monkey
Figure 3. Coimmunostaining of SCP3 and TRF2 in adult monkey ovaries compared with testis. SCP3 lateral filaments and the telomere perinuclear distribution at the termini of the SCP3 filaments in meiocytes likely at the pachytene stage are found in testis but not in adult monkey ovaries. H&E shows histological sections. Inset, magnified image of a neo-meiocyte showing SCP3 axis filaments (red) attached with telomeres
(green). Scale bar 5 20 lm.
Methods) in adult monkey testis, but none in all immunostained sections from adult monkey ovaries for each age
group, substantially different from those of testis by statistical
analysis (SAS). Only fragmented and punctuate SCP3 staining
was found in oocyte nuclei, resembling postnatal day 10 and
adult mouse ovaries (supporting information Fig. S9). Immunohistochemistry using anti-SCP3 antibody identified SCP3 in
germ cells at various stages as well as in OSE cells, but the
morphology of these SCP3-positive structures in adult monkey ovaries differed from that of testes and fetal ovaries-no
clear SCP3 containing filamentous structures could be seen
(supporting information Fig. S10). Immunohistochemistry
does not permit resolution of SCP3-bearing filamentous structures, and thus cannot image meiotic synapsis and homologous pairing. Further, we performed additional experiments
by coimmunostaining of TRF2 to indicate telomeres and
MVH/VASA to indicate germ cells. Again, meiocytes with
perinuclear distribution of telomeres were evident in adult testis, but not in adult monkey ovaries (supporting information
Fig. S11). Together, the results of these experiments are not
consistent with neo-oogenesis in adult monkey ovaries.
Proliferative Somatic Stem Cells Found in Adult
Monkey and Mouse Ovaries
While searching for GSCs in adult monkey ovaries, we unexpectedly found cells with characteristics of somatic stem cells
in the adult ovary. LAMIN A is expressed exclusively in differentiated cells, including Sertoli, theca, epithelial cells as
well as germinal vesicle oocytes of mouse testis and ovaries
(supporting information Figs. S2, S12). In the OSE and ovarian cortex of mouse postnatal day 10 and adult ovaries, some
cells stained for PCNA, while others stained both for PCNA
and LAMIN A, suggestive of a differentiated state. Granulosa
cells from growing follicles also were positive for PCNA, but
not for nuclear LAMIN A, whereas oocytes all expressed
LAMIN A. As a positive control for GSCs, monkey spermatogonia stained strongly for PCNA and negatively for LAMIN
Yuan, Zhang, Wang et al.
2545
Figure 4. Proliferative somatic stem cells identified by coimmunostaining of LAMIN A with PCNA in ovaries of monkeys at different age
compared with adult testis from a 5-year-old monkey. Proliferative spermatogonia are strongly stained by PCNA without LAMIN A in the
nuclear envelope (white arrows), in contrast to strong LAMIN A positive but PCNA-negative staining in Sertoli cells and basal membrane cells.
Most cells in the ovarian cortex are strongly stained by nuclear envelope LAMIN A, but few PCNA strongly positive cells are negative for
LAMIN A (white arrows). Scale bar 5 20 lm.
A, whereas spermatocytes stained diffusely and weakly for
PCNA, but not for LAMIN A (Fig. 4; supporting information
Fig. S13).
Rare cells in the cortex or OSE of monkey ovaries stained
strongly positive for PCNA and lacked LAMIN A (Fig. 4), but
these cells did not stain for cytoplasmic DAZL (Fig. 2), so
these cells most likely were somatic stem cells, not GSCs. Primary oocytes in ovaries from young and middle-aged monkeys
diffusely stained for PCNA and exhibited punctuate staining for
KI67, and were negative for LAMIN A (supporting information
Figs. S13, S14). Most OSE cells, ovarian cortex, and the single
theca cell layer of primordial and primary follicles strongly
stained for LAMIN A, but not for PCNA or KI67, consistent
with their differentiated state. In contrast, granulosa cells from
growing follicles exhibited proliferative, stem cell-like properties, as evidenced by staining strongly for PCNA and KI67, but
negative for LAMIN A. KI67 and LAMIN A costaining of ovaries from monkeys of different ages revealed rare LAMIN Anegative, KI67-positive cells in the cortex and tunica albuginea
(arrows, supporting information Fig. S15). Consistently, mouse
ovaries contained rare proliferative cells with stem cell properties in the cortex, as shown by BrdU incorporation and LAMIN
A-negative staining (supporting information Fig. S16). Nuclei
of these cells in monkey ovaries were smaller (4–5 lm) than
testis GSCs (6 lm). Since these cells did not stain for DAZL,
they are likely somatic stem cells, not GSCs.
To test whether these cells originated from hematopoietic
stem cells (HSCs), we performed immunocytochemistry using
anti-CD45 and -CD34 antibodies. As expected, HSCs exhibited CD45 and CD34-positive staining in their cell membranes, but the monkey ovarian cells showed no specific
membrane staining for CD45 and CD34 (supporting information Fig. S17). Background staining for CD45 did not colocalize with KI67 staining. Together, these data suggest that rare
www.StemCells.com
proliferative stem cells exist in the cortex of adult ovaries,
but that these stem cells are not of germ cell origin, and
therefore they are not GSCs.
Age-Associated Ovarian Senescence in Monkey
Despite the extensive search, we failed to find compelling evidence for GSCs and early meiocytes in adult monkey ovaries.
We hypothesized that follicular renewal should be evident in
adult ovaries, if GSCs existed and neo-oogenesis took place
in the adult monkey. Hence, we examined follicle reserve by
counting follicles, measuring serum sex steroid hormone levels, and analyzing expression of genes related to senescence
in ovaries of monkeys at various ages.
Ovaries from old monkeys contained only rare follicles.
The numbers of primordial, primary, and secondary follicles
were reduced markedly in ovaries of old monkeys (18–19
years) compared with those of young (3–4 years) and middleaged (7–8 years) monkeys (Fig. 5A, 5B). Only few follicles
and no mature or antral follicles could be found in ovaries
from old monkeys. The number of primordial, primary, and
secondary follicles was significantly greater in ovaries from
3- to 4-year-old than from 7- to 8-year-old monkeys. Fewer
antral follicles appeared in ovaries from 3- to 4-year-old than
in ovaries from 7- to 8-year-old monkeys.
Levels of serum progesterone P4 were higher in the 18–
19-year-old compared to other monkeys. Levels of testosterone (T) were increased from young (3–4 years) to middle-age
(7–8 years), but dropped significantly in old monkeys (18–19
years). Similarly, levels of estradiol E2 were higher in 7–8year-old than in younger and older monkeys (Fig. 5C), consistent with their increased number of antral follicles
(Fig. 5B). Levels of T and E2 were much lower in monkeys
at 18–19 years of age. Follicle depletion, with reduced levels
2546
Germline Stem Cells and Neo-Oogenesis in Monkey
Figure 5. Ovarian ageing as evidenced by follicle depletion and decreased levels of sex hormones in monkeys with increasing age. (A): Representative morphology of primordial and primary, secondary, and antral follicles in the ovaries of monkeys at various reproductive ages (young,
Y, 3–4 years; middle-age, M, 7–8 years; and old, O, 18–19 years). For young and middle-age monkey ovaries, (a) indicates primordial and primary follicles, (b) secondary, and (c) antral follicles. For old monkey ovaries, (a) indicates rare primary follicles, (b) secondary, and (c) the
absence of antral follicles. Scale bar 5 100 lm. (B): Number of ovarian follicles at various developmental stages from monkeys at different
reproductive ages. (C): Levels of serum steroid hormones progesterone, testosterone, and estradiol from monkeys at various age. (D): Relative
expression levels of p21 and p16 in the monkey ovaries. n 5 3 (mean 6 SEM). Different superscripts shown above the bars indicate significant
differences (p < .05).
Yuan, Zhang, Wang et al.
2547
Figure 6. mRNA and protein levels of genes required for proliferation, meiosis, and germ cell development in monkey ovaries compared with
other tissues by RT-PCR analysis (A–G) and Western blot (H). (A) Markers for primordial germ cells formation, migration, and specification;
(B) markers of double-strand breaks; (C) markers for homologous pairing and meiotic synapsis; (D) markers of germ cells; (E) markers for cell
proliferation; (F) gene related to follicular development; (G) housekeeping gene and negative control. GAPDH was coamplified as an internal
control. MOCK, mock transcribed RNA samples. Y, young monkey (age 3–4 years); M, middle-aged monkey (7–8 years); O, old monkeys (18–
19 years). Liver and kidney served as negative controls for meiosis and germ cell development. (H) Protein expression required for cell proliferation and germ cell development by Western blot using antibodies against PCNA, TRF2, LAMIN A, VASA, and DAZL. b-ACTIN served as loading control.
of E2 and T, corroborated with an age-related reduction of
ovarian function in aging monkeys. Furthermore, expression
of genes associated with cell senescence, p21 and p16 [66,
67], was higher in ovaries from old than from young and
middle-aged monkeys (Fig. 5D). These data further support
the notion that the absence of GSCs and neo-oogenesis is
associated with ovarian aging in monkeys.
Furthermore, if GSCs and neo-oogenesis persist in adults,
their ovaries should express genes encoding proteins specific to
premeiotic or early prophase stages of oogenesis [7]. Early
germ cell development is controlled by a number of genes,
including NANOS3 [68], PRDM1 (also known as Blimp1) [69],
PRDM14 [70], and DAZL [71]. Msh5, Dmc1, and Scp1–3 are
crucial for homologous pairing and synapsis. PRDM9 [9, 72]
and Spo11 are involved in double-strand break formation.
To confirm the previous findings obtained by flow cytometry and immunofluorescence, we performed PCR analysis
and validated that stem cell and early meiosis genes are
expressed in testis, but not or at minimal levels in adult ovaries. PRDM1 was expressed in monkey ovaries of all ages,
but NANOS3 and PRDM14 only at low or minimal levels in
adult monkey ovaries unlike testis (Fig. 6A). Expression of
genes for homologous recombination, SPO11, PRDM9,
DMC1, and REC8, was low or undetectable in ovaries from
monkeys of all ages, and SCP3 or SCP1 mRNA levels were
very low or absent in young and middle-aged monkey ovaries, in contrast to testis (Fig. 6B, 6C). These results supported the immunocytochemistry data, and together show that
activities necessary for meiotic synapsis from leptotene to
pachytene and recombination at prophase I unlikely take place
www.StemCells.com
in adult monkey ovaries. Genes for germ cell development
(OCT4, DPPA3, and VASA) (Fig. 6D) and cell proliferation
(TERT and KI67) (Fig. 6E) were expressed at higher levels in
young and middle-age than in old monkey ovaries.
Notably, NOBOX, involved in follicular development [73],
was expressed in young and middle-aged monkey ovaries, but
its expression was very low or absent in testis and ovaries from
old monkeys (Fig. 6F), consistent with the active follicular
development in young and middle-aged ovaries, but not in testis or old monkey ovaries (Fig. 5). Moreover, expression of
DAZL, VASA, and KI67 was detected in young and middleaged monkey ovaries, but only minimally in ovaries from old
monkeys. At the protein level, PCNA was significantly
enriched in testis, and reduced greatly in ovaries from old monkeys. TRF2 protein also was highly expressed in testis but
much reduced in monkey ovaries (Fig. 6H). By contrast,
LAMIN A levels were high in adult ovaries. Notably, DAZL
and VASA protein levels were remarkably higher in testis than
in ovaries. Liver served as a negative control did not express
DAZL and VASA, as expected (data not shown), in agreement
with the PCR data. These data are not consistent with GSCs
and early germ cell formation in adult monkey ovaries.
DISCUSSION
We are unable to find evidence for proliferative GSCs and
germ cell renewal in adult monkey and mouse ovaries. The
lack of neo-oogenesis is further substantiated by the
Germline Stem Cells and Neo-Oogenesis in Monkey
2548
Table 1. Comparison of markers for GSCs and neo-oogenesis in testis and ovaries
GSC markers
Male/female
Monkey
Adult testis
Ovary
Young
Middle-age
Old
Mouse
Fetal ovary
Adult testis
Neo-oogenesis markers
Telomere TRF2
perinuclear
distribution
DAZL
VASA
KI67
PCNA
LAMIN A
DAZL/LIN28
VASA
SCP3 lateral
filaments
11
11
2
1/1
2
1
1
11
11
1
1
1
1
1
1
1/2
1/2
1/2
2/2
2/2
2/2
2
2
2
2
2
2
2
2
2
1
1
2
1
1
2
1
11
1
11
2
2
1/NA
1/NA
2
2
1
1
N/A
N/A
1
1
1
1
N/A: TRF2 antibody was not specific to mouse cells, or NA data not available. 11, strong staining; 1, positive staining; 2, negative staining; 1/2, few small cells negative staining.
Abbreviation: GSC, germline stem cell.
universal observation of robust ovarian failure in reproductively aged monkeys confirmed here. Continuous meiosis
and germ cell renewal, as occurs in testes, relies on a constant supply of proliferative GSCs, but our experiments
show no evidence of these in ovaries from mice or monkey.
Consistently, ovaries from female rhesus monkeys
approaching or undergoing the menopausal transition (about
20 years of age) demonstrated evidence of ovarian senescence, with decreasing numbers of primordial follicles [43],
concomitant hormone changes, and a molecular signature of
cell senescence. Ovaries from old monkeys contain very
few follicles, in association with their reduced levels of
estrogen and testosterone. These data are consistent with
ovarian senescence in older monkeys, similar to that
observed in women, and absence of neo-oogenesis which
presumably contributes to the ovarian senescence. However,
our data cannot exclude the possibility that dormant GSCs
may exist in adult females and are activated to replicate by
ovotoxic damage [74, 75].
We did find evidence of proliferative stem cells in the
cortex of adult ovaries, but these were somatic rather than
GSCs. Interestingly, granulosa cells also showed proliferative, stem cell-like properties, consistent with recent findings
[76]. These proliferative somatic stem cells in the adult ovaries exhibit strong positive staining for PCNA, but negative
for LAMIN A (Fig. 4). GSCs in the adult testis are positive
for SOX2 and LIN28, but negative for nuclear LAMIN A.
However, cells positive for SOX2 or LIN28 were not found
in adult monkey ovaries, regardless of PCNA-positive staining (supporting information Fig. S6A, S6B). It is unlikely
that the proliferative somatic stem cells found in adult monkey ovaries are positive for markers of SOX2 and LIN28. In
the ovarian cortex, the small nuclei with diameters of 4–5
lm were found, but were negative for markers of hematopoietic stem cells (CD45 and CD34). These cells might be
related to very small embryonic-like stem cells found in
adult human tissues and organs, for example, bone marrow
[77, 78]. Similar population of cells was recently found in
adult human ovaries [24, 79]. It is not excluded that these
small putative “somatic” stem cells are the prestage of
GSCs. The nature of these somatic stem cells in the ovary
remains to be determined, but presumably they replenish
granulosa cells and ovarian cortex, both highly proliferative
in the ovary. The lack of new meiocytes, marked decline in
follicle number, and elevated molecular signature of senescence in the aging ovary suggest that these cells lack signifi-
cant capacity to undergo neo-oogenesis and folliculogenesis
in vivo.
Together, GSCs, defined as proliferative stem cells that
stain for nuclear PCNA, KI67, and LIN28 and cytoplasmic
DAZL but not for LAMIN A, are readily found in testis but
not in ovary (Table 1). Neo-oogenesis as marked by SCP3
lateral filaments associated with perinuclear telomere distribution at termini also readily appears in adult testes but not
in ovaries, yet these decisive structures marked for homologous pairing in early meiocytes were not demonstrated, but
only SCP3 punctuate staining shown in previous studies [14,
15]. We found SCP3 punctuate staining in the primary
oocytes.
CONCLUSIONS
The well-established paradigm of female reproduction is that
the number of oocytes and the pool of follicles are fixed at
birth and continuously decline to the point when few
oocytes are available after menopause, and the supply of follicles dictates the length of her reproductive lifespan [37,
80]. The oocytes themselves orchestrate and coordinate ovarian follicular development [81]. By rigorous searching using
a variety of experimental approaches, we do not demonstrate
GSCs or neo-oogenesis in adult monkey ovaries, although
our data also cannot rule out the possibility that experimental conditions could reprogram ovarian adult somatic stem
cells to germ cells, and also the possible existence of
LAMIN A-positive female GSCs cannot yet be refuted, yet
they do support the existing paradigm that germ cells and
follicle reserve are not replenished in vivo by neo-oogenesis
in adult ovaries [8, 9, 11, 16, 18, 82–84]. They also raise
the distinct possibility that prior studies may have confounded a mixture of proliferative somatic stem cells and
primary oocytes, which do normally reside in the ovary,
with GSCs.
ACKNOWLEDGMENTS
We thank Shouquan Zhang, Xinglong Zhou, Yan Geng, Lixin
Feng, Yifei Liu, Xiangdong Tang, Jing Li, Yuanli Chen, and
Yahui Ding for help with the materials, experiments, or discussion. This work was supported primarily by the China
Yuan, Zhang, Wang et al.
National Basic Research Program (2010CB94500 and
2011CBA01002 to L.L., 2012CB911202 to N.L.,
2011CB964801 to T.C.), and Natural Science Foundation of
Tianjin (12JCZDJC24800, L.L.).
2549
DISCLOSURE OF POTENTIAL
CONFLICTS OF INTEREST
The authors indicate no potential conflicts of interest.
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