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Endocrinology 147(8):3789 –3796
Copyright © 2006 by The Endocrine Society
doi: 10.1210/en.2006-0394
Origin and Ontogeny of Mammalian Ovarian Neurons
W. Les Dees, J. K. Hiney, N. H. McArthur, G. A. Johnson, G. A. Dissen, and S. R. Ojeda
Department of Veterinary Integrative Biosciences (W.L.D., J.K.H., N.H.M., G.A.J.), College of Veterinary Medicine, Texas
A&M University, College Station, Texas 77843-4458; and Division of Neuroscience (G.A.D., S.R.O.), Oregon National
Primate Research Center/Oregon Health and Science University, Beaverton, Oregon 97006-3448
Mammalian ovaries contain sympathetic neurons expressing
the low affinity neurotropin receptor (p75NTR). To date neither the role these neurons might play in ovarian physiology
nor their embryological origin is known. Immunohistochemistry was used to detect postnatal changes in distribution and
number of both p75NTR-positive and tyrosine hydroxylasepositive neurons in rhesus monkey ovaries. Pig fetuses were
used to map the pathway of ovarian neuronal migration during embryonic development. Antiserum to p75NTR revealed
the presence of isolated neurons and neurons clustered into
ganglia in 2-month-old monkey ovaries. After 8 months, the
neurons exhibited well-developed processes, and other than
being more extensively interlaced, the localization and morphology did not change after 2 yr of age. Total number of
p75NTR-positive neurons present decreased gradually between 2 months and 12 yr of age and declined markedly with
F
reproductive aging. Conversely, the subpopulation of neurons
immunoreactive to anti-tyrosine hydroxylase increased significantly at puberty and then declined with the loss of reproductive capacity. By d 21 of fetal life in the pig, p75NTR
neurons had migrated medially from the neural crest to form
the paraaortic autonomic ganglia. Some neurons migrated
ventrally from the ganglia and then continued ventrolaterally
to enter the genital ridge. By d 27, neurons had entered the
developing ovary, and by d 35, the migration was complete
with neurons demonstrating immunoreactivity to NeuN, a
neuron-specific marker. Results demonstrate that p75NTR-expressing ovarian neurons originate from the neural crest and
that a catecholaminergic subset is associated with pubertal
maturation of the ovary and subsequent reproductive
function. (Endocrinology 147: 3789 –3796, 2006)
OR MANY YEARS it has been known that the mammalian ovary is innervated by sympathetic and sensory
neurons extrinsic to the gland. In addition to this extrinsic
innervation, the ovary also contains an intrinsic innervation
provided by neurons residing within the gland. Over a century ago, using a potassium bichromate-osmium tetroxide
stain, neuron-like cells were described in ganglia within the
medulla of the human ovary (1); however, this finding was
challenged some 40 yr later (2). This controversy remained
for another 60 yr, until the early 1990s, when we described
unequivocally the presence of neurons in nonhuman primate
ovaries (3, 4). Specifically using immunocytochemistry, we
observed in ovaries from adult rhesus and Japanese macaque
monkeys, the presence of neuronal-like cell bodies that were
approximately 20 ␮m in diameter and expressed the lowaffinity neurotropin receptor (p75NTR). In addition to their
morphological appearance, these cells also expressed neuron-specific enolase and the high-molecular-weight neurofilament peptide, neurofilament-200, conclusively identifying them as neurons. A subpopulation of these were also
immunoreactive to tyrosine hydroxylase (TH), revealing
their catecholaminergic phenotype and thus their sympathetic lineage. The neurons were observed either isolated or
in small, discrete ganglia within both the cortex and medulla
of the ovary and were also associated with the blood vasculature and thecal layers of developing follicles.
Subsequent studies demonstrated the presence of neurons
in ovaries from rats of the Wistar line (5, 6) and fetoneonatal
human ovaries (7). The presence of the vertebrate neuronspecific nuclear protein (NeuN) in these cells further identified them as neurons (6). As in the monkey ovary, some of
the neurons in both rat and human ovaries proved to be
sympathetic as defined by their immunoreactivity to antibodies to TH (6, 7). Others contain neuropeptide Y, confirming their sympathetic nature (6).
Although these findings establish the existence of an intrinsic network of ovarian neurons, nothing is known about
their embryological origin and their potential role in ovarian
function. If these neurons contribute to regulating ovarian
endocrine activity, one would expect to detect plastic
changes in their chemical phenotype, distribution within the
gland, and/or their population size at critical times during
the gland’s life span. Major functional changes in primate
ovarian function occur during peripubertal development,
the menstrual cycle, and during menopause. In the present
study, we used postnatal rhesus monkey ovaries to show that
whereas the total size of this neuronal population declines
with age, the number of TH-containing neurons increases at
puberty and then declines precipitously by the end of reproductive life. In addition, we used embryonic pig ovaries
to demonstrate the mammalian intrinsic ovarian neurons are
derived from the neural crest.
First Published Online May 25, 2006
Abbreviations: NeuN, Neuron-specific nuclear protein; RTU, ready to
use; TBS, Tris-buffered saline; TH, tyrosine hydroxylase.
Endocrinology is published monthly by The Endocrine Society (http://
www.endo-society.org), the foremost professional society serving the
endocrine community.
Materials and Methods
Rhesus monkey ovaries
Both ovaries from rhesus macaques (Macaca mulatta) ranging between
2 months and 28 yr of age were used to assess the postnatal ontogeny
of primate ovarian neurons. The ovaries were obtained through the
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Endocrinology, August 2006, 147(8):3789 –3796
Oregon National Primate Research Center necropsy program from monkeys killed for a variety of reasons or animals ovariectomized for studies
conducted by other researchers. The ovaries were assigned to six developmental groups: infantile (2–3.7 months of age; n ⫽ 3 pair), juvenile
(8 months to 2 yr; n ⫽ 4 pair), peripubertal (3–3.5 yr; n ⫽ 3 pair), adult
(8 –12 yr; n ⫽ 3 pair), presenescent (17 yr; n ⫽ 1 pair), and senescent
(20 –28 yr; n ⫽ 3 pair). All ovaries were cleaned of adherent tissue, halved
and fixed by immersion in Zamboni’s fixative for 24 h at 4 C. After
fixation, they were rinsed overnight in 0.01 m phosphate buffer (pH 7.4)
at 4 C, frozen in 2-methylbutane cooled in liquid nitrogen, and then
stored in air-tight plastic bags at ⫺80 C before sectioning. Ovaries of
infantile and peripubertal monkeys had all stages of follicular development but showed no signs of ovulation. Adult ovaries showed all
stages of follicular development, including corpus luteum. Presenescent
and senescent ovaries showed atretic follicles and predominantly connective and interstitial tissue.
Embryonic pig ovaries
Due to the difficulty of obtaining fetal tissue from nonhuman primates and because we identified a population of p75NTR-positive neurons in the adult pig, we used embryos from this species to define the
migratory pathway of these neurons into the fetal ovary. Surgical procedures involving pigs were approved by the Texas A&M University
Institutional Agricultural Animal Care and Use Committee. Sexually
mature cross-bred gilts (Yorkshire ⫻ Landrace dams and Duroc ⫻
Hampshire sires) of approximately 8 months of age and weighing 100
kg were observed daily (0700 h) for estrous behavior through direct
exposure to intact boars. Gilts exhibiting at least two estrous cycles of
normal duration (18 –21 d) were bred to cross-bred boars (Yorkshire ⫻
Landrace dams and Duroc ⫻ Landrace sires). Pregnant gilts were hysterectomized on d 21, 27, 35, or 60 of gestation. Briefly, gilts received an
im injection of Telazol (1 mg/kg body weight) to induce anesthesia,
followed by administration of isofluorane (1–5%) via inhalation during
surgery. The uterus was removed by midventral laparotomy. Fetuses
were then removed, killed in a CO2 saturated environment, and immersion fixed in 4% paraformaldehyde for 72 h at 4 C. The fetal tissues
were then rinsed for 48 h in 0.05 m Tris-buffered saline (TBS) (pH 7.4)
at 4 C and embedded in paraffin.
Immunohistochemistry
Immunohistochemical staining of monkey ovaries was performed on
15-␮m frozen sections that were floated in 0.01 m TBS (pH 7.4), as we
described previously (4). A typical experiment consisted of approximately 10 sections from each of the ovaries representing one of the above
age groups. The sections from each ovary were separated such that half
were stained for the presence of p75NTR and the other half for TH. This
was repeated two to three times to obtain 10 –15 sections for p75NTR and
the same number for TH from different parts of the ovary. In each case,
after three 10-min washes in TBS, the sections were floated for 20 min
in TBS containing 0.1% H2O2 to inhibit endogenous peroxidases, followed by a 1-h incubation at room temperature in washing buffer (TBS
with 0.5% Triton X-100) containing 3% normal horse or goat serum to
reduce background during p75NTR and TH staining, respectively. Sections
were then incubated with either a 1:10,000 dilution of a monoclonal
antibody raised against the human p75NTR (Neomarker, Inc., Fremont,
CA) or a 1:500 dilution of a polyclonal antibody to TH (Eugene Technology, Allendale, NJ) for 24 h at 4 C and then for 3 h at 37 C. For controls,
either buffer or normal rabbit serum replaced the specific antisera. The
sections were then rinsed in washing buffer, incubated in biotinylated
horse antimouse or goat antirabbit IgG (IgG; Vector Laboratories,
Burlingame, CA) for p75NTR and TH, respectively, at a dilution of 1:250
for 1 h at room temperature, washed, and then incubated in avidin-biotin
complex for 75 min at room temperature. Sections were then rinsed in
TBS and washed three times in 1 m sodium acetate/0.2 m imidazole
buffer (pH 7.2–7.4). They were then exposed for 2–5 min to a solution
containing 2.5% nickel ammonium sulfate, 0.05% 3,3-diaminobenzadine-HCl, and 0.005% H2O2, yielding a black reaction product. The
reaction was stopped in 1 m sodium acetate/0.2 m imidazole buffer, and
the sections were washed in TBS and mounted on gelatin-coated slides.
Finally, the sections were dehydrated in graded alcohols, cleared in
Histoclear (VWR Scientific, Philadelphia, PA); coverslipped, and viewed
Dees et al. • Primate Ovarian Neurons
with a E400 microscope (Nikon, Tokyo, Japan) equipped with a Nikon
DXM 1200 video camera using the ACT-1 version 2 software for image
acquisition. Some immunocytochemically stained sections were counterstained with methyl green to show the surrounding ovarian
histology.
The p75NTR immunohistochemical staining of 21-, 27-, and 35-d-old
fetal pig tissue was conducted directly on gelatin-coated glass slides
containing four to eight 15-␮m paraffin sections made through the thoracic and lumbar regions. The sections were deparaffinized, rehydrated
in Histoclear, and descending concentrations of alcohol ending in water.
The sections underwent antigen unmasking by microwaving them for
20 min in 10 mm Na citrate (pH 6.0) and then allowed to cool for 20 min.
All sections were stained immunohistochemically as above except that
the p75NTR antibody dilution was used at 1:5000. NeuN immunostaining
was used to specifically identify neurons in 35-d fetal tissue as above as
well as ovaries collected from 60-d-old pig fetuses. The protocol used
was the same as above with the following exceptions. Sections were
incubated for 1 h in ready-to-use (RTU) normal horse blocking serum
and then incubated overnight at 4 C with NeuN antiserum (7.5 ␮g/ml;
Chemicon, Temecula, CA). The following day, the sections were washed
with TBS and then incubated with the RTU secondary antibody for 30
min at room temperature. The sections were then washed with TBS for
5 min then incubated for 30 min in RTU streptavidin/peroxidase reagent
at room temperature. After washing in TBS, the sections were exposed
to the 3,3-diaminobenzadine-HCl/Ni solution for 5 min. The tissues
were then washed with water and then dehydrated, cleared, coverslipped, and viewed as described above.
Quantitation of postnatal ovarian neuronal density
The numbers of p75NTR and TH-positive ovarian neuronal perikarya
from monkeys ranging from 2 months to 28 yr of age were counted by
two investigators. A total of 10 –15 sections per ovary were evaluated.
Depending on the age of the ovary and size of the section, 15– 40 fields
of view, at a magnification of ⫻40, were assessed per section. The
number of neurons per field was recorded, and then the total number
of neurons counted was divided by the number of fields assessed to
determine the mean number of neurons per field per section. Data were
assigned to one of the above six postnatal age groups to determine the
mean (⫾sem) number of neurons per field for that age group. The
differences between age groups were then assessed by ANOVA, followed by post hoc testing using the Student-Newman-Keuls multiple
range test. These data are presented as the mean (⫾sem) number
of p75NTR or TH neurons per field, and differences were considered
statistically significant if P ⬍ 0.05.
Results
Postnatal ovarian neuronal development in the
rhesus monkey
In infantile ovaries from 2 to 3.7 months of age, a layer of
p75NTR-immunopositive cells was observed surrounding follicles of various sizes (Fig. 1A). Counterstaining the tissue
with methyl green confirmed that the positive immunoreactivity was localized to the thecal cell layer of developing
follicles (Fig. 1B). Other p75NTR-positive staining in these
ovaries revealed neurons with their short processes extending from the soma. These neuron-like cells are mostly clustered into discrete ganglia between small follicles (Fig. 1C).
There are also neurons present that are isolated but only a
few in number at this age. Most of them are round to spherical in shape, localized within both the medulla and cortical
regions of the gland, and, as in adult ovaries (3, 4), are most
often associated with the blood vasculature.
By 8 months of age, the ovarian neurons had already
attained their adult-like morphological appearance. Specifically, the perikarya of these neurons were irregular to spherical in shape and exhibited well-developed unipolar, bipolar,
Dees et al. • Primate Ovarian Neurons
Endocrinology, August 2006, 147(8):3789 –3796
3791
or multipolar processes extending from them (Fig. 2, A–C).
These neurons were observed scattered individually
throughout both the medulla and cortex, with most of them
being in the cortical region. By 2 yr of age, the neuronal
processes were longer and appeared to reach outward toward adjacent neurons (Fig. 2D). Other than more extensive
network of processes, the localization of ovarian p75NTR immunoreactivity and the morphology and distribution of the
ovarian neurons within the interstitial area as well as around
blood vessels and developing follicles did not change after 2
yr of age. The number of ovarian neurons present, however,
underwent dramatic age-related changes as discussed
below.
Quantitation of postnatal ovarian neuronal density
Assessment of the number of ovarian p75NTR immunoreactive neurons throughout the life span of the rhesus monkey
revealed marked age-related changes. Figure 3 demonstrates
that the greatest number of immunoreactive neurons were
present during the infantile period of development, with a
gradual decline occurring thereafter. An initial reduction in
the number of neurons (P ⬍ 0.001) occurred between the
infantile (2– 4 months of age) and early juvenile period (8
months to 2 yr of age). The number of neurons continued to
gradually decline (P ⬍ 0.01) between 2 yr of age and adulthood (8 –12 yr of age). Between 12 yr of age and the end of
reproductive life (17 yr of age), another striking decline (P ⬍
0.001) was noted in the number of neurons present, which
FIG. 1. p75NTR-positive cells in ovaries from an infantile (2 month
old) monkey. A, p75NTR immunoreactivity is observed in elongated
cells (arrowheads) surrounding individual follicles (f). B, Methyl
green counterstaining shows that the p75NTR immunoreactivity is
present in thecal cells (arrows) but not granulosa cells. C, Isolated
p75NTR neuron-like cells with their processes are also observed (arrowheads), but the vast majority of them are clustered in ganglia
between the developing follicles in both medulla and cortex (arrows).
Bar, 50 ␮M (A and C); 25 ␮M (B).
FIG. 2. p75NTR-positive neurons in ovaries from juvenile monkeys.
A–C, Ovarian neurons by 8 months of age have perikarya of spherical
or irregular shape and exhibit long processes extending from them. D,
Neurons from a 2-yr-old ovary connected via long processes (arrows)
that suggest the formation of an intraovarian neuronal network. Bar,
10 ␮M (A–C); 7.5 ␮M (D).
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Dees et al. • Primate Ovarian Neurons
FIG. 5. p75NTR-positive neurons in the adult pig ovary. Note that pig
ovarian neurons (arrows) have the same morphological appearance as
rhesus monkey ovarian neurons depicted in Fig. 2. Bar, 10 ␮m.
FIG. 3. Postnatal ontogeny of p75NTR-positive ovarian neurons from
infantile development to old age in rhesus monkeys. Note the gradual
decline in the number of immunoreactive p75NTR neurons over prepubertal and adult years and the abrupt loss of detectable neurons at
the end of reproductive life. Bars represent mean (⫾SEM). a vs. b, P ⬍
0.001; b vs. c, P ⬍ 0.05; c vs. d, P ⬍ 0.001; d vs. e, P ⬍ 0.05.
was even more evident in monkeys 20 yr old and older. At
this time, very few neurons were observed.
In contrast to this persistent, constant, age-related decline
in the total number of ovarian neurons, the fraction of catecholaminergic neurons, as assessed by their TH immunoreactivity, increased (P ⬍ 0.001) at the expected time of puberty (Fig. 4), i.e. 3–3.5 yr of age (8). After puberty, the
numbers of TH-containing neurons remained elevated
throughout the years of peak reproductive activity, decreasing markedly (P ⬍ 0.001) between 12 and 17 yr of age. After
20 yr of age, the neurons had essentially disappeared from
the ovary.
therefore, we used this species as the animal model to assess
the embryological origin of mammalian ovarian neurons.
It has been known for many years that sympathetic neurons migrate from the neural crest to form bilateral autonomic ganglia, referred to as the primary sympathetic chain,
along the dorsolateral aspects of the aorta (9 –11). Figure 6
shows a schematic drawing of the neuronal migratory pathway from the neural tube/spinal cord region to the autonomic ganglia. The neuronal migration continues from the
autonomic ganglia to the genital ridge as revealed by the
following experiments. At 21 d of fetal life (length of gesta-
Embryonic origin of ovarian neurons
To conduct these studies, we used pig fetuses because of
the near impossibility of obtaining early monkey fetal tissues.
As in the monkey, the adult pig ovary also contains p75NTRpositive neurons (Fig. 5). Although they appeared to be fewer
in number, the pattern of localization and their morphology
was the same as the ovarian neurons described in primates;
FIG. 4. Postnatal ontogeny of ovarian neurons containing TH in the
rhesus monkey ovary. Note the increased number of TH-positive
neurons detected around the time of puberty (3–3.5 yr) in rhesus
females. Also, note that the number of TH neurons remained elevated
during the years of productive reproductive life and then declined
markedly after 12 yr of age. Bars represent mean (⫾SEM). a vs. b, P ⬍
0 001; b vs. c, 0.05; c vs. d, P ⬍ 0.001; d vs. e, P ⬍ 0.05.
FIG. 6. Schematic drawing showing the ovarian neuronal migratory
pathway from the neural crest to the genital ridge in a 21-d pig fetus.
It has been known for many years that neurons within the autonomic
ganglion just dorsolateral to the aorta are of neural crest origin. We
now show that a population of these neurons emerge bilaterally from
the ganglia and continue to migrate ventrally along the lateral borders of the aorta. The neurons eventually merge medially beneath the
aorta and then continue with a ventral migration until they diverge.
At this point, most of the neurons continue to migrate ventrally and
enter the developing gut on the midline, whereas other neurons take
a lateral route to enter the dorsal aspect of the genital ridge, located
on the medial surface of the developing mesonephric kidney. Note that
as the fetus develops, the ovary emerges as an outpocket of the developing genital ridge. The arrows denote the pathway of neuronal
migration toward the genital ridge, and the stars represent the neurons. nt, Neural tube; n, notochord; ag, autonomic ganglion; a, aorta;
gr, genital ridge; mk, mesonephric kidney; g, gut.
Dees et al. • Primate Ovarian Neurons
Endocrinology, August 2006, 147(8):3789 –3796
3793
tion 114 d), p75NTR-positive neurons in pig fetuses had migrated medially to form the autonomic ganglia associated
with the aorta. Some of these neurons had migrated beyond
the ganglia, in a ventral direction along the lateral border of
the artery (Fig. 7A). This occurred bilaterally, and the migrating neurons from both sides converged at the midline,
beneath the ventral border of the aorta. At this point, most
of these p75NTR-positive neurons continued with their ventral pattern of migration, entering the dorsal aspect of the
developing gut (Fig. 7, B and C); however, a subgroup of the
neurons can be seen migrating laterally, entering the most
dorsal aspect of the thickened epithelium forming the genital
ridge on the medial surface of the mesenephros (Fig. 7, B and
C). The epithelial cell lining in this region showed very little
to no immunoreactivity (panel C), suggesting that most of the
p75NTR immunoreactivity in the genital ridge at this time is
present in migrating neurons.
In a 27-d-old fetus, the developing ovary is a rounded
structure that develops as an outgrowth from the genital
ridge in close association with the medial surface of the
kidney. At this time, p75NTR immunoreactive cells were
abundant at the pole of entry into the ovary (Fig. 8, A and B).
Again, the adjacent epithelial lining at the interface of the
ovary and developing kidney appeared to express little or no
immunoreactivity, suggesting that most p75NTR-positive
cells entering the ovary at this stage are neurons. Some
p75NTR immunoreactive cells had penetrated beyond the entry pole, taking residence within the ovarian tissue (Fig. 8A),
suggesting that entry into the ovary began before d 27.
By 35 d of embryonic development, the ovary is fully encapsulated, the migration into the gland is complete, and
p75NTR-positive cells can be observed dispersed throughout
the gland (Fig. 9A). Higher magnification reveals that
whereas some of these p75NTR immunoreactive cells are
dispersed within the interstitial tissue, others are near small
follicles (Fig. 9, B and C). Specific immunolocalization of
nuclear NeuN in ovaries at both 35 (Fig. 9D) and 60 (Fig. 9E)
d of embryonic development demonstrates that some of the
cells present are indeed neurons.
Discussion
In the present study, we used pig fetuses to demonstrate
that p75NTR-expressing neurons, previously shown to be
present in monkey (3, 4), human (7), and rat (5, 6) ovaries
originate from the neural crest during embryonic development. Using rhesus monkeys, we also showed that during
postnatal development ovarian p75NTR-positive neurons undergo remarkable plastic changes as a population, first decreasing gradually in number between early postnatal development and adulthood and then dramatically by the end
of reproductive life. This decrease notwithstanding, when
catecholaminergic neurons are segregated from the overall
population by TH immunostaining, it becomes apparent that
their number increases at puberty instead of declining. The
TH-immunopositive neurons continue to decrease precipitously at the end of reproductive life after the developmental
pattern of the entire population. In addition to these changes
in population size, the neurons also show distinct changes in
both morphology and distribution. At 2– 4 months of age,
FIG. 7. Immunohistochemical demonstration of ovarian neuronal
migration from the autonomic ganglia to the genital ridge in a 21-d
pig fetus. A, p75NTR-positive neurons in the autonomic ganglia are
located at the dorsolateral aspect of the aorta. Note that some neurons
are observed migrating ventrally from the ganglia along the lateral
border of the aorta (arrows). B, Neurons merge beneath the aorta and
continue to migrate ventrally (white arrows). Note the neurons diverge eventually at this level, either migrating ventrolaterally (black
arrows) as they enter the genital ridge or continuing ventromedially
to enter the gut. C, Higher magnification of the area shown in B,
specifically depicting the immunopositive neurons (arrows) entering
the dorsal aspect of the genital ridge. a, Aorta; ag, autonomic ganglion;
g, gut; gr, genital ridge. Bar, 10 ␮M (A); 25 ␮M (B); 10 ␮M (C).
they had short processes and were mainly clustered into
discrete ganglia between follicles, with some isolated neurons present in both cortical and medullary regions of the
gland. By 8 months of age, the neurons exhibit perikarya of
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Dees et al. • Primate Ovarian Neurons
FIG. 8. Immunohistochemical demonstration of ovarian neurons entering the developing ovary of a 27-d pig fetus. A, Dense p75NTRpositive neuronal staining at the pole of entry into the gland (arrows).
Note that although the majority of cells are entering the gland at this
time, some staining deeper within the ovary (arrowheads) indicates
their initial arrival had occurred before d 27. B, Higher magnification
of the area shown in A, depicting the immunopositive neurons at the
pole of entry into the developing ovary. o, Ovary. Bar, 100 ␮M (A); 10
␮M (B).
oval to irregular shape and well-developed processes, as
previously described in adult rhesus monkey ovaries (4).
Between 8 months and 2 yr of age, the processes were more
extensive and appeared to form connections with other neurons, although true connections would need to be confirmed
by electron microscopy, for example.
Ovarian neurons have been observed in all human and
nonhuman primates assessed thus far; however, they have
been observed in only one strain of rat. Whereas ovarian
neurons are present in Wistar rats (5), they are not present in
Sprague Dawley or Long Evans rats (6). Additionally, the
number of neurons is very small in the rat (⬃15 per ovary)
(5), compared with several hundred per ovary in the monkey, depending on the age of the animal (present study). The
only human ovaries studied to date have been from the
fetoneonatal glands (7). An assessment of the number of
human ovarian neurons was not performed due to limited
availability of tissue, but the neurons were mostly isolated
instead of clustered into ganglia. Most of them were located
within the medulla, with only a few neurons present in the
cortex, even in 10-month-old ovaries. By comparison, we
describe here the presence of numerous ganglia in monkey
ovaries during fetal and neonatal development as well as a
large population of cortical neurons by 8 months of age. This
difference could represent a species variation or, perhaps,
reflect an earlier development of the ovarian cortex in nonhuman primates. With regard to the pig; in this study we
demonstrate the presence of neurons in adult ovaries and
report that their morphology and localization in cortex and
medulla are similar to those ovarian neurons described
above in the other species.
FIG. 9. Immunohistochemical demonstration of ovarian neurons in
the ovary of 35- and 60-d pig fetuses. A, The ovary is completely
encapsulated, the neuronal migration is complete, and the p75NTR
immunoreactivity is dispersed throughout the gland. B and C, Presence of several neurons (arrows) as well as apparent thecal cells
surrounding a small follicle (arrowheads). D and E, NeuN-immunopositive cells (arrows) in 35- and 60-d-old fetal ovaries, respectively,
demonstrating that these cells are neurons. f, Follicle; o, ovary. Bar,
100 ␮M (A); 10 ␮M (B); 40 ␮M (C–E).
The function of ovarian neurons is not yet known, but to
gain insights into their role in ovarian physiology, we assessed their postnatal ontogeny throughout the lifetime of
the rhesus monkey. We first determined the number of ovarian neurons expressing p75NTR and found that these neurons
were more abundant in neonatal ovaries. Subsequently their
numbers declined gradually until the monkeys were about
12 yr of age, which is near the end of peak reproductive
performance in this species (Oregon National Primate Research Center fertility records, unpublished data). Between
12 and 17 yr of age, there was a marked decline in the number
of these neurons, and by 20 yr of age, their presence in the
ovary was rare. The fact that some of these ovarian neurons
in every species studied to date are capable of producing
catecholamines (4, 6, 7, 12, 13) suggests that they may be able
Dees et al. • Primate Ovarian Neurons
to influence steroidogenesis (14 –18). Therefore, we determined whether the number of TH-positive neurons changes
with age and found they increased markedly at the time of
puberty, remaining elevated during the years of reproductive activity before declining dramatically during the presenescent and senescent years.
This developmental pattern of expression suggests that
ovarian neurons contribute to the pubertal activation of ovarian function as well as maintaining the activity of the gland
during adulthood. Inferential support for this conclusion is
provided by the fact that Wistar rats, whose ovaries contain
neurons (6) and have a longer reproductive life span than
Sprague Dawley rats (19), which lack these neurons (6). It has
been demonstrated that subsets of intrinsic ovarian sympathetic neurons contain not only TH (4) but also neuropeptide
Y (6). Because both TH and neuropeptide Y-containing fibers
extensively innervate the ovary (20 –22) from neurons outside the gland, it appears that the intrinsic ovarian neurons
may act coordinately with the extrinsic innervation to influence ovarian physiology. Further assessment of the function(s) of these ovarian neurons will no doubt be important
for gaining a better understanding of mechanisms controlling normal ovarian physiology but may also lead to a better
understanding of specific ovarian malfunctions, whether
caused by genetic, toxicological, or other types of insults.
Selective ablation of ovarian neurons by genetic or pharmacological means may be required to define the roles they may
play in the gland.
Whereas resolution of this issue awaits the development
of specialized tools, we used a morphological approach to
address another important question: the origin of ovarian
neurons. In this study, we described the pathway of neuronal
migration to the genital ridge using the developing fetal pig.
The dorsal rims of the neural plate that meet during tube
closure form the neural crest, a specialized population of
migratory cells that then give rise to many regions of the
developing embryo. It has been known for many years that
neurons of the autonomic nervous system are made up entirely of cells that have migrated out of the neural crest (9, 10,
23). Specifically, sympathetic neurons migrate from the neural crest to form a primary sympathetic chain along the
dorsolateral border of the aorta (9 –11). Some neurons of this
chain persist as the primary sympathetic ganglia, but the
majority of neurons migrate from the paraaortic area using
segmental branches of the aorta as routes of migration (24,
25). Interestingly, many years ago it was shown that neurons
in the walls of the thoracic and abdominal viscera migrated
to those regions from the neural crest and did not differentiate from mesodermal or endodermal cells (9, 25). We now
demonstrate, using a 21-d-old pig fetus, a population of
p75NTR-positive neurons within the paraaortic sympathetic
ganglia that migrate in a ventral direction. Most of these
neurons continued on their ventral migration to enter the
dorsal aspect of the gut; however, we noticed that a small
population of these cells diverge just dorsal to the gut and
migrate laterally, entering the dorsal aspect of the genital
ridge developing on the medial border of the mesonephric
kidney. The ovary develops as an out growth of this genital
ridge, and we observed p75NTR-positive cells entering the
ovary on d 27, with the migratory process to the ovary
Endocrinology, August 2006, 147(8):3789 –3796
3795
complete by d 35 of fetal development. Importantly, some of
these intraovarian cells were confirmed to be neurons as
demonstrated by their content of NeuN.
In summary, the present study documents the presence
and developmental changes in the size of a population of
p75NTR-immunoreactive neurons in the rhesus monkey
ovary. Furthermore, we demonstrate that the size of a catecholaminergic subpopulation of these cells increases at the
time when ovarian reproductive competence is attained and
then decreases dramatically by the end of the reproductive
life span. Our results also demonstrate that ovarian neurons
migrate to the genital ridge from the paraaortic sympathetic
ganglia and that their origin, therefore, is the neural crest.
Acknowledgments
The authors thank Dr. R. C. Burghardt for the use of the Image
Analysis Facility (Department of Veterinary Integrative Biosciences,
Texas A&M University) and Sarah Burdick, Grace Schnell, and Maria E.
Costa for technical assistance. Special thanks to Dr. Frederick A. Pereira
(Baylor College of Medicine, Houston, TX) for his assistance with the
embryology of the fetal pig.
Received March 27, 2006. Accepted May 17, 2006.
Address all correspondence and requests for reprints to: Dr. W. Les
Dees, Department of Veterinary Integrative Biosciences, College of Veterinary Medicine, Texas A&M University, College Station, Texas 778454458. E-mail: [email protected].
This work was supported by National Institute of Health Grants
AA07216 and AA00104 (to W.L.D.); ES01906 (to the Texas A&M University Center for Environmental and Rural Health); HD-24870, National Institute of Child Health and Development through cooperative
agreement U54 Hd18185 as part of the Specialized Cooperative Centers
Program in Reproduction Research; and RR00163 for the operation of the
Oregon National Primate Research Center (to S.R.O.).
Disclosure statement: W.L.D., J.K.H., N.H.M., G.A.J., G.A.D., and
S.R.O. have nothing to declare.
References
1. Winterhalter EH 1896 Ein sympathisches ganglion im menschlichen ovarium.
Arch Gynakol 51:49 –55
2. Dahl W, Flaskamp W 1937 Innervation der weiblichen geschlechtsorgane. In:
LR Muller, ed. Vegative nervensystem. Madrid: Labor; 719 –736
3. Dees WL, Schultea TD, Hiney JK, Dissen GA, Ojeda SR The rhesus monkey
ovary contains a developmentally regulated network of nerve growth receptor
immunoreactive neurons in addition to its extrinsic innervation. Program of
the 74th Annual Meeting of The Endocrine Society, San Antonio, TX, 1992, p
59
4. Dees WL, Hiney JK, Schultea TD, Mayerhoffer A, Danilchik M, Dissen GA,
Ojeda SR 1995 The primate ovary contains a population of catecholaminergic
neuron-like cells expressing nerve growth factor receptors. Endocrinology
136:5760 –5768
5. D’Albora H, Barcia JJ 1996 Intrinsic neuronal cell bodies in the rat ovary.
Neurosci Lett 205:65– 67
6. D’Albora H, Lombide P, Ojeda SR 2000 Intrinsic neurons in the rat ovary: an
immunohistochemical study. Cell Tissue Res 300:47–56
7. Anesetti G, Lombide P, D’Alborda H, Ojeda SR 2001 Intrinsic neurons in the
human ovary. Cell Tissue Res 306:231–237
8. Terasawa E, Fernandez DL 2001 Neurobiological mechanisms of the onset of
puberty in primates. Endocr Rev 22:111–151
9. Van Campenhout E 1930 Contributions to the problem of the development of
the sympathetic nervous system. J Exp Zool 56:295–320
10. Detwiler SR 1937 Observations upon the migration of neural crest cells, and
upon the development of the spinal ganglia and vertebral arches in Amblystoma. Am J Anat 61:63–94
11. Thiery JP, Duband JL, Devouvee A 1982 Pathways and mechanism of avian
trunk neural crest migration and localization. Dev Biol 93:324 –343
12. D’Albora H, Anesetti G, Lombide P, Dees WL, Ojeda SR 2002 Intrinsic
neurons in the mammalian ovary. Microsc Res Tech 59:484 – 489
13. Mayerhofer A, Smith GD, Danilchik M, Levine JE, Wolf DP, Dissen GA,
Ojeda SR 1998 Oocytes are a source of catecholamines in the primate ovary:
evidence for a cell-cell regulatory loop. Proc Natl Acad Sci USA 18:10990 –10995
14. Adashi EY, Hsueh AW 1981 Stimulation of ␤2-adrenergic responsiveness by
3796
15.
16.
17.
18.
19.
Endocrinology, August 2006, 147(8):3789 –3796
follicle-stimulating hormone in rat granulosa cells in vitro and in vivo. Endocrinology 108:2170 –2178
Aguado LI, Petrovic SL, Ojeda SR 1982 Ovarian ␤-adrenergic receptors during
the onset of puberty: characterization, distribution, and coupling to steroidogenic responses. Endocrinology 110:1124 –1132
Hernandez ER, Jimenez JL, Payne DW, Adashi EY 1988 Adrenergic regulation of ovarian androgen biosynthesis is mediated via ␤2-adrenergic thecainterstitial cell recognition sites. Endocrinology 122:1592–1602
Dyer CA, Erickson GF 1985 Norepinephrine amplifies human chronic gonadotropin-stimulated androgen biosynthesis by ovarian theca-interstitial cells.
Endocrinology 116:1645–1652
Ratner A, Weiss GK, Sanborn CR 1980 Stimulation by ␤2-adrenergic receptors
of the production of cyclic AMP and progesterone in rat ovarian tissue. J
Endocrinol 87:123–129
vam Saal FS, Finch CE, Nelson JF 1994 Natural history and mechanisms of
reproductive aging in humans, laboratory rodents, and other selected verte-
Dees et al. • Primate Ovarian Neurons
20.
21.
22.
23.
24.
25.
brates. In: Knobil E, Neill JD, eds. The physiology of reproduction. 2nd ed. New
York: Raven Press; 1213–1314
McDonald JK, Dees WL, Ahmed CE, Noe BD, Ojeda SR 1987 Biochemical and
immunocytochemical characterization of neuropeptide Y in the immature rat
ovary. Endocrinology 120:1703–1710
Schultea TD, Dees WL, Ojeda SR 1992 Post-natal development of sympathetic
and sensory innervation in the rhesus monkey ovary. Biol Reprod 47:760 –767
Lawrence Jr IE, Burden HW 1980 The origin of the extrinsic adrenergic innervation to the rat ovary. Anat Rec 196:51–59
Yntema CL, Hammond WS 1947 The development of the autonomic nervous
system. Biol Rev 22:344 –359
Tello JF 1925 Sur la formation des chaines primaries et secondaires du grand
sympathique dans l’embryon de poulet. Trab Lab Invest Biol Univ Madrid
23:1–28
Van Campenhout 1931 Le developpement du systeme nerveux sympathique
chez le poulet. Arch Biol (Paris) 42:479 –507
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