NEUROENDOCRINOLOGY
Rapid Impact of Progesterone on the Neuronal
Growth Cone
Laura Olbrich,* Lisa Wessel,* Ajeesh Balakrishnan-Renuka, Marion Böing,
Beate Brand-Saberi, and Carsten Theiss
Institute of Anatomy and Molecular Embryology (L.O., L.W., A.B.-R., M.B., B.B.-S., C.T.) and Institute of
Anatomy, Department of Cytology (C.T.), Ruhr-University Bochum, Universitätsstrasse 150, 44780
Bochum, Germany
In the last two decades, sensory neurons and Schwann cells in the dorsal root ganglia (DRG) were
shown to express the rate-limiting enzyme of the steroid synthesis, cytochrome P450 side-chain
cleavage enzyme (P450scc), as well as the key enzyme of progesterone synthesis, 3␤-hydroxysteroid
dehydrogenase (3␤-HSD). Thus, it was well justified to consider that DRG neurons similarly are able
to synthesize progesterone de novo from cholesterol. Because direct progesterone effects on
axonal outgrowth in peripheral neurons have not been investigated up to now, the present study
provides the first insights into the impact of exogenous progesterone on axonal outgrowth in DRG
neurons. Our studies including microinjection and laser scanning microscopy demonstrate morphological changes especially in the neuronal growth cones after progesterone treatment. Furthermore, we were able to detect a distinctly enhanced motility only a few minutes after the start
of progesterone treatment using time-lapse imaging. Investigation of the cytoskeletal distribution
in the neuronal growth cone before, during, and after progesterone incubation revealed a rapid
reorganization of actin filaments. To get a closer idea of the underlying receptor mechanisms, we
further studied the expression of progesterone receptors in DRG neurons using RT-PCR and immunohistochemistry. Thus, we could demonstrate for the first time that classical progesterone
receptor (PR) A and B and the recently described progesterone receptor membrane component 1
(PGRMC1) are expressed in DRG neurons. Antagonism of the classical progesterone receptors by
mifepristone revealed that the observed progesterone effects are transmitted through PR-A and
PR-B. (Endocrinology 154: 3784 –3795, 2013)
n recent years, glial and neuronal cells in the central
nervous system (CNS) and peripheral nervous system
(PNS) were shown to express several kinds of steroidogenic enzymes and synthesize steroids de novo from cholesterol (1–5). Because sensory neurons and Schwann cells
in the dorsal root ganglia (DRG) express the rate-limiting
enzyme of steroid synthesis, cytochrome P450 side-chain
cleavage enzyme (P450scc), as well as the key-enzyme of
progesterone synthesis, 3␤-hydroxysteroid dehydrogenase (3␤-HSD), it is well justified to consider that DRG
neurons similarly are able to synthesize progesterone de
I
novo from cholesterol (4, 6, 7). In recent years, progesterone was shown to have many nonreproductive, neuroprotective, and neuroregenerative effects in the CNS and
PNS, which offer new opportunities for clinical interventions (8 –11). Traditionally, progesterone has been considered to act via genomic mechanisms by binding to the
classical progesterone receptor (PR) A and B. But other
nongenomic mechanisms have recently been described in
varying neuronal tissues, mediated by different progesterone receptors. Besides classical progesterone receptors,
another receptor referred to as ventral midline antigen,
ISSN Print 0013-7227 ISSN Online 1945-7170
Printed in U.S.A.
Copyright © 2013 by The Endocrine Society
Received February 21, 2013. Accepted July 23, 2013.
First Published Online August 2, 2013
* L.O. and L.W. contributed equally.
Abbreviations: CLSM, confocal laser scanning microscopy; CNS, central nervous system;
DRG, dorsal root ganglia; GFP, green fluorescent protein; 3␤-HSD, 3␤-hydroxysteroid
dehydrogenase; NFM, neurofilament M; NGF, nerve growth factor; PGRMC1, progesterone receptor membrane component 1; PNS, peripheral nervous system; PR, progesterone
receptor; P450scc, cytochrome P450 side-chain cleavage enzyme; RFP, red fluorescent
protein.
3784
endo.endojournals.org
Endocrinology, October 2013, 154(10):3784 –3795
doi: 10.1210/en.2013-1175
doi: 10.1210/en.2013-1175
also known as progesterone receptor membrane component 1 (PGRMC1), was shown to be expressed in the developing rodent spinal cord (11). However, up to now, the
function of progesterone in DRG neurons is not fully understood, nor has the expression of progesterone receptors
in this tissue been investigated in detail.
Interestingly, PGRMC1 was discussed as playing an
important role in the regulation of axonal guidance (12).
Additionally, PGRMC1 was shown to have an age-dependent expression pattern in the spinal cord, with high
mRNA and protein levels exclusively during embryonic
development (12). Thus, an involvement of progesterone
in the formation of the peripheral neuronal circuit by regulation of axonal pathfinding can be assumed.
Axonal navigation is crucial for a proper formation of
neuronal connections during development. The neuronal
growth cone plays a key role in this axon navigation, acting as a sensory motile machine. Influenced by different
guidance signals, it leads the axon to the final target and
allows the formation of proper synaptic connections.
Nerve growth factor (NGF), which was first described by
Cohen et al in 1954 (13), is well established to be one of
various potent guiding agents for the neuronal growth
cone, employing its receptors trkA and p75NTR (14, 15).
The purpose of the present study was to investigate and
compare the effects of short- and long-term progesterone
incubation with those of NGF treatment on the neuronal
growth cone. We particularly focused on actin and neurofilament dynamics using red fluorescent protein (RFP)actin– and green fluorescent protein (GFP)-neurofilament
M (NFM)–microinjected chicken DRG for time-lapse imaging. Furthermore, we studied the distribution of progesterone receptors in DRG neurons and specifically antagonized progesterone receptors with mifepristone to
verify the observed effects.
endo.endojournals.org
3785
L-glutamine (Sigma-Aldrich; G7513), and 0.1% gentamicin (Sigma-Aldrich; G1397) enriched with NGF-7S (50 ng/mL; SigmaAldrich; N0513), progesterone (10nM; Sigma-Aldrich; P8783),
and/or mifepristone (1␮M; Sigma; M8046) alone or in varying
combinations. These concentrations are in accordance with previous studies (16 –18). The explants were treated for 3 days in
vitro and fixed afterward.
Dissociated DRG cell cultures
Dissociated cell cultures of DRG neurons have been obtained
as previously described (19). Dissection was performed as described above; DRG were transferred into prewarmed (37°C)
dissociation medium (0.05% trypsin, 1:250 in calcium/magnesium-free saline) and stirred with a Teflon-covered magnetic stirring bar. Agitation was executed for 5 minutes, and supernatant
containing the dissociated cells were transferred into a centrifuge
tube containing 20 mL MEM, 10% horse serum, 1% L-glutamine, and 0.1% gentamicin or penicillin to block trypsinization. This protocol was repeated 3 times for a total of 20 minutes.
The obtained suspension was centrifuged for 10 minutes, and the
resulting pellet was resuspended in 4 mL fresh nutrient medium,
before 1 ⫻ 105 cells/mL of the cell suspension were cultured on
glass coverslips for up to 1 week.
Immunohistochemistry
Fixation was carried out with 4% paraformaldehyde followed by a 0.1% Triton X-100 incubation in PBS for 10 minutes
for permeabilization. After washing, probes were treated with
10% (wt/vol) goat serum (Sigma; G9023, 1:50 in PBS) for 30
minutes to block nonspecific binding sites. Again, interim washing steps followed before incubation with anti-NFM (1:500 in
PBS, AB1987; Chemicon) or antiprogesterone receptor (A/B)
(SAB4502184, 1:200 in PBS; Sigma) was done overnight at 4°C.
Next, fluorescein isothicyanate-conjugated secondary goat antirabbit antibodies (1:500 in PBS, F6005; Sigma-Aldrich) were
applied for 1.5 hours at room temperature. For the staining of
actin filaments, phalloidin-rhodamine (1:40 in PBS, P1951; Sigma-Aldrich) was used for 30 minutes. Finally, cell cultures were
rinsed in PBS and coverslipped in mounting medium (Dako;
S302380 –2).
Reverse transcription PCR
Materials and Methods
DRG explants
Tissue was handled according to the Tenets of Helsinki and
The Guiding Principles in the Care and Use of Animals (DHEW
Publication, NIH 80 –23). DRG explants were obtained from
10-day-old chicken embryos. Preparation of DRG was performed under visual control by means of a binocular microscope,
and probes were transferred into ice-cold Hanks’ solution. Before collecting samples in nutrient medium, connective tissue was
removed and ganglions were divided into 2 even halves. Cultivation was accomplished in nutrient medium on custom-made
glass coverslips (diameter 32 mm; Kindler) covered with rat-tail
collagen in a CO2 incubator (5% CO2, 37°C, 90% humidity) in
double-coverslip Maximov slides. Ingredients of the used nutrient medium include MEM (Sigma-Aldrich; M2279), supplemented with 10% horse serum (Biochrom; product S9135), 1%
Total RNA was isolated from chicken DRG using TRIzol
(Invitrogen) reagent extraction method. One microgram of total
RNA was reverse transcribed using the QuantiTect reverse transcription kit (QIAGEN) to prepare first-strand cDNA. For all
PCRs, an aliquot of the cDNA solution corresponding to 50 ng
of the total RNA was used as the template in 20 ␮L reaction
mixture. PCR master mix including the cDNA solution was prepared to ensure equal amount of template was submitted for all
reactions. The PCR was carried out using the Taq DNA polymerase kit (QIAGEN). PCRs without a template were used as
negative control (H2O control). Primers used were cPR forward 5⬘-GGAAGGGCAGCACAACTATT-3⬘ and reverse 5⬘GACACGCTGGACAGTTCTTC-3⬘ (product size, 83 bp) (20),
cPGMRC1 forward 5⬘-AACAATAGGCCTGACCGTTG-3⬘
and reverse 5⬘-ccatatatccagcccccttt-3⬘ (product size, 120 bp), and
cGAPDH forward 5⬘-GAGGGTAGTGAAGGCTGCTG-3⬘ and
reverse 5⬘-CCACAACACGGTTGCTGTAT-3⬘ (product size, 200
3786
Olbrich et al
Progesterone and the Growth Cone
bp) (21). A 2.5% agarose gel was used for proper resolution of the
PCR products.
Western blot
The tissue of chicken brain was lysed in RIPA buffer with
protease inhibitor cocktail (Sigma-Aldrich) and mixed with 0.25
vol 5⫻ sodium dodecyl sulfate (SDS) sample buffer. Then, 20 ␮L
of the sample was subjected to 7% (wt/vol) SDS-PAGE, followed
by protein transfer onto a nitrocellulose membrane (0.45 ␮m;
Macherey-Nagel) using the wet blotting system mini Trans-Blot
Cell (Bio-Rad) and 48mM Tris, 78mM glycine, 0.02% SDS (wt/
vol), and 20% (vol/vol) methanol as transfer buffer. Blotting
membranes were blocked with 3% (wt/vol) low-fat milk powder
dissolved in PBS supplemented with 0.05% (vol/vol) Tween 20.
This was followed by overnight incubation with the primary
anti–PR-A/B (SAB4502184; Sigma-Aldrich; dilution 1:1000)
antibody in the same solution. Primary antibody was detected
with a horseradish peroxidase-conjugated secondary antibody
(antirabbit IgG, horseradish peroxidase-linked; Cell Signaling
Technology; dilution 1:2000) and immunodetection was done
using ECL detection reagent (Amersham Biosciences).
Analysis of neurite outgrowth and growth cone
morphology
After culturing DRG pieces for 3 days in vitro, analysis of the
extent of neuritic outgrowth in 7 different medium conditions
was performed with controls lacking any stimulating factors,
NGF, progesterone, progesterone plus NGF, mifepristone, mifepristone plus progesterone, and mifepristone plus progesterone
plus NFG. With the aid of confocal laser scanning microscopy
(CLSM) (Zeiss LSM 510) in combination with Zeiss ⫻10 lenses,
neuritic length was measured. The analysis of neuritic outspread
was performed in a standardized pattern, as 8 loci were defined
surrounding the centrally located DRG piece in a clockwise manner. The maximal neuritic extension was measured using the
screen ruler JR Screen Ruler Pro (Spadix Software). Therefore, 8
loci per DRG piece were surveyed, with 8 independent pieces per
condition, acquiring 64 datasets per condition, resulting in a
total number of 448 along all 7 conditions. Likewise, treated
samples were used for growth cones survey, all growth cones
were analyzed by CLSM in combination with Zeiss ⫻40 oil immersion lenses (Plan-Neofluar; numerical aperture 1.3), measuring the growth cone area as well as growth cone circumference. The base width was defined as the maximum distance
perpendicular to the axonal axis measured 2 ␮m from the beginning of the axonal enlargement forming the growth cone.
Microinjection
Transfection of cytoskeletal vector insertion into single neurons of dissociated DRG cultures was performed by targeting the
cell nucleus according to the microinjection technique performed
by Meller (22, 23). Briefly, 2 ␮L vector suspension in distilled
water was back-filled in sterile glass capillaries (diameter 0.2–
0.5 ␮m, Femtotips; Eppendorf). By means of an inverse microscope equipped with phase-contrast optics (Zeiss), microinjection was performed using a pressure injection tool (Eppendorf),
which maintained a constant pressure of 5–15 hPa on the tip
before the injection and during injection with up to 15 to 30 hPa
over 0.2 seconds (constant pressure ⫽ 5–15 hPa, injection pressure ⫽ 15–30 hPa, injection time ⫽ 0.2 seconds). During micro-
Endocrinology, October 2013, 154(10):3784 –3795
injection, cultures were kept at 37°C on a tempered stage and
rinsed in fresh medium lacking any stimulating factors after the
treatment. Distribution and expression of cytoskeletal proteins
such as actin and neurofilaments throughout distances of several
hundred micrometers along the prolongations of DRG neurons
were feasible after 24 hours incubation. Different vectors were
used, such as pLifeAct-TagRFP (LifeAct, 60102; Ibidi), GFPNFM (24) and the combination of LifeAct plus GFP-NFM, all
suspended in distilled water.
Time-lapse imaging
Subsequent to the postincubation period of 24 hours, timelapse imaging of cytoskeletal proteins was performed with the
aid of CLSM (Zeiss LSM 510 Meta). Using Rose chambers enabled us to use 40-long distance-apochromatic lens (Plan-Neofluar; numerical aperture 1.1), therefore achieving the best resolution. Temperature and gas were maintained using CTI
controller 3700 digital, O2 controller, Tempcontrol 37–2 digital,
and the Incubator Soxygen (Zeiss). Cell cultures retained active
growth cones and normal organelle movement and cytoskeletal
turnover for hours. Immediately before starting imaging, nutrient medium was exchanged with medium enriched with different
factors: 50 ng/mL NGF, 10nM progesterone, progesterone plus
NFG, or 1␮M mifepristone plus progesterone. Intervals of 60
seconds were defined for time-lapse studies, in total capturing the
growth cone motility for approximately 2 hours. By means of
z-stack mode, the whole extension of the growth cone was covered, additionally avoiding bleaching and cytotoxic effects by
decreasing laser power.
Statistics
All in all, 448 neurites and 308 growth cones were stained and
analyzed. The statistical significance was evaluated by use of the
software Statistica (release 10; StatSoft). A one-way ANOVA
with additional post hoc analysis (Scheffé test) was conducted to
verify the quantitative analysis in progesterone-treated DRG versus controls.
Results
The effects of progesterone on the morphology of
outgrowing neurons
After cultivation for 3 days, primary chicken DRG samples grew radially into the periphery and showed characteristic morphological properties, including the centrally
located DRG piece as the origin of numerous axons
spreading radially into the periphery (Figure 1). Neurofilaments were prominently expressed within the neurites
and could be used to visualize the extension of cell processes throughout hundreds of micrometers. Due to the
dense organization of the organotypic DRG cultures, individual neuronal somata could not be detected in the
center of the cell culture, but with the aid of CLSM, we
were able to identify and analyze the vast number of cell
processes (Figure 1A). Controls were obtained by incubating samples in nutrient medium, lacking any supple-
doi: 10.1210/en.2013-1175
endo.endojournals.org
3787
Figure 1. A and B, Immunostaining of NFM in chicken DRG using confocal LSM after cultivation for 3 days in nutrient medium lacking any
stimulating factor (A) and with addition of 50 ng/mL NGF (B). C and D, Outgrowth and cross-linking were significantly enhanced by adding 10nM
progesterone (C) and progesterone plus NGF (D). E, Treatment with progesterone in combination with 1␮M mifepristone led to morphological
features with slightly decreased cell prolongations. F, Quantitative analysis of neurite length subsequent to treatment revealed highly significant
differences compared with controls: **, ⫽ P ⬍ .001. Scale bar, 500 ␮m. Abbreviations: mif, mifepristone; prog, progesterone.
3788
Olbrich et al
Progesterone and the Growth Cone
mented growth factors but horse serum. In concordance
with other studies (18, 25) limited neuritic outgrowth was
detectable, whereas treatment with 50 ng/mL NGF (Figure
1B), 10nM progesterone (Figure 1C), and progesterone
plus NGF (Figure 1D) augmented the outspread, extending several hundreds of micrometers with ramified neuronal cell processes. Neurons exposed to NGF showed
many neurites extending into the periphery and forming
dense networks (Figure 1B). Incubation with progesterone
(Figure 1C) or progesterone plus NGF led to a slightly
enhanced neuritic outgrowth (Figure 1D). In contrast, after blocking progesterone receptors with 1␮M mifepristone, the radial outgrowth was comparable to controls
(Figure 1E), because mifepristone completely antagonized
progesterone-induced outgrowth. This inhibition was selective for progesterone-induced outgrowth but did not
affect progesterone-independent outgrowth because studies with 1␮M mifepristone, 10nM progesterone, and 50
ng/mL NGF were comparable to samples treated exclusively with NGF. Furthermore, after treating neurons
solely with 1␮M mifepristone, no difference to controls
was obvious. These findings suggest a negligible effect of
endogenous progesterone on neuritic outgrowth.
The quantitative analysis of 8 independent cultures for
each condition verified these observations of neuritic outgrowth as dependent on NGF, progesterone, and progesterone plus NGF (F6,127 ⫽ 6.113; P ⬍ .01; Figure 1F).
Control DRG explants (n ⫽ 8) showed an average outgrowth of 1198 ⫾ 352 ␮m (64 neurites per condition),
whereas NGF incubation (n ⫽ 8) increased the mean outgrowth length by 46% (average of 64 neurites 1721 ⫾ 243
␮m, P ⬍ .001 compared with controls). Again in comparison with controls, treatment with progesterone likewise
led to a considerably augmented outspread (n ⫽ 8) being
increased by 40% (64 neurites, average 1673 ⫾ 290 ␮m,
P ⬍ .001), which was being exceeded by an enhancement
of 51% by simultaneous incubation with progesterone
plus NGF (n ⫽ 8; mean length of 64 neurites 1803 ⫾ 311
␮m, P ⬍ .001 compared with controls). However, neuritic
outgrowth did not differ significantly between NGF, progesterone, and progesterone plus NGF treatment. Incubation with progesterone and simultaneous blocking of
progesterone receptors with mifepristone (n ⫽ 8) showed
that the progesterone-induced outgrowth was completely
antagonized, resulting in samples comparable to controls
(64 neurites, average 1152 ⫾ 410 ␮m). Interestingly, the
descriptive impression of samples incubated exclusively
with mifepristone was confirmed, because no significant
difference from controls was detectable (64 neurites, average 1083 ⫾ 326 ␮m). Samples treated with mifepristone
plus progesterone plus NGF showed morphometric prop-
Endocrinology, October 2013, 154(10):3784 –3795
erties similar to samples treated solely with NGF (64 neurites, average 1664 ⫾ 368 ␮m).
Analysis of growth cone shape and cytoskeletal
alterations
Higher magnification of growth cones in the periphery
of the neurites revealed the typical cytoskeletal composition. Whereas densely packed neurofilaments were located centrally, thin actin filaments were detectable in the
periphery, reaching up to the cell membrane and forming
cellular protrusions. Even though scattered neurofilaments occasionally extended into the periphery of the
growth cone, lamellipodia were formed by a dense actin
meshwork and filopodia were constructed of tight actin
bundles reaching into the periphery.
Distinct cytoskeletal alterations were examined using
CLSM to study nuanced variations in the morphology. All
studies were based on comparisons with controls (Figure
2A), showing clear growth-enhancing effects after 50
ng/mL NGF incubation (Figure 2B) and also 10nM progesterone treatment (Figure 2C). Morphologic changes
were detectable as an alteration of growth cone shape with
a clear shift of cytoskeletal components. Here we observed
an increasing amount of lamellipodia and filopodia with
the result of an augmented growth cone circumference and
area. In comparison with controls, these changes were obvious after NGF incubation and even more prominent in
progesterone-treated neurons. These lamellipodia and
filopodia were characterized by densely packed actin filaments. Thus, the shift of the actin to neurofilament ratio
was in favor of actin filaments, leading to a considerably
enlarged peripheral zone. Interestingly, NGF and progesterone led to different phenotypical presentation, because
progesterone seemed to generate a lamellipodia-like phenotype, whereas NGF led to an increased amount of filopodia extension. Incubation with a mixture of progesterone
plus NGF dramatically increased the size of growth cones,
characterized by very long filopodia and wide lamellipodia, with a vast number of actin filaments (Figure 2D),
without affecting the distribution of neurofilaments. All
effects of progesterone on growth cone size could be inhibited by specifically blocking progesterone receptors
with 1␮M mifepristone (Figure 2E). In line with the neuritic outgrowth mifepristone-only treatment led to growth
cone morphology comparable to controls, therefore indicating negligible effects of endogenous progesterone. Additionally, the observed stimulating effect of NGF incubation was not affected by mifepristone. Morphometric
analysis of growth cone circumference and growth cone
area verified these morphological findings (F6,127 ⫽ 6.113;
P ⬍ .01; Figure 3, A and B). Progesterone incubation led
to an increase in growth cone circumference and growth
doi: 10.1210/en.2013-1175
endo.endojournals.org
3789
Figure 2. Changes in cytoskeletal properties within the neuronal growth cone. A, Controls showed typical distribution of neurofilaments (anti–
NFM-FITC) and actin (phalloidin-tetramethylrhodamine-5-(and 6)-isothiocyanate (5(6)). B and C, Incubation with NGF (B) and even more
pronounced with progesterone (C) led to an enlargement of the peripheral zone, with additional lamellipodia and filopodia. D and E, These effects
were enhanced by adding the combination of progesterone plus NGF (D) but inhibited by blocking with mifepristone (E). Scale bar, 5 ␮m.
cone area (n ⫽ 9 DRG, 20 growth cones; mean circumference 85 ␮m, mean area 56 ␮m2) compared with controls (n ⫽ 9 DRG, 20 growth cones; mean circumference
53 ␮m, mean area 33 ␮m2), resulting in a significant enlargement of 60% for circumference (P ⬍ .001) and 70%
for area (P ⬍ .001). Again, NGF (n ⫽ 9 DRG, 20 growth
cones) displayed a similar effect, resulting in an average
circumference of 69 ␮m and average area of 44 ␮m2, being
significantly increased compared with controls (both P ⬍
.001). The effects on growth cone morphology were again
Figure 3. Quantitative analysis of morphological changes subsequent to 3 days incubation with 50 ng/mL NGF, 10nM progesterone,
progesterone plus NGF, 1␮M mifepristone, mifepristone plus progesterone, and mifepristone plus progesterone plus NGF reveal highly significant
morphometric changes in both circumference (A) and area (B) compared with controls; n ⫽ 20 per condition. Error bars represent SD. **, P ⬍
.001. Abbreviations: mif, mifepristone; prog, progesterone.
3790
Olbrich et al
Progesterone and the Growth Cone
Endocrinology, October 2013, 154(10):3784 –3795
increased by incubation with a mixture of progesterone
plus NGF (n ⫽ 9 DRG, 20 growth cones; circumference 85
␮m; mean area 63 ␮m2) which means an increase of 60%
for circumference (P ⬍ .001) and 91% for area (P ⬍ .001)
compared with controls. The quantitative analyses confirmed that specific inhibition with mifepristone (n ⫽ 9
DRG, 16 growth cones) resulted in parameters of growth
cone circumference and area that were similar to controls
(mean circumference 53 ␮m, mean area 34 ␮m). Mifepristone treatment alone did not affect growth cone morphology (n ⫽ 9 DRG, 16 growth cones; mean circumference
53 ␮m, mean area 35 ␮m2), whereas incubation with mifepristone plus progesterone plus NGF revealed a morphology
comparable to NGF treatment (n ⫽ 9 DRG, 16 growth
cones; mean circumference 75 ␮m, mean area 45 ␮m2). Distribution of PR-A/B over the entire surface of the neuronal
growth cone was visualized by CLSM (Figure 4A). Specificity
of the PR-A/B antibodies used in chicken was shown by
Western blot experiments (Figure 4B). In addition, expression of classical progesterone receptors (PR-A/B) as well as
PGRMC1 was proven by RT-PCR analysis.
Progesterone immediately affects the growth cone
Approximately 24 hours after microinjection, expression of RFP-actin along with GFP-NFM was detectable
throughout the entire neuron, and even prominent within
the neuronal growth cone (Figure 5). Time-lapse imaging
Figure 4. A, CLSM verified the distribution of PR-A/B (green) within
growth cones. The actin cytoskeleton is stained with phalloidintetramethylrhodamine-5-(and-6)-isothiocyanate (5(6)) (red). Scale bar,
5 ␮m. B, Specificity of PR-A/B antibodies was proven by Western blot.
C, RT-PCR of chicken DRG revealed a moderate expression of classical
progesterone receptors (PR-A and PR-B) and PGRMC1.
Figure 5. Time-lapse imaging of cytoskeletal dynamics within the
growth cone. Microinjected and double-transfected primary DRG
neurons were observed for approximately 2 hours. Distribution and
dynamics of GFP-NFM and RFP-actin within the same neuron were
analyzed. Growth cones were motile for the entire period of
observation, palpating the environment. Although growth cones in
controls (A) showed static movement, samples treated with 10nM
progesterone (B) showed highly accelerated filopodia and lamellipodia
turnover and started growing forward. Scale bar, 10 ␮m.
doi: 10.1210/en.2013-1175
could be performed for many hours, because samples displayed active and motile neurons with apparently normal
organelle movement. In controls, the growth cones stayed
maneuverable (Figure 5A and Supplemental Movie 1,
published on The Endocrine Society’s Journals Online
website at http://endo.endojournals.org), showing a constant turnover of neurofilaments, being localized in the
central region of the growth cone, and actin filaments,
forming the peripheral zone. Especially actin filaments
showed a constant motion and turnover, forming new
filopodia, representing motile cell protrusions, apparently
palpating the environment by constantly changing their
shape. Besides this, lamellipodia were identifiable by their
veil-like structure, not changing their shape as quickly as
the filopodia, but still showing dynamic alterations. In
addition to this, within the growth cone, an actin turnover
was observed that was increased before the outspread of
the growth cone, which seemed to initiate a new protrusion, whereas neurofilaments were likely to follow new
actin branches once they were getting solid, thus representing the phase of consolidation.
Controls showed constantly palpating growth cones,
scanning the environment without prominent changes of
their position. In contrast, the addition of 10nM progesterone (Figure 5B and Supplemental Movie 2) induced
morphological changes within minutes, leading to a clear
forward movement of the axonal growth cone. This was
obvious as solid accelerations of changes in growth cone
morphology with a rapid turnover of filopodia and lamellipodia. These progesterone-treated growth cones displayed increased cytoskeletal motility within a few minutes and started growing out after approximately 30
minutes incubation. However, the combination of progesterone and NGF showed no additional effect in growth
cone motility (not shown).
To verify these results, we measured the circumference
over time in 8 growth cones for each condition (F4,179 ⫽
22.663; P ⬍ .01; Figure 6). By adding NGF, progesterone,
or the mixture of both, the average circumference motility
increased significantly from 1.5 ␮m/min (controls, n ⫽ 8)
to 2.7 ␮m/min (NGF, n ⫽ 8), an increase of 77% (P ⬍
.001), and 3.1 ␮m/min (progesterone plus NGF, n ⫽ 8), an
increase of 103% (P ⬍ .001). Interestingly, progesterone
alone showed the largest enhancement of growth cone
motility (n ⫽ 8; 3.9 ␮m/min) with an increase of 154%
compared with controls (P ⬍ .001); however, this is not
significantly different from progesterone plus NGF. The
effects of progesterone were completely blocked by the
incubation of progesterone together with the classical progesterone receptor inhibitor mifepristone (n ⫽ 8; 1.5
␮m/min).
endo.endojournals.org
3791
Figure 6. Quantitative analysis of growth cone circumference-motility.
Sizing the same growth cones for several minutes revealed highly
significant (**, P ⬍ .001) differences within a few minutes after
exchanging control medium with medium supplemented with NGF,
progesterone, progesterone plus NGF, or progesterone plus
mifepristone. Abbreviations: mif, mifepristone; prog, progesterone.
Discussion
After it was shown that neuronal and glial cells of different
brain regions express steroidogenic enzymes and produce
progesterone de novo from cholesterol, progesterone and
its impact on neuronal tissues are of increasing scientific
and therapeutic interest. Therefore, extensive studies investigated the influence of progesterone on various neuronal tissues during the last two decades. The temporal
correlation between dramatic morphological changes during the embryonic period and endogenous production of
progesterone in this period suggests an involvement of
progesterone in the physiological maturation process of
neuronal circuits. Indeed, it was shown that progesterone
and other neuroactive steroids are involved in many physiological processes of the developing and adult brain.
Thus, progesterone is, for example, involved in the modulation of neurogenesis, neuronal development, mood,
and cognition (17, 26 –30). But in addition to its involvement in such physiological neuronal processes, progesterone was shown to additionally have neuroprotective, neuroreparative, antidegenerative, and antiapoptotic effects
in both PNS and CNS (32–36). In the PNS, progesteroneassociated positive effects on peripheral neuropathic pain
(37– 40), protective effects after nerve crush injury (34),
and promyelinating effects (11) play a dominant role in
current research. Nevertheless, neuroprotective and promyelinating effects of progesterone in DRG were previously discussed; up to now, explicit effects of exogenous
progesterone stimulation on the morphology of DRG neurons with special focus on axonal outgrowth have not
been described (4).
Because proper maturation and function of all neuronal
circuits requires axonal growth cones to guide axons to
their final targets, we analyzed progesterone effects in
3792
Olbrich et al
Progesterone and the Growth Cone
DRG neurons with special focus on the morphology and
motility of axonal growth cones. Here we especially concentrated on changes in the cytoskeletal distribution after
progesterone and NGF stimulation.
Progesterone modulates growth cone shape and
cytoskeletal composition
As the evidence of cytochrome P450scc and 3␤-HSD
expression within embryonic DRG neurons suggests, progesterone indeed promotes explicit structural changes in
evaluated samples. DRG neurons revealed a significantly
higher outgrowth and enhanced axonal sprouting after
treatment with progesterone. Remarkably, progesterone
especially increased area and circumference of the growth
cones compared with untreated controls. Because sex steroids are likely to be effective regulators of tissue organization through regulation of the cytoskeleton (43), we
assumed an involvement of cytoskeletal proteins in the
transmission of observed progesterone effects. The cytoskeleton in general is formed by 3 different types of filaments interacting in a dynamic network: actin filaments,
microtubules, and intermediate filaments. The cytoskeleton of the neuronal growth cone in particular can be divided into 3 domains called central, transit, and peripheral
region, characterized by a certain distribution and organization of cytoskeletal proteins (44). Actin, which is especially located in the peripheral zone, is arranged either
in a meshwork-like manner, constituting lamellipodia, or
in bundled actin filaments reinforced by individual microtubules composing filopodia (45). These structures are
partly rooted in the adjacent transit zone, containing an
arc of actomyosin that is crucial for contractile motions
(46). The central region is made up of microtubules, neurofilaments, and actin filaments (45).
Indeed, significant changes in this characteristic cytoskeletal distribution were detectable after incubation with
progesterone. For instance, a modification in the cytoskeletal composition toward an enlarged actin-containing peripheral region and a higher amount of lamellipodia and
finger-like filopodia could be visualized. In contrast, neurofilaments within the central zone revealed only slight
alterations in their distribution. Corresponding with additional studies, the shift of the actin to neurofilament
ratio seemed to be in favor of actin polymerization, indicating actin as the effector of the observed progesteroneinduced rearrangements (43). Thus, it is likely that progesterone promotes the increase in growth cone area and
circumference by direct or indirect interactions with the
actin cytoskeleton.
Endocrinology, October 2013, 154(10):3784 –3795
Progesterone has a direct and rapid effect on the
actin cytoskeleton
In accordance with these morphological changes especially in the peripheral region after progesterone administration, it has been described that actin is indispensable
for a cytoskeletal response to guidance cues in culture and
in model organisms because the axon does not seem to be
able to alter direction once it has started growing (47–52).
This capability of turning is crucial for the growth cone’s
exploration of the environment (44, 45). Fast actin remodeling is a key component of proper neuronal outgrowth, but
there are relatively few detailed high-resolution studies of
cytoskeletal and morphological rearrangements (34 –38,
44). Nevertheless, it has been described that the application
of chemoattractants leads to an increased protrusion, an accumulation of F-actin, and an augmented barbed-end density within the growth cone near the locus of application (38).
Such observations have been reported for instance for vascular endothelial growth factor (18, 25, 58). Furthermore,
studies by Argiro and colleagues (59) showed a direct correlation between growth cone shape and motility. These authors assumed that a reduced growth cone size is accompanied by slower neurite extension, probably due to a decreased
protein synthesis in the cell body or a reduced axonal transport of proteins toward the leading edge (59). In consequence, a stimulating factor with enlarging effects on growth
cone size might stimulate growth cone motility as well.
Taken together, different studies and our results to date suggest an increased growth cone motility after incubation with
progesterone (38, 59).
On this basis, we performed live cell studies investigating
the effects of stimulating factors on the average movement of
growth cones as well as the cytoskeletal response. In the present experimental setup, we observed RFP-tagged filamentous actin (LifeAct) (60) and GFP-tagged neurofilaments
(24) in individual growth cones over a period of several
hours, revealing the rearrangements of the cytoskeleton.
Moreover, we focused on the temporal dimension to determine whether the morphological changes after progesterone
treatment are mediated by fast cytoskeletal reorganization or
due to slow changes within the cellular system. In fact, progesterone leads to a highly significant increase of the circumference dynamics. Interestingly, the observed results show
for the first time that the reorganization of the growth cone
starts within a very few minutes after the onset of the incubation with progesterone. Thus, a direct interaction of progesterone with the actin cytoskeleton is probable, because
progesterone administration results in such a rapid and measurable reorganization of the actin-dependent peripheral
zone of the growth cone. We therefore anticipate different
coexisting mechanisms of progesterone’s interaction with
the actin cytoskeleton.
doi: 10.1210/en.2013-1175
Progesterone and NGF do not have additive
effects on growth cone morphology
In a next step, the molecular background of the observed progesterone effects needed to be found. Because
these effects are very similar to those seen after neurotrophin incubation, a connection between progesterone and
neurotrophins might be possible. Indeed, Lanlua et al (61)
revealed an up-regulation of NGF receptors, trkA and
p75NTR, in DRG neurons due to progesterone injection.
NGF was shown to be an attractive cue for neuronal
growth cones in numerous studies, favoring actin polymerizations and stabilizations by interaction with actin
binding proteins like actin depolymerizing factor/cofilin
(63– 65). Thus, an up-regulation of the amount of NGF
receptor would increase the growth cone´s sensitivity for
NGF and in turn increase the growth cone’s area and circumference (18, 53, 57, 62– 66). Furthermore, ventral
midline antigen/VEM1, a certain isoform of PGRMC1,
was discussed as being involved in axonal pathfinding by
the regulation of surface availability of guidance receptors
(12, 67). Regarding these studies, we further directly compared progesterone and NGF effects on growth cone morphology. Here, no additive effects of progesterone and
NGF were detectable, even if the growth cone morphology
revealed comparable morphological changes after single
progesterone or NGF incubation. Such an additive effect
would be expected if progesterone effects would indeed be
mediated by an increase of NGF sensitivity due to progesterone stimulation. But because we could not detect additive effects, a common signal mechanism of progesterone and NGF is more probable than a progesteroneinduced increase of the amount of NGF receptor.
Progesterone effects in DRG neurons are
completely antagonized by mifepristone
Next we focused on the identification of progesterone
receptors expressed in DRG. Thus, we could reveal the
expression of classical progesterone receptors in this tissue
on both protein as well as mRNA level. We therefore compared DRG neurons, which were treated with progesterone, whereas classical progesterone receptors were antagonized with mifepristone at the same time. Indeed
progesterone-induced effects were no longer detectable
when progesterone treatment was combined with mifepristone. Thus, an involvement of these receptors in the
mediation of observed progesterone effects is highly probable. Typically, these classical progesterone receptors
PR-A and PR-B, which are ligand-activated transcription
factors, are seen to be expressed in the soma, bound to
chaperones (68). Upon progesterone binding, receptors
change their confirmation, dissociate from chaperone proteins, dimerize, and translocate to the nucleus (68). Within
endo.endojournals.org
3793
the nucleus, the PR complex is known to modulate the transcription through interaction with specific progesterone response elements (68, 69). But up to now, there is only sparse
information about the underlying mechanisms between PR
activation and observed morphological changes. Because we
further demonstrated that progesterone has an increasing
effect on neuronal outgrowth and motility, associated with
changes in actin cytoskeleton of the neuronal growth cone, a
link between the genomic mechanism and the observed
changes in growth cone motility and observed rearrangements of the actin cytoskeleton needed to be found. Okabe
and Hirokawa (31) already demonstrated in 1991 that an
anterograde flux of actin monomers increases polymerization of actin within growth cones. Thus, a higher amount of
actin monomers in the growth cone might lead to increased
rearrangements of the actin cytoskeleton. Therefore, it is conceivable that the observed effects are due to an increased
protein biosynthesis, for example, of actin mRNA or proteins involved in actin synthesis (41) induced by progesterone
through (epi)genetic mechanisms of classical progesterone
receptors.
But the presented data concurrently indicate rapid
changes in the growth cone morphology after progesterone administration. These results suggest an additional,
faster, fairly nongenomic effect of progesterone on the
actin cytoskeleton. Indeed, progesterone receptors have
recently been identified to mediate rapid, nongenomic signal transduction as well, activating kinase cascades within
the cytoplasm (42, 54). These nongenomic progesteroneassociated mechanisms seem to include the recruitment of
different molecular cascades, such as G proteins, tyrosine
kinases, and c-Src (43). These cascades probably allow
faster rearrangements of the actin cytoskeleton through
engaging different actin binding proteins and thereby increasing cell motility. Thus, progesterone receptors probably do not just induce the well-known genomic mechanisms but may additionally activate alternative pathways
that do not require modulation of protein synthesis. In
regard to these considerations, we did not detect just the
classical progesterone receptors in DRG but also
PGRMC1, which is well discussed as mediating rapid progesterone effects in other neuronal tissues (55, 56, 70).
Thus, an involvement of this membrane-associated progesterone binding protein in the transmission of the observed direct and rapid progesterone effects in DRG neurons need to be studied in future studies. Taken together,
different genomic and nongenomic mechanisms of progesterone interactions with the actin cytoskeleton might
coexist in DRG neurons, allowing direct, permanent, and
rapid changes in the neuronal growth cone. But additional
studies are needed to get more detailed information about
3794
Olbrich et al
Progesterone and the Growth Cone
the underlying molecular basis of both genomic and nongenomic mechanisms.
For the first time, we were able to demonstrate direct effects of exogenous progesterone on DRG neurons. Progesterone was shown to promote axonal outgrowth of DRG
neurons by increasing area, circumference, and motility of
the axonal growth cones. Furthermore, an involvement of
the actin cytoskeleton in the transmission of progesterone
effects was proven. Even if it was shown that some of the
observed progesterone effects are mediated through classical
progesterone receptors, underlying detailed mechanisms
needed to be examined in further studies.
Acknowledgments
We thank C. Grzelak, A. Lodwig, and T. Nguyen for excellent
technical work as well as A. Lenz for secretarial work.
Address all correspondence and requests for reprints to: Dr
Carsten Theiss, Faculty of Medicine, Institute of Anatomy and
Molecular Embryology, Ruhr-University Bochum, 44780 Bochum, Germany. E-mail: [email protected].
We gratefully thank FoRUM (Forschungsförderung Ruhr
Universität Bochum Medizinische Fakultät) and RUB (Ruhr Universität Bochum) for financial support (F670 –2009). L.O. especially thanks the Heinrich und Alma Vogelsang-Stiftung for
financial support in line with a graduation scholarship.
Disclosure Summary: L.O., L.W., A.B.-R., B.B.-S., and C.T.
have nothing to declare.
References
1. Mellon SH, Deschepper CF. Neurosteroid biosynthesis: genes for
adrenal steroidogenic enzymes are expressed in the brain. Brain Res.
1993;629(2):283–292.
2. Compagnone NA, Bulfone A, Rubenstein JL, Mellon SH. Steroidogenic enzyme P450c17 is expressed in the embryonic central nervous system. Endocrinology. 1995;136(11):5212–5223.
3. Furukawa A, Miyatake A, Ohnishi T, Ichikawa Y. Steroidogenic
acute regulatory protein (StAR) transcripts constitutively expressed
in the adult rat central nervous system: colocalization of StAR, cytochrome P-450SCC (CYP XIA1), and 3␤-hydroxysteroid dehydrogenase in the rat brain. J Neurochem. 1998;71(6):2231–2238.
4. Schaeffer V, Meyer L, Patte-Mensah C, Mensah-Nyagan AG. Progress in dorsal root ganglion neurosteroidogenic activity: basic evidence and pathophysiological correlation. Prog Neurobiol. 2010;
92(1):33– 41.
5. Do Rego JL, Seong JY, Burel D, et al. Neurosteroid biosynthesis:
enzymatic pathways and neuroendocrine regulation by neurotransmitters and neuropeptides. Front Neuroendocrinol. 2009;30(3):
259 –301.
6. Guennoun R, Schumacher M, Robert F, et al. Neurosteroids: expression of functional 3beta-hydroxysteroid dehydrogenase by rat
sensory neurons and Schwann cells. Eur J Neurosci. 1997;9(11):
2236 –2247.
7. Patte-Mensah C, Kibaly C, Boudard D, et al. Neurogenic pain and
steroid synthesis in the spinal cord. J Mol Neurosci. 2006;28(1):17–31.
Endocrinology, October 2013, 154(10):3784 –3795
8. Baulieu EE, Schumacher M, Koenig H, Jung-Testas I, Akwa Y. Progesterone as a neurosteroid: actions within the nervous system. Cell
Mol Neurobiol. 1996;16(2):143–154.
9. Ghoumari AM, Dusart I, El-Etr M, et al. Mifepristone (RU486)
protects Purkinje cells from cell death in organotypic slice cultures
of postnatal rat and mouse cerebellum. Proc Natl Acad Sci U S A.
2003;100(13):7953–7958.
10. He J, Evans CO, Hoffman SW, Oyesiku NM, Stein DG. Progesterone and allopregnanolone reduce inflammatory cytokines after
traumatic brain injury. Exp Neurol. 2004;189(2):404 – 412.
11. Schumacher M, Guennoun R, Ghoumari A, et al. Novel perspectives
for progesterone in hormone replacement therapy, with special reference to the nervous system. Endocr Rev. 2007;28(4):387– 439.
12. Runko E, Kaprielian Z. Expression of Vema in the developing mouse
spinal cord and optic chiasm. J Comp Neurol. 2002;451(3):289 –299.
13. Cohen S, Levi-Montalcini R, Hamburger V. A nerve growth-stimulating factor isolated from Sarcom as 37 and 180. Proc Natl Acad
Sci U S A. 1954;40(10):1014 –1018.
14. Levi-Montalcini R, Angeletti Pu. Essential role of the nerve growth
factor in the survival and maintenance of dissociated sensory and sympathetic embryonic nerve cells in vitro. Dev Biol. 1963;7:653– 659.
15. Sofroniew MV, Howe CL, Mobley WC. Nerve growth factor signaling, neuroprotection, and neural repair. Annu Rev Neurosci.
2001;24:1217–1281.
16. Sakamoto H, Ukena K, Tsutsui K. Effects of progesterone synthesized de novo in the developing Purkinje cell on its dendritic growth
and synaptogenesis. J Neurosci. 2001;21(16):6221– 6232.
17. Sakamoto H, Ukena K, Tsutsui K. Dendritic spine formation in
response to progesterone synthesized de novo in the developing Purkinje cell in rats. Neurosci Lett. 2002;322(2):111–115.
18. Olbrich L, Foehring D, Happel P, Brand-Saberi B, Theiss C. Fast
rearrangement of the neuronal growth cone’s actin cytoskeleton
following VEGF stimulation. Histochem Cell Biol. 2013;139(3):
431– 445.
19. Meller K. The reaggregation of neurons and their satellite cells in
cultures of trypsin-dissociated spinal ganglia. Cell Tissue Res. 1974;
152(2):175–183.
20. Ocón-Grove OM, Maddineni S, Hendricks GL 3rd, Elkin RG,
Proudman JA, Ramachandran R. Pituitary progesterone receptor
expression and plasma gonadotrophin concentrations in the reproductively dysfunctional mutant restricted ovulator chicken. Domest
Anim Endocrinol. 2007;32(3):201–215.
21. Li X, McFarland DC, Velleman SG. Effect of Smad3-mediated transforming growth factor-beta1 signaling on satellite cell proliferation
and differentiation in chickens. Poult Sci. 2008;87(9):1823–1833.
22. Meller K. Axoplasmic transport of horseradish peroxidase in single
neurons of the dorsal root ganglion studied in vitro by microinjection. Cell Tissue Res. 1992;270(1):139 –148.
23. Meller K. Cryo-electron microscopy of myelin treated with detergents. Cell Tissue Res. 1994;276(3):551–558.
24. Theiss C, Napirei M, Meller K. Impairment of anterograde and
retrograde neurofilament transport after anti-kinesin and anti-dynein antibody microinjection in chicken dorsal root ganglia. Eur
J Cell Biol. 2005;84(1):29 – 43.
25. Foehring D, Brand-Saberi B, Theiss C. VEGF-Induced Growth
Cone Enhancement Is Diminished by Inhibiting Tyrosine-Residue
1214 of VEGFR-2. Cells Tissues Organs. 2012;196(3):195–205.
26. Bäckström T, Andreen L, Birzniece V, Björn I, Johansson IM, Nordenstam-Haghjo M, Nyberg S, Sundström-Poromaa I, Wahlström
G, Wang M, Zhu D. The role of hormones and hormonal treatments
in premenstrual syndrome. CNS Drugs. 2003;17(5):325–342.
27. Mani SK, Blaustein JD. Neural progestin receptors and female sexual behavior. Neuroendocrinology. 2012;96(2):152–161.
28. Liu L, Wang J, Zhao L, Nilsen J, McClure K, Wong K, Brinton RD.
Progesterone increases rat neural progenitor cell cycle gene expression and proliferation via extracellularly regulated kinase and pro-
doi: 10.1210/en.2013-1175
29.
30.
31.
32.
33.
34.
35.
36.
37.
38.
39.
40.
41.
42.
43.
44.
45.
46.
47.
48.
49.
gesterone receptor membrane components 1 and 2. Endocrinology.
2009;150(7):3186 –3196.
Su C, Cunningham RL, Rybalchenko N, Singh M. Progesterone
increases the release of brain-Derived neurotrophic factor from glia
via progesterone receptor membrane component 1 (Pgrmc1)-dependent ERK5 signaling. Endocrinology. 2012;153(9):4389 – 4400.
Sakamoto H, Ukena K, Tsutsui K. Effects of progesterone synthesized de novo in the developing Purkinje cell on its dendritic growth
and synaptogenesis. J Neurosci. 2001;21(16):6221– 6232.
Okabe S, Hirokawa N. Actin dynamics in growth cones. J Neurosci.
1991;11(7):1918 –1929.
Schumacher M, Guennoun R, Mercier G, et al. Progesterone synthesis and myelin formation in peripheral nerves. Brain Res Brain
Res Rev. 2001;37(1–3):343–359.
Schumacher M, Guennoun R, Robert F, et al. Local synthesis and
dual actions of progesterone in the nervous system: neuroprotection
and myelination. Growth Horm IGF Res. 2004;14(Suppl A):S18 –
S33.
Roglio I, Bianchi R, Gotti S, et al. Neuroprotective effects of dihydroprogesterone and progesterone in an experimental model of
nerve crush injury. Neuroscience. 2008;155(3):673– 685.
Kipp M, Amor S, Krauth R, Beyer C. Multiple sclerosis: neuroprotective alliance of estrogen-progesterone and gender. Front Neuroendocrinol. 2012;33(1):1–16.
Giatti S, Boraso M, Melcangi RC, Viviani B. Neuroactive steroids,
their metabolites, and neuroinflammation. J Mol Endocrinol. 2012;
49(3):R125–R134.
Cermenati G, Giatti S, Cavaletti G, et al. Activation of the liver X
receptor increases neuroactive steroid levels and protects from diabetes-induced peripheral neuropathy. J Neurosci. 2010;30(36):
11896 –11901.
Kim MJ, Shin HJ, Won KA, et al. Progesterone produces antinociceptive and neuroprotective effects in rats with microinjected lysophosphatidic acid in the trigeminal nerve root. Mol Pain. 2012;19:
8:16.
Verdi J, Jafari-Sabet M, Mokhtari R, Mesdaghinia A, Banafshe HR.
The effect of progesterone on expression and development of neuropathic pain in a rat model of peripheral neuropathy. Eur J Pharmacol. 2013;699(1–3):207–212.
Dableh LJ, Henry JL. Progesterone prevents development of neuropathic pain in a rat model: timing and duration of treatment are
critical. J Pain Res. 2011;4:91–101.
Wei Q, Zhu G, Cui X, Kang L, Cao D, Jiang Y. Expression of
CCT6A mRNA inchicken granulosa cells is regulated by progesterone. Gen Comp Endocrinol. 2013;189:15–23.
Simoncini T, Genazzani AR. Non-genomic actions of sex steroid
hormones. Eur J Endocrinol. 2003;148(3):281–292.
Giretti MS, Simoncini T. Rapid regulatory actions of sex steroids on cell
movement through the actin cytoskeleton. 2008;73(9 –10):895–900.
Dent EW, Gupton SL, Gertler FB. The growth cone cytoskeleton in
axon outgrowth and guidance. Cold Spring Harb Perspect Biol.
2011;3(3).pii:a001800
Dent EW, Gertler FB. Cytoskeletal dynamics and transport in growth
cone motility and axon guidance. Neuron. 2003;40(2):209 –227.
Schaefer AW, Kabir N, Forscher P. Filopodia and actin arcs guide the
assembly and transport of two populations of microtubules with
unique dynamic parameters in neuronal growth cones. J Cell Biol.
2002;158(1):139 –152.
Marsh L, Letourneau PC. Growth of neurites without filopodial or
lamellipodial activity in the presence of cytochalasin B. J Cell Biol.
1984;99(6):2041–2047.
Lafont F, Rouget M, Rousselet A, Valenza C, Prochiantz A. Specific
responses of axons and dendrites to cytoskeleton perturbations: an
in vitro study. J Cell Sci. 1993;104(Pt 2):433– 443.
Dent EW, Kalil K. Axon branching requires interactions between
dynamic microtubules and actin filaments. J Neurosci. 2001;21(24):
9757–9769.
endo.endojournals.org
3795
50. Bentley D, Toroian-Raymond A. Disoriented pathfinding by pioneer neurone growth cones deprived of filopodia by cytochalasin
treatment. Nature. 1986;323(6090):712–715.
51. Chien CB, Rosenthal DE, Harris WA, Holt CE. Navigational errors
made by growth cones without filopodia in the embryonic Xenopus
brain. Neuron. 1993;11(2):237–251.
52. Kaufmann N, Wills ZP, Van Vactor D. Drosophila Rac1 controls
motor axon guidance. Development. 1998;125(3):453– 461.
53. Dent EW, Barnes AM, Tang F, Kalil K. Netrin-1 and semaphorin 3A
promote or inhibit cortical axon branching, respectively, by reorganization of the cytoskeleton. J Neurosci. 2004;24(12):3002–
3012.
54. Fu XD, Simoncini T. Non-genomic sex steroid actions in the vascular system. Semin Reprod Med. 2007;25(3):178 –186.
55. Bashour NM, Wray S. Progesterone directly and rapidly inhibits
GnRH neuronal activity via progesterone receptor membrane component 1. Endocrinology. 2012;153(9):4457– 4469.
56. Bali N, Arimoto JM, Iwata N, et al. Differential responses of progesterone receptor membrane component-1 (Pgrmc1) and the classical progesterone receptor (Pgr) to 17␤-estradiol and progesterone
in hippocampal subregions that support synaptic remodeling and
neurogenesis. Endocrinology. 2012;153(2):759 –769.
57. Marsick BM, Flynn KC, Santiago-Medina M, Bamburg JR, Letourneau PC. Activation of ADF/cofilin mediates attractive growth cone
turning toward nerve growth factor and netrin-1. Dev Neurobiol.
2010;70(8):565–588.
58. Ruiz de Almodovar C, Coulon C, Salin PA, et al. Matrix-binding
vascular endothelial growth factor (VEGF) isoforms guide granule
cell migration in the cerebellum via VEGF receptor Flk1. J Neurosci.
2010;30(45):15052–15066.
59. Argiro V, Bunge MB, Johnson MI. Correlation between growth
form and movement and their dependence on neuronal age. J Neurosci. 1984;4(12):3051–3062.
60. Riedl J, Crevenna AH, Kessenbrock K, et al. Lifeact: a versatile
marker to visualize F-actin. Nat Methods. 2008;5(7):605– 607.
61. Lanlua P, Decorti F, Gangula PR, Chung K, Taglialatela G, Yallampalli C. Female steroid hormones modulate receptors for nerve
growth factor in rat dorsal root ganglia. Biol Reprod. 2001;64(1):
331–338.
62. Glebova NO, Ginty DD. Heterogeneous requirement of NGF for sympathetic target innervation in vivo. J Neurosci. 2004;24(3):743–751.
63. Kuruvilla R, Zweifel LS, Glebova NO, et al. A neurotrophin signaling cascade coordinates sympathetic neuron development
through differential control of TrkA trafficking and retrograde signaling. Cell. 2004;118(2):243–255.
64. Gallo G, Lefcort FB, Letourneau PC. The trkA receptor mediates
growth cone turning toward a localized source of nerve growth
factor. J Neurosci. 1997;17(14):5445–5454.
65. Gallo G, Letourneau PC. Neurotrophins and the dynamic regulation
of the neuronal cytoskeleton. J Neurobiol. 2000;44(2):159 –173.
66. Marsick BM, San Miguel-Ruiz JE, Letourneau PC. Activation of
ezrin/radixin/moesin mediates attractive growth cone guidance
through regulation of growth cone actin and adhesion receptors.
J Neurosci. 2012;32(1):282–296.
67. Runko E, Kaprielian Z. Caenorhabditis elegans VEM-1, a novel
membrane protein, regulates the guidance of ventral nerve cordassociated axons. J Neurosci. 2004;24(41):9015–9026.
68. Brinton RD, Thompson RF, Foy MR,et al. Progesterone receptors:
form and function in brain. Front Neuroendocrinol. 2008;29(2):
313–339.
69. Truss M, Beato M. Steroid hormone receptors: interaction with
deoxyribonucleic acid and transcription factors. Endocr Rev. 1993;
14(4):459 – 479.
70. Thomas P. Characteristics of membrane progestin receptor alpha
(mPR␣) and progesterone membrane receptor component 1 (PGMRC1) and their roles in mediating rapid progestin actions. Front
Neuroendocrinol. 2008;29(2):292–312.