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Three‑dimensional
and two‑dimensional
relationships of gangliogenesis
with folliculogenesis in mature
mouse ovary: a Golgi–Cox staining
approach
Mohammad Ebrahim Asadi Zarch1,7, Alireza Afshar2,7, Farhad Rahmanifar3,7,
Mohammad Reza Jafarzadeh Shirazi1*, Mandana Baghban4, Mohammad Dadpasand1,
Farzad Mohammad Rezazadeh1, Arezoo Khoradmehr2, Hossein Baharvand5,6 &
Amin Tamadon2*
The present study was set out to investigate two‑dimensional (2D) and three‑dimensional (3D)
evaluations of ovarian nervous network development and the structural relationship between
folliculogenesis and gangliogenesis in mouse ovaries. Adult mice ovarian tissue samples were
collected from follicular and luteal phases after cardiac perfusion. Ovarian samples were stained by a
Golgi–Cox protocol. Following staining, tissues were serially sectioned for imaging. Neural laments
and ganglia were present in the ovaries. In both 2D and 3D studies, an increase in the number and
area of ganglia was seen during the follicular growth. The same pattern was also seen in corpora lutea
development. However, in some cases such as ratio of ganglia number to follicle area, the ratio of
ganglia area to follicular area, 2D ndings were dierent compared with the 3D results. 3D analysis
of ovarian gangliogenesis showed the possible direct eect of them on folliculogenesis. Golgi–Cox
staining was used in this study for 3D evaluation in non‑brain tissue. The results of 3D analysis of the
present study showed that, in some cases, the information provided by 2D analysis does not match
the reality of ovarian neuronal function. This conrmed the importance of 3D analysis for evaluation of
ovarian function.
Follicles are basic units of mammalian ovary. e development of rodent’s follicles begins at neonatal period,
the stage at which primordial follicles are formed1. Each primordial follicle has an oocyte which is held at rst
prophase of meiosis and is covered by attened granulosa cells layer2. Aer female maturation the estrous cycle
starts. rough this cycle, primary, secondary, antral and preovulatory follicles develop from primordial follicles3.
At this stage, most of the antral follicles undergo atretic degeneration and a few of them, under stimulation of
follicle-stimulating hormone (FSH) and luteinizing hormone (LH), become preovulatory follicles2,4. Aer that,
due to follicle response to LH hormone, the follicle ovulates. e remaining cell transforms and forms corpus
luteum4. e ovarian cycle in mouse strains is called estrous cycle. is cycle includes four stages: proestrus and
OPEN
The Persian
Department of Basic Sciences, School of Veterinary
Department of Obstetrics and Gynecology, School of Medicine, Shiraz
Department of Stem Cells and Developmental Biology, Cell Science
Department of
These authors contributed equally:
*email: jafarzd@shirazu.ac.ir;
amintamaddon@yahoo.com
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estrus as follicular phase and metestrus and diestrus as luteal phase5. Ovulation and corpus luteum formation
occurs in estrus stage. Presence of corpus luteum is vital for progesterone secretion 6,7. So, due to lack of preg-
nancy, corpus luteum undergoes degeneration and progesterone secretion reduces at proestrus stage. Hence,
next estrous cycle starts8.
e mammalian’s ovary is regulated by hormonal factors and direct neuron eects9. Several studies have
demonstrated that in mouse strains there are distinct populations of neurons, both internal and external neurons.
It has been shown that the chemical phenotypes of ovarian neurons of some mouse strains are sympathetic,
similar to primates10. Due to results of previous studies, it is now well established that ovarian neurons are
derived from neural crest cells, which form with complete ovarian maturation and subsequent reproductive
functions11. Noradrenergic nerves are expressed in the ovary near birth12. It has been conclusively shown that
the total number of neurons in puberty increases and then decreases12. External nervous system of mouse ovary
has many roles. Several studies have shown its role in developmental process, cyclic stages, pregnancy, and aging
process13–15. ese nerves and ganglia are responsible for ovarian estradiol secretion15. e number of internal
neurons in neonatal ovaries was lower than that of adult ovaries, some of which form ganglia and networks16.
Some neurotransmitters such as neurotrophins have an important role in follicle growth. For example,
reduction of brain-derived neurotrophic factor (BDNF) and neurotrophin-4 (NT-4) can cause folliculogenesis
disorder17. Also, these nerves and ganglia can take part in pathological conditions such as polycystic ovary syn-
drome (PCOS)18. Based on the previous studies, hypothetically assumed that the ganglia may have an important
role in mice ovary function. In spite of the fact that many researchers have utilized the two-dimensional (2D)
methods for evaluation of ovarian nervous system, so far no three-dimensional (3D) analysis research has been
performed on ovarian ganglia using image processing techniques and non-immunostaining methods. e pur-
pose of the present study was to perform three-dimensional evaluation of ovarian ganglia network development
and their structural relationship with folliculogenesis in the mice ovary. Golgi–Cox staining was performed in
the ovary for imaging of ganglia.
Results
Ganglia and ovarian structures in Golgi–Cox staining. In the present study, Golgi–Cox staining was
used for the rst time to identify the ganglia network of mouse ovaries. Also, with the help of this method
of staining and serial cryo-sectioning technique and three-dimensional reconstruction of ovarian slices, the
parameters of gangliogenesis relationship with ovarian structures were compared. Briey, in 2D images, the gan-
glia structures were stained black and the ovarian tissue was stained brown (Fig.1). Conversely, in 3D images,
the ganglia structures were recolored red and the ovarian tissue color was changed to transparent for 3D recon-
struction (Fig.2, Video S1).
In addition, the neural network was detectable in this color in black, but with continuous lament structures
in 2D (Fig.1A) and 3D images (Fig.2A). e ganglia were also recognizable as a network of tree dendrites
from a cell body between the theca and granulosa cell layers around all types of follicles (Fig.1B,C). In corpora
lutea, these ganglia were scattered throughout the corpus luteum structure (Fig.1A). e size, the shape and the
number of branches were structurally dierent between ganglia but all of them were multipolar (Fig.1B,C). e
ganglia of the follicular phase ovaries (Fig.1A,B) and the luteal phase ovaries (Fig.1C,D) were measured and
compared. e number of the ganglia at luteal phase ovary seems to be lower than the follicular phase ovary
(Fig.1A,D).
Aer reconstruction of ovarian structures by 3D method, scattering of ganglia between follicles and corpus
luteum was observed (Fig.2B). e segmentation of the ganglia aer the segmentation of the follicular structures
made it possible to image the spatial relationship between both ganglia networks and reproductive structures
(Fig.2C). e spot algorithm for measuring follicles and corpora lutea completely segmented the ovarian struc-
tures (Fig.2B,D). e cell algorithm also segmented the network structures of the ganglia (Fig.2C). Ganglia
and neurons were observed in all parts of the ovarian tissue. Nerve tissue density especially neural laments was
higher in the medulla of ovaries than the cortex.
Follicular growth and increase of ganglia number. In the 2D study, the total number of ganglia
increased during follicular growth (p < 0.05; Fig.3A,B). In contrast with luteal phase ovary, in follicular phase
ovary, the total number of ganglia in the antral follicles was higher than in the secondary follicles and atretic
antral follicles (p < 0.001 and p = 0.001, respectively; Fig.3A,B). Investigating changes in the number of ganglia
relative to increasing follicle area in the 2D study, it was observed that the ratio of ganglia number to follicle
area in secondary follicles was higher than antral follicles and atretic antral follicles (p = 0.023 and p = 0.022,
respectively, Fig.3C). However, the number of ganglia in secondary follicles in the follicular and luteal phases
was not signicantly dierent (p > 0.05, Fig.3D), but the number of ganglia in antral follicles in follicular phase
was higher than luteal phase (p = 0.03, Fig.3E).
On the other hand, in the 3D study, the total number of ganglia increased during follicular growth, as well
as 2D study (p < 0.05; Fig.3F). Indeed, the total number of ganglia in the antral follicles was higher than the sec-
ondary and atretic antral follicles in both luteal and follicular phase ovaries (p < 0.01 and p < 0.001, respectively;
Fig.3F,G). Furthermore, the ratio of ganglia number to follicle area in the secondary follicles and the antral
follicles was not dierent, which was in contrast with the 2D study ndings (p > 0.05; Fig.3H). In addition, this
ratio in the antral follicles was higher than atretic follicles, unlike the 2D study analysis (p = 0.026; Fig.3H).
Follicular growth and increase of ganglia area. In 2D study, the total area of ganglia increased during
follicular growth (p < 0.05; Fig.4A,B). Total area of ganglia in the antral follicles was higher than the secondary
follicles in both luteal and follicular phase ovaries (p = 0.48 and p < 0.001, respect ively; Fig.4A,B). In addition,
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the ratio of ganglia area to ganglia number between the antral follicles and the secondary follicles was not dif-
ferent (p > 0.05, Fig.4C). However, the ratio of ganglia area to area of structures in the secondary follicles was
higher than the antral follicles (p > 0.05, Fig.4D). In addition, the ratio of ganglia area to area of structures in
the secondary follicles was higher than the atretic antral follicles (p = 0.034, Fig.4D). Furthermore, the area of
ganglia in the secondary follicles in luteal phase ovary was higher than the follicular phase ovary (p = 0.001,
Fig.4E). Also, the area of ganglia in the antral follicles in luteal phase ovary was higher than the follicular phase
ovary (p = 0.006, Fig.4F).
In the 2D study, there were positive correlations between increase in ganglia area and increase in ganglia
number; increase in ganglia area and increase in ovarian structures’ area; and increase in ganglia number and
increase in ovarian structures’ area (p = 0.0001, Table1). In addition, in the secondary follicles, there were posi-
tive correlations between increase in ganglia area and increase in ganglia number; increase in ganglia area and
increase in secondary follicles’ area and increase in ganglia number and increase in secondary follicles’ area
(p = 0.0001, Table1). Moreover, in the antral follicles and the atretic antral follicles, there was also positive cor-
relation between these three groups (p = 0.0001, Table1).
In the 3D study of luteal phase ovaries, the total area of ganglia in antral the follicles was higher than the
secondary and atretic antral follicles (p < 0.01; Fig.4G). On the other hand, in the 3D study of follicular phase
ovaries, the total area of ganglia increased during follicular development (p < 0.05; Fig.4H). Area of ganglia in the
antral follicles was higher than the secondary follicles, as well as the 2D study (p < 0.001, Fig.4H). In contrast with
the 2D study, the ratio of ganglia area to follicular area in the antral follicles was not dierent with the secondary
follicles (p > 0.05, Fig.4I). Additionally, the ratio of ganglia area to follicular area in the antral follicles was higher
Figure1. Mouse ovarian sections were stained with Golgi–Cox method. (A) Two-dimensional follicular phase
ovary. (C) Distribution of ganglia around follicular phase ovarian follicles. (D) Distribution of ganglia around
luteal phase ovarian follicles. e ganglia (red arrow head), and neural laments (blue arrow head) are stained
black. Secondary follicles (SF), Antral follicles (AF) and corpus luteum (CL) are also seen (all images are gray
scale format).
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than the atretic antral follicles, which it was not seen in 2D study (p = 0.009, Fig.4I). However, the ratio of ganglia
area to ganglia number in the atretic antral follicles was higher than the secondary follicles (p = 0.017, Fig.4J).
In the 3D study, the same as the 2D study, there were positive correlations between increase in ganglia area
and increase in ganglia number; increase in ganglia area and increase in ovarian structures’ area; and increase
in ganglia number and increase in ovarian structures’ area (p = 0.0001, Table2). In addition, in the secondary
follicles as well as the 2D study, there were positive correlations between increase in ganglia area and increase
in ganglia number; increase in ganglia area and increase in secondary follicles’ area; and increase in ganglia
number and increase in secondary follicles’ area (p < 0.05, Table2). In contrast with the 2D study in the antral
follicles, there were no correlations between increase in ganglia area and increase in ganglia number; increase
in ganglia area and increase in antral follicles’ area; and increase in ganglia number and increase in antral fol-
licles’ area (p > 0.05, Table2). In addition, in contrast with the 2D study in the atretic antral follicles there were
no correlations between increase in ganglia area and increase in ganglia number; increase in ganglia area and
increase in atretic antral follicles’ area; and increase in ganglia number and increase in atretic antral follicles’
area (p > 0.05, Table2).
Corpus luteum development and increase of ganglia parameters. In the 2D study, the total area
and number of ganglia increased during corpus luteum development (Figs.2A,B,3A,B). Specically, the total
number of ganglia in the corpora lutea was higher than the antral, and secondary and atretic antral follicles in
luteal phase ovary (p < 0.001, p = 0.002 and p < 0.002, respectively Fig.3A). Additionally, the total number of gan-
glia in the antral follicles was higher than the corpus luteum in follicular phase ovary (p = 0.036, Fig.3B). In both
luteal and follicular phases, number of ganglia in corpora lutea was higher than the secondary follicles (p < 0.001
and p = 0.002, respectively; Fig.3A,B).
In 2D study, the ratio of ganglia number to area of structures and also the ratio of ganglia area to ganglia
number in the corpora lutea and the antral follicles was not dierent (p > 0.05, Figs. 3C,4C). Also, the ratio of
ganglia area to area of structures in the corpora lutea was more than the atretic antral follicles (p = 0.025, Fig.4D).
Moreover, number of ganglia in the corpora lutea in luteal and follicular phases was not dierent (p > 0.05,
Fig.5A). In contrast, the area of ganglia in the corpora lutea in luteal phase ovary was higher than follicular
phase ovary (p = 0.01, Fig .5B).
In the 2D analysis, there were positive correlations between area of ganglia and number of ganglia in the
corpora lutea (p = 0.003, Table1). Also, positive correlations between area of ganglia and area of structure and
number of ganglia and area of structure in the corpora lutea were observed (p = 0.004 and p = 0.007, respectively;
Table1).
Figure2. ree-dimensional (3D) reconstruction and segmentation of mice ovary structures aer Golgi–Cox
staining for detection of ovarian structures relationship with ganglia networks. (A) Whole tissue imaging and
3D reconstruction of mice ovary aer Golgi–Cox staining (red color represent the ganglia); (B) ovarian follicles
and corpora lutea segmentations in the whole tissue 3D reconstructed ovary. e blue spots represent Antral
follicles, yellow spots represent secondary follicles, purple spots represent corpus luteum and brown spots
represent atretic antral follicles. (C) Ovarian follicles, corpora lutea and ganglia segmentations. Ganglia are
shown with white cells. (D) Ovarian follicles and corpora lutea segmentations. All scale bars in four images are
300µm. All images are processed and drawn by Imaris soware.
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In 3D analysis of luteal phase ovaries, the total number of ganglia in the corpora lutea was higher than the
secondary and atretic antral follicles (p < 0.001, Fig.3F). In addition, in the 3D study of follicular phase ovaries,
Figure3. Comparisons of means and standard errors of variables in the three-dimensional (3D) and two-
dimensional (2D) studies show the relationship of follicular growth and increase in ganglia number in the
mouse ovary. Number of ganglia in luteal phase (A) and follicular phase (B) phases in follicles and corpora lutea
in the 2D study. (C) Number of ganglia to area of structures ratio in luteal phase in the 2D study. (D) Number
of ganglia in secondary follicles in follicular and luteal phase ovary in the 2D study. (E) Number of ganglia in
antral follicles in follicular and luteal phase ovaries in the 2D study. (F) Number of ganglia in follicles and corpus
luteum in the 3D study. (G) Number of ganglia to area of structures ratio in the 3D study (*p < 0.05, **p < 0.01,
***p < 0.001).
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total number of ganglia in the antral follicles was higher than the corpora lutea in follicular phase (p = 0.001,
Fig.3G) e number of ganglia in corpora lutea was higher than the secondary and the atretic antral follicles
in follicular phase ovary (p < 0.001 and p = 0.002, respectively, Fig.3G), same as the 2D analysis of luteal phase
ovary. e 3D study results of luteal phase ovaries were almost similar to the 2D study. In the 3D analysis of
luteal phase ovaries, the total area of ganglia in the corpora lutea was higher than the secondary, antral and
atretic antral follicles (p < 0.001, p = 0.003 and p < 0.001, respectively; Fig.4G). In both the 2D and 3D study of
follicular phase ovaries, total area of ganglia in the antral follicles was higher than the corpora lutea (p = 0.013
and p = 0.025, respectively, Fig.4B,H) and higher than secondary follicle (p < 0.001, Fig. 4H). Moreover, the
total number and total area of ganglia in the corpora lutea were higher than the atretic antral follicles (p = 0.002
Figure4. Comparisons of means and standard errors of variables in the three-dimensional (3D) and two-
dimensional (2D) studies to show the relationship of follicular growth and increase in ganglia area in the mouse
ovary. Area of ganglia in luteal phase (A) and follicular phase (B) phases in follicles and corpora lutea in the 2D
study. (C) Area of ganglia to number of ganglia ratio in luteal phase in the 2D study. (D) Area of ganglia to area
of structures ratio in luteal phase in the 2D study. (E) Area of ganglia in the secondary follicles in follicular and
luteal phases in the 2D study. (F) Area of ganglia in the antral follicles in follicular and luteal phases in the 2D
study. (G) Area of ganglia in follicles and corpora lutea in the 3D study. (H) Area of ganglia to area of structures
ratio in the 3D study. (I) Area of ganglia to number of ganglia ratio in the 3D study (*p < 0.05, **p < 0.01,
***p < 0.001).
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and p < 0.001, respectively, Figs.3F,4H). Furthermore, the ratio of ganglia area to ganglia number between the
corpora lutea and the antral follicles was not dierent (p > 0.05, Fig.4J). In contrast, the ratio of ganglia area
to ganglia number in the corpora lutea was higher than the secondary follicles (p = 0.004, Fig. 4J). e area of
ganglia to area of structures ratio in the corpora lutea was higher than the secondary and the atretic antral fol-
licles (p = 0.006 and p = 0.001, Fig.4I). In contrast, this ratio between the corpora lutea and the antral follicles
was not dierent (p > 0.05, Fig.4I).
In the 3D analysis, there were positive correlations between area of ganglia and number of ganglia in the
corpora lutea (p = 0.02, Table2), area of ganglia and area of structure (p < 0.001, Table2) and number of ganglia
and area of structure (p = 0.02, Table2).
Discussion
In the present study the gangliogenesis during folliculogenesis and their relationship was shown in both 2D and
3D evaluation of mice ovaries. We found that during folliculogenesis the area and number of ganglia in the fol-
licular wall increased. Also, the 3D study results revealed that although the number of ganglia did not increase
by development of the secondary follicles to the antral follicles, proportionally, considering the increase of
volume and area of follicles during folliculogenesis, the area of ganglia increased from the secondary follicles to
the antral follicles. As a result, proliferation and hypertrophy of ganglia were observed during folliculogenesis
in mice ovary. ese phenomena were observed in both follicular and luteal phases. Besides, during follicu-
lar development, their function increased, too19. erefore, due to their enhanced function, secretion activity
increased, subsequently19. Furthermore, in a 3D evaluation of mouse ovary it has been shown that angiogen-
esis happened during folliculogenesis20. On the other hand, previous studies have shown that there is some
Table 1. Correlation coecient of follicular area and ganglia development including ganglia number and area
in dierent follicular phases and corpus luteum in two-dimensional ovary analysis.
Structures
Ganglia area-ganglia number Ganglia area-structure area Ganglia number-structure area
Correlation coecient p-value Correlation coecient p-value Correlation coecient p-value
Ovarian structures 0.971 0.0001 0.924 0.0001 0.925 0.0001
Secondary follicle 0.880 0.0001 0.920 0.0001 0.882 0.0001
Antral follicle 0.956 0.0001 0.904 0.0001 0.983 0.0001
Corpus luteum 0.955 0.003 0.950 0.004 0.932 0.007
Atretic antral follicle 0.993 0.0001 0.976 0.0001 0.993 0.0001
Table 2. Correlation coecient of follicular area and ganglia development including ganglia number and area
in dierent follicular phases and corpus luteum in three-dimensional ovary analysis.
Structures
Ganglia area-ganglia number Ganglia area-structure area Ganglia number-structure area
Correlation coecient p-value Correlation coecient p-value Correlation coecient p-value
Ovarian structures 0.985 0.0001 0.978 0.0001 0.979 0.0001
Secondary follicle 0.966 0.008 0.948 0.014 0.878 0.05
Antral follicle 0.532 0.468 0.566 0.434 0.429 0.571
Corpus luteum 0.927 0.023 0.997 0.0001 0.918 0.028
Atretic antral follicle 0.540 0.460 0.667 0.333 0.857 0.143
Figure5. Comparisons of means and standard errors of number of ganglia in the two-dimensional studies to
show the relationship of corpora lutea function and increase in ganglia number (A) and area (B) in the mouse
ovary.
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relationship between internal nervous network and vascular system in ovary functions and both of them take
part in ovarian secretion18,21–23. ey indicated that vessels and nerves have an important role in folliculogenesis
and ovulation18,21–23. Consistent with our ndings on the relationship between ovarian function and ganglio-
genesis, the same ndings about other neuronal cells in mice10,24, monkeys and humans9,25 demonstrated that
ovarian internal neuronal laments increased in sexual maturation. Furthermore, distribution, morphology,
and chemical phenotype of ovarian intrinsic nervous system in guinea pigs increase in adult animals compared
with neonates16. Comparing previous ndings with our results, it can be speculated that follicular function has
a positive relationship with gangliogenesis, intrinsic neuronal network and vasculogenesis. Another result of
current study showed that the estrous ovary had higher number of ganglia of antral follicles than luteal phase
ovary, however, their area was higher in luteal phase than follicular phase ovary. ese results suggest that during
follicular phase there is ganglia proliferation around the antral follicles. In addition, it is further conducted that
at the luteal phase the ganglia undergo hypertrophy and the area of the ganglia increase.
On the other hand, data of the current 3D study demonstrated that the number of ganglia to area of structures
ratio in the secondary and antral follicles and corpora lutea were not dierent. As the area of the follicles enlarges,
which indicates an increase in the number of granulosa and theca cells vascularization26, the number of ganglia
has increased to such an extent that the ratio of the ganglia number to the area of the follicles and corpora lutea
remains constant. is indicates a constant ovarian structure volume-dependent gangliogenesis. e number
of ganglia to area of structures ratio remains high during follicular development. Due to the increase in ovarian
follicular metabolism27 and in hormonal activity28, to ensure the interaction of ovarian structures and ganglia
remain constant, the area and volume of ganglia, as well as ganglia number increase.
During follicular development, some follicles undergoes atresia29. e specic criteria for follicles undergo
atresia, which it called atretic follicles, is pyknotic nuclei30. However, other criteria for atretic follicles recognition
is follicular deformity and they don’t have rounded shape29. As the Golgi–Cox staining only stain the cells of
nervous system31, the pyknotic nuclei in granulosa cells around the follicles are not recognizable. In the present
study, the atretic antral follicles were distinguished by their shapes’ deformity and non-rounded shape from antral
follicles. Totally, in the follicular phase ovary, the total number and area of ganglia in atretic antral follicles were
lower than antral follicles in both 2D and 3D studies. e follicles in follicular phase became almost mature and
on the other hand the rest of the follicles undergo atresia29. is showed that the as the atretic follicles undergoes
degeneration and their cells reduced, their ganglia also reduced in number and area. However, in the luteal phase
ovary, there was no signicant dierence between antral follicles and atretic antral follicles which it could be
because of the less maturation of antral follicles in luteal phase ovary29,32.
e corpora lutea has the highest value of ganglion area and number compared with the other ovarian struc-
tures in luteal phase. is result was observed at both 2D and 3D analysis of luteal phase ovaries. Along with
these results, the study of mice estrous cycle showed that progesterone secretion of the corpora lutea increased in
luteal phase33. In early luteal phase, area of luteal tissue and its function increased, which is known as luteogenesis
phase34. In addition, results from rat ovary showed that the corpora lutea formed at early luteal and progesterone
secretion started35. ese results indicated that corpora lutea in luteal phase has a high function and produces
progesterone. In order to reach this function, corpora lutea needed more secretory cells. On the other hand,
ganglia of the corpora lutea decreased during its atretic degradation. As the ovary was in follicular phase, the
results showed that the antral follicle has higher value of ganglia’s area and number than the corpora lutea. Due
to lack of pregnancy, the corpora lutea begin degradation and at the follicular phase are no longer observed8,36.
erefore, it can be resulted that during degradation of the corpora lutea, its ganglia undergo degradation too.
e 3D results conrmed our 2D ndings and showed degradation of ganglia during degradation of the corpora
lutea. e results of previous studies demonstrated that the corpora lutea has higher vessels and angiogenesis37,
which shows its higher activity rather than the other ovarian structures. Due to the fact that the nervous system
of ovary has a role in regulation of mammalian ovary function, steroidogenesis and ovulation38 and ganglia may
have roles in progesterone secretion, also, the higher number of ganglia in corpora lutea at luteal phase was seen
in comparison with regressing corpora lutea in the follicular phase.
To the best of our knowledge, this is the rst time that 3D morphological parameters of ganglia in the ovar-
ian follicles and the corpora lutea of mice were determined. In contrast with the 2D study, in the 3D study, the
number of ganglia to area of structures ratio in the secondary and the antral follicles was not dierent. is nd-
ing showed that during follicular growth, due to preserving the function of follicles, this ratio always remained
constant, which cannot be seen by the limitations of the 2D analysis. In some other ndings of our study, the
results of the 2D study were dierent from the 3D study and the 2D analysis could not give us a proper aspect of
the view of reality compared to the 3D analysis. In line with our ndings, even results of the 2D and 3D ultra-
sound techniques in ovary showed that the 2D analysis could not detect all data of ovarian structures and the
3D analysis was realistic39. In addition, in a histomorphological evaluation of rodents’ brain, the 3D analysis was
more accurate in analyzing all parts of brain rather than the 2D analysis.
In the present study, although the cost of immunohistochemistry method for whole tissue imaging of neuronal
networks of brain40 and dierent organs makes this approach unconventional, Golgi–Cox method was performed
for nervous tissue labeling and 3D imaging. Golgi staining is one of the oldest staining methods for nervous
system imaging and was developed by Camillo Golgi at 187341. is method has been shown to be eective due
to neuronal morphology imaging such as dendritic and axonal arborization and spines detection42. ere are
three methods of Golgi staining: Golgi–Cox, Rapid Golgi and Golgi–Kopsch43,44. While all of these methods
have some dierent advantages and disadvantages, Golgi–Cox method is better than the others for dendritic
trees analysis due to less background density44. In this method neurons, especially dendritic trees stain clearly
without noise44. Four types of ganglia exist in ovary of young and postnatal adult rats, including mesovarial,
Hilar, medullary, and cortical ganglia24. In the present study, Golgi–Cox method based on the previous study
“Golgi–Cox staining step by step”45 with slight modication was performed.
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In conclusion, the present study demonstrates the positive relationship of gangliogenesis during folliculo-
genesis in mouse ovary. Furthermore, gangliogenesis has been shown in corpus luteum development. Ovarian
ganglia as an independent part of ovarian nervous system is likely to have an important role in folliculogenesis
and luteogenesis. Furthermore, 3D analysis instead of conventional 2D approach, in addition to Golgi–Cox
staining of ovary can be used for study of this physiological phenomena in ovary.
Methods
Animals. e ovaries of 12 non-pregnant adult BALB/c mice were used in the present experimental study.
e mice weighed 35 ± 2g and were 49days old. Mice had free access to food and water and were kept in labo-
ratory cages in standard conditions at 22°C and 12h light/dark cycle. Complete ovarian sample was used for
imaging and statistical analysis.
Briey, aer the mice were anesthetized, 0.9% NaCl solution was perfused in le ventricle of the mouse hearts.
en the ovaries were removed from the mice, and the pattern of nervous system distribution with intra-ovarian
origin and their relation to folliculogenesis was determined by cross-sectional imaging and image analysis of the
ovarian tissues. According to the protocol, the initial experiments were performed so as to repeat the dierent
times of the samples in Golgi–Cox solution, showing the stability of this staining and the most appropriate result.
Estrous cycle evaluation. e vaginal smear test was performed to detect estrous cycle. Using 100µl of
physiological salt solution, aspiration of vaginal canal was done. Aspiration was considered about 4 times on
each mouse. e contents of the sampler’s head were then drained onto the slide and coated with cover slips.
Finally, based on previous study5,46, using light microscopy estrous cycle phases for mouse was determined.
Briey, predominant cornied squamous epithelial cells observation indicated follicular phase. However, at
luteal phase, the cornied squamous epithelial cells was not (or rarely) observed. At luteal phase, the most pre-
dominant cells were leukocytes.
Cardiac perfusion and ovary collection. e mice were anesthetized by chloroform-impregnated cot-
ton. en dissecting the heart, the right atrium was cut and 0.9% saline solution was injected into the le ven-
tricle. e saline solution slowly entered the circulatory tract. e perfusion was then performed with a syringe
containing 10mL of 4% formalin until the tissues became pale. Aer this, the ovaries were removed.
Golgi–Cox staining. Staining was done by a modied Golgi–Cox technique as described by Zaqout etal.45.
is modied method includes three main solutions.
Solution A: 5% (w/v) solution of potassium dichromate (K2Cr2O7; UNI-CHEM, Serbia).
Solution B: 5% (w/v) solution of mercuric chloride (HgCl2; UNI-CHEM, Serbia).
Solution C: 5% (w/v) solution of potassium chromate (K2CrO4; UNI-CHEM, Serbia).
All three solutions were kept in glass bottles at room temperature in the dark. In this condition, these solutions
can be used for a long time. For preparation of impregnation solution 25mL of solution A was slowly mixed with
25mL of solution B. 20mL of solution C was carefully added to previous mixture. In the nal step, 50mL of
dd-H2O was added to the mixture of all three solutions. Final solution was covered with aluminum foil and was
kept at room temperature in completely dark condition for 48h. Aer that, the reddish-yellow precipitation was
formed. e supernatant solution was gently collected by glass pipet (avoiding the reddish-yellow precipitant in
supernatant collection). e ovary tissue was transferred into glass bottle containing Golgi–Cox solution. Aer
24h, the tissue was transferred into fresh Golgi–Cox solution. e glass bottle was kept in the dark at room
temperature for 14days.
Ovary cryo‑sectioning. Firstly, cryoprotectant solution was prepared. For cryoprotectant preparation,
30g of sucrose was dissolved in 100mL ddH2O (30% sucrose solution). Aer tissue impregnation in Golgi–Cox
solution, tissues were transferred in cryoprotectant solution. e samples were kept in this solution at 4°C in
dark for 24h. Aer that, cryoprotectant solution was refreshed and the samples were kept in new solution in
the same conditions for 5days. en samples were washed with ddH2O and xed on cryo-holder by cryo-glue.
Finally, the samples were transversely sectioned on a cryo-holder at a temperature of − 25°C and a thickness of
30μm. e serial sections were transferred onto gelatin coated slides for developing stage on staining.
Developing step of Golgi–Cox staining. e developing step of Golgi–Cox staining was performed. In
detail, slides were kept in ddH2O twice for 2min each time. For dehydration of sections, slides were placed in
50% ethanol for 5min. Aer that, sections were transferred into ammonia solution (3:1 ammonia to ddH2O)
for 6min. en slides were placed in ddH2O twice, each for 2min. At the next step, samples were kept in 5%
sodium thiosulfate solution for 10min in dark condition. Aer repeating the ddH2O step twice (1min each),
samples were dehydrated with ascending percentage of ethanol (70%, 95%, and 100% ethanol each for 5min).
Aer dehydration step, tissues were placed in xylol for 10–15min until tissues were completely cleared. In the
nal step, slides were mounted by Entellan glue. Sections were kept in the dark until imaging.
Ovarian tissue imaging. An optical microscope (Nikon, E200, Japan) with a 40 × magnication and a
Dino Capture camera (AnMo Electronics Corporation, New Taipei City, Taiwan) were used. Whole ovarian
serial sections images were captured for each ovary. e images were provided in 2600 × 1950 pixels with TIFF
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format. e numbers and area of follicles, corpus lutea, ganglia and neuronal laments were calculated. e
criteria for assortment of follicles and corpus luteum in 2D analysis was described in Azarnia etal.47 and for 3D
analysis it was described in Feng etal.48. In addition, the assortment of the atretic antral follicles were described29.
2D analysis of ovarian follicular and neuronal structures. ImageJ soware used for 2D image
analysis of ovarian structures. Firstly, imported images converted to 8-bit images using “Image type” option in
“Image” panel. en scale of all images was set using “Set scale” option in “Analyze” panel. In order to analyze
single follicle, the area of each follicle containing its surrounded ganglia and a few neuronal laments was manu-
ally cropped. For this purpose, the border of each follicle with its ganglia were selected by “Oval” tool in main
menu of ImageJ soware (FigureS1A). Aer that, the selected area was duplicated and analysis was continued.
e specied structures (ganglia and neural laments) were measured by “threshold” algorithm. e size of
threshold eld was adjusted by dragging the threshold border, manually. By adjusting the signal threshold in the
soware, the shape of the ganglia and the neuronal laments around the follicles were detected. Using “Analyze
particles” in analyze panel, the number and area of particles were measured (FigureS1B). In this stage, laments
in a few crops were deleted manually using “delete” option in “ROI manager”. Finally, data were measured by
measure option in ROI manager and data were saved as an excel le with CSV format (FigureS2A).
3D analysis of follicular and neuronal structures. First, using ImageJ soware, serial images were
combined as a TIFF series image using “Images to Stack” tool. e TIFF series image was saved as stack with
TIFF format. en, using Imaris soware (V 7.4.2, ImarisX64, Bitplane AG), 3D reconstruction was performed.
Specically, aer serial TIFF image was imported, the dimension of image was corrected in z-stack according to
thickness of tissue slices using “Image properties” in “Edit” panel. Firstly, the whole image was reconstructed. In
order to reconstruct follicles and corpora lutea, “Spot” algorithm was used in “Surpass” panel. is procedure
was done manually in order to cover follicle area and diameter. ree “Spot” algorithms were used for three
dierent ovarian structures: secondary follicles, antral follicles and corpora lutea. en, the whole nervous net-
work was reconstructed by “Cell” algorithm in “Surpass” panel. e data of each algorithm was extracted from
“Statistics” in “Preferences” in “Edit” panel. In order to delete the data of neuron laments, the biggest ganglion
in whole image was isolated by “Crop 3D” in “Edit” panel and the area of this ganglia was examined by “Cell”
algorithm. e areas higher than area of biggest ganglia were deleted, as well as their counts. e number and
total area of neuronal laments were calculated by subtracting the total number and area of whole image from
the number and area of ganglia. For follicle and corpus luteum analysis, each follicle and its surrounding gan-
glia were isolated by “Crop 3D” in “Edit” panel. “Cell” algorithm was used for ganglia reconstruction in crops.
Only two neuronal laments were detected in these crop 3Ds and their data were manually deleted. e data
of area and number of ganglia from each crop were extracted from “Statistics” in “Preferences” in “Edit” panel
(FigureS2B).
Data analysis. Aer data extraction from ImageJ and Imaris soware, the data of area and number of struc-
tures (ganglia and neuronal laments) obtained from image analysis were entered into Excel soware. IBM SPSS
Statistics 26 (SPSS for Windows, version 26, SPSS Inc., Chicago, Illinois, USA) soware was used for statistical
analysis. e mean dierences between follicular groups were analyzed by one-way ANOVA and post hoc Tukey
test. e correlation of the total ganglia area and number and the area of structures (follicles and corpora lutea)
with each other in four dierent groups including total ovarian structures, secondary and antral follicles and
corpora lutea were analyzed by Pearson correlation. All data were expressed as the mean ± standard error of the
mean, and p value was considered less than 0.05 for statistical signicance. GraphPad Prism (v7.0a, GraphPad
Soware, Inc., San Diego, CA, USA) soware was used for drawing the graphs. e table format was “Grouped”
and the data input was the mean of area and number of ganglia and standard error of the mean.
Statement of ethics. All experimental protocols were approved by the Shiraz University Ethics Commit-
tee (project number: 97gcu4m148075). All methods were carried out in accordance with the World Medical
Association Declaration of Helsinki. is study was carried out in compliance with the ARRIVE guidelines
(http://www.nc3rs .org.uk/page.asp?id=1357). Eorts were made to minimize animal suering and to reduce the
number of animals used.
Received: 24 November 2020; Accepted: 19 February 2021
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Author contributions
M.E.A.Z., A.A., F.R., F.M.R., M.B. and A.K.: data collection, and manuscript writing. A.T., H.B., and M.R.J.S.:
idea conception and study design. M.D., A.A., and A.T., statistical analysis. A.T., H.B., and M.R.J.S: review and
proof-reading. All authors approved of the nal version.
Funding
is study was nancially supported by the Shiraz University (Grant number: 97gcu4m148075).
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Competing interests
e authors declare no competing interests.
Additional information
Supplementary Information e online version contains supplementary material available at https ://doi.
org/10.1038/s4159 8-021-84835 -0.
Correspondence and requests for materials should be addressed to M.R.J.S.orA.T.
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