Neuroscience Vol. 45, No. 3, pp. 663--{í70, 1991 0306-4522/91$3.00 + 0.

00
Printed in Great Britain PergamonPress pIe
© 1991 IBRO

SEXUAL DIMORPHISM IN ACCESSORY OLFACTORY

BULB MITRAL CELLS: A QUANTITATIVE GOLGI STUDY
A. A. CAMINERO,S. SEGOVIAand A. GUILLAMÓN*
Departamento de Psicobiología, Universidad Nacional de Educación a Distancia, Ciudad Universitaria
s/n, 28040 Madrid, Spain

Abstract- The purpose of the present study was to identify the existence of sexual dimorphism in the
dendritic field of accessory olfactory bulb mitral cells in rats and to investigate the effects of male
orchidectomy and female androgenization on the day of birth upon this dendritic field. The rapid Golgi
method was used to conduct a quantitative study of various characteristics of the dendritic field of
accessory olfactory bulb mitral cells.
The results indicated greater values for males than females for the following characteristics: (i) somatic
area; (ii) degree of branching in the dendritic field; (iii) total dendritic length; and (iv) dendritic density
around the neuronal soma. Orchidectomy of males, as well as androgenization of females, on the day of
birth inverted these differences.

According to extensive experimentation, the acces-
sory olfactory bulb (AOB), a vomeronasal system
(VNS) structure,":" has been shown to receive affer-
ents from various structures incIuding the
vomeronasal organ (VNO),3,35,37 the bed nucIeus of
the accessory olfactory tract (BAOT),13 the medial
(Me) and corticoposteromedial (C3) amygdaloid nu-
cIeus,13,44 the bed nucIeus of the stria terrninalis
(BNST),13,14 the medial and dorsal raphe nuclei" and
the locus coeruleus (LC).59 On the other hand, the
AOB sends efferents to the BAOT, the Me, the C3,
and to the medial division of the BNST (BN-
STM).9,13,14,17,18,52,70
The dendritic trees of the AOB
mitral cells receive VNO input by way of the
vomeronasal nerve (VN)3 and also receive synapses
from the AOB periglomerular and the granular
cells.29,36,48These mitral cells are al so AOB efferent
neurons; inforrnation sent by the AOB to other VNS
nucIei depends on the integration of signals found
wi thin these cells.36,48
The VNS is a sexuaIly dimorphic neural cir-
cuit12,25,55which plays an important function in the
control ofreproductive physiology and behaviour.P:"
Much of the Iiterature also reveals that the AOB is
related to reproductive behaviour in both mal e and
female rats4,5,28,42,53and that it is sexualIy dimor-
phic.49,56,57,68
The AOB volume is greater in males than
females.v-" Furthermore, males have a greater num-
ber of mitral cells" and dark and light granule ceIls
*To whom correspondence should be addressed.
Abbreuiations: AOB, accessory olfactory bulb; BAOT, bed
nucleus of the accessory olfactory tract; BNST, bed
nucleus of the stria terminalis; BNSTM, medial division
of the BNST; C3, corticoposteromedial; LC, locus
coeruleus; Me, medial; POA, preoptic area; SNB, spinal
nucleus of the bulbocavernosus; TP, testosterone propi-
onate; VN, vomeronasal nerve; VNO, vomeronasal
organ; VNS, vomeronasal system.
than females." The expression of these morphologi-
cal sex differences is dependent on the gonadal steroid
level early after birth.49,56,57,68
Studies have al so indi-
cated that the AOB receives afferents from sexuaIly
dimorphic structures incIuding the VNO,54 the
BAOT,12 the BNST,16,25 the MeIO,38-40,60and the Lc.24
To our knowledge, however, no studies showing
sexual dimorphism in the AOB dendritic field have
been carried out.
Several reports in the Iiterature indicate the influ-
ence of gonadal steroids on the dendritic field in the
amphibian" and avian15,33,34cIasses. In mammals,
sexual dimorphism in the dendritic field pattern has
been described for the hamster preoptic area
(POA).2.20 In rats, sex and experience appear to
interact in the appearance of the dendritic field of
hippocampal granular cells" and pyramidal ceIls in
the visual cortex." In white-footed mice, photo-
period-induced fluctuations in gonadal function were
associated with shrinkage of somata of motoneurons
of the spinal nucleus of the bulbocavernosus (SNB).
In addition, gonadectomy of male mice also reduced
the size of the soma and dendritic trees of these
motoneurons.'? Testosterone and estradiol have been
reported to selectively enhance neurite growth and
arborization in the organotypic cultures of the mouse
hypothalamus and POA.61-64 In aIl of these cases, the
data show that sexual dimorphism in the dendritic
field appears to be due to the action of gonadal
steroids.
Because of the existence of sex differences in the
volume ofthe AOB layer" and the number" ofmitral
cells, and other factors noted below, we decided to
investigate the possible existence of sexual dimor-
phism in the dendritic field of AOB mitral ceIls along
with the effects of male orchidectomy and female
androgenization on the day of birth. The other
factors leading to this direction of study incIude

663
664 A. A. CAMINERO et al.

findings that the AOB receives afferents from sexually three factors [sex, treatment and repeated measures (soma
dimorphic nuclei,IO,16,24,25,38-40,60
especially from the distance)) in the analysis of the dendritic density around the
neuronal soma, In addition, t-tests were performed for post
VNO.54 Other studies showed that gonadal steroids
61 hoc comparisons, To analyse the number of branches, as
can infiuence the expression of the dendritic field. -64 wel! as dendritic length and dendritic density around the
neuronal soma, the data were transformed into square
EXPERIMENTAL PROCEDURES roots. This analysis uses each animal, rather than each
Forty rat pups (20 males and 20 females) from the Wistar neuron, as the unit of analysis.
strain were divided into four groups immediately after birth.
These groups were: (i) 10females androgenized with a single RESULTS
injection of 1.25 mg testosterone propionate (TP) on the day
of birth (DI); (ii) 10 males orchidectomized on DI; (iii) 10 The number of dendritic shafts ansmg from the
females injected once each with the TP vehicle (sesame oil); soma of AOB mitral cells is similar for the four
and (iv) 10 males sham-operated (abdominal incision). For groups studied: 61 % showed three dendritic shafts,
these treatments, a11subjects were anaesthetized by cooling.
34% showed two dendritic shafts and 5% showed
At 60 days after birth, a11the rats were deeply anaesthetized
with an intraperitoneal injection of natrium pentobarbital four dendritic shafts. The distribution of AOB mitral
and then perfused transcardia11y with an isotonic saline cells with two, three or four dendritic shafts was
solution fo11owedby 10% formaldehyde neutralized to pH consistent throughout the nucleus and no preferential
7.2. After the brains were removed, the olfactory bulbs were zones were detected, Camera lucida drawings of
stained using the rapid Golgi method,"? and were then
sectioned into 200-¡.Im-thick transverse slices. The AOB representative AOB mitral cells of the four groups
mitral ce11swere located using the parameters described by studied are shown in Fig. 1, These illustrate the sexual
Ramon y Cajal in his earlier cytoarchitectonic studies of the dimorphism found in the dendritic fields of the mitral
AOB.46,47After excluding the poorly stained and damaged cells. The results with respect to the quantitative
neurons, we selected a total of 414 neurons for quantitative
study were as follows,
examination of the somatic area (114 from the control
males, 100 from control females, 81 from orchidectomized
males and 119 from androgenized females). In addition, 160
Somatic area
neurons from the dendritic field (40jgroup) were also chosen A statistically significant fírst-order interaction of
for quantitative examination. An homogeneous sample of sex by treatment was found (F(l,36)) = 14,184;
a11of the nuclear regions of the AOB mitrallayer from al!
of the animal s studied was used to obtain the various groups P < 0,0007), The AOB mitral cells of the control
of neurons. males have a greater somatic area than those of the
The purpose of the quantitative study was to characterize control females (P < 0,004), as can be seen in Table 1.
different aspects of the dendritic field including soma tic Orchidectomy of males on DI decreased the somatic
area, and a series of topological and metric parameters of
area of the AOB mitral cells in comparison with that
the dendritic field. A semiautomatic image analysis system
(VID S III, Shirehil! Industrial Estate, Saffron Walden, of the control males (P < 0,002), while the somatic
Essex, U,K,) coupled to a Leitz Diaplan microscope was area of orchidectomized males was almost the same
used to calculate the soma tic area, To determine the limits as that of the control females (P > 0,9), Androgeniza-
of the somata at the sites where the dendrites and axon tion of females on DI increased the somatic area of
emerged, a smooth are was drawn between the free edges of
each side. The are appeared to be a continuous extension of the AOB mitral cells to almost that of the control
the cel! body profile. males (P > 0,1), which was significantly greater than
Measurement of the topological and metric parameters of that of the control females (P < 0,05),
the dendritic field was performed using a computerized Sex differences also occurred in the frequency
system.' Input to the system treats the neuron three-dimen-
distribution of the AOB mitral cell soma tic area, as
siona11yas a dichotomic tree. It had been precoded since the 2
neurons had been drawn and measured with a camera lucida shown in Table 2, X comparisons showed a signifi-
coupled to an optic microscope (Leitz Diaplan). The neur- cant difference between the control males and females
ons were represented in graph form so that the spatial (X2(6) = 15,224; P < 0,02), The orchidectomized males
coordinates of relevant points could be taken (Le, soma, presented a frequency distribution of somatic are a
infiection and intermediate points, and bifurcation and
terminal points) according to centrifugal order.6,65,66This that did not differ significantly from the control
analytical method allowed us to obtain the fo11owing females (X 2(7)= 8,83; n.s.) and a frequency distri-
dendritic parameters: (i) average number of branches bution that did differ significantly from the control
per neuron in each group; (ii) bifurcation angles (the males (X
angle between the two daughter dendritic branches),1l,65,66
\7) = 18.434; P < 0,01), TP females showed a
frequency distribution of somatic area that did not
(iii) total dendritic length of branches; (iv) length of the
dendritic field (distance between centre of ce11body and differ significantly from that of the control males
terminal point of branches); and (v) dendritic density (X2(6) = 3,79; P > 0.1), but that did differ significantly
around the neuronal soma, The latter parameters were from that of the control females (X2(6) = 13,177;
determined according to an analysis technique developed by P < 0,05). Thus, the early postnatal treatments not
Sholl." This procedure characterizes the ce11ulardendritic
field by quantifying the dendritic intersection points through only were effective in abolishing or inverting sex
imaginary spheres. In our study, these points were drawn at differences, but they actually mimicked the pattern
regularly increasing radii every 20 ¡.1mfrom the neuronal observed in intact control male and female rats.
body.
The data were statistica11y analysed using: (i) variance Number o/ branches
analysis with two factors (sex and treatment) for analysis of
a11parameters; (ii) chi-square (X ) test to compare relative
2 For the dendritic parameter of number of
frequencies of the soma tic area; (iii) variance analysis with branches, a first-order interaction of sex by treatment
 Sex differences in accessory olfactory bulb mitral cells
Fig. 1. Camera lucida drawing of the AOB mitral cells in the four groups studied: (A) control males; (B)
control females; (C) orchidectomized males; (D) androgenized females. The arrows indicate the axon.
in the number of branches of AOB mitral cells was
found to be statistically significant (F(I,36) = 37.051;
P < 0.0001). However, an analysis of variance
showed main effects of sex (F(I,36) = 8.919; P < 0.005)
and treatment to be statistically significant. These
effects depend on the drama tic increase in the total
number of branches in the TP females (Table 1). The
AOB mitral cells of the control males had a larger
total number of branches than those of the females
(P < 0.05). The orchidectomized males showed a
lower total number ofbranches than that found in the
control males (P < 0.006), but a similar number to
the control females (P < 0.1). However, the AOB
mitral cells of the TP female had a larger total
number of branches than the control females
(P < 0.0001) and the control males (P < 0.002).
Bifurcation angles
In terms of bifurcation angles, a main effect of
treatment (F(I,36) = 7.954; P < 0.008) was observed,
although no significant interaction between sex and
treatment was found (Table 1). No sex differences
were found for this parameter.
Total dendritic length
A main effect of the variable treatment was also
obtained (F(l,36) = 4.142; P < 0.05), as can be seen in
665
Table 1. This effect was dependent on an increase in
dendritic length for the TP females. Sex also inter-
acted significantly with treatment (F(l,36) = 25.392;
P < 0.0001). Control males showed longer dendrites
than control females (P < 0.01), while androgenized
females had longer dendrites than control females
(P < 0.0001) and control males (P < 0.02). On the
other hand, orchidectomized males had dendritic
length values which were equal to those of the control
females (P > 0.1) and which were also lower than
those for the control males (P < 0.05).
Dendritic field length
A significant main effect of sex (F(l,36) = 34.428;
P < 0.0001) and a significant interaction of sex by
treatment (F(l,36) = 76.764; P < 0.0001) were obtained
for the parameter of dendritic field length. The results
for each of the groups studied are shown in Table l.
Greater lengths were observed for the control males
than for the control females (P < 0.08). The length of
the dendritic field in androgenized females was also
greater than in both the control females (P < 0.0001)
and the control males (P < 0.0001). Male orchidec-
tomy decreased the length of the dendritic field
compared to the control males (P < 0.0001), as well
as the control females (P < 0.001). Thus, the main
effect of sex seen in the analysis of variance appears
to be caused by the dramatic effect on the length of
666 A. A. CAMINERO el al.

dendritic field of orchidectomized and androgenized

animals.

Dendritic density around the neuronal soma

In terms of dendritic density in the area of the
soma, an analysis of variance indicated the existence
of a statistically significant interaction of sex by
treatment (F(I,36) = 12.859; P < 0.0003). Greater den-
dritic density values were obtained for males than
females (P < 0.05). Androgenization of females on
DI increased dendritic density around the neuronal
soma, but no difference from control males was
observed (P > 0.1). Androgenized females showed a
difference frorn control females of P < 0.002. Or-
chidectomy of males on DI produced a decrease in
the dendritic density around the neuronal soma com-
pared with control males (P < 0.05), but no differ-
ence from control females was found (Table 1).
A second-order interaction of sex by treatment and
by distance from the soma was also found to be
significant (F(14,504) = 2.104; P < 0.02). Control males
showed greater dendritic density around the neuronal
soma than control females, as can be seen in Fig. 2.
However, this sexual dimorphism was statistically
significant only for a distance of between 140 and
200 iut: (P < 0.05) from the soma. Orchidectomy of
** males on DI produced a decrease in dendritic density
** * * *o **'<:f"
*\O *r-. 0'\
.-q"f')
around the neuronal soma between 160 and 300 usx:
"'o:::j"('f")('f")NO""Í"

~-;-;N~~ from the soma (P < 0.05), while the androgenized

\ON,......;OOl/")("I")
females showed greater dendritic density around the
neuronal soma than control females. These results
were statistically significant for distances of 100 and
140 f1.m, and between 180 and 300 utx: (P < 0.05).

DISCUSSION

Our quantitative Golgi study of AOB mitral cells

shows the existence of sexual dimorphism in various
parameters including somatic area, number of
branches, total dendritic length and dendritic density
around the neuronal soma. These examples of sexual
dimorphism are controlled by the early postnatal
gonadal environment since DI male orchidectomy
and female androgenization were effective at inducing
changes in the sex differences found in the genetic
male and female rats.
There are several reports in the literature showing
sexual dimorphism in the dendritic field. Gregory"
described sex differences in the somatic volume of the
pyramidal cells of the somatosensory cortex of the
rat. In his study, male pyramidal cells showed a
greater value for volume than that of females. Gorski
et al.20 found that the size of the neurons of the
sexually dimorphic nucleus ofthe POA was greater in
male than in female rats. Similar findings were re-
ported for neurons in the rat SNB,8 and for the
robustus nucleus of the zebra finch archistriatum.P:"
In white-footed mice, exposure to short daylengths
resulted in shrinkage of somata of motoneurons of
the SNB.19 In addition, gonadectomy of male mice
 Sex differences in accessory olfactory bulb mitral cells 667

Table 2. Frequency distribution of soma tic area of the accessory olfactory

bulb mitral cells in the four groups studied
Somatic Control Control Orchidectomized Androgenized
area (¡lml) males females males females
0-50 0.00 0.00 1.25 0.00
50-100 2.63 11.00 16.04 2.52
100-150 29.82 39.00 28.39 32.77
150-200 28.94 30.00 33.33 36.13
200-250 22.80 15.00 13.58 15.96
250-300 10.55 3.00 7.40 9.24
300-350 4.38 1.00 0.00 3.37
350-400 0.87 0.90 0.00 0.00

also reduced the soma size of these motoneurons
compared witb intact mice or castrates tbat were
provided witb testosterone capsules."
Our results for dendritic brancbing also agree witb
tbose reported in several otber studies. Tbese include
studies on the bamster POA,20the ventrolateral POA
of Macaca fascicularis.: tbe robustus nucleus of the
arcbistriatum of the zebra fincb15,26 and the vocal
production nucleus of Xenopus laeois." Greater den-
dritic brancbing in males compared to females was
reported for all of tbese neural structures. In white-
footed mice, the gonadectomy of males reduced the
dendritic length and the dendritic density around the
soma of motoneurons of tbe SNB, compared with
intact mice or castrates that were provided with
testosterone capsules."
In terms of dendritic field lengtb, for the control
animals the longest length of the males compared to
females was almost statisticalIy significant (P < 0.08).
After orchidectomy of the males and androgenization
of the females on DI, these differences increased and
inverted.
In our study, males showed bifurcation angles that
were similar to females. Bifurcation angles have been
associated with a preference of direction of the
dendritic field.f For this reason, and based on our
results, the lack of differences between males and
females regarding tbis parameter indicates that the
preference direction of the dendritic field of the AOB
mitral celIs is similar for both sexes. This is logical if
we keep in mind that the principal afferent input to
tbe mitral celIs comes from the VN, which accesses

~
ºt3
UJ

'"
a:
UJ
1- -- CONTROL MALES
~ __ CONTROL FEMALES
o
a:
3
UJ
CD --f3- ORCHIDECTOMIZED MALES
~
2
z -e-- ANDROGENIZED FEMALES
~
2

O
o 40 BO 120 1&:l 200 240 280 320
DISTANCE FROM SOMA (¡m)

6
8 e
~
5
w
5
§w
12 ~ 4
~ ~
~ 3 ~
a::
w
CD
¡¡¡
2 2
~
2
z
2
=>
z

I ~
O O
O 40 00 120 1&:l 200 2«) 200 320 O 40 BO 120 100 200 240 2BO 320
D1STANCE FROM SOMA{jIn} DISTANCE FROM SOMA!".,}

Fig. 2. Distribution of dendritic density of the AOB mitral cells around the neuronal soma in the four
groups studied. (A) Comparison between control males and control females. (B) Comparison between
control males and orchidectomized males. (C) Comparison between control females and androgenized
females.
668 A. A. CAMINERO et al.

the nucleus by way of the dorsal portion. It is also
significant that the dendrites of the mitral cells end in
various glomeruli.v-"
Bifurcation angles have not been analysed in other
studies of sexual dimorphism. Nevertheless, they
seem to be rather consistent for neuron types studied
for this parameter, Factors such as the environment
of the pyramidal neurons of the cerebral cortex" or
the state of development of Purkinje cells!' do not
affect bifurcation angles. Paldino and Púrpura" have
published the only report in the literature that shows
modification of the bifurcation angles parameter.
They found a decrease over time of the angles of the
pyramidal cells of the human hippocampus.
Soma tic growth is promoted by gonadal steroids,
according to Toran-Allerand.v+' While working in
organotypic cultures taken from the fetal rat POA
and hypothalamus, she found that testosterone and
estradiol enhance neuronal size including soma and
neurite branching and length. Our results concerning
AOB mitral cells suggest that testosterone (and/or its
metabolites) increases the dendritic field of these cells
in control male rats shortly after birth. Furthermore,
male orchidectomy decreased the dendritic field of
mitral cells, while TP administration enhanced the
dendritic field of these cells in genetic females.
The establishment of the dendritic field during
early development is also influenced greatly by affer-
ent input. Both neuronal afferent and environmental
stimulation are major processes at this
time.7,22.30,31,45,50,51 We have shown that sexual dimor-
phism exists in the number of neurons of several
vomeronasal structures which send direct afferents to
the AOB. A major direct neural input to the AOB
mitral cells comesfrom the VNO bipolar neurons.'-"
According to a pattern of sexual dimorphism, males
had a greater number of bipolar neurons than females
in the rat VNO.54 As a result, a greater number of
VNO efferents in males might contribute to the
greater dendritic field of their AOB mitral cells, since
orchidectomy of males on DI decreased the number
of VNO bipolar neurons." In the present study the
same treatment also decreased the AOB mitral den-
dritic field. Moreover, the androgenization of females
on DI, which increased VNO bipolar neurons, also
increased their AOB mitral dendritic field.
CONCLUSION
The greater dendritic field of AOB mitral cells in
male compared to female rats reported in the present
study agrees with results fram previous studies in
which we found that males have a greater number of
mitral'" and granule (dark and light) AOB cells.? In
addition, greater values for volume of the AOB as a
whole and the mitral cell layer were obtained for
males as compared with females, thereby agreeing
with Segovia et al.56 The present results contribute to
an understanding of the basis for sexual dimorphism
in the AOB and support the concept that the VNS of
the rat brain is sexually dimorphic."
Acknowledgements-This work was supported by a DGI-
CYT Grant PB87-0621. We would like to thank the Depart-
mento de Investigación y Desarrollo de Telefónica (Madrid,
Spain) and especially Gerardo Abella and Alfonso Vicente
for helping with this research. We would also like to
acknowledge M. de Amaral, Luis Carrillo, Luis Troca and
Gonzalo Moreno for their technical assistance, and Mary
Frances Litzler for her editorial help.

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(Accepted 14 May 1991)