Jan 4, 1983 - CHARLES. J. WYSOCKI,1. YAIR ... Dr. Charles. J. Wysocki, ...... Bronson,. F. H. (1979). The reproductive ecology of the house mouse. Q Rev.
BIOLOGY
OF
REPRODUCTION
28,
Male Vomeronasal
917-922
(1983)
Organ Mediates J.
CHARLES
WYSOCKI,1
Female-Induced YAIR
Monell
KATZ
Chemical
Pbiladelpbia,
and
Senses
Surges in Mice
Testosterone RONALD
BERNHARD
Center
Pennsylvania
19104
ABSTRACT In many species, the odor of a female can elicit a surge in plasma testosterone radioiminunoassays of plasma samples obtained from mice lacking a specific structure, the vomeronasal organ, indicate an absence of the expected surge expozre to an anesthetized female. We conclude that the male’s vomeronasal female primer pheromone which subsequently induces a testosterone surge.
1978;
INTRODUCTION
Chemical
substances,
by
emitted
Doty, 1977;
are
animals
neuroendocrine havior in
especially capable
reflexes recipients
and influencing (see contributions
1976; Mllller-Schwarze Mliller-Schwarze and
the
1980;
to
be
mediated
by
changes
levels, fects
thereby producing slow, upon both physiology Signaller effects, on the other
fined
as
vous
system,
being
mediated thereby
long-lasting and
directly permitting
physiology
immediate
responses to odors. Both primers and signallers are detected by nasal chemoreception, but recent studies have increasingly the
than the detection
vomeronasal
classical of and
olfactory subsequent
network,
in to
the
reviews and
of
mice.
female-induced
central receiving extent, (Winans
olfactory
network (Jacobson’s bulb (AOBJ
Accepted Received
Winans,
1975;
Krettek
the
main
olfactory
the
many of vomero-
by report
Keverne, of
in
1979;
Lehman
and
that an intact for the expres-
testosterone
surges
in
experiments were conducted. that males lacking a vomeronasal
conclude
a surge in testosterone (DHT) in response
and to a
female.
the sex
AND
METHODS
Organ, and
and
4, 1983. 9, 1982. 1Reprint requests: Dr. Charles J. Wysocki, Chemical Senses Center, 3500 Market St., phia, PA 19104.
(RIA)
Radioimmunoassay
other
For Experiments heparinized syringes
nervous system [CNSI structures efferents from the AOB), to a large is independent of the olfactory system and Scalia, 1970; Raisman, 1972; and
olfrom
Two
MATERIALS
vomeronasal
accessory
Scalia
(see
male
odors.
The
nose,
have either been implicated for the regulation of reproductive
organ fail to exhibit 5a-dihydrotestosterone
rather
system, responses
to
level. Functionally, which comprise
1979;
sion We
an
the
information
Winans, 1982). We now report vomeronasal organ is necessary
behavioral
implicated
in
Organ, communicate olfactory bulb while
epithelium
Wysocki,
are dethe ner-
within
anatomical separation is maintained CNS structures receiving efferents
nasal network or are crucial
ef-
Sensory
found
convey
from the bulbar the CNS structures
behavior.
hand, by
olfactory
1981a,b).
structure
receptors
bulb. This in other
in hormone
Winans,
network,
(vomeronasal) accessory
factory
Breipohl, 1982). These as yet unidentified compounds have tentatively been grouped according to the response elicited from conspecifics as either primer or signaller (Bronson, 1979). Primer effects are generally conceived
this distinct
Jacobson’s with the
Mozell,
Silverstein,
and
of
anatomically
bein
and
Kevetter
receptors
sex odors, of eliciting
in a male. Steroid nasal chemosensory in testosterone after organ detects the
ether
anesthesia.
3000
X g for
brought NaCI),
blood was collected in puncture under deep samples were centrifuged at
Blood 25
Procedures
1 and 2, by cardiac
min
to
obtain
plasma.
The
latter
was
up to a final volume of 0.5 ml (with 0.15 M transferred to teflon-stoppered tubes and extracted once with 10 ml acid-washed diethylether and once with 10 ml petroleum ether (b.p. 35-56#{176}C). The combined extracts were dried in a warm (50#{176}C) sand bath under a gentle stream of nitrogen and then subjected to separation on 8 mm (i.d) Sephadex LH-20 columns (2.5 g, 8.0 ml), eluted according to Zemecnik et al. (1977). The eluants from the individual fractions (Table 1) were dried and taken up in 1 ml of 0.1 M Na-phosphate in saline, pH 6.9, contain-
Price,
January
June
Monell
Philadel-
917
918
WYSOCICI
TABLE
1. Fractionation
.
and RIA cross-reactivity
values
ET AL.
of steroids. % Recovery . . of tritiated
.
Fraction
Ml
Steroids
no.
eluted
eluted
standard
1
0-2.5
2
2.5-5.5
3
5.5-9.5
.
Cross-reactivity Steroid
.
%
tested
---
---
--
Progesterone
98
Androstenedione
96
Androstenedione 5a-Androstanedione Progesterone
9.5-14.0
14.0-24.0
24.0-33.0
aND
not
bSteroids
0 0
0 68.7
5a-Androstanedione ND Testosterone 5a-Dihydrocesterone
-
0i%
97 91
Testosterone
94
5a-Dihydrotestosterone
87
Estrone Corticosterone
86 96
Corticosterone Estrone
29.3 1.5 19.7 0 3.0 -
7pdioIb
78
ND
---
---
Estradiol-17(3
94
Cortisol
72
Cortisol Estradiol-17(3
not included
gelatin
and
thimerosal.
0.01%
cortisol-21-hemisuccinyl-thyroglobulin
lin
-
0 0
in the present experiments.
6-(O-carboxymethyl)-oxime-thyroglobulin 7-3Hlestrone, 85 Ci/minol; HI cortisol,
-
determined.
Aliquots
93
anti-corticosterone-2
cortisol,
5A19721A
desk-top
computer;
validation
data
(100
(DHT), rabbit anti-DHT-lo-carboxyethyl-thioetherBSA and [1,2,4,5,6,7,16,17-3H)DHT, 190 Ci/mmol; estradiol-1 7(3, rabbit anti-estradiol-1 7j1-6-(O-carboxymethyl)-oxime-BSA and [2,4,6,7,16,1 73 HI estradiol17(3, 152 Ci/mmol; estrone, rabbit anti-estrone-
rabbit
-
0 0 0
Dehydroepiandrosterone 5a-Dihydrotestosterone Dehydroepiandrosterone
---
uI) of the buffered solutions were analyzed for their steroid contents by RIA techniques using specific steroid antibodies. Separation of free from bound steroids was affected by dextran-coated charcoal methodology. The following antibodies and tritiated steroids were used: progesterone, rabbit anti-progesterone1 1o-hemisuccinyl-bovine serum albumin (BSA) and (1 ,2,6,7H) progesterone, 90 Ci/mmol; androstenedione, rabbit anti-androstenedione-7cs-carboxyethylthioether-BSA and [1 ,2,6,7HI androstenedione, 114 Cilmmol; testosterone, rabbit anti-testosterone-7cscarboxyethyl-thioether-BSA and [1,2,6,7,16,17#{149} H) testosterone, 150 Ci/mmol; 5a-dihydrotestosterone
6,7-i
-
5oAndrostanedioneb
HP-981 ing
Cross-reaction
Dehydroepiandrosteroneb
5a-Androstane-3o,1 5 6
binding
-
Testosterone
4
.
at 50%
and
[2,4,6,
rabbit and
anti[1,2,
Ci/mmol;
and corticosterone, 1-hemisuccinyl-thyroglobu-
and (1,2,6,7-5Hlcorticosterone, 89 CiJmmol. Estradiol-1 7(3 antibody was kindly donated by Dr. D. T. Armstrong, London, Ontario, Canada; all other antibodies were produced by Miles-Yeda, Rehovoth, Israel. Radioactive steroids were purchased from New England Nuclear, Boston, MA, and ,ere purified on Sephadex LH-20 columns prior to use. Steroid standards were supplied by Steraloids, Wilton, NH. RIA raw data were analyzed by a Hewlett-Packard
have been Experiment
published
elsewhere
(Katz
et
al.,
1982).
I
The first experiment was conducted to establish the baseline plasma testosterone and DHT levels in male mice lacking a vomeronasal organ and to determine if these levels deviated significantly from those of intact males. Individually housed adult DBA/2J male mice received heterosexual encounters in the form of repeated 3-min exposures to female mice over 8 consecutive days (see Nyby et al., 1977 for details). These males then underwent either removal of the vomeronasal organ (VNX) or a sham surgical procedure. To prevent death from the inhalation of blood, surgery was conducted with the mouse in a suspended prone position. After an incision was made in the palate, the vomeronasal organ was detached from the incisive bones by gentle dorsal pressure. The vomer bone was then fractured and the entire vomeronasal organ was removed from the nasal cavity through the incisive foramen. The wound was sealed with a cyanoacrylate-based glue and the animal was allowed to recover (see Wysocki et al., 1982 for more details).
Absence
of
a
confirmed by dissection. all steps but vomer bone Removal does not Although determined unaffected unpublished
Three
vomeronasal
The
organ
was
later
sham surgery included and organ removal. organ in this manner
fracture vomeronasal the primary olfactory epithelium. not done in these experiments, we have that olfactory mediated behaviors remain by this surgery (Wysocki et al., 1982; also observations). weeks elapsed between surgery and the
of the endanger
ORGAN
VOMERONASAL
AND
collection of blood for the testosterone and DHT RIA’s. During this period, the mice appeared in good health and had participated in a behavioral experiment which included exposures to other male and female mice, the last of which occurred 1 week prior to blood collection (Wysocki et al., 1982). No females were encountered during the 7 days prior to the collection of blood for RIA. Experiment
2
was conducted to determine if removal of the vomeronasal organ affected the responsiveness of the male to female-generated cues which normally would elicit a testosterone surge in nonrefractory males (Macrides et al., 1974, 1975). As in Experiment 1, two surgical groups were used (Sham and VNX), but in this experiment, the males were exposed to females prior to withdrawal of blood. This
experiment
Individually housed adult male DBA/2J mice, with numerous previous heterosexual encounters, underwent either the sham or VNX surgical procedure. Complete removal of the vomeronasal organ was later confirmed by dissection. Two weeks were allowed for recovery. All mice survived and appeared in good health. On the test day, each male was presented with an anesthetized (Nembutal) adult female mouse for 3 min in his home cage (during which time behavioral observations were recorded; Wysocki et al., 1982) followed by a 27-mm exposure to the same female in
her home cage. Each female served as a stimulus for 3 males, 2 males of one treatment type and one male of the other. Pairings were counterbalanced. Blood samples were drawn after the 3Omnin exposure period. RIA’s for cotrisol, corticosterone, progesterone, sndrostenedione, estrone, and estradiol-1 7(3, as well as
for
testosterone
and
DHT,
were
conducted.
RESULTS Experiment
TESTOSTERONE
trone,
and
found
(Fig.
of shown
represent
by the recent
her
odors.
The
independent ment
testosterone and DHT Fig. la. These values levels in male mice,
in
endogenous
unstimulated or
the
data
groups
groups
did
t not
plasma levels of (t(17)=1.29, O.20
