TIMING IS EVERYTHING: HORMONAL CONTROL OF THE DEVELOPING SPINAL
NUCLEUS OF THE BULBOCAVERNOSUS (SNB) IN MICE
By
Aaron M. Welsch

A THESIS
Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of
Neuroscience-Master of Science
2017

PUBLIC ABSTRACT
TIMING IS EVERYTHING: HORMONAL CONTROL OF THE DEVELOPING SPINAL
NUCLEUS OF THE BULBOCAVERNOSUS (SNB) IN MICE
By
Aaron M. Welsch
Hormones are responsible for much of our development and normal day to day
functioning. From muscle growth, to aging, and our sleep wake cycle, hormone act
throughout our body to maintain homeostasis. Testosterone is a hormone found in
much higher concentrations in males than females, and thus acts predominately to
promote “male-typical” development. Two conditions that arise when this signaling
doesn’t function properly are androgen insensitivity syndrome (AIS) and congenital
adrenal hyperplasia (CAH). In the case of AIS, a person produces normal amounts of
testosterone, however, tissues, such as muscle or neurons, do not respond
appropriately. As a result, a person may be genetically male, but show physical traits of
a female. Conversely, CAH results in the body producing either too much or too little of
a desired hormone. This can lead to abnormal development and infertility. In both
cases, these developmental challenges can lead to difficulties throughout life. As a
result, it is important to study how testosterone acts to promote development in order to
understand how we can treat cases when something goes wrong. Here, I looked at the
importance of when testosterone acts to promote normal masculinization. I show that
cell number and cell size are influenced by testosterone at different times immediately
after birth, suggesting they are controlled in different ways. This is useful going forward,
as it gives us a better idea of when hormones are influencing development.

ABSTRACT
TIMING IS EVERYTHING: HORMONAL CONTROL OF THE DEVELOPING SPINAL
NUCLEUS OF THE BULBOCAVERNOSUS (SNB) IN MICE
By
Aaron M. Welsch
In rats there is a sensitive period for the masculinization of soma size in motoneurons of
the spinal nucleus of the bulbocavernosus (SNB), which extends beyond the sensitive
period for masculinization of SNB cell number. The current study aimed to assess such
sensitive periods in the SNB of mice. Female mice were exposed to testosterone
propionate (TP) or vehicle during one of three potential sensitive periods of
development; late embryonic (E 16-18), early postnatal (PN 1, 3, 5), or late postnatal
(PN 7, 9, 11). Testosterone (T) was then provided to the females in adulthood via
Silastic capsules to half of the animals in each group to serve as “activational”
background from which organizational effects of the perinatal hormones could be
assessed. Early neonatal TP treatment significantly increased the adult number of SNB
motoneurons in females, and also increased the size of both the somata and nuclei of
the motoneurons in adulthood. As expected, adult T treatment also significantly
increased the size of SNB somata and nuclei in all groups, but not the number of SNB
cells. There was no significant interaction of perinatal and adult hormone treatment on
cell number, soma size, or nuclear size. These results begin to define the sensitive
periods in which androgens masculinize the SNB system in mice, where genetic tools
are available to perturb mechanisms of androgen action on SNB development.

This thesis is dedicated to everyone who has supported me on my journey to this point.
Thank you to Anna for all your support and encouragement. Thank you to my parents,
all of my family, my friends, and everyone else who has helped get me to where I am
today.

iii

ACKNOWLEDGEMENTS

Thank you to Drs. S. Marc Breedlove and Cynthia Jordan for providing me the
opportunity to pursue my research interests in their lab. In addition, the support and
encouragement from everyone in the Breedlove/Jordan lab is much appreciated and I
value the time we spent together. Thank you to my committee members, Dr. Joe
Lonstein and Dr. Greg Swain for your time and feedback and my project developed.
Thank you to the MSU Neuroscience Program for allowing me to develop professionally
and discover my passion for teaching and outreach. Thank you to all of my fellow NSP
graduate students for providing a community that demanded hard work in the lab, and
encouraged enjoying time out of it. I’m grateful for the many friendships developed in
East Lansing that I will take with me going forward. Thank you to Anna for listening
when I needed to vent, supporting me when I wasn’t sure, encouraging me when I got
stuck, and distracting me when I needed a break. I cannot imagine taking on this
challenge without you. Thank you to everyone who sacrificed their time and/or
schedule to help accommodate my needs throughout school. Finally, thank you to
everyone who has helped make MSU such a fun and fulfilling place to live and attend
school. Go Green!

iv

TABLE OF CONTENTS

LIST OF TABLES………………………………………………...…………………………….vi
LIST OF FIGURES…..……………………………………………………………………...…vii
KEY TO ABBREVIATIONS……………..……………………………………………………viii
CHAPTER 1……………………………………………………………………………………..1
INTRODUCTION……………….…………………………………………………...…..1
MATERIALS AND METHODS…………..………………………..…………….……..6
RESULTS………………………………..……………………………………..……....10
DISCUSSION……………………………………..…………………………………...12
APPENDICES……………………………………………………………………………….....15
APPENDIX A: Tables………………………………………………………………….16
APPENDIX B: Figures……......……………………………………………………….19
REFERENCES…………………………………………………….………………………..…26

v

LIST OF TABLES

Table 1: Samples sizes for each of the measured variables. Y-axis labeled as Perinatal
Treatment + Adult Treatment. …………………………………….…………………….…..16
Table 2: Body weights. Average body weight (g) at sacrifice presented as (mean ±
SEM). ………………...……….………………………..……………………………………....17
Table 3: Anogenital distances. Average anogenital distances (mm) at sacrifice
presented as (mean ± SEM)...……………………………………………………………..…18

vi

LIST OF FIGURES

Figure 1: Plasma testosterone levels. Females treated with Testosterone in adulthood
had significantly higher levels of plasma testosterone than those given Blank capsules,
independent of perinatal treatment (p<0.0001). There was no significant effect of
perinatal treatment on this measure (p=0.366) and no interaction between the two
factors (p=0.157)……………………..……….………………………………………….....…19
Figure 2: Average number of SNB cells. There was no significant main effect of adult
hormone treatment on SNB cell number (p=0.820). Females given TP during the Early
Postnatal period had significantly more SNB cells than females in the Control group
(p=0.04). There was no interaction between the two factors (p=0.321)………………...20
Figure 3: Average cell sizes. Females treated with Testosterone in adulthood had
significantly larger SNB motoneuron cell bodies than those given Blank capsules,
independent of perinatal treatment (p<0.0001). In addition, females given TP during the
Early Postnatal period had significantly larger SNB cells than females in the Control
group (p=0.007). There was no interaction between the two factors (p=0.200)…….…21
Figure 4: Average nuclear sizes. Females treated with Testosterone in adulthood had a
significantly larger cell nuclei in SNB motoneurons than those given Blank capsules,
independent of perinatal treatment (p<0.001). The effect of perinatal treatment was just
under the level of significance (p=0.061). There was no interaction between the two
factors (p=0.596)………………..…………………………………………………………..…22
Figure 5: Thionine stained section and SNB cells. (a) Representative image of a
thionine-stained lumbar spinal cord section showing the location of the SNB (black
arrow), DLN (red arrow), and RDLN (white arrow), along the depth of the “U” the white
matter (yellow arrow) makes on the dorsal side of the central canal in the region of the
SNB. (b) Arrows indicate two SNB cells found just ventral to the central canal of the
same slice shown in (a)……………………………...........................................................23
Figure 6: The difference between a section with many SNB cells and a section with 1
SNB cell. (a) Shows a section with a cluster of SNB cells (circled) located in the white
matter just ventral of the central canal. (b) Shows a section with only 1 SNB cell. Inserts
show that representative sections from similar regions of the spinal cord, given the
similar depth of the white matter
“U”……….………………………………………………………………………………………24
Figure 7: Examples of traced SNB cell bodies and nuclei. Cell body and nuclei sizes
were measured by tracing the outline of the cell body (black trace) and nuclei (white
trace) of 20 cells/animal, distributed throughout the length of the spinal cord section
containing SNB cells………………………………………………………………………..…25

vii

KEY TO ABBREVIATIONS

AIS

Androgen Insensitivity Syndrome

CAH Congenital Adrenal Hyperplasia
SNB Spinal Nucleus of the Bulbocavernosus
TP

Testosterone Propionate

T

Testosterone

BC

Bulbocavernosus

LA

Levator Ani

AR

Androgen Receptor

Tfm

Testicular Feminization Mutant

viii

CHAPTER 1

INTRODUCTION

The spinal nucleus of the bulbocavernosus (SNB) is a group of sexually dimorphic
motoneurons in the lower lumbar spinal cord of both rats (S M Breedlove & Arnold,
1980) and mice (Zuloaga, Morris, Monks, Breedlove, & Jordan, 2007). These
motoneurons innervate the bulbocavernosus (BC) and levator ani (LA) striated perineal
muscles, which act to control penile reflexes important for male copulatory behaviors
(Sachs, 1982). Initially shown to accumulate radiolabeled testosterone or its
metabolites, SNB cells are more numerous in males than females, and this sex
difference can be manipulated by altering neonatal, but not adult, androgen treatment
(S. Marc Breedlove, Jacobson, Gorski, & Arnold, 1982; S M Breedlove & Arnold, 1981).
Further, in genetically male rats with an inactivating mutation of the androgen receptor,
the number of SNB cells is similar to that seen in females. This finding led to the
conclusion that “the sexually dimorphic nature of the SNB depends on neither the adult
hormone state nor the presence of a Y chromosome, but on the interaction of
androgens with their receptors early in development” (S M Breedlove & Arnold, 1981).

In order to affect the developing animal, hormones such as androgens must not only be
present, but they must be present when the animal is sensitive to any changes in
hormone levels. This organizational effect of hormones usually occurs during a short
critical period around the time of birth and acts to permanently alter the behavior or

1

neuroendocrine functioning of the animal (PHOENIX, GOY, GERALL, & YOUNG,
1959). Studies examining how hormones affect development can only be useful if we
first understand when during development the hormones are effective, thus guiding
experimental design. In the case of the SNB system, the hormone of interest is
testosterone (T), acting via the androgen receptor (AR). Masculinization of the rodent
brain is traditionally believed to be mediated by aromatized metabolites of T (T is
converted to 17-β estradiol via aromatase, which then acts on estrogen receptors
(Naftolin, Ryan, Davies, Petro, & Kuhn, 1975)). However, work with testicular
feminization mutant (Tfm) rodents and the AR antagonist, flutamide, show that T can
also act directly on ARs to promote masculinization of certain brain regions (Zuloaga,
Puts, Jordan, & Breedlove, 2008). Similarly, sexual differentiation of the SNB is
regulated by androgens acting via the AR (Swift-Gallant & Monks, 2017). Therefore,
the SNB system is ideal to study the timing of androgens’ effects as androgens, and
androgens alone, are required for system survival. Given this circumstance, studying
SNB system survival in females treated with testosterone at varying potential critical
periods can tell us when testosterone is required for maintenance of the motoneurons,
as the lack of T in females early in life normally leads to the cell death that is
responsible for establishing a sex difference in SNB morphology (Nordeen, Nordeen,
Sengelaub, & Arnold, 1985; Sengelaub & Forger, 2008).

In addition to their presence within the motoneurons in the central nervous system and
skeletal muscle in the periphery, androgen receptors have been found in other tissue
types, suggesting additional potential sites of action. Using immunocyctochemistry and

2

reverse transcriptase-PCR, AR protein and mRNA has been shown in the sciatic nerve
of adult rats (Jordan, Price, & Handa, 2002). More specifically, endoneurial and
perineurial fibroblasts showed AR staining, whereas epineurial fibroblasts showed no
signs of AR staining. Finally, at least within the sciatic nerve, Schwann cells do not
seem to be a potential site of action, as they showed no specific AR stain, further
suggesting they are not the source of AR mRNA (Magnaghi et al., 1999). Androgen
receptor has also been observed in the prostate of male rats (Prins, Birch, & Greene,
1991). Using immunohistochemistry, luminal epithelial cells demonstrated strong AR
positive staining, whereas basal epithelial cells were AR negative. Taken together,
these studies remind us to consider the potential effects of direct T action on tissue
types beyond SNB motoneurons and BC-LA skeletal muscle. This is an important
caveat to keep in mind as research moves toward knockout lines to assess the
importance T-AR interactions in different tissue types.

The historical understanding of testosterone’s mechanism of action involves T entering
the cell, being converted to dihydrotestosterone via 5α-reductase, and binding with AR
in the cytoplasm. Following phosphorylation and dissociation from heat-shock proteins,
the AR then translocates to the nucleus where it dimerizes, binds to DNA, and can alter
gene transcription and translation (Gottlieb, Lombroso, Beitel, & Trifiro, 2005). This was
sufficient when we believed T only acted on AR around the cell soma. As cell types in
the periphery have been shown to be AR positive, there may be an additional
mechanism of action for how T exerts its affects without translocation to the nucleus.
One way this may occur is by activating MAPK signaling cascades that modulate

3

intracellular calcium levels (Heinlein & Chang, 2002). In addition to occurring in tissuetypes lacking functional AR, these cascades lead to changes that occur too rapidly to
involve gene transcription, further indicating a nongenomic effect. Further, physiological
levels of T added to Sertoli cells can induce mitogen-activated protein kinase signaling
pathways that lead to spermatogenesis within Sertoli cells (Fix, Jordan, Cano, & Walker,
2004), again indicating T can act on the AR in non-genomic ways.

In rats, adult hormone manipulation has varying effects on SNB cell number and cell
size. Whereas adult hormone manipulation has no effect on the number of SNB cells,
the size of these same neurons is sensitive to changes in hormone levels in adulthood,
thus suggesting these two measures of SNB masculinity are regulated by different
mechanisms (S. Breedlove & Arnold, 1983). These reports show that the critical period
for the organizational masculinization of SNB cell size extends beyond the critical period
for the masculinization of SNB cell number, indicating that, indeed, critical periods of
development exist and also that these hormone-dependent changes do not all occur
within the same window.

While these critical periods are established in rats, most genetic models today are more
readily available in mice. Thus, it is worthwhile to investigate whether similar critical
periods of development exist in mice, and if so, when those periods are. For example,
Cre/loxP technology can be used to render the AR gene dysfunctional in a desired
tissue type and/or at a particular point in development. Studying the resulting changes
by disrupting the AR gene in different tissues could allow researchers to draw

4

conclusions about the importance of androgens and the AR in that type of tissue.
However, if the critical period of development occurs before the disruption of AR in
those tissues, such studies could be misleading.

5

MATERIALS AND METHODS

Adult wild-type C57BL6J male and female mice from Jackson Laboratories were
housed in a temperature-controlled vivarium with a 12/12 light/dark cycle with food and
water available ad libitum. All procedures were conducted in compliance with NIH
guidelines and approved by the Michigan State University IACUC.

For breeding, a male was placed into a cage housing two females for no longer than 24
hours. During the next 14 days, the females were gently handled daily.

Embryonic

day 0 (E0) was defined as the day the parental animals were paired. After two weeks,
pregnant dams were readily identified and randomly assigned to one of four treatment
groups. Pups were exposed to testosterone propionate (TP) during one of three
developmental periods: prenatal exposure (E16-18), early neonatal exposure (postnatal
(PN) days 1, 3, and 5), or late neonatal exposure (PN 7, 9, and 11), or control animals
given only sesame oil vehicle. Prenatal injection days (E16-18) were selected to
provide maximum time to develop before treatment, while minimizing stress on the dam
before birth. Early and late postnatal injection periods were selected based on known
critical periods in rat androgen sensitivity. Prenatal hormone treatment was delivered
by subcutaneous injections of the pregnant dam with 0.5 mg of TP in 50 ul sesame oil
vehicle, daily. Postnatally, 0.1 mg of TP in 25 ul vehicle was injected subcutaneously to
all pups in a litter on alternate days. These dosages were reduced from the 2mg TP
(prenatal) and 1mg TP (postnatal) given to rats in order to account for the size
difference between the species. 25 ul vehicle was determined to be the maximum

6

volume injectable into a newborn pup to avoid leakage. To control for the stress of
handling/injections, all animals received an oil injection each day, with TP present only
as defined by the treatment groups. Prenatal TP treatment hindered pup survival, so
those pups were cross-fostered on PN day 1, the first light period after birth. Any crossfostered female siblings were placed into different adult treatment groups in order to
minimize the potential confounding effect of cross-fostering. All pups were weaned on
PN day 21 and group-housed with same sex siblings (2-4/cage). On PN day 68, the
female mice were anesthetized via isoflurane inhalation and subcutaneously implanted
with a Silastic capsule (1.57mm inner diameter, 3.18mm outer diameter, effective
release length of 6 mm, total tube length of 16mm) containing nothing (blank) or free T,
to provide an “activational” background from which the organizational effects of perinatal
hormones could be detected. Following capsule implant, all mice were treated with 0.1
mL ketoprofen (1 mg/mL) analgesic and monitored daily for 10 days. The Silastic
capsule served to control the testosterone release rate, ensuring stable delivery
throughout the duration of treatment.

Thirty days after capsule implants, animals were sacrificed with sodium pentobarbital
and perfused intracardially with saline followed by buffered formalin. The spinal cord
from lumbar segment 4 through sacral segment 1 was removed and postfixed in
buffered formalin for at least 30 days. Spinal cords were trimmed and frozen-sectioned
in the transverse plane at 30 um thickness and alternate sections were mounted on gelsubbed microscope glass slides, then Nissl-stained with thionine for SNB motoneuron
number and size measurements. Animal weight (g) was recorded just prior to sacrifice.

7

Anogenital distance (AGD) was measured as the distance (mm) between the anus and
the penis/vaginal opening, where a longer AGD is considered more masculinized.
Blood was collected at sacrifice for the plasma testosterone (nmol/L) assay.

Cell counts were conducted prior to size measurements in order to allow for the
distribution of SNB cells to be visualized. Once counts were complete, 20 SNB cells
distributed throughout the rostrocaudal extent of SNB cells were selected for soma and
nuclear size analysis. In order to be selected, a cell had to exhibit a clear nuclear
profile. Cell soma and nuclei were traced using MetaVue software, and data were
exported to Microsoft Excel to calculate averages within an animal.

Sample sizes for each of the measured variables was as follows (Perinatal
Treatment+Adult Treatment- N). Body Weight; Control+Blank- 10, Control+T- 10,
Prenatal+Blank- 7, Prenatal+T 9, Early Postnatal+Blank- 10, Early Postnatal+T- 14,
Late Postnatal+Blank- 15, Late Postnatal+T- 8. Anogenital Distance; Control+Blank10, Control+T- 10, Prenatal+Blank- 6, Prenatal+T 9, Early Postnatal+Blank- 10, Early
Postnatal+T- 14, Late Postnatal+Blank- 15, Late Postnatal+T- 8. Plasma Testosterone;
Control+Blank- 8, Control+T- 7, Prenatal+Blank- 5, Prenatal+T 7, Early
Postnatal+Blank- 8, Early Postnatal+T- 8, Late Postnatal+Blank- 10, Late Postnatal+T5. SNB Cell Number; Control+Blank- 9, Control+T- 9, Prenatal+Blank- 7, Prenatal+T 8,
Early Postnatal+Blank- 9, Early Postnatal+T- 13, Late Postnatal+Blank- 13, Late
Postnatal+T- 7. Cellular Size; Control+Blank- 9, Control+T- 9, Prenatal+Blank- 7,
Prenatal+T 8, Early Postnatal+Blank- 9, Early Postnatal+T- 13, Late Postnatal+Blank-

8

14, Late Postnatal+T- 7. Nuclear Size; Control+Blank- 9, Control+T- 9, Prenatal+Blank7, Prenatal+T 8, Early Postnatal+Blank- 9, Early Postnatal+T- 13, Late Postnatal+Blank14, Late Postnatal+T- 7. (Table 1).

Statistical analysis consisted of 2-way Analysis of Variance (ANOVA) with factors of
perinatal treatment (prenatal, early postnatal, and late postnatal) and adult treatment
(testosterone and blank). Significant main effects of perinatal treatment were further
analyzed using Bonferroni’s post hoc tests. All graphs and/or tables present the mean ±
SEM based on N = the number of animals in the group. Significance was considered at
p<0.05.

9

RESULTS

Body Weight. Adult testosterone treatment significantly increased body weight
(F(1,82)=15.66, p<0.0001), (Blank, 22.21 g ± 0.24 (SEM), Testosterone, 23.54 ± 0.24).
In addition, early postnatal TP-treated females weighed significantly more at sacrifice
than all other perinatal treatment groups (F(3,82)=14.96, p<0.0001). There was no
interaction between the perinatal and adult treatments on body weight (F(3,82=1.25,
p=0.296). (Table 2).

Anogenital Distance. Adult testosterone treatment slightly lengthened anogenital
distance (F(1,81)=5.98, p=0.017), (Blank, 4.23 mm ± 0.12, Testosterone, 4.67 ± 0.12).
Prenatal TP treated females had a more masculine anogenital distance at sacrifice than
all other perinatal treatment groups (F(3,81)=15.15, p<0.0001). There was no
interaction between the two factors (F(3,81)=1.185, p=0.321). (Table 3).

Plasma Testosterone Levels. As expected, females given testosterone capsules in
adulthood had significantly higher plasma levels of the hormone than females given
blank capsules, independent of perinatal treatment (F(1,57)=68.33, p<0.0001), (Blank,
5.58 nmo/L ± 1.42, Testosterone, 22.63 ± 1.5). There was no effect of perinatal
treatment on plasma T concentrations (F(3,57)=0.50, p=0.683), nor any interaction
between the two factors (F(3,57=1.91, p=0.14). (Figure 1).

10

Konigsmark-adjusted SNB Cell Number. As previously reported, SNB cell number was
unaffected by adult hormone treatment (F(1,74)=0.052, p=0.820), (Blank, 42.61 ± 3.26,
Testosterone, 43.67 ± 3.31). However, TP treatment during the early postnatal period
masculinized SNB cell number, compared to control females (F(3,74)=4.975, p=0.004).
There was no interaction between the two factors (F(3,74)=1.455, p=0.235). (Figure 2).

Cellular Size. As previously reported, adult testosterone treatment increased SNB cell
size, independent of perinatal treatment (F(1,75)=26.446, p<0.0001), (Blank, 395.79 ±
13.04, Testosterone, 491.69 ± 13.33). In addition, TP treatment during the early
postnatal period also increased cell size, as compared to control females
(F(3,75)=4.342, p=0.007). There was no interaction between the two factors
(F(3,75)=1.588, p=0.200). (Figure 3).

Nuclear Size. Adult testosterone treatment also increased the size of the nuclei of SNB
cells, independent of perinatal treatment, compared to blank treated females
(F(1,75)=16.678, p<0.001), (Blank, 124.49 ± 4.69, Testosterone, 151.86 ± 4.79). There
was a marginally significant main effect of perinatal treatment (F(3,73)=2.576, p=0.061),
but no interaction between the two factors (F(3,75)=0.633, p=0.596). (Figure 4).

11

DISCUSSION

Here I present evidence that there are unique critical periods for different aspects of
masculinization of the SNB system in mice. The number of SNB cells can be
masculinized by early postnatal TP treatment, however, this critical period is over
around PN day 7, as late postnatal TP treatment of female mice yields no difference in
cell number, compared to control. In addition, SNB number is not affected by adult
testosterone treatment in mice, suggesting that this aspect of the SNB system is
determined within the first week of life. SNB cell size is also masculinized by TP during
the early postnatal period, however, this variable continues to remain sensitive to
testosterone into adulthood, as adult females treated with testosterone for a month
before sacrifice had larger SNB cells than animals given blank capsules. This sensitivity
to testosterone in adulthood suggests that the mechanism of action for cell size is
different than that for cell number. While the size of SNB motoneuronal nuclei can still
be enlarged by adult testosterone treatment, this variable was unaffected by perinatal
treatment, suggesting nuclear and cell size masculinization may be controlled in
different ways during the first weeks of development. It should be noted, however, that
the effect of perinatal treatment on nuclear size was just under the level of significance
(p=0.061), and therefore it is possible that larger sample sizes would have revealed an
effect.

Maternal behavior can have an effect on SNB system survival. In particular, changes in
pup licking behavior can lead to changes in SNB cell number, size, and dendritic length,

12

with decreases in licking leading to decreases in variable (Lenz & Sengelaub, 2006;
Moore, Dou, & Juraska, 1992). Further, cross-fostering has been shown to increase
pup licking (Van Der Veen, Abrous, Ronald De Kloet, Piazza, & Koehl, 2008). If
decreased pup licking leads to fewer and smaller SNB cells, it is reasonable to ask if
increased licking can lead to more and larger SNB cells. If so, one could argue that
pups prenatally treated with TP and then cross-fostered would show an increase in SNB
cell number and size due to cross-fostering alone. There may even be evidence of this
in our analysis, as prenatally TP-treated females that received blank capsules in
adulthood had increased SNB cell numbers, similar to those of females treated with TP
neonatally.

While cross-fostering only 2 out of our 8 groups was not ideal, it was

deemed necessary to achieve our desired group numbers. While it is possible this may
have affected our findings, it is likely our handling/injection schedule would have
masked any effect of cross-fostering simply due to the increased handling required to
inject pups. That said, in order to eliminate this variable in any future studies, it would
be advised to cross-foster all pups.

As previously shown in rats, the sensitive period for the masculinization of soma size in
motoneurons extends beyond the sensitive period for masculinization of SNB cell
number, suggesting these variables are controlled by separate mechanisms. The goal
of this project was to examine if similar critical periods of development exist in mice, and
if so, when those periods are. I have shown that androgen exposure masculinizes cell
number and soma size when given during the same early postnatal window, however,
only cell and nuclear sizes are sensitive to the activation dose of androgens given in

13

adulthood, thus suggesting different mechanisms regulate the development of these
aspects of the SNB system, as seen in rats. In addition, I provide new evidence that
soma size and nuclear size may be masculinized by different mechanisms, as perinatal
androgen treatment was able to masculinize soma, but not nuclear, size.

In addition to furthering our understanding of the mechanisms involved in
masculinization of the nervous system, this study also provides useful information on
when the critical period windows are open. I found that masculinization of the SNB
system extends after birth, and is complete by PN day 7. These data are important to
remember when working with genetic tools that alter the function of the androgen
receptor. For example, these results indicate that Cre/loxP-induced AR dysfunction
must occur before birth if it is to effectively prevent androgens from masculinizing the
SNB system.

14

APPENDICES

15

APPENDIX A:

Tables

Control + B
Control + T
Prenatal + B
Prenatal + T
Early Postnatal +
B
Early Postnatal +
T
Late Postnatal +
B
Late Postnatal +
T

Body
Weight
10
10
7
9
10

Anogenital
Distance
10
10
6
9
10

Plasma
T
8
7
5
7
8

Cell
Number
9
9
7
8
9

Cell
Size
9
9
7
8
9

Nuclear
Size
9
9
7
8
9

14

14

8

13

13

13

15

15

10

13

14

14

8

8

5

7

7

7

Table 1: Samples sizes for each of the measured variables. Y-axis labeled as Perinatal
Treatment + Adult Treatment.

16

Control

Blank
21.68 ± 0.47

Testosterone
22.25 ± 0.47

Prenatal
Early Postnatal
Late Postnatal

20.79 ± 0.56
23.59 ± 0.47
22.77 ± 0.38

22.84 ± 0.5
25.49 ± 0.4
23.56 ± 0.53

Table 2: Body weights. Average body weight (g) at sacrifice presented as (mean ±
SEM).

17

Control

Blank
3.97 ± 0.23

Testosterone
4.02 ± 0.23

Prenatal
Early Postnatal
Late Postnatal

5.23 ± 0.3
3.87 ± 0.23
3.97 ± 0.19

5.82 ± 0.24
4.67 ± 0.2
4.17 ± 0.26

Table 3: Anogenital distances. Average anogenital distances (mm) at sacrifice
presented as (mean ± SEM).

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APPENDIX B:

Figures

Figure 1: Plasma testosterone levels. Females treated with Testosterone in adulthood
had significantly higher levels of plasma testosterone than those given Blank capsules,
independent of perinatal treatment (p<0.0001). There was no significant effect of
perinatal treatment on this measure (p=0.366) and no interaction between the two
factors (p=0.157).

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Figure 2: Average number of SNB cells. There was no significant main effect of adult
hormone treatment on SNB cell number (p=0.820). Females given TP during the Early
Postnatal period had significantly more SNB cells than females in the Control group
(p=0.04). There was no interaction between the two factors (p=0.321).

20

Figure 3: Average cell sizes. Females treated with Testosterone in adulthood had
significantly larger SNB motoneuron cell bodies than those given Blank capsules,
independent of perinatal treatment (p<0.0001). In addition, females given TP during the
Early Postnatal period had significantly larger SNB cells than females in the Control
group (p=0.007). There was no interaction between the two factors (p=0.200).

21

Figure 4: Average nuclear sizes. Females treated with Testosterone in adulthood had a
significantly larger cell nuclei in SNB motoneurons than those given Blank capsules,
independent of perinatal treatment (p<0.001). The effect of perinatal treatment was just
under the level of significance (p=0.061). There was no interaction between the two
factors (p=0.596).

22

Figure 5: Thionine stained section and SNB cells. (a) Representative image of a
thionine-stained lumbar spinal cord section showing the location of the SNB (black
arrow), DLN (red arrow), and RDLN (white arrow), along the depth of the “U” the white
matter (yellow arrow) makes on the dorsal side of the central canal in the region of the
SNB. (b) Arrows indicate two SNB cells found just ventral to the central canal of the
same slice shown in (a).

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Figure 6: The difference between a section with many SNB cells and a section with 1
SNB cell. (a) Shows a section with a cluster of SNB cells (circled) located in the white
matter just ventral of the central canal. (b) Shows a section with only 1 SNB cell. Inserts
show that representative sections from similar regions of the spinal cord, given the
similar depth of the white matter “U”.

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Figure 7: Examples of traced SNB cell bodies and nuclei. Cell body and nuclei sizes
were measured by tracing the outline of the cell body (black trace) and nuclei (white
trace) of 20 cells/animal, distributed throughout the length of the spinal cord section
containing SNB cells.

25

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